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STUDIES ON THE BIOLOGY OF THE NEUROACTIVE AMINES 


A MORPHOLOGIC AND PHARMACOLOGIC APPROACH 


Herbert Y. Meltzer 


A thesis presented to the faculty of the 
Yale University School of Medicine 
in partial fulfillment of the 
requirements for the degree of 
Doctor of Medicine 
1963 


JUN1963 ) 

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The author gratefully acknowledges 
the indulgence and support of Drs. 
R.J. Barmett, N.J. Giarman, D.X. 
Freedman, and J.P. Green who per¬ 
mitted this young investigator to 
explore his own ideas in their 
excellent laboratories. This the¬ 
sis is dedicated to the memory of 
my late father. Hr. David Meltzer. 




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CONTENTS 




PREFACE 1 

INTRODUCTION TO MAST CELL STUDIES 3 

Outline of Experimental Investigations H 

The Mast Cell 7 

Morphology of the Mast Cell 8 

The Pharmacology of the Mast Cell 13 

Mast Cell Heparin and Its Particu- 13 

late Nature 

Mast Cell His tamine and Its Particu- llj. 

late Nature 

Mast Cell 5-hydroxytryptamine and 15 

its Particulate Nature 
The Release of Mast Cell Amines 15 

Other Constituents of Mast Cells 21 


Synthesis aid Storage of Amines 21 

Function of the Mast Cells 22 

Reserpine 23 

Chlorpromazine 26 

The Dunn-Potter Mouse Mastocytoma 2? 


METHODS 29 

Growth of Ascites Cells 29 

Growth of Tissue Culture Cells 29 

Reserpine Treatment 29 

Chlorpromazine Treatment 30 

Light and Phase Microscopy 30 

Electron Microscopy 30 

Isolation of Subcellular Components 

from the Ascites Cells 31 

Isolation of Subcellular Components 

from the Tissue Culture Cells 33 

Determinati cn of 5-HT 34 

Determination of Histamine 34 

Determination of Heparin 35 

Determination of Succinoxidase 35 



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Attempts to Demonstrate Monoamine Oxidase 35 

Activity in the Dunn-Potter Mastocytoma 

Technique of Cotzias and Greenough 35 

Technique of Weissbach 36 

Attempt to Demonstrate Histaminase Activity 

in the Dunn-Potter Mastocytoma 36 

RESULTS AND DISCUSSION 

The Ascites Dunn-Potter X-l-C Mastocytoma 37 

Cell 

The Origin of the Mast Cell Granules 38 

The Tissue Culture Dunn-Potter X-l-C 

Mastocytoma Cell l\2 

Effects of Reserpine on the Tissue 

Culture Mastocytoma Cells lp 8 

Effects of Chlorpromazine on the Tissue 

Culture Mastocytoma Cells 58 

The Large Granule Fraction of the Tissue 

Culture Cells 60 

Isolation of Mast Cell Granules from the 

Ascites Cells 61 

Monoamine Oxidase and Histaminase 65 

SUMMARY 67 

ELECTRON MICROGRAPHS 70 

INTRODUCTION TO MONOAMINE OXIDASE STUDIES 100 

MATERIAL AND METHODS 110 

EXPERIMENTAL RESULTS 112 

Effect of Pyridine Aldoxime Dodecyliodide 112 

(PAD) on the MAO Activity of Homogenates 
of Liver and Brain of Various Species 

Effect of PAD on MAO Activity of Washed 113 

Rat Liver Mitochondria and Comparison 
of PAD with other Inhibitors of MAO 

Effect of Preincubation on the Inhibition lilj. 

of Rat Liver Mitochondrial MAO by PAD 





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Effect of Dialysis on t he Inhibition of 115 

Rat Liver Mitochondrial MAO by PAD 

Effect of PAD on Solubilized MAO 116 

Ability of PAD to Inhibit MAO in vivo 117 

Effect of PAD on the Levels of 5-HT in 118 

Rat Brain 

Effect of PAD on the Catecholamine Levels 119 
of Rat Brain 

Effect of PAD on 5-hy droxytryptophan de- 119 
carboxylase of rat kidney in vitro 

Effect of PAD on 5-HT of Mast Cells 119 

Effect of PAD and JB-516 on levels of 5-HT 121 
in Rats After Administration of Reserpine 

Effect on Rats of Pretreatmerib with PAD 122 

followed by Reserpine and Pretreatment 
with Reserpine Followed by PAD. 

Effects of Various Quaternary Ammonium 123 

Derivatives on MAO in vitro 

In vivo and In vitro Studies with Thiamine 123 
and MAC 

Effect of Dodecyl Iproniazid on MAO in 124 

vitro 

Ability of Dodecyl Iproniazid Iodide to 125 

Inhibit MAO in vitro 

Effect of Dodecyl iproniazid on the Levels 125 
of 5-HT in dat Brain 

DISCUSSION 126 

PAD as an Inhibitor of Monoamine Oxidase 126 

PAD and Amine Release 131 

Dodecyl Iproniazid Iodide 134 

The Effect of Thiamine on Monoamine 135 

Oxidase 

SUMMARY 139 

BIBLIOGRAPHY 142 










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PREFACE 

The biology of the neuro-active amines has occupied 
a large portion of the imagination and energy of pharma¬ 
cologists and physiologists investigating the vertebrate 
nervous system in the last decade* The basis for this 
concentration of interest are studies which implicate these 
amines in neurotransmission and, beyond that, through many 
derangements in their disposition from synthesis to destruct¬ 
ion, in mental and nervous disorders* The investigations 
described and discussed in this thesis explore certain aspects 
of this class of compounds and the tissues which possess them. 

In brief outline, with several simplifications, the life 
cycle of a neuro-active amine 5-hydroxytryptamine 

(5-HT), histamine, norepinephrine (RE)_7 is! synthesis by 
decarboxylation of its precursor amino acid, storage in 
membrane-bound granules, release in response to various 
stimuli, action at the receptor site, and then either degreda- 
tion by one or more enzymes or reabsorption by a neurosecretory 
cell for re-use* 

The investigations in this work were concerned with: 

(i) the fine structure of the Dunn-Potter murine mast cell 
tumor grown in tissue culture and as ascites cells in the 
mouse peritoneal cavity; the origin of the amine granules 
of these cells and how they get the amines they contain; the 
mechanism of amine release by reserpine; the morphologic 
correlates of chlorpromazine action; the isolation of the 
amine-containing granules from the ascites mastocytoma cells; 
the absence of monoamine oxidase (MAO), an enzyme that has 



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been implicated in the degredaticn of several biogenic 
amines, and histaminase, the enzyme which oxidizes histamine, 
in these cells; {ii} the synthesis and study of a new class 
of inhibitors of MAO; and (iii) the relationship between the 
vitamin, thiamine, and MAO, The studies on the mastocytoma 
cells and the MAO studies will be presented separately for 
the sake of convenience and clarity. 


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INTR0DI3CTI ON 

The discoveries that histamine is found in the mast 

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cells of a myriad of species, that 5-hydroxytryptamine is 

2 3 

found in the mast cells of the rat and mouse, * and that 
these amines are largely bound in granules^ made this cell 
of great interest to the student of amine biology. It 
meant that the mast cell, along with the cells of the 
adrenal medulla and the blood platelets could serve as a 
model system for the amine biology of the vertebrate nervous 
system whose complexity makes a direct experimental approach 
extremely difficult. Thus, infonnation about amine synthesis, 
destruction, binding, release, regulation, and the influence 
of hormones and drugs is much more easily obtained from tbs 
mast cell than from the vertebrate nervous system. And with 
tbs economy of mechanism that nature practices, it seems 
reasonable to hope that the information obtained from the 
mast cells would be of value in understanding many of the 
features of amines in the nervous system. This has indeed 
proven true. Investigators throughout the world have ex¬ 
plored the mast cells with particular attention to their 
amines. It need not be stressed that the mast cell has great 
intrinsic interest in its own right. 



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Outline of Experimental Investigations 


The Yale Medical School Department of Pharmacology has 

been very active in the study of mast cells. The Dunn-Potter 

£ 

mouse mastocytoma which has continued to produce histamine, 

5-HT and heparin has been and is being studied by many people 
in that department. The author felt that these biochemical 
and pharmacologic studies could be greatly aided if the fine 
structure of these cells as seen at the level of resolution 
of the electron microscope could be established, because some 
of the studies undertaken have obtained a level of sophistication 
where function could not be divorced from structure. Indeed, 
even the simpler experiments could be more readily interpreted 
if the cells 1 morphology was known, for truly "one (electron 
microscopic) picture is worth a thousand words," 

The Dunn-Potter mastocytoma can be grown as a solid tumor, 
as ascites cells in the mouse peritoneal cavity, or in tissue 
culture as a cell suspension. My first endeavour was to es¬ 
tablish the fine structure of the ascites cell. It was then 
decided to use the tissue culture cells for studies using 
pharmacologic techniques and electron microscopy. The tissue 
culture cells are a pure population of mastocytoma cells 
whereas the ascites cells obtained from the mouse peritoneal 
cavity are really a mixture of mastocytoma cells, fibroblasts 
and leuokcytes. The tissue culture cells are more readily 
treated with drugs than the ascites cells and there is no 
interference from the host animal. The tissue culture cells 
were found to have a morphology significantly different from 



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the ascites cells and indeed, from any other mast cell 
previously described, but I decided to continue my studies 
with this cell form, fully realizing that it is a very 
atypical cell. The tissue culture cells have been investigated 
by Furano who attempted to separate the amine-containing 
granules from the mitochondria of these cells by density 
gradient centrifugation. I sought to check his separation 
by electron microscopy but was unable to reproduce this work. 
Instead, an unseparated large granule fraction of these cells 
was observed in the electron microscope. 

Several studies were undertaken with drugs which release 
mast cell 5-HT because it was felt that if the process could 
be visualized in the electron microscope valuable information 
about the mechanism of amine release would be forthcoming. It 
was hoped that such basic questions as whether amine release 
requires disruption of the cell and release of granules or 
whether it can occur without loss of cytoplasmic granules or 
other visible cytologic change could be answered. I hoped to 
obtain information about the mode of action of the drugs used, 
reserpine and chlorpromazine. 

Of special interest were pictures of ascites mastocytoma 
cells which show what are thought to be early forms of the 
mast cell granules. These suggested a new theory for the 
origin of these granules. 

Several studies of these mast cells performed by the 
author prior to the electron microscope studies will be 
reported briefly because they are of interest in interpreting 
some of the electron microscopic findings; (i) separation 
by density gradient centrifugation of subcellular components. 


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particularly the amine -containing granules and the mito¬ 
chondria of the ascites mastocytoma cells; (ii) the 
demonstration of the absence or extremely low activity in 
the Dunn-Potter mastocytoma of MAO and histaminase* 





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7 


The Mast Cell 

The discovery of the mast cell Is attributed to 
Paul Ehrlich who, in 1877» while investigating the staining 
of various tissues with aniline dyes, noted in the connective 
tissues of various species, granule-containing cells which 
had the property of staining more violet or redder than the 

*7 

rest of the cells (i#e# metachromasia)* He named these 
cells "Mastzellen n or well-fed cells because their cytoplasm 
was so densely crammed with granules# Ehrlich described the 
light microscope morphology, staining properties and dis¬ 
tribution of the mast cells, but nothing of note elucidating 
their physiology or function was accomplished for seventy 
years. In 1937> Jorpes in Sweden noted that heparin, whose anti¬ 
coagulant properties attracted great interest, stained meta- 

o 

chromatically with toluidine blue*' 1 Jorpes was then able to 
show that the most cell content of various tissues was pro¬ 
portional to its concentration of heparin#^' Another big 
advance was the finding by Rocha e Silva that after anaphylactic 

Q 

shock both heparin and histamine were released from dog liver, 7 

This led to a number of studies which firmly established the 

presence of histamine in mast cells,^ Many other constituents 

of mast cells have since been found, including 5-hydroxytryptamine 

2 3 

but the latter only in the mast cells of the mouse and rat# 9 



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Morphology of the Hast Cells 

The morphology of the mast cell as seen with the light 

or phase microscope has been reviewed by Asboe-Nansen.^ 0 

Mast cells may vary within wide limits in size and shape 

depending on many environmental as well as intrinsic factors* 

They may be flat, spherical, spindle-shaped, stellate or 

filiform. The rounded mast cells have a diameter of 8-15 

microns, while the elongated ones may be twice as long. The 

nucleus is usually centrally located, about Ij.-6 microns in 

size and round or oval. Cells with two nuclei are occasionally 

seen but segmented nuclei have never been found. 

According to Asboe-Hansen, mast cell granules are almost 

always globular-shaped, rarely oval. Elliptic or rod-shaped 

granules have been seen and attributed to pressure exerted by 

adjacent granules. Early investigators found the granules to 

be 0.2-0.1| microns in size, although one investigator, using 

freeze-dried mast cells from the rat mesentery found granules 

ranging in diameter from 0.3-1.0 microns. 

Little else could be determined about the morphology of 

the mast cell, with the light microscope. The first electron 

micrographs of mast cells and normal mast cell granules were 

11 ** 12 

reported by Asboe-Eansen and Koksal. The techniques of 
electron microscopy were not very advene ed at this time and 

little new information was obtained from these pictures. 

•* / 

Koksal, however, did demonstrate a definite boundary to the 

12 


mast cell granules. 



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9 


The first electron micrographs of mast cells utilizing 

13 

Palade»s osmium tetroxide fixative and thin-sectioning 
were obtained by Bloom _et al 0 in Sweden.*^ The tissue 
studies was a dog mastocytoma which contained large amounts 
of heparin and histamine* The cells had the typical granular 
appearance of non-neoplastic mast cells* They were about 
10 microns in size, round or oval in shape* The nucleus 
occupied from one-fourth to one-third of the cell volume and 
varied in shape from round to kidney configuration. The 
nucleus was surrounded by the usual nuclear membrane and had 
one or two nucleoli* The cytoplasm was filled with granules 
about 0.7 microns in size. Mitochondria with the usual fine 
structure were noted in mast cells for the first time. Some 
cells had large vacuoles in the cytoplasm. The cell surface 
had a great number of small filamentous cytoplasmic protrusions 
about 0.6 microns long and 0.08 microns wide. 

Excellent electron micrographs of noimal mouse mast cells 

15 

were published by G.E. Rogers in 1956. Phase microscopy and 
various histochemical techniques were used to demonstrate that 
certain granule-containing cells seen in the dorsal skin of 
three day-old mice were mast cells. The mast cell granules 
were quite electron dense and irregular in shape. Their size 
varied from 0.3 to 1.0 microns but were usually 0.5 microns 
wide. The number of granules per cell varied considerably but 
unlike most other mast cells that have been studied, did not 
occupy a large area of the cytoplasm of the cells. Each granule 
is usually located in a clear area in the cytoplasm which has 
no definite limiting membrane. The interior of the granule has 


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the appearance of a network of filamentous elements# The 
cytoplasm contained an extensive well-organized agranular 
reticulum but a sparse endoplasmic reticulum# Mitochondria, 
both rounded and rod-shaped, with internal cristae were seen. 

Other good electron micrographs of normal mast cells 

t „ 16 

were published by Smith and Lewis in 1957* They found 
that the mast cells from the spleen, spleen and liver 
capsules, mesentery, skin and peritoneal fluid of both the 
hamster and rat all had the same appearance# These cells were 
organized somewhat differently than the cells just described 
in that the granules were packed more closely together and the 
mitochondria appeared chiefly in groups in granule-free areas 
adjacent to the nucleus. However, elongated mitochondria 
were occasionally seen sandwiched in between the granules# An 
endoplasmic reticulum was also apparent in mast cell cytoplasm 
for the first time. The granules were round, oval, or irregu¬ 
larly shaped, 0.5-1*0 microns in size# High power views of 
them showed a reticular structure# They varied in osmophilia 

and thus some were much darker than others. 

17 

Hagen, Barmett and Lee published excellent electron 

l8 

micrographs of the Furth mouse mastocytoma in 1958. The 
structure of these cells was again much different from any 
of those already described. The Furth mastocytoma possessed 
nuclei which tended to be irregularly shaped. The nucleus 
was bounded by double membrane which had frequent gaps or 
pores and was continuous with membranes of the agranular 
reticulum# The nuclei usually possessed one or more nucleoli. 


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The plasma membrane is very irregular. Small vesicles and 
cytoplasmic organelles are frequently seen adjacent to it. 

In many places a discrete membrane was not visible and cells 
were indistinctly demarcated from one another. "The cytoplasm 
is very complex. It contains a light homogenous matrix in 
which are embedded many cytoplasmic organelles, some of which 
are bizarre in form. Many small irregular mitochondria 
with numerous cristae were scattered throughout the cytoplasm. 
There was a very extensive, complex agranular reticulum, 
varying in diameter from 200A° to 1,000A^, often with dense 
homogeneous material in the vesicles. The endoplasmic 
reticulum was seen much less often. The cells contained 
double-membraned vesicles as well as smaller vesicles and 
dense granules within one large vesicle. Large mastocytoma 
granules were frequently seen, usually scattered irregularly 
in the cytoplasm, but sometimes concentrated in one region 
of the cell. The granules, which varied in size and density, 
were occasionally seen in vacuole-like spaces. 

Hibbs et al. recently published electron micrographs of 

19 

normal human mast cells. These cells do not look very 
complex and are akin to those in noxmal hamster and rat 
cells’^ and the dog mastocytoma.^ The cells are round or 
oval in shape and contain numerous widely spaced granules 
which are about 0*75 microns in diameter. High power micro¬ 
graphs reveal that "each granule consists of lamellar groups, 
some in the foira of scrolls, in close association with parti¬ 
culate material." The cells had single regular nuclei and an 


active cell membrane. 


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It is important to point out that all the mast cells 
just described, the only ones so far studied with the electron 
microscope, all were fixed in osmium tetroxide, dehydrated in 
alcohol and embedded in methacrylate, and all showed granules * 

The author studied the fine structure of the Dunn-Potter 
mouse mastocytoma^* It is of interest to review the light 
microscope description of the tumor at its inception* It was 
noted about the solid tumor that: "No masses of intensely 
granulated cells closely resembling normal mast cells have 
ever been found in the transplanted tumors* The ascites 
tumor cells, stained with Wright *s stain, were frequently 
found to have a ring or donut-shaped nucleus. The granules 
in a given cell were of the same size, but there was a great 
variation in the size of granules in different cells* The 
granules were not evenly distributed throughout the cell but 
tended to be clumped in one portion of it* The cytoplasm of 
less differentiated cells, with large round nuclei occuping 
most of the volume of the cell, usually contained small 
delicate granules. It is thus clear, that the Dunn-Potter 
mastocytoma had few granules even at its inception. 

Distribution of Tissue Mast Cells 

Mast cells are found in greatest abundance in the sub¬ 
cutaneous connective tissue, lung, mesentery, scrotum, uterus 
and thymus of mammals* The parenchyma of most organs have 
few mast cells but the fibrous tissue of their capsules and 
the connective tissue around small blood vessels Is often 





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13 


rich in them. The central nervous system has almost no mast 
cells but the connective tissue sheaths of peripheral nerves 
have many. Hast cells have been found in the tissues of almost 
all classes of animals in which they have been looked for. 

The Pharmacology of the ^ast Cell 

Selected aspects of the pharmacology of mast cells, 
normal and neoplastic, will be reviewed. Particular attention 
will be paid to studies dealing with heparin, histamine and 
5-HT, the sequestering of these substances within granules, 
and the effects of amine-releasing agents on these granules 
and the mast cells. 

Mast Cell Heparin and Its Particulate Nature 

Erik Jorpes, a Swedish biochemist, first proposed that 

20 

the mast cells produced or contained heparin. This 

hypothesis was based on the observation that there is a good 

proportionality between the number of mast cells and the 

amount of heparin in many tissues. Oliver et al. found that 

a dog mastocytoma contained fifty times as much heparin as 

21 

normal dog liver. Jorpes* observation that heparin, like 

the mast cell granules, is metachromatic, was the first 

evidence that heparin was contained within the mast cell 
22 

granule. Koksal later isolated mast cell granules from 

mouse connective tissues and showed them to contain a 

23 

heparin-like anti-coagulant. The synthesis of heparin by 

2Il 

mast cells has recently been directly demonstrated. Hagen, 




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Barmett, and Lee demonstrated the presence of heparin the 

specific granules which were clearly distinct from mitochondria 

17 

in the Furth mastocytoma. 

Mast Cell Histamine and Its Particulate Nature 

Q 

The finding by Rocha e Silva et al. that both histamine 

and heparin were liberated from the isolated dog liver during 

anaphylactic shock 3e d J.F. Riley and others to Investigate 

whether histamine too was produced and stored in the mast cell,'** 

Utilizing the chemical histamine liberators discovered by 

oti 

Macintosh and Paton, Riley demonstrated that stilbamidine 

and d-tubocurarine caused disruption of mast cells. However, 

these drugs quickly killed the animals and It was possible that 

the observations were cftte to agonal changes. Fawcett, using 

mast cells in rat mesenteiy, demonstrated that if the mesentery 

was depleted of mast cells by prior treatment with distilled 

water, intraperitoneal injections of compound lp 8/80 produced 

no histamine release, indicating that the mast cells were the 

source of histamine, f Quantitative studies suggested that 

the amount of histamine in the mast cells must have been 

27 

extraordinarily high. 1 

Simultaneously, evidence was obtained through centrifugation 
studies that histamine is particulate bound rather than free 
in the cytoplasm .* The large granule fraction rich in 
histamine was found to contain many mast cell granules, 
identifiable by their metachromatic staining, and to contain 
few mitochondria indicating that the granules which contained 





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15 


histamine were not mitochondria. Hagen et_ al. demonstrated 
that in the Furth mastocytoma, histamine was bound in granules 
which were morphologically and chemically distinct from mito¬ 
chondria.^ 

Mast Cell 5-HT and Its Particulate Nature 

The presence of 5-HT in rat mast cells was discovered 

by Benditt et al^. in 1955« The only other species whose 

mast cells have definitely been found to possess 5-HT is 

the mouse.^ The presence cf 5-HT in the Dunn-Potter mouse 

mastocytoma and the skin lesions of two people with urticaria 

pigmentosa (which is characterized by an excess of mast cells 1 ) 

30 

was noted by Sjoerdsma et~ al. 5-HT was not found in the mast 

cells of the human spleen. Very recently, Enerback claimed 

he demonstrated 5-HT in mast cell granules in human carcinoid 

tumors.-^ Presumably, the mast cells took up excess 5-HT 

produced by enterochromaffin cells of the carcinoid tumor. 

The presence of 5-HT in the granules of the Furth mastocytoma 

17 

wasd «ionstrated by Hagen et al. ' 

The Release of Mast Cell Amines 

It is not our aim to review the enormous amount of 
literature on the release of mast cell amines. Several aspects 
of this problem, hot/ever, are pertinent to our work: the re¬ 
lease of amines as compared to the release of granules; the 
effect of release on cell morphology; the relationship between 
histamine release and 5-HT release; and the mechanism of the 
release process. 




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r- • a :.or \ e.. . .Cox . : : : • • •'• • 


• 81 



16 


Mot a et al«^ demonstrated that Compound I 1 . 8 / 8 O, a con¬ 
densation produce of p-methoxyphenylethylmethylamine and 
formaldehyde which had been shown by Paten to liberate 

O ) 

histamine, ^ produced a generalized disruption and release 
of granules from mast cells of rat mesentery. Parekh and 
Glick found that lj. 8/80 did not cause any release of amines 
from intact mast cells short of absolute destruction of the 
cells. 3$ 

Similar findings were obtained by Bloom et aj. who 
followed the morphological effects of more potent histamine 
liberators on the dog mastocytoma previously described. Com¬ 
pound 1 ^ 8/80 and stilbaraidine produced "vacuolation and 
dissolution of mast cell granules as well as disintegration 
of the cell* w The drug-induced vacuoles increased rapidly 
in size and number while the mast cell granules decreased 
in number. Some vacuoles contained granules and those near 
the periphery of the cell often ruptured, discharging their 
contents. The vacuoles were surrounded by a distinct wall 
- which consisted of a double membrane.-' 

However, Fawcett showed that the extent of degranulation 
and histamine release in the rat mesentery was proportional 
to the amount of ij.8/80 present. He demonstrated that after 
injecting a Tyrode»s solution containing Ij.8/80 int rap eri tone ally 
into rats the mast cell granules were still present in the 
mesentery, but in the extracellular matrix surrounding each 
mast cell; histamine was now present in the Tyrod$*a solution 
in the peritoneum indicating that J 4 . 8 / 8 O had produced both 


61 


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17 


degranulation and release of histamine from the cells.^ 

It was not an all-or-none phenomenon In which the plasma 
membrane ruptured and all of the granules were released as 
part of a general destruction of the cell. The cells seemed 
to survive partial loss of their granules; they then underwent 
cell division and gradually regenerated their normal content 
of granules. 

There is still a third correlation between histamine 
release and cell morphology. G.P. West has observed that if 
histamine liberators of low potency are used "histamine re¬ 
lease can occur in a tissue without damage to its mast cell 
population." This is based upon light microscope studies.-^ 
Reserpine is one of the histamine liberators which West found 
released 5-HT maximally from rat skin but had little anatomic 
effect on mast cells as observed by light microscopy. 

D.E. Smith reported that substantial release of histamine from 
mast cells can occur without degranulation in the presence of 
suitable concent rat ions of protamine sulfate and toluidine 
blue and considered this process closer to the physiologic 
release process than the massive degranulation produced by 
lj. 8/80 or stilbamidine 

It would seem that the effect of the releasing agents 
on cell morphology is a highly variable phenomena depending 
upon which agent and pa?haps which type of mast cells are 
studied. It also appears that the amines may be released 
without releasing the granules and evidence will be cited 
shortly that the reverse is true a3 well. 


VI 


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18 


The release of 5-HT and histamine is often not 
parallel when mast cells which contain both these amines 
are exposed to releasing agents, Paratt and West in¬ 
vestigated the effects of a series of releasing agents 
on the histamine and 5-HT content of the skin of rats: 

Polymyxin B produced extensive degranulation but little 
loss of 5-HT; morphine and Compound 48/80 produced 
equivalent depletion of both histamine and 5-HT along 
with widespread degranulation and disruption of mast 
cells; reserpine released more 5-HT than histamine and 
produced modest mast cell degranulation only after very 
large doses were administered,^ Moran et al« confirmed f 
that 4^/80 releases equal percentages of 5-HT and histamine 
in rat mast cells,^ 1 A good deal of evidence was put forth 
by these authors in support of the idea that the release 
of 5-HT and histamine from the rat mast cells occurs by 
the same mechanism: identical time course of release of 
the two amines by 48/80, lack of selective release of either 
amine alone by any of the releasing agents tested (in contrast 
to Paratt and West*s findings), comparable inhibition of release 
by chemical and thermal means and similar pH dependence,^' 1 
Reserpine in concentrations of 150 micrograms/ml did not 
release either histanine or 5-HT,^' 1 Reserpine was found to 
be more active in releasing 5-HT than histamine from the 
brain, lungs and intestines of guinea pigs and rabbits,^ 2 

Reserpine significantly decreased both 5-HT and histamine 

31 

in the solid form of the Dunn-Potter mastocytoma. It 


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19 


releases 5-HT but not histamine from the same tumor grown 

I A 

as ascites cells* But it releases both amines at ex¬ 
tremely low concentrations from the same tumor cells grown 
in tissue culture *^ ! 

The question of the chemical basis of the action of 

the histamine liberators has been investigated extensively. 

One of the first suggestions and still widely held is that 

the histamine releasers displace histamine from its binding 

site, presumably heparin. This is based on the strong 

affinity of most histamine liberators for heparin.^ Many 

studies showing that inhibitors of various enzymes block 

the release process have prompted the theory that the 

histamine liberators act by stimulating the action of an 

enzyme which brings about the release. ^ This has been 

persuasively disputed by van Arsdel and Brey who found no 

clear cut relationship between the enzyme inhibitors and 

blockade cf [}B/8 0»s act ion. ^ Enzymatic processes have 

also been implicated in anaphylactic histamine release from 

tissues since several substances which inhibit enzyme action 

91 

also inhibit histamine release and mast cell degranulation, 
both of which have been shown to occur during antigen-antibody 
reactions 

One of the important questions concerning the mechanism 
of release is whether the releasing agents act predominantly 
on the granules or on some other cell constituent. Certain 
evidence suggests it is a direct action on the granule: 

48/80 and octylamine release histamine more rapidly from 


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20 


isolated granules from guinea pig lung than from lung 
minces;^ similar findings have been noted with regard 
to the release of histamine from granules in dog liver." 
McIntosh cites considerable evidence that the release of 
histamine involves rupture of the granule membrane: 
histamine release from isolated particles can be brought 
about by purely physical procedures which might be expected 
to disrupt a surface membrane such as suspension in hypotonic 
solutions, freezing and thawing, as well as surface-active 
agents such as saponin, bile salts, lysolecithin and the 
detergent Tween-20. McIntosh quotes Grossberg and Garcia 
i Arocho who suggested that “the final step, in any series 
of reactions leading to the release of histamine would be 
a change in the properties of the particle-cytoplasm inter¬ 
face, permitting the histamine to diffuse out. This change 
can be thought of most simply as a rupture or increase in 
the permeability of a membrane enclosing the particDe. 
McIntosh believes that an increase in permeability of the 
granules is brought about by the histamine liberators them¬ 
selves even though these bases are only weakly surface-active 
in dilute solution."^ As Paton has pointed out, one must 
consider the possibility that there is no one single 
mechanism of histamine release because there are so many 
types of histamine releasers: sensitizing compounds, toxin 
and venoms, proteolytic enzymes, surface-active agents, large 

J I £ 

molecules, histamine liberators, and monobasic compounds. ‘ 


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21 


Other Constituents of Mast Celia 

In addition to mucopolysaccharides such as heparin, 
and the amines, many other constituents have been found 
in mast cells: to name a few, albumen, glycoprotein, 

phospholipids, acid and alkaline phosphatase, lipase and 

11 

cytochrome oxidase. The enzymatic capacity of mast cells 

to synthesize amines from their precursor amino acids was 

( 

reported by Hagen and Lee who demonstrated the presence of 

5-hydroxytryptophane and histidine decarboxylase in the mast 

cell non-particulate cytoplasm.^ The absence of histaminase 

and the presence of monoamine oxidase in the Furth mastocytoma 

57 

has been reported by Hagen. 

i 

Synthesis and Storage of Amines 

Two of the leading unsolved questions of amine biology 

are the origin of the amine granules and how the amines get 

to the granules where they are stored. Cell fractionation 

studies have established that the amino acid decarboxylases 

which produce the amines from their precursors are present 

in the cell sap, that is, they are not bound to any structure 

58 59 

as heavy as the endoplasmic or agranular reticulum. * The 

amines, then, are fomed in the cell cytoplasm and rnusb be 

conveyed to the granules or incorporated during formation of 

the granules. However, enzymes such as the mitochondrial 

monoamine oxidase ^ or non-particulate O-methyltransferase^ 1 
62 

or histaminase could inactivate these amines after synthesis 
and before storage. The explanations that have been offered t*> 
this dilemma will be cited in the discussion of our findings 




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22 


which permit us to postulate a new theory that explains the 
origin of the granules and the protection of the amines by 
a unified mechanism. 

Function of the Hast Cells 

The important question of the function of the mast cells 

I 

is still far from settled, but we are in a far better position 
today to offer hypotheses than Ehrlich was in 1877* The 
presence of heparin, an anti-coagulant, and the location of 
tremendous numbers of mast cells along blood vessels, stimu¬ 
lated the idea that the cells are involved in endogenous anti¬ 
coagulation or in clearing lipids from blood, both well es- 

20 

tablished functions of exogenous heparin. The presence 
of the mucopolysaccharides heparin and hyaluronic acid in 
the mast cells suggested that the cells were important in 
laying down the ground substance of connective tissue* 

Recent work has tended to substantiate this thesis. The 
release of amines from mast cells has been shown by Riley 
to stimulate the mesenchymal cells to phagacytose and digest 
metachromatic material from nearby mast cells. The connective 
tissue cells then produce more mucopolysaccharide and new 
ground substance. J A mixture of heparin and histamine is 
required to produce local stimulation of mesenchymal activity: 
histamine alone is inadequate*^ Wound healing is speeded 
by heparin. 

Histamine released from mast cells may also be important 
in the formation of bradykinin from one of the plasma globulins. 
This peptide has already been shown to possess a potent vasodi¬ 
lator property, to increase capillary permeability, to cause 



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23 


6k 

the accumulation of leukocytes, and to produce pain. ~ 

It is well established that both histamine and 5-ET 
increase capillary perraeability* This has led to the 
suggestion that after injury the mast cell releases 
histamine and (in the rat and mouse) 5-HT and that these 
are among the factors involved in the hyperemia and in¬ 
creased vascular permeability which occurs as a response 
66 

to injury# Draper and Smith showed that in rats whose 

peritoneura had bean depleted of mast cells by an intra- 

paritoneal injection of distilled water, there was an 

increased extravasation from capillar-ie s of the peritoneum 

following a passively induced antigen-antibody reaction* 

This led them to postulate that roast cells may contribute 

to the initiation of the inflammati on which follows antigen- 

67 

antibody reactions* ' 

Reserpine 

Reserpine and chlorpromazine are the drugs whose 
effects on mast cells morphology was investigated with the 
electron microscope by the author* 

The similarity in the effects of reserpine and 5-HT 
on mice were the initial clues that the effects of reserpine 
were due to amine release*^® it was later shown that re¬ 
serpine increased the excretion of a metabolite of 5-HT, 

68 

5-hydroxyindoleacetic acid* Direct measurement of 5-HT 

levels after reserpine treatment demonstrated that it 

lowered the 5-HT content of intestine, brain, platelets of 

6ft 

many species and rat mast cells* Reserpine also releases 
catecholamines from tissues including the brain, heart. 



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68 

ganglia, arteries and blood* It lowers histamine levels 

68 

in rabbit blood but not in other rabbit tissues. Re- 

serpine is more effective in releasing 5-HT than histamine 

from the mast cells of rat skin* 51 and the Dunn-Potter 
39 

mastocytoma. Studies with the latter tumor grown as free 

cells in tissue culture by Giarman et al. showed that re- 

-9 

serpine at 10 M produced approximately $0% release of 5-FT 
and 10 M, 100% release. 

Since these amines are particulate bound in many tissues, 
the effect (s) of reserpine on these particles in vitro in an 
isolated state is of great interest. Various histamine liberators 
such as 1,10-diaminododecane, propamidine and Compound ip8/80 have 
been shown to release histamine from isolated mast cell granules. 
However, the release process differed from that observed in vivo 
in that the time course of the reaction was a 1st order re¬ 
action, proportionate to the concentration of both granules 
and releasing agent. The explosive release seen in intact 
tissues did not occur."^*'^ Walaszek and Abood found that 

reserpine released 5-HT from an isolated large granule fraction 
71 

of rat brain, Whittaker, however, found that reserpine did 

not release 5-HT from isolated brain particulate nor did it 

suppress uptake of 5-HT by these particles. However, particles 

depleted of 5-HT could be obtained from animals treated with 
72 

reserpine. Van Euler found that the effects of reserpine 
on NE-containing particulated isolated from splenic nerves 
varied with the concentration of reserpine at concentrations 
of 1-10 micrograms/ml it prevented the spontaneous release of 




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25 


HE but in higher doses (100 mlcrograms/ml) it enhanced 
73 7b 

release* * These studies indicate that reserpine can 
exert a direct effect on the amine-containing granules in 
vitro * But this does not necessarily mean that the same 
is true in vivo or that this is the sole mechanism operating 
in vivo * 

Other studies indicated that in. vivo reserpine can 
either release the amine granules from the cells which contain 
them or disrupt them intracellularly* Marks et al. did a 
quantitative count of the 5-HT containing granules of enter- 
ochrohmaffin cells with the light microscope before and after 
reserpine treatment in vivo and found that the extent of de¬ 
granulation at eight and sixteen hours par ailed the changes in 
5-HT content* Bum et aJ found that reserpine decreased the 
number of chromaffin cells(^complete degranulation) and de¬ 
creases the number of granules in the remaining cells in the 

76 

skin of cat*s tail and the nictitating membrane.' Electron 
microscopic studies were carried out by De Robertis and his 
colleagues using the rat pineal gland which has been shown to 
contain high concentrations of catechol amines, 5-HT and 
H-acety1-5-methoxytryptamine.De Robertis found that the 
pinealocytes of rats have "club-shaped perivascular expansions" 
containing vesicles of various types, some of which had dense 
granules that might contain the osmophilic amines known to be 
present in the pineal. Reserpine was observed to produce almost 
complete disappearance of the heterogeneous vesicles containing 
dense granules between two and forty-eight hours after a single 
injection. The time course of regeneration of the granules 







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26 


over an eight day period corresponds strikingly with the 
recovery of 5-ET and ME in the brain of rabbits after treat¬ 
ment with reserpine. 

Chlorpromazine 

The other drug whose effects on mast cells were studied, 
was chlorpromazine, a veiy effective tranquilizer. Two 
factors prompted the choice of this particular pyschotropic 
agent* First, recent work in these laboratories demonstrated 
that chlorpromazine, like reserpine, releases 5-HT and histamine 

00 

from the Dunn-Potter mastocytoma. Chlorpromazine also re¬ 
leases 5>-HT from, platelets, by a mechanism which appears to be 

S1 

different than that of reserpine. Secondly, although most 
of the biochemical work with chlorpromazine has been concerned 
with its effects on various enzyme systems (to be reviewed 
later), a current theory of chlorpromazine * s mode of action 
considers its effectson various membranes with the cell, ^ 
particularly those of the mitochondria,®"' as being of central 
importance. Spirtes and Guth have shown that chlorpromazine 
inhibits the water imbibition of isolated mitochondria pro- 

oq fill 

duced by thyroxine, detergents and phosphate. 9 It in¬ 
hibits the uptake of 5-HT by platelets,the uptake of 

86 

adrenaline in vivo from the blood into tissues, the 

in vitro release of acetylchoine from isolated storage 

' 8 ? 

organelles, the metrazol-enhanced passage cf dyes into 

act 

the brain, 0 the absoiption of drugs from subcutaneous 

89 90 

tissues, the intestinal absorption of lipids and sugars, 

91 

and the release of brain amines by reserpine. All these 





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effects might be explained by the accumulation of chlor- 
promazine which is lipid soluble in the lipoprotein of 
the cell membranes where it might alter permeability 

92 

either directly or through various enzymatic processes. 7 
The electron microscope might be of great value in demon¬ 
strating a morphologic effect of chlorpromazine on cell 
membranes if such exists. Roizin et al. believe that they 
have demonstrated with the electron microscope that chlor¬ 
promazine increased the osmophilia and reduced the size of 

mitochondria of nerve cells and neuroglia in biopsy speci- 

93 

mens of rat brain and monkey cerebral cortex. 

The Dunn-Potter ^ouse Mastocytoma 

The spontaneous appearance of mast cell neoplasms in 

914- 

mice and other animals has been noted many times. ^ However, 

only two of these have been successfully transplanted and 

found to maintain their mast cell characteristics in suc- 

cessive generations .^ 9 ~ 9 9 The present investigation is 

concerned with one of these tumors, the P-815 mouse masto- 

5 

cytoma reported by Dunn and Potter in 1957# A good deal of 

information about this tumor has already been reviewed in 

this Introduction. This tumor arose in a mouse of DBA f/2 

strain whose skin had been repeatedly treated with raethyl- 

cholanthrene. The morphology of the original tumor at the 

light microscope level has already been described (p.12). 

Sjoerdsma et al. established that this tumor produced 

31 

histamine and 5-HT. An ascites line of these cells was 



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28 


established by intraperitcneal injection of a tumor brei." 
Schindler et al. developed a medium that permits growth of 
the cells in vitro; the medium contains high concentrations 
of folic acid and undialyszed horse serum in addition to the 
usual constituents for tissue culture. Pure cell lines were 
established by isolating single cells and allowing them to 
multiply on feeder layers of chick embryo fibroblasts. A 
cell line called the T-Line which produced much larger 
amounts of 5-KT and histamine than the original line was 
established. Prom the T-Line two other lines were established 
by two successive cloning procedures, the X-l and X-2 lines, 
each descendent from a single cell and thus each genetically 
pure. The X-l is a near-teraploid line, the X-2, a near¬ 
diploid* Both these clones produce even more 5-HT and histamine 

than the T-line* The cells grown in tissue culture contain more 

96 

amines than those grown in mice. 7 With the solid mastocytoma, 
70-85$ of the amines and heparin was found in the fractions 
containing unbroken cells, nuclei, mitochondria and raicrosomes. 
These fractions were found to adsorb l\.0% of added 5-HT, hista¬ 
mine and heparin following addition to the homogenates of 

quantities of these compounds of comparable magnitude to those 

96 

originally present. This points up the well known difficulty 
of interpreting data from homogenization and centrifugation 
studies* Not only does the homogenization release granular 
bound material, the centrifugation procedures can cause 
factitious distribution of the released material by selective 
adsorption. 





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29 


METHODS 

Growth of ascitic cells 

The ascites line of the Dunn-Potter mastocytoma was 


carried by innoculating approximately 1 X 10^ mastocytoma 


cells in isotonic saline intraperitoneally into DBA mice* 
After a period of seven days, the animals were killed by 
cervical dislocation, the peritoneum was exposed, and the 
cells were washed out of the peritoneal cavity with isotonic 
saline. Aliquots were taken for 5-HT assays* The cells were 
then centrifuged into a pellet and prepared for electron 
microscopy. 

Growth of tissue culture cells 

The same cell line that was carried in the ascitic 

fluid was grown as a cell suspension in vitro in a special 

g-> 

tissue culture medium particularly rich in folic acid, ^ 

The cells were not allowed to reach a concentration greater 



When sufficient cells were present, they 


than 


were harvested by centrifugation. Aliquots were taken for 
5-HT assay. The number of cells present was determined by 
the use of a Coulter counter. The cells were then centrifuged 
into a pellet and prepared for electron microscopy, 

Reserplne Treatment 

The tissue culture cells were incubated at 37°C for 
2l± hours with reserplne at concentrations of 10~^M or 10"^M. 
Controls containing no reserplne were studied simultaneously. 
Aliquots were taken for 5-HT assay and for cell counts. 















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30 


The reserpine-treated cells and the controls were then pre¬ 
pared for electron microscopy* 

Chi or promazine Treatment 

The tissue culture cells were incubated for 12 hours at 
3 7°C with chi or promazine at a concentration of 1 x lO^Ii. 
Controls containing no chlorpromazine were studied simul¬ 
taneously* Aliquots were taken for 5-HT assay and for cell 
counts* The chlorpromazine-treated cells and the controls 
were then prepared for electron microscopy. 

Light and Phase Microscopy 

The mast cells were observed with the light and phase 
microscope in the unfixed condition, after fixation with 1$ 
osmium tetroxide, and after staining with 0*1$ Azure A in 
30$ alcohol (for metachromasia). 

Electron Microscopy 

Review of the literature on the electron microscopy of 

cultured or ascitic cells revealed that the best results have 

been obtained when the cells were centrifuged into a pellet 

and treated as a small tissue mass rather than by working with 

qj qg 

a loose suspension of cells. , Preliminary studies con¬ 

firmed this about the mastocytoma cells* Therefore, for all 
the electron microscopic work, the ascitic cells and tissue 
culture cells were centrifuged into a pellet from a 0*30M 
sucrose media and the pellet was then gently disrupted to 

produce many small fragments. These were then fixed, de- 

99 

hydrated, and embedded according to standard techniques. 





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31 


which briefly are: fixation for 60 minutes in 1% osmium 
tetroxide buffered with veronal acetate to pH7.3> dehy¬ 
dration by a series of graded alcohols to absolute alcohol, 
embedding in prepolymerized methacrylate ( 80 /£ methyl, 20 % 
butyl) after saturating the tissue with this unpolymerized 
mixture, and polymerizing overnight at 60°C using dibenzoyl 
peroxide as catalyst* The condition of the cells was checked 
during these procedures by light and phase microscopy* In 
several instances, the cells were embedded in Epon according 
to the method of Luft*^ 0 Thin sections were cut on a 
Porter-Blum microtome and mounted on grids containing a 
collodion film. The sections were then observed with an 
Akashi electron microscope. 

Isolation of Subcellular Components From the Ascites Cells 

The aim of this work was to isolate a purified preparation 
of mast cell granules uncontaminated by mitochondria* Such a 
preparation would be of great value in characterizing the 
granules. 

The ascites cells were chosen because they represented 
a source of mast cells easily available and relatively un¬ 
contaminated by other cell types. The first problem was to 
rupture the cell membrane and release the cytoplasmic in¬ 
clusions. This proved to be quite a difficult problem. 

Sonic oscillation, high speed oscillation with small glass beads 
and freezing and thawing proved inferior to homogenization with 
the Potter-Eveljhem homogenizer. 10-15 minutes of homogeniza¬ 
tion at high speeds at li-°C ruptured over G0% of a 10% suspension 
of mast cells in 0«3M sucrose* Undoubtedly some constituents 



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32 


originally present in the granules were released by this 
prolonged homogenization* 

The presence of 5-HT, histamine and heparin were used 
as markers for the amine granules* Succinoxidase was used 
as a marker for the mitochondria* 

Differential centrifugation of the sib pens icn of the 
homogenized mast cells at various speeds and lengths of 
time was inadequate to separate the amine granules from 
the mitochondria* 

Therefore, the technique of density gradient centri¬ 
fugation was tried.104-106 with this technique, a solution 
containing the particles to be separated is layered on top 
of a column of sucrose solutions of increasing density in 
a plastic test tube which can be centrifuged in a "swing-out” 
rotor at high speeds. Particles with different sedimentation 
rates are separated into discrete zones which can then be 
separated from one another by a variety of techniques. 

A great deal of work was necessary before the optimal 
conditions for separating the granules and mitochondria 
of the ascites mastocytoma cells were found. Even then 
not all the mitochondria could be separated from the granules 
indicating that some mitochondria have the same sedimentation 
rate as the granules. The following procedure gave the most 
successful separation of granules and mitochondria. 

Mice containing Dum-Potter X-l-C ascites cells were 
given S 3£- sulfate sixteen hours prior to isolation of the 
cells. The cells were washed several times with isotonic 
saline and then a 10$ suspension in 0.3M sucrose was 


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homogenized in a Potter-Eveljhem apparatus at 4° c until 
the majority of cells were broken. Unbroken cells, nuclei 
and debria were removed from the homogenate by centrifuga¬ 
tion at 700g for 10 minutes. The supernatant was decanted 
and a large granule fraction was separated from it by- 
centrifuging at 20,000g for 30 minutes. The resulting 
precipitate was suspended in 5 cc*s 0.3M sucrose, 
homogenized gently with the Potter Eveljhem homogenizer 
and layered gently over the sucrose density gradient. 

This density gradient consisted of lOcc* of 1.7M 
sucrose, 5 cc of 1.2M sucrose and 5«cc of 0 o 8m sucrose 
each layered carefully on top of one another so as to 
minimize mixing. The large granule fraction in 0.3M 
sucrose was layered over the 0.8M sucrose and the fluid 
column in a clear plastic test tube was centrifuged for 
60 minutes at 25,000 rpm in a Spinco -SW 25 rotor. 

The centrifugation procedure produces several bands 
of particulates at varying densities. A Spinco tube-cutting 
device was used to cut the tube so as to be able to separate 
each band cleanly. Aliquots of these fractions were then 
used for analytical purposes. On one occasion one fraction 
was subjected to density gradient centrifugation on a second 
gradient in an attempt to obtain a better separation. 

Isolation of Subcellular Components from the Tissue Culture Cells 

The same techniques that were just described were later 

used by Furano to separate these components of the cultured 

6 

mastocytoma cells. We attempted to reproduce this work so 
as to obtain isolated granules for electron microscopy but 



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were unsuccessful* All the cytoplasmic material that was 
layered on top of the gradient traversed the entire gradient 
and was found in pellet form on the bottom of the test tube* 

This pellet was used for electron microscopy, nevertheless* 

Determination of 5>-HT 

5-HT was determined spectrofluorometrically by the 
method of Udenfriend et ali°^ 5-HT was extracted into 
aqueous butanol from an alkaline-borate buffer solution 
saturated with sodium chloride* It was then extracted into 
dilute hydrochloric acid in the presence cf heptane which is 
miscible with butanol and drives the 5-HT into the hydro¬ 
chloric acid* The quiantity of 5-HT present in the hydro¬ 
chloric acid is then determined by the fluorescence in an 
Amino spectro-fluorometer, when activated at 295 millimicrons 
and read at 550 millimicrons in a concentrated hydrochloric 
acid solution* A series of standards was carried through the 
same extraction procedure to quantify the fluorescence. 

Determination of Histamine 

Histamine was determined spectrofluorometrically by the 
method of Shore et al* A O.IN hydrochloric acid extract of the 
mast cells was made alkaline with sodium hydroxide and saturated 
with sodium chloride* It was then extracted into n-butanol. 

This was then shaken with heptane and hydrochloric acid to 
transfer histamine to the hydrochloric acid. Histamine was 
then coupled with orthophthaldialdehyde in an alkaline medium 
and the resulting fluorophore is measured at l\$0 millimicrons 
after activation at 360 millimicrons* Appropriate standards 
and controls were run through the same procedure. 





35 


Determination of Heparin 

Heparin was determined by using radioactive S^ -sulfate 
as a tag. These mast cells can incorporate S^' -sulfate 
into heparin. The amount incorporated was related directly 

qA 

to the amount of heparin in the cells.' The radioactive 
sulfate was injected intreperitoneally into rats one day 
before sacrificing the animals. Heparin was extracted from 
the sample to be analyzed by placing the sample together with 
an excess of pancreatin in a dialysis bag. It was then dialyzed 
against tris buffer pH 8.4 for twenty four hours and against 
running tap water for twelve hours. The mixture was centrifuged 
and the radioactivity of the supernatant was used as a measure 
of the amount of heparin present. The radioactivity of an 
aliquot of this material was measured in a Tri Garb liquid 
scintillation spectrometer by the usual means. 

Determinati on of Succinoxidase 

Succinoxidase was determined by the method of Slater. 10 *" 

The oxidation of succinate was coupled to the reduction of 
potassium ferricyanate which was quantitated spectrometrically. 
Sufficient potassium cyanide was added to inhibit cytochrome 
oxidase. Succinoxidase was used as a marker for the mito¬ 
chondria. 

Attempts to Demonstrate Monoamine Oxidase Activity in the 

Sunn-Potter Mastocytoma. 

107 

1* Techniques of Cotzias and Greenough 

The tissue to be assayed for MAO activity is incubated 
with tyramine or 5-ET as a substrate in a phosphate buffer 
at pH 7.4 at 3?°C for 30 minutes. The reaction is stopped 






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36 


with sodium carbonate. The ammonia liberated by the 
deamination of tyramine is transferred to a dilute hydro¬ 
chloric acid solution by bubbling air through a test tube 
containing the reaction mixture connected by tubing to the 
hydrochloric acid solution. The ammonia in the hydrochloric 
acid solution is then determined with Nessler’s reagent. 30 ^ 
Appropriate controls without substrate or with heat inactivated 

enzyme are always used. Known monoamine oxidase inhibitors 

109 

such as iproniazid were also used to check if the ammonia 
liberated was due to monoamine oxidase activity. 

The solid mastocytoma tumor as well as the ascites cells 
were used as the enzyme source. The cells were disrupted by 
homogenizaticn, sonication, lysozyme, or freezing and thawing 
in order to liberate the enzyme. Mitochondria isolated from 
the equivalent of a gram of tissue were used in some experiments. 
2. Technique of Weissbach et al 

Because the previous methods failed to demonstrate any 
monoamine oxidase activity, a more sensitive assay technique 
was tried. This involved measuring the disappearance of 
kynuramine, a substrate of monoamine oxidase, spectrophoto- 
metrically. Purified preparations of mastocytoma mitochondria 
from up to one gram of tissue was used as the tissue source. 

Attempts to Demonstrate Diamine Oxidase Activity in the Dunn - 

Potter Mastocytoma 

Diamine oxidase, the enzyme which oxidizes histamine in 
112 

mammalian tissues, was sought by adapting the techniques 

of Cotzias and Greenough previously described, using histamine 

107 

as a substrate. 






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37 


RESULTS AND DISCUSSION 

The Ascitic Dum-Potter X-l-C Mastocytoma Cell 

The ascitic fom of the Durm-Potter mastocytoma was 
the first form of this tumor the author studied with the 
electron microscope. The cells are either round or elongated 
and vary from ten to twenty microns in size (Pigs 1-3). The 
nuclear-cytoplasmic ratio is greater in the rounded cells 
than the elongated cells; the nuclei usually have one or two 
nucleoli and are surrounded by a double membrane. 

The numerous long, thin pseudopodia of the plasma 
membrane, which in some places are forming vacuoles, indicates 
a high degree of surface activity in the elongated cells* The 
plasma membrane is quite distinct and is intact. The cytoplasm 
of the cell contains numerous constituents. Scattered throughout 
the cell are numerous oval-shaped granules, varying in size from 
about 0.05 to 0.2 microns. The granules are thus smaller than 
those of the other mast cells previously described. The granules 
vary in osmophilia, some being quite dense with an entirely solid 
matrix, while others are lighter and have clear areas within. 

Each granule has a distinct limiting membrane. A number of 
these large granules contained numerous much smaller granules 
and vesicles. Similar granules and vesicles were scattered 
throughout the cytoplasm. Round and elongated mitochondria 
with the usual structure can be seen. Vacuoles found at the 
cell surface appear to move deeper into the cell and degenerate. 
Deeply osmophilc amorphous lipid inclusions are present. Abun¬ 
dant smooth-raembraned agranular reticulum is present but little 
endoplasmic reticulum can be seen. 





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38 


The ascites mastocytoma cells, even though they possess 

granules are quite different in appearance from normal mouse 
1E> 16 

mast cells, (p* 9 )> rat mast cells,(p« 10 ), human mast 

19 17 

cells (p # ll), and even the Furth mouse mastocytoma. (p* 10 ). 

They resemble somewhat the dog mastocytoma cell described by 

Bloom et al (p.9) in their general configuration, distribution 

of granules, and active plasma membrane. 

Thus, electron microscopy reveals that there is a tremendous 
species variation in mast cells although this is not as evident 
in light microscope studies. Indeed, the ascites mast cells 
look like ’’typical" mast cells with the light microscope in 
that they appear full of met achromatic granules other than in 
the area occupied by the nucleus. The explanation of the 
difference between the light and electron microscope appearance 
may be that in the electron microscope we look at a two- 
dimensional view of very thin horizontal sections which contain 
granules in only a few areas whereas in the light microscope 
we see the "composite cf a series of thin sections" which 
collectively contain granules in all areas of the horizontal 
planes contained in the thin sections so that when viewed from 
above, as in the light microscope, one gets the inpression that 
the non-nuclear cytoplasm is occupied only by granules. This 
presupposes that the rest of the material in the cell is 
essentially transparent. 

The Origin of the Mastocytoma Granules 

Two of the most interesting and important unanswered 
questions of amine biology concern the origin of the amine- 
containing granules and how the amines get from the cell 



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39 


cytoplasm where they are synthesized from their precursor 

amino acids by the decarboxylating enzymes'^*'^ to the 

granules where they aie stored and yet avoid degredation 

by the various cytoplasmic or mitochondrial enzymes capable 

59,61 

of inactivating these compounds. It has been suggested 

that the amines in the granules are those which have been 

111 

able to escape degredation, a seemingly wasteful mechanism, 
or that the amines are bound in some fashion which prevents 

them from being destroyed while passing from clear cytoplasm 

. n 112 
to granules. 

Pictures of the ascites mastocytoma cells and certain 
of the atypical granule-containing tissue culture cells pro¬ 
vide the basis for a new theory of the origin of the granules 
of these cells and how they come to contain amines. As pre¬ 
viously mentioned, these cells contain an extensive agranular 
reticulum (which includes the Golgi apparatus) consisting 
mainly of tubules and sacs seen sometimes 3n profile as linear 
arrays parallel to one another but mostly 3n cross section as 
microvesicles and microgranules (which are interpreted as 
microvesicles containing protein, heparin and amines (Pig. 1-3 
13,114.)* In some areas of the cell, these microvesicles were 
closely associated with one another but not membrane-bound 
(Fig.3,G2, G3). These were most commonly seen in the region 
of the Golgi apparatus (Fig. 3), Elsewhere there are membrane 
bound particulates, of the same size and shape as mature amine 
granules (Figs. 1,2) which contain microvesicles and micro¬ 
granules (Fig. 3 9 GI 5 Fig. 14, Gl). In other particulates of 
this general nature, the microvesicles and micro-granules 


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appear to be clumping together (Fig* 01; Fig* 13, 01; Fig. 
14, G2, 03). In the final stage of this process, itfhich I 
believe to be that of granular maturati cn, the microgranules 
and microvesicles have coalesced within the membrane-delimited 
area* The membrane of the final granule is probably a dilated 
part of the agranular Golgi reticulum which has detached itself 
from this system. 

I further propose that the amines, after being synthesized 
in the clear cytoplasm gain access to the lumen of the vesicles 
and cistemae of the Golgi reticulum, part of which is then 
incorporated into the granules as well as used to form their 
limiting membrane in the manner just described. This would 
protect the amines from any degredative enzymes In the mito¬ 
chondria or cell sap as the amines become granular bound. 

Green and Day have shown that 30 % of the 5-HT and histamine 

of the solid Dunn-Potter mastocytoma was found in the micro- 

96 

somal fraction. 7 This could represent amines in transit to 
the granules if the proposed theory is correct* 

The granules of normal mouse mast cells have been noted 
to have a "filamentous structure," - those of normal rat and 
guinea pig mast cells, a "reticular structure,and those 

19 

of normal human mast cells, to consist of "lamellar groups." 

It has been claimed that the inner part of the granules of the 

113 

adrenal medulla is in the form of very small granules. 

«• I 

The studies of Ball cited by Hagen and Barmett ’ demonstrate 

that the granules of the adrenal medulla possess an enzyme 

characteristic of the raicrosomes, which are partly made up of 

11 ^ 

agranular reticulum. ^ 


This led them to speculate that the 


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protein matrix cff the adrenal medullary granules originates 
in the endoplasmic reticulum (where all proteins are syn¬ 
thesized). Hagen, Barrnett and Lee noted that the large 
mastocytoma granules of the Furth mastocytoma occurred in 
vacuole-like spaces in close relationship to the vesicular 
system of agranular reticulum. They suggest that this "may 
indicate a probable role of the reticulum in the development 
of the granules or the laying down of its contents."^ 

Numerous studies with other cell types which produce 
granules containing secretory material other than amines, 
suggest that their granules also originiate in, if not from, 
the agranular reticulum, with which the agranular reticulum 
is in direct continuity. Wellings, Siegel, Dalton and Felix 
in a series of papers have extensively studied melanocytes 
in this regard, demonstrating the progressive maturation of 

melanin granules within the agranular reticulum.^*6-119 

T 20 

Similar mechanisms have been claimed for plasma cells 

123 

and lactating mouse mammary cells. A morphological 

and functional association of the reticulum of the pancreatic 

acinar cell 5n the development of zymogen granules has been 

122 

described by Siekevitz and Palade. 

This hypothesis that the granules cf the Dunn-Potter 
mastocytoma cells are made from segments of agranular 
reticulum containing sequestered amines synthesized in the 
free cytoplasm may be considered an instance of Essner f s and 
Novikoff's thesis that; "Cytomembranes are in a dynamic state 
of flux, movement and transformation in the living cell and 
smooth surfaced derivatives of the endoplasmic reticulum become 
fashioned into the Golgi membranes as the Golgi membranes are 


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123 

being refashioned into those that delimit secretory vacuoles*” 

Whether this mechanism is a common one for making amine 

granules cannot yet be answered* The studies cited concerning 

the fine structure of the granules of other mast cells ^*^,19 

113 

and the adrenal and the chemical nature of the adrenal 
granules'*’^' suggests that it may be a widely employed system. 

This hypothesis if readily testable by looking for ad¬ 
ditional enzymatic activities and constituents common to both 
the granules and the agranular reticulum of the ascites cells, 
both of which can be isolated in reasonably pure states by 
procedures which the author developed* These studies are in 
progress. Electron microscopic autoradiography with radio¬ 
active histamine, 5-HT or heparin would be a very elegant way 
to test the hypothesis. 

The Tissue Culture Dunn-Potter X-l-C Mastocytoma Cell 

It was thought that distinct advantages would accrue from 
working with cells from tissue culture rather than with ascites 
cells aspirated from the mouse peritoneal cavity. The aspirate 
is really a mixture of neoplastic mast cells, fibroblasts and 
leuokocytes. It was thought that the cultured cells would pro¬ 
vide pure populations of identical mast cells which could be 
readily obtained in large quantities. Also the tissue culture 
cells could be easily treated with known concentrations of drugs 
for known periods of time without the interference provided by 
the mouse* Similar considerations have made the tissue culture 
cell the most frequently investigated foau of this mastocytoma. 

It was thus of great interest to learn that, unlike the 
ascites cells, the tissue culture cells contained few or no 



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granules* (Pigs* 4 - 7 )# While several tissue culture cells 
definitely possessed granules (Fig* 8), the great majority 
possessed no cytoplasmic particulates that could be called 
granules* Those granules that were present in the cultured 
cells were much smaller than those of the ascitic cells 
(Pig* 8)* This is the only cell that has been described 
which is functionally a mast cell but which contains no 
granules. Whether or not it should be called a mast cell 
is a question of semantics. But the fact that is amines 
are not in granules is obviously of paramount importance 
in interpreting experiments on amine biology in these cells. 

The tissue culture cells have a relatively undifferentiated 
structure. They have one or two irregular nuclei with one or 
more large nucleoli, abundant mitochondria, an extensive 
agranular reticulum, and pinocytotic vesicles. In these 
respects, save for more mitochondria and the absence of 
granules and vacuoles, they are quite like the ascites cells. 

The absence of granules in the tissue culture cells sets 
them apart from the ascites cells and belies their descent 
from the same cell . The ascites cell line as presently main¬ 
tained is frequently renewed from the tissue culture cells. 
Clearly the environment in which these cells divide and grow 
exerts a great influence cn the cells phenotype, on which 
part of its genetic inheritance is expressed. 

The X-l-C cell line is derived from a sirgle cell and 
is presumably genetically pure. This has led investigators 
to believe that a population of these tissue culture cells 
was homogeneous. But, with the elctron microscope, many 
atypical cells were seen. 



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As was mentioned, a few cells were seen which were 
identical in all respects, save for the appearance of 
membrane bound inclusions of smaller size than mitochondria, 
but lacking an internal structure, which could be amine 
granules or degenerated mitochondria (Pig* 8). Just why 
these cells and no others produce granules cannot yet be 
answered. 

A few cells were seen which contained bizarre ”multi- 
vesiculated dense bodies” which look very much like the lyso- 
somes described by de Duve (Pig. 9).^^ These were seen very 
infrequently in the ordinary tissue culture mastocytoma cells. 

The most interesting atypical cell was seen in a re- 
serpine-treated group of cells. The basic morphology of this 
cell could not be ascribed to the effects of reserpine since 
it was the only one of tens of thousands of reserpine-treated 
cells that had this appearance. This cell bore a striking re¬ 
semblance to the normal mouse mast cells described by Rogers 
(p, 9, Pigs. 10-12). Both cells possess about the same number 
of granules, each surrounded by a clear area and located at 
the periphery of the cell. The cytoplasm of both cells is 
relatively undifferentiated. In the reserpine-treated masto¬ 
cytoma cell, some of the granules are being released through 
the cell membrane. This will be discussed shortly. The 
similarities between this "atypical” neoplastic mouse mast 
cell and the normal mouse mast cell prompt the speculation 
that the genetic material of the neoplastic cell line still 
possesses the information to produce a cell near-normal in 
appearance on occasion. 




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45 


Another veiy interesting variant which was seen only 

once was a cell three or four times as large as large as 

any other cell, with four nuclei, and a cytoplasm densely 
k 

packed with granules of varying sizes and density, vesicles 
and mitochondria. (Figs. 13, 14)• Significantly, this 
tissue culture cell which possessed granules demonstrated 
what I interpret as early forms of granules, made up of 
agranular reticulum, and identical to those seen more 
frequently in the ascites cells. 

A number of cells were seen whose cytoplasn. was 
occupied mainly by large clear area which could have been 
granules with a sparse matrix. (Fig. 15). These cells re¬ 
sembled the normal rat and hamster mast cells described 

16 

by Smith and Lewis. 

It can be stated with conviction that the failure to 

see granules in most of the cultured cells in electron 

micrographs is not an artifact of preparation. The same 

technique of osmium tetroxide fixation, alcohol dehydration 

and embedding in methacrylate or epon have been successfully 

used by all investigators who have published electron mico- 

14-17,19 

graphs of mast cells. The same technique demonstrated 

granules in the ascites Dunn-Potter mastocytoma cells. In 
addition, the fine structure of the tissue culture cells 
was excellently preserved. 

It is possible, but very unlikely, that all the amines 


are present in the granules of the few cells with granules 
and that the great majority of cells possess neither granules 



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46 


or amines. This would necessitate an enormously high 
concentration of amines In these granules, approximately 
100,000 times the concentration of anines in the ascites 
tumor cells based on the amine concentration of each cell 
type, the percentage of cells with granules and the number 
of granules per cell. 

It is clear then that most cf the amines in these cells 
are not granular bound unless they are present in mitochondria. 
Certain evidence makes this unusual possibility at least worth 
considering, even though every amine-containing cell studied 
to date has amine granules which are distinct from mitochondria. 
First, these cells are unusually rich in mitochondria; whereas 
the ascites cells possess few mitochondria and many granules, 
the reverse is true of the tissue culture cells. Second, 
density gradient centrifugation of the tissue culture cell 
large granule fraction, which sedimented between 700g and 10,000g 
in thirty minutes and contained 53$ of the 5-ET of the cultured 
mast cells gave results compatible with the amines being in the 
mitochondria: 49$ of this fractions 5>-ET was in a sub-fracticn 
which contained 60$ of its succinoxldase activity (located in 

■j 1 O 

the mitochondria) while another 43$ of the 5-HT was found 



hich contained 24 * 5 $ of the 





ASXdl 


were in the same sub-fractions inplying that they might well 
be located in the same sub-cellular particle. My attempts to 
check this by electron microscopy will be reported and discussed 
in full, later, but it should be mentioned here that they are 
consistent with the proposition that the amines are located in 


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the mitochondria* The data on the cultured cells just pre¬ 
sented should be contrasted with the author’s studies using 
density gradient centrifugation of the large granule fraction 
of the ascites cells which were done several years before the 
work just presented. With the ascites cells, the author was 
able to obtain one sub-fraction of the large granule fraction 
which contained £6$ of the total succinoxidase activity and 
no 5-KT at all, while another sub-fraction had 100$ of the 
5-HT and histamine and only 28$ of the succinoxidase activity. 
This is at least suggestive that in the ascites cells there 
are amine storage granules which are not mitochondria and 
indeed it is the ascites cells which do contain many granules 
when viewed in the electron microscope. Some mitochondria 
have been found to contain phosphatides and cerebroside 

11 

sulfate, both of which have been implicated in amine binding. 
The chief objection to 5-ET being stored in mitochondria would 
be that the enzyme which oxidizes 5-ET, monoamine oxidase (MAO) 
is a mitochondrial enzyme. ^ The author*s prolonged studies 
with sensitive MAO assay techniques failed to find any MAO 
activity in the Dunn-Potter mastocytoma. The same is true 
for histaminase, the enzyme which oxidizes histamine. Thus, 
both these amines could be stored in mitochondria, whereas 
this would be impossible for the usual cell which contains 
biogenic amines and the enzymes which degrade them. 

It is very hard to conceive of the mitochondria being 
used to store amines. As was mentioned, all amine-containing 
cells studied to date have amine granules distinct from 


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mitochondria* The role of the mitochondria in energy 
metabolism hardly makes it suitable to act as a storage 
particle. The mechanism of amine storage I just postulated 
for the Dunn-Potter ascites cells would be ruled out. Why 
the tissue culture cells should store their amines in mito¬ 
chondria is not clear. Much more work will have to be done 
to settle this problem. At this point, all we can say is that 
the experimental evidence available concerning the tissue 
culture cells is consistent with the possibility that in these 
cells the mitochondria contain hist amine and 5-ET but that past 
experience with other amine-containing cells makes this a highly 
unlikely possibility. 

It is possible that the amines or any complexes they may 
be bound in, are free in the cytoplasm, Furano*s finding of 
the particulate nature of the amines of these cells^ may be 
spurious and represent cytoplasmic amines adsorbed on particles 
after homogenization. The absence of monoamine oxidase and 
histaminase in these cells which I demonstrated would permit 
the amines to be non-sequestered. 

Effects of Reserpine on the Tissue Culture 

Mastocytoma Cells 

It was a difficult decision to decide between using the 
ascites cells which possess granules and the tissue culture 
cells which possess essentially none to investigate the 
process of amine release with reserpine. I decided to use 
the tissue culture cells because this is the cell that several 
other investigators are using to investigate the release process 




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49 


with biochemical and pharmacologic techniques* The oppor¬ 
tunity to correlate their data with morphologic changes in 
the cell was decisive* 

After being treate d with reserpine at concentrations 

-7 

of 10 for twenty-four hours, the tissue culture mastocytoma 
cells were found to contain less than 0*1 gamma of 5-HT per 

Q 

10° cells, less than 1 ;% of the control levels* Similarly, 

the cells had lost the greater part of their histamine con- 

-9 

tent. The cells treated with 10 M reserpine had approximately 

$0% of their original levels cf 5-HT and histamine at the end 

69 

of this time. When these cells were looked at with the 
electron microscope after suitable preparation, a number of 
different reactions to the drug treatment were noted. They 
may represent different stages of the release process so that all 
cells at one time may have gone through the stages seen. The 
cells were observed only at the end of twenty-four hours when 
there was no further measurable release at both concentrations 
of the drug studied. It is obviously of interest to study the 
cells at earlier times when the process is not yet complete. 

Thus, the cells to be described are cells who have already 
released their amines, some perhaps with the cellular changes 
that brought about still present, others with the cell not 
showing any unusual features, but lacking amines. 

The outstanding change in cell morphology noted was the 
development of vaiying numbers of "multivesiculated dense 
bodies" in a large percentage of the cells. (Pigs. 16,17). 

Some cells possessed as many as thirty of these bodies (Fig.17). 


•xc 




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They are 0,2 microns or oval in shape, and characteristically 

densely osmophilic with internal discrete clear areas. The 

appearance of these bodies suggests that they are lysosomes, 

the enzyme-containing particulates de Duve discovered in liver 

121l 

and subsequently found in a number of the tissues. The 
multivesiculated dense bodies were almost never found in the 

untreated cells. More were found in the cells treated with 

-9 -7 

10 M than in those treated with 10 M reserpine, perhaps 

because the release process was incomplete at the lower con¬ 
cent rat ion. 

The chemical nature of these particles together with 
what is known about amine-binding and release suggests that 
the appearance of these particles in reserpine-treated, 
amine-releasing cells is not simply coincidental; rather 
evidence can be marshalled for the theory that reserpine 
brings about amine release in these mastocytoma cells by 
"inducing" the formation of lysosomes, whose enzymatic com¬ 
ponents could serve as the actual means of bringing about 
amine release. 

First, let us consider the nature of the lysosomes. 

Their discovery is credited to de Duve who found he could 
separate by- centrifugation a subcellular fraction from liver, 
rich in acid phosphatase and poor in mitochondrial enzymes. 
These particles have since been found to contain at least ten 
hydrolases which have an acid pH optimum: acid phosphatase, 
the cathepsins A,B, and C, phosphoprotein phosphatase, 
mannosidase, acid ribonuclease, acid deoxyribonuclease, 

12k 

B-galactosidase, B-glucuronidase and arylsulfatase A and B. 



51 


As will be indicated, several of these enzymes are strong 

candidates for a role in the release of amines* 

The lysosomes cf the liver are fairly polymorphic. Some 

"appear to be solid and are surrounded by a single membrane; 

others show one or more internal cavities, sometimes lined 

with a broad layer of denser material, or contain clumps of 

such material." Most of t hem have a fine granular structure. 

Lysosomes of other tissues have somewhat different size and 

126 

shape. They have been found in practically all tissues. 

If we are correct in considering the "multivesiculated dense 
bodies" as lysosomes, this will be the first identification 
of these structures in mast cells. Enzymatic studies are in 
progress to definitely identify these bodies as lysosomes. 

These cannot be reported as yet. However, additional morpho¬ 
logic evidence will be presented subsequently which tends to 
support their identification as lysosomes. 

To understand the proposal that lysosomal enzymes are 
important in amine release, it is first necessaxy to review 
some of what is known about amine binding. The most information 
available on this subject pertains to the catecholamines stored 
in the granules of the adrenal medulla. All evidence points 

to an analagous mechanism existing in all anine-containing 

111 

cells, including the mast cells. 

Hillarp et_ al . has shown that in the adrenal medulla 

127 

the sympathomimetic amines are found in granules which 

126 113 

others have shown are bounded by a membrane. 9 Hillarp 
showed that amines, adenosine phosphates, proteins and lipids 
represent practically the entire content of solids in the 


; if. 


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52 


129 

granules* He and his colleagues also showed that the 

granules of the adrenal medullary cells store catechol 

amines and ATP in nearly equivalent amount s ^30,131 and 

that stimulation of the medulla in v ivo causes a drop in 

the ATP that is proportional to the decrease in catechol 
132 133 

amines* 9 Sheep treated with reserpine released 

amines and adenosine phosphates from their adrenal medulla 

13k 

at the same rate* Hillarp believed he had demonstrated 

13*0 

ATPase activity 3n the adrenal medullaiy granules* This 

finding was disputed by others who claimed Hillarp*s granule 

fraction was contaminated with mitochondria and that pure 

136 

fractions of granules ccntain no ATPase* Nevertheless, 

the presence of ATPase in the granules is still an open 

question because of the difficulty of demonstrating this 

enzyme* Hillarp suggested that amine release is brought 

about by the dephosphorylation of ATP to which the amines 

are probably bound, by the ATPase of the granules which is 

activated, either directly or indirectly, by the agent which 

139 

triggers release* 

The lysosomal enzymes migjit work Independent of, or 

together with granular ATPase or obher intragranular 

mechanisms of release* Let us consider an independent mode 

of action first* The action of the lysosomal enzymes might 

be to degrade the anion which the amines are bound to - in 

1 111 

the mast cell this is probably either heparin or ATP* * 

The lysosomes possess both sulfatases and phosphatases'^' 
although it has not been proven that heparin and ATP are 



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53 


substrates of these enzymes. In the tissue culture masto¬ 
cytoma cells where the amines may be in the cytoplasm, the 
action of these enzymes on the amine complexes could occur 
directly; in cells where amines are in granules, the enzymes 
would first have to enter the granules. The lysosomal 
enzyme (s) might increase the permeability of, or otherwise 
alter strategic membranes with the cell, perhaps either the 
granule membrane or the cell membrane. These membranes are 
basically lipoprotein in nature and the lysosomes possess the 
necessary equipment (phosphatases, proteinases) to bring about 
their disruption. After disruption, amine release may occur 
simply by diffusion because a previously impermeable barrier 
has now been removed, or contact of lysosomal degredative 
enzymes and the anions bound to the amines may be promoted. 
Another possibility is that disruption of the granule membrane 
may change conditions with the granule, such as the pH or the 
concentration of various ions within the granule and thus 
modify the activity of the enzymes present in these granules. 

As has already been mentioned, there is evidence for an ATpase 
in the granules. 9 One of the important questions re¬ 
garding the significance of granule ATPase in the release of 
amines was the mechanism by which it might be activated at 
the time of release. The change in local conditions that 
might occur as the result of the action of lysosomal enzymes 
may be the answer. 

It might be argued that the appearance of lysosomes in 
these cells is an indication of cell death because the lysosomes 


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119 


Against this is 


have been implicated in this process, 
the finding that the reserpine-treated cells will resynthesize 
their amines, divide and grow when placed in fresh media. 

It is clear that no such lysosomal-mediated explanation 
of the mode of action of reserpine is possible when one is 
dealing with isolated amine particles in vitro . But it is 
not at all necessary that these two phenomena have a common 
mechanism. It has already been pointed out that in vitro 
reserpine possesses certain properties such as an inhibition 
of amine release which are never seen in vivo . 

It is quite possible, of course, that the increase in 
lysosoraes following reserpine treatment, has nothing to do 
with amine release. Other morphologic differences between 
some few reserpine-treated cells and untreated cells were 
seen. These will now be described. 

At the very outset of these experiments on the neoplastic 
mouse mast cells, I hoped to see the process of granule re¬ 
lease in reserpine-treated cells. The almost invariable 
absence of granules in these highly dedifferentiated cells 
generally frustrated this desire. How surprising it was, 
then, to f ind one cell, which had been treated with reserpine, 
in which a number of granules were passing through or had 
already traversed the cell membrane, and whose general 
morphology bore a striking resemblance to that of a normal 
mouse mast cell as described by Rogers (Pigs, 10-12 p.9).^ 

A possible interpretation would be that this cell had re¬ 
gained some of Its normal characteristics and was responding 
as a normal granule-filled mast cell would, that is, with 














55 


granule release. Some re serpine-treated typical tissue 
culture cells were seei which had cytoplasmic inclusions 
passing through the cell membrane (Fig, 18). This was 
occasionally seen in untreated cells, I am presently 
investigating the effects of reserpine on the ascite masto¬ 
cytoma cells which do possess granules, 

Reserpine treatment produced a group of cells which 
suggested another mode of amine release. These cells had 
large me mb ran© -bound areas with a very sparse matrix. (Fig. 
19)« In several of these cells, these areas were located 
at the cell surface with a partially disrupted segment of 
the plasma me rib rane as one of its borders. Other such 
areas were often seen deeper within these cells. It is 
possible that discharge of cellular contents had taken 
place at the cell surface, leaving behind amorphous mem¬ 
brane-bound inclusions that then detached themselves from 
the cell surface to migrate back into the middle of the cell. 
What makes this of particular interest is that De Robertis 
and Vaz Ferreira described a similar occurrence in the 

adrenal medulla of rabbits after stimulabicn of the spalnchnic 
137 

nerve. Their observations suggested that the secretion of 
the catechol amines involved an increase in size but a de¬ 
crease in electron density of the granules and their attach¬ 
ment to the cell surface. The granules then released their 
contents at the cell surface and were left as folded membranes 
attached to the cell surface. Furthermore, Wellings and 
De Ome observed that in lactating mouse mammary cells vacuoles 


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56 


containing milk protein granules appeared to open at the 
cell surface and release their contents into the alveolar 
lumen. Similar methods of secretion have been observed in 
many other tissues. An analogous process might be happening 
in the tissue culture mastocytoma cell, differing in that a 
whole region of the cell enclosed within a membrane, probably 
part of the agranular reticulum is being discharged. If the 
amines are located in the cytoplasm of these cells this would 
effectively accomplidi amine release. This cell type was 
not seen in any of the untreated cells. It Is unlikely that 
it represents vacuole formation at the cell surface, although 
a secondary result of such a process might be imbibition of 
extracellular fluid. 

At least a half of the cells seen after treatment with 

•» W Q 

both 10 7M and 10 7 M showed no morphologic difference from 

untreated cells, despite the fact that the cells treated with 

-7 

10 M possessed less than 1% of their normal amine concen¬ 
tration, This indicates that release can occur without any 
visible change in the cells morphology, or that the changes 
which brought about or accompanied release in these cells 
was no longer evident. It is impossible to make a choice 
between these two interpretations, although the latter 
possibility seems more likely. 

It would be nice to fit all these findings inb o a 
single interpretation that would encompass them all. Since 
the untreated cells are clearly polymorphic, we are not 
really required to do so, Reserpine may act differently on 



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57 


each different cell type present. This clearly seems to 
be the case in the cells with granules where reserpine 
produced release of the granules. It is thus possible 
that these findings, the induction of what appear to be 
lysosomes, the release of granules, and the release of 
cell cytoplaan from merti>rane-bound areas are phenomena 
seen only in these atypical neoplastic cells. Nevertheless, 
they do suggest new experiments which should and can be done 
on other tissues such as the adrenal medulla and the central 
nervous system. 

Some suggestions may be offered about the origin of 
and additional activities of the "multivesiculated dense 
bodies" which both tend to support that they are lysosomes 
and indicate how reserpine promotes their formation. At 
the same time they contribute in a small way to what is 
known about the origin of and the activity of the lysosomes. 
In Pigs. 16 and 17, in the midst of areas packed with the 
multivesiculated dense bodies, we see membrane-bound 
vacoules filled with additional segments of membrane and 
clumps of osmophilc matter. They appear to be early stages 
of the osmophilic dense bodies. In Pig. 17, we see the 
dense bodies seeming to ingest mitochondria. This phenomenon 
has been described previously by Novikoff^^^ and the re¬ 
sulting inclusion has been called a "cytolysome". 1 ^ These 
micrographs suggest that the lysosomes are formed in dic¬ 
tations of the agranular reticulum, as suggested by Novi- 
koff^ 0 by ingesting cellular material and that once formed 



58 


the lysosoraes can incorporate other cell contents such as 
mitochondria. 

Effects of Chlorpromazine on the Dunn-Potter Tissue 
Culture Mastocytoma Cells 

Until recently most of the experimental studies of the 

biochemical basis for chlorpromazine»s central nervous system 

depressant or tranquilizing action were cpncemed with its 

activity as an inhibitor of respiratory enzymes or oxidative 

phosphorylation In vitro effects could be demonstrated 

on these systems only by using concentrations of the drug 

hundreds of times greater than those achieved in vivoj^ 

Recent experiments which were reviewed in the Introduction 

(p. 91-92) have led to the hypothesis that the primary effect 

82 

of chlorpromazine is on various cell membrane phenomena, 
particularly those of the mitochondria.®^~^ 

Studies with the mast cells treated with chlorpromazine 
at 10-£ M for twelve hours demonstrate a morphologic de¬ 
rangement of mitochondrial structure which seems to result 
from t he action of this drug. (Pigs, 20-22). In the un¬ 
treated mastocytoma cells the outer limiting membrane of 
the mitochondria is smooth contoured and continuous, whereas 
the inner one folds inward periodically to foim the incom¬ 
plete septa that form the cristae. The two leaves of the 

crista usually run straight and parallel and are separated 

, 11+2 

by a narrow interspace of uniform width (Pigs. J+-7, 23). 

The mitochondria of the chlorpromazine treated cells fre¬ 
quently have highly disrupted outer membranes. Cristae are 









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59 


seldom perpendicular to the limiting mitochondrial membrane 
but are instead parallel or come off at irregular angles* 
Some mitochondria appear compartmentalized by crista running 
in a number of directions. Other mitochondria consist of 
whorls of membranes, like the myelin of a nerve sheath. 

There are numerous elongated unusually shaped mitochondria 
in t he treated cells. There are very few normal appearing 
mitochondria in most of the treated cells (Figs, 20-22), 

This is to be contrasted with the appearance of "multi- 
vesiculated dense bodies” in about a quarter of the cells 
after reserpine treatment. 

The morphological effects of chiorpromazine on mito¬ 
chondrial membranes could be due to the same chemical 
changes which produce inhibition of mitochondrial swelling 
in hypotonic solutions, etc, (p, 91-92), ^5-93 or re _ 
verse could be true and the chemical effects might be due 
to the moiphological effects which have some other origin. 
These morphological effects could explain the inhibition of 
mitochondrial respiratoiy enzymes which are known to be de¬ 
pendent upon an intact mitochondrial structure and to in¬ 
volve a series of enzymes located in precise order along 
the cristae,^^ The bizarre mitochondrial forms produced 
by chlorpromazine should interfere with these delicate 
functions to a great extent. On the other hand, the enzy¬ 
matic inhibition could be the cause of the morphologic 
aberrations. Regardless, this work has established an 




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60 


effect of chlorpromazine on mitochondrial membranes that 
is well suited for experimental analysis, 

Giarman has established that this dose of chlorproma¬ 
zine release significant amounts of 5-HT from the tissue 
culture cells.As was discussed previously, it is 
possible that the tissue culture cells store their amines 
in mitochondria. If so, the release of amines by chlor¬ 
promazine may be related to the mitochondrial structural 
changes just discussed as cause or effect. The increased 
disruption of the plasma menbrane of the mastocytoma cells 
may also be involved. There was no evidence of lysosome 
formation, granule release or release of cytoplasm in the 
chlorpromazine-treated cells, as was seen in the reserpine- 
treated cells. 

The Large Granule Fraction of the Tissue 

Culture Cells 

A large granule fraction was isolated from the tissue 
culture cells after homogenization in 0.3M sucrose. This 
fraction was layered on a density gradient according to the 
method of Furano but no separation was achieved* The only 
tissue evident after centrifugation was cn the bottom of the 
centrifuge tube. This material was prepared for electron 
microscopy which revealed that it consisted largely of dis¬ 
torted mitochondria (Fig. 2l|). Very few granules were noted. 
It is clear that these cells possess very few if any granules. 






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61 


Isolation of Mast Cell Granules from the 

'Aa'cTte'j Cells "~”™' 

The purpose of this work was to isolate the amine 
and heparin containing granules of these cells so that 
accurate chemical studies of their nature could be carried 
out. These studies were carried out before electron micro¬ 
scopy revealed that the ascites cells, but not the tissue 
culture cells possessed such granules, so the choice was 
a fortuitous one* 

Initially, the cells were vigorously homogenized 
with the Potter-Eveljhem apparatus, followed by removal 
of unbroken cells, nuclei and debris by centrifugation. 

A large granule fraction is then sedimented from this 
cell and nucleus free homogenate by centrifugation and 
the distribution of amines, heparin and succinoxidase 
between the large granule fraction and supernatant de¬ 
termined. The results of one experiment are given in 
Table 1. They are representative of many similar experi¬ 


ments 




' 


- 

■ 

, 

l 

, % 

* : • 

p. r ;• jcg v b$>; K.tb© s ns.clrJ «... xtoltfo- .. 





62 

Table 1 


The Distribution of Histamine, 5-HT and Heparin in 
Fractions Derived from Mastocytoma Cells by Differential 
Centrifugati on 



r~ ” — 

Nucleus-free 

Homogenate 

Large Granule 
Fraction 

Supernatant 

Histamine 

63 

53 

' 15 

5-HT 

180 

66 

51 

Heparin 

100,000 

67,500 

31,500 

Succinox¬ 

idase 

7,600 

5,u>o 

1,750 


Histamine and 5-HT are expressed as micrograms* Heparin 
is expressed as total counts* Succinoxidase is expressed 
as change in optical density under the assay conditions 
described in the section on Methods* 

This demonstrates that the greater part of the hista¬ 
mine, heparin and succinoxidase is particulate in nature* 

Only about 35$ of the 5-HT of the nucleus free homogenate 
was found in the large granule fraction* Some 28$ was in 
the supernatant. 37$ of the 5-HT could not be accounted 
for and is presumed to be n on-enzymatic ally destroyed since, 
as will be discussed shortly, no enzyme system capable of 
degrading 5-HT has ever been found in these cells. Values 
for the amount of 5-HT which could be recovered in the 
particulate fraction varied between 30 and lj.5$ in numerous 
experiments* But the 37$ of t he 5-HT of the nucleus free 
homogenate which could not be accounted for, as well as 
some or all of the supernatant 5-HT might originally have 
been in the granules. Undoubtedly, much 5-HT is released 
from the granules during the arduous process of homogenization 
needed to disrupt these cells* The 5-HT must be more easily 








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63 


removable by mechanical methods from the granules than 
heparin or histamine. Hagen, Barmett and Lee have shown 
the granules of the Furth mastocytoma easily lose their 
amines in isolation procedures.*^ Of the 5-HT of the 
nucleus-free homogenate which could be accounted for 
either in the particulate form or in the supernatant, 

60 % was particulate (Tabl& 1). 

The large granule fraction is then subject to density 
gradient centrifugation as previously described. After 
centrifugation, two distinct bands of tissues are evident; 
(Text Fig. 1) the first is at the border of the 0.8 m and 
1.2M sucrose solutions and the second is at the border of the 
1.2M and 1.8M sucrose solutions. There is a small pre¬ 
cipitate at the bottom of the tube. 


Text Fig. 


1 



Five fractions are taken with the aid of the tube 
cutting device already described and aliquots analyzed for 
5-HT, histamine, heparin and succinoxidase. The results 
are described in Table 2. 












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64 

Table 2 


Distribution of 5-HT, Histamine, Heparin and 
Succinoxidase in tbs Five Fractions Obtained 
From Density Gradient Centrifugation of the 
Large Granule Fraction. 


Fraction 5-HT 

Histamine 

Heparin 

Succinoxidase 


Units $ 

Units $ 

Units 

$ 

Units 

%. 

1 

! 

0 0 ^ 

0 

0 

15,000 

26 

700 

16 

2 

0 0 

0 

0 

1 

0 

0 

2400 

56 

3 

63 100 

12 

100 

r 

30,000 

53 

1100 

28 

4 

0 0 

0 

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7,600 

13 

0 

0 

5 

0 0 

0 

0 

4,5oo 

8 

0 

0 


5-HT and histamine are expressed as micrograms* 
Succinoxidase is expressed as charge in optical 
density under the assay conditions already 
described* Heparin is expressed as total counts* 

All of the 5-HT and histamine and 53$ of the heparin 
is in fraction three, which is the material which did not 
sediment below 1*7M sucrose* This fraction contained 28$ 
of the succinoxidase activity as well. Fraction 2 con¬ 
tained 56$ of the succinoxidase activity and no 5-HT, 
histamine, or heparin. 

Fraction 2 was resuspended over a second density 
gradient consisting of 2.5cc 0.8M sucrose, 5«0cc 1.5M 
sucrose, 7*5cc 1.6M sucrose and 5*0cc of 1.7M sucrose 
aid centrifuged for an hour at 25,OOOg. A single band 
was evident at the level of the 1.5M sucrose layer. This 
was found to contain nearly all of the histamine, heparin. 















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65 


5-HT and succinoxidase that had been layered over the gradient 
indicating no further separation was achieved by the second 
gradient* 

This should not be constructed as an Indication that in 
the ascites cells, the amine-containing particles are identical 
with some cf the mitochondria* Had this second gradient pro¬ 
duced a fraction with succinoxidase activity, but containing 
no amines, we could have definitely ruled out the identity 
of these particles* The only fair interpretation of the data 
is that there is In these cells a population of mitochondria 
which behaves Identically to amine-containing particulates 
during cerbain centrifugati on procedures. Electron micro¬ 
scopy of the intact cells suggests that the amine granules 
and mitochondria are different* Electron microscopy of the 
isolated fractions will be done shortly and should help 
settle the problem* At the present stage of purification, 
the amine-containing particles in fraction 3 are not suf¬ 
ficiently uncontaminated by mitochondria to permit the precise 
studies needed to characterize the granules chemically. Other 
techniques will be employed to improve the separation. 

Monoamine Oxidase and Histaminase (Diamine Oxidase ) 

The attempts to detect the presence of these enzymes by 

liberation of ammonia from substrates such as tyramine, 5-HT 

107 

and histamine by the method of Cotzias and Greenough met 
with no success. In a few experiments on monoamine oxidase, 
small amounts of armonia were produced, but the production 



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66 


was not inhibited by known monoamine oxidase inhibitors 
such as iproniazid. No evidence for histaminase was 
found. 

The sensitive spectrophotometric assay for monoamine 
oxidase"** 1 ^ failed to reveal any enzyme present in mitochondria 
from large numbers of the ascites, as well as the solid tumor 
cells. It could be calculated from the number of cells used 
that if they do possess this enzyme, they contain less than 
l/300th the amount present in mouse liver. 

The absence of both MAO and histaminase in these cells 

is to be contrasted with the presence of MAO and absence of 

17 

histaminase in the Furth mastocytoma. It would be very 
interesting to determine if noimal mast cells possess these 
enzymes. Green points out that cells which contain biogenic 
amines, almost invariably contain enzymes which inactivate 
them.^^^ Thus, these amines must normally be protected from 
the degredative enzymes, usually by granule sequestration. 

The absence of these enzymes in the Dunn-Potter mastocytoma 
cells allowes the cell wide latitude in its handling of the 
amines, and it thus is less surprising that the tissue culture 
form of this tumor possesses no granules, but may store its 
amines either in t he mitochondria or the free cytoplasm as 
previously discussed. No catabolism of histamine or 5-HT 
by any enzyme system has ever been found in this tumor, 
though it has been extensively looked for.^ 0 *^ This opens 
up the interesting question of what prevents the cells from 
continually accumulating amines. 


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67 

summary 


1, The basic structure of the Dunn-Potter mastocytoma 
X-X-C cells grown in tissue culture and the mouse peri¬ 
toneum (Ascitic cells) as seen in the electron micro¬ 
scope, is described* These cells are 7-15 millimicrons 
in size, possess in common 1-2 nuclei usually irregular 
in shape, a plasma membrane, a well-developed agranular 
reticulum, but sparse endoplasmic reticulum and lipid 
inclusions* The ascites cells possess large vacuoles 
which do not appear in the tissue culture cells* 

2 0 The ascites cells possess varying numbers of oval 
granules about 0*05 and 0*2 microns. They have few raite- 
chondri a. 

3 # The tissue culture cells possess few or no granules 
and large numbers of mitochondria* The possible storage 
of amines in these cells in mitochondria is discussed. 

The polymorphism of the tissue culture cells is described, 

!}.« A new theory for the origin of the amine granules and 
how they get the amines they contain, is proposed and 
supported* Collections of microvesicles and micogranules 
of the agranular reticulum appear to be reorganizing within 
dilitations of the agranular reticulum into mature granules* 
Chemical assays and evidence from the literature are ad¬ 
vanced in supports of this interpretation, 

5* It is proposed that the amines after synthesis by the 
cytoplasmic decarboxylases are secreted into tbs granular 
reticulum which then is formed into the granules. 













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68 


6. Treatment of the tissue culture cells with re- 

—7 ~9 

serpine at 10"'M and 10 M for 2k hours, which re¬ 
leases approximately 100^ and 50 % of the histamine 
and 5>-HT of these cells respectively results in the 
formation of ’’osmophilic dense bodies” in many of the 
cells* It is proposed that these osmophilic dense 
bodies aie lysosomes. A number of possible mechanism 
whereby the lysosomes might bring about amine release 
are discussed. 

7# The release of granules in an unusual reserpine- 
treated cell which strikingly resembled normal mouse 
mast cells is described. 

8. The possible relationship to amine release of certain 
membrane-enclosed clear areas of cytoplasm in the re- 
serpine-treated cells is discussed, 

9, Gross derangement of the structure of the mito¬ 
chondria of chiorpromazine-treated tissue culture cells 
is illustrated. 

10. The morphology of the large granule fraction of the 
tissue culture cells is described. It consists mainly 
of mitochondria, 

11, The separation of the mitochondria and amine granules 
of the tissue culture cells by density gradient contri- 
fugation is described. One fraction contained $6% of the 
mitochondrial enzyme activity and no histamine, heparin 



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69 


or5-HT# Another fraction ccntained 100$ of the 5-HT 
and histamine, 53$ of the heparin, but only 28$ of the 
mitochondrial enzyme activity# 

12 0 The absence of monoamine oxidase and histaminase 
in t he Dunn-Potter mastocytoira is discussed* 


.... "ic . 

tl -:.a: ;' 


* - 

. '■ : ■■ • ’’ » 

, 


70 


Fig. 1: A typical ascites Dunn-Potter mastocytoma 

cell showing the granules (G) which are easily 
distinguishable from the mitochondria (M). 

The nucleus (N) is in the upper left-hand 
corner* Vesicles of the agranular-reticulum (AR) 
are scattered throughout the cytoplasm. The 
highly active cell surface ingesting extra- 
cellur fluid is apparent. (Xl5,000). 

Fig. 2 ; High magnification picture of ascites masto¬ 
cytoma cell showing membrane-bound granules (G) 
some cf which can be seen to be made up of still 
smaller granules and vesicles. Nucleus (N) with 
a double membrane and abundant agranular reticulum 
(AR) are apparent. (X35*000). 

Fig. 3: An ascites mastocytoma cell. In the cytoplasm 

adjacent to the nucleus (N) are aggregates of micro¬ 
granules and microvesicles (Gl, G2, G3). The 
vesicles in Gl are clearly enclosed within a mem¬ 
brane. The similarity between these micro-granules 
and microvesicles and those of the agranular reticu¬ 
lum (AR) (see also Fig. 2) is clear. In Gl, G2, and 
G3,, some of the microbodies appear to have clumped 
together, producing areas resembling the mature 
granules seen in Fig. 2. Gl, G2 and G3 could be 
early foims of the mast cell granules, thus indi¬ 
cating the agranular reticulum as the origin of 
the granules. (X30,000). 





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LIBRARY 


MVDD D EUVEK.V 





















71 

Pig. 1 



















c- 


















72 

Pig. 2 




























73 

Fig. 3 





















Figs, k, 5 


Fi£^6: 


Fig. l i 


FA g . * . 8 : 


7^4- 

Two typical tissue culture mastocytoma 
cells* Nucleus (N) with double membrane 
(NM) is evident as are a great many mito¬ 
chondria (M). A few cytoplasmic inclus¬ 
ions 3eem to lack cristae and could be 
either degenerated mitochondria or granules 
(A). The border (B) between two adjacent 
cells is evident in Fig, as is a large 
lipid body (Li), (Pig, Ij. Xl8,000; Fig. 5, 

X16,000). 

One of the few tissue culture cells 
in which the endoplasmic reticulum (ER) 
was seen. 

High power picture of tissue culture 
mast cell with cytoplasm filled with 
numerous mitochondria (M), agranular 
reticulum (AR), and two lipid bodies 
(Li), Note absence of granules. (Xl|-3,000) * 

Several tissue culture cells closely 
adherent to one another with small, membrane- 
bound granules (G) scattered through the 
cytoplasm and easily distinguishable from 
the mitochondria (M). 






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Pig. 8 
































80 


Fig# 9: A tissue culture cell with many very 

bizarre inclusions (L). They resemble 
somewhat the vacuolated dense bodies 
(probably lysosomes) seen after reserpine 
treatment of these cells# (X13>000). 

Fig# 10 : A tissue culture mastocytoma cell 

treated with reserpine. Note the granules 
(G), some of which are within the cell and 
surrounded by a c3e ar area of cytoplasm, 
and other granules which have been or being 
extruded at the cell surface in the upper 
right-hand corner# No nucleus is present 
but this may be due to the plane of section¬ 
ing# Mitochondria (M) and lipid bodies (L) 
are present# Note the difference between 
this cell and the other tissue culture cells 
(Figs. Ip—7) and the similarity to a normal 
mouse mast cell (Fig. 12). (X12,000). 

Fig# 11 : A high power shot cf the same cell seen 

in Fig# 10. The granules (G) are seen more 
clearly as well as the extrusion of granules 
at the cell surface# (X30,000). 

Fig# 12 : A normal mouse mast cell. Note how few 

granules are present and the clear area around 
"each granules." Nucleus (N) and mitochondria 
are apparent. Compare with Fig. 10 (X7,000). 






08 


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85 


Fig* 13 : 


Portion of an unusual mastocytoma cell, 
much larger than any other cell, with four 
nuclei (N). Mitochondria (M), granules (G), 
and lipid bodies (Li) are seen. Many of 
the granules are made up of microgranules 
(Gi, G2, G3). Note the similarity between 
these granules and those of the ascites 
cell* (Figs. 2, 3). Small granules (SG) 
are scattered throughout the cytoplasm. 


(X12,000)• 


Fig * ll; : Another region of the same cell seen 

in Fig. 13. Gl, G2 and G3 are granules 
whose fine structure is clearly evident. 
Gl| is a mature granule. Numerous small 
granules, mitochondria, and agranular 
reticulum are scattered throughout the 
cytoplasm. (X12,000). 

Fig* lli : An unusual tissue culture cell with 

large clear areas in the cytoplasm and 
two large lipid inclusions (Li). The 
clear areas (G) probably represent 
granules. Mitochondria (M) are located 
between the granules. (Xl8,000). 










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Fig. 16 : Portion of a tissue culture cell treated 

-9 i 

with reserpine at 10 M for 24 hours. Numerous 
vacuolated dense bodies are seen (L), Large 
vacuoles (V) containing smooth membranes and 
osmophilic matter are evident. The vacuolated 
dense bodies could be formed from the material 
in these vacuoles. The cell membrane is in¬ 
tact. (xi5, ooo). 

Fig. 17 : Another cell filled with vacuolated dense 

bodies (L). The relationship of these to mito- 
choldria (M) is evident at the areas where the 
mitochondria are marked. The mitochondria 
appear to be undergoing dissolution in con¬ 
nection with the formaticn or activity of 
the vacuolated dense bodies. (Xlf?,000) # 

Fig. 18 : Two tissue culture cells treated with re¬ 

serpine at 10"% for 24 hours. The arrow in¬ 
dicates a granule which is being extruded from 
or budded from one cf the cells. A granule (G) 
is apparent in the second cell. (X20,000). 

Fig. 19 : Tissue culture cell treated with reserpine 

-9 i 

at 10 M for 24 hours. Membrane -bound amorphous 
cytoplasmic areas (I-IV) at the cell surface and 
deeper within the cytoplasm may have been pro¬ 
duced by d3s charge of cytoplasmic constituents 
at the cell surface. Gaps in plasma membrane 
are evident in areas I, IV, V. (Xl5,000). 








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Figs* 20-22 : Tissue culture cells treated with 

chlorpromazine at 10 M for 12 hours* 
Practically all the mitochondria present 
have grossly distorted internal structures. 
Note that the outer membrane of some of 
the mitochondria is partially absent 
as well. Some of the mitochondrial mem¬ 
branes have a whirled configuration, 

(Fig. 20, X20,000; Fig. 21, X25,000; 

Fig. 22, X15,000). 

Fig* 23 : Normal mitochondria of tissue culture 

cells for purposes of comparison with cells 
treated with chlorpromazine. Note two 
parallel membrane s of the cristae which 
are perpendicular to the outer mito¬ 
chondrial membrane. See also Figs. 4-8* 
(X30,000). 

Fig. 24 *• The large granule fraction from the 

ascites cells consisting mainly of battered 
mitochondria. A few granules are evident 
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(X20,000). 





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INTRODUCTION 


The first evidence for the ocidative deamination of 

amines by living tissues was the discovery in 1928 of a 

!liE> 

liver enzyme which oxidized tyramine, ^ Further studies 

in the 1930's on the enzymes which metabolize amines through 

oxidative deamination led to the concept that were was a 

single enzyme which degraded diverse monoamines such as 

tyramine, the catecholamines, and aliphatic amines, and 

another which oxidized diamines such as putrescine, the 

former being named monoamine oxidase (MAO), and the latter, 

146-148 

diamine oxidase, ' It was the idea that MAO could be 

related to central nervous system function that made it 

such an interesting enzyme to investigate. In 1940, Mann 

and Quastel observed that amphetamine inhibited MAO in vitro 

and suggested that the stimulant effect of amphetamine might 

be due to inhibition of MAO in vivo by blocking the formation 

of certain amine degradative products which they believed had 

lli 9 

a depressant effect# However, no inhibition could be 
demonstrated in vivo after administration of sufficient 
amphetamine to produce stimulation. The next stimulus to 
work in this area was the finding by Zeller et al,, 
that isoniazid and iproniazid, "hydrazines" that were active as 



l 


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Ott i 


Iproniazid 

anti-tuberculous agents and which clinically had the "side- 

15>1 

effect" of being central stimulants, were potent inhibitors 












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101 


of MAO in vitro and in vivo . Subsequent studies demonstrated 
that reserpine pretreatment produced marked excitation rather 
than depression when given to animals pretreated with ipron¬ 
iazid and that it no longer possessed its capacity to lower 

1^3 

brain 5-HT and catecholamine levels in these animals, 9 
These observations led to the testing of iproniazid as an 
antidepressantThe success of this clinical trial and 
the assumption that the mode of action of iproniazid was 
the inhibition of MAO attracted a great many investigators 
to this area. Since this finding, hundreds of basic and 
clinical studies of MAO and its inhibitors have been carried 
out in an effort to explore the relationship of this enzyme, 
its substrates, and its inhibitors, with mental illness, 
affective states such as depression and stimulation, angina 
pectoris and many other conditions. 

A possible explanation of the anti-depressant action of 

the monoamine oxidase inhibitors (MA0I) was soon forthcoming 

with the finding that the MA0I caused the accumulation of 

brain 5-HT and catecholamines, presumably by preventing their 
1^-157 

breakdown. The accumulation of these amines which are 

believed to function as neurotransmitters, was, and is be¬ 
lieved by some to exert an ant i -dep re ssant effect per se. It 
should be mentioned that rigidly controlled clinical studies 
on the efficacy of the MA0I as anti-depressants have not always 
yielded unequivocal results; and because of the toxicity of these 
compounds, they are now used infrequently as compared to imi- 
pramine (Tofranil) which is not a MA0I and which most studies 




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102 


have shown to be a more effective and less toxic mood 

. 1^9 

elevator). 

The multiplicity of effects of the hydrazines on other 
enzymes and on various parameters of amir© biology other than 
just accumulation of amines, have greatly complicated the ex¬ 
planation of the anti-depressant action of these compounds. 

The hydrazines have been shown to inhibit diphosphopyridine 
nucleotidase,* 1 * 00 spermine oxidase,guanidine deaminase 
succinic dehydrogenase ,-^3 his tarn ina se, and even the de¬ 

carboxylase which brings about amine synthesis from precursor 
amino acids (by interfering with coenzyme of the decarboxylase, 
pyridoxal - 5' - phosphate), " 9 The MA0I potentiates the 

•I L’-i 

action of various pharmacologic agents, f including the bar- 
168 

bit urates. The hydrazines have been shown to inhibit the 

169 

release by reserpine of 5-HT from rat brain, ' as well as 

inhibiting the release of NE from various storage sites, 

Schanberg and Giarraan have shown that phenylisopropyl-hydrazine 

produces an increase in the proportion of amines which are 

’’bound” (within granules) rather than free (in the cytoplasm). 

Iproniazid increases the amount of both forms of the amine 

proportionately and does not change its distribution,* 1 *^ 

172 

Dubnich et al, have shown that 5-HT levels in rat brain 
continue to increase in response to doses of phenethylhy- 
drazine which exceeded the dose required for the complete 
inhibition of MAO, This was though to be due to the blockade 
of amine release. Others have shown that the various MA0I 
increase the amount of rat brain adenonsine triphosphate and 


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103 


suggest this augmented energy supply is the reason for 

I 73 

the anti-depressant effect* Gey and Pletscher have 

shown that MAOI can produce increased blood lactic acid 
17k 

levels. There are many studies which demonstrate 

peripheral effects of the MAOI such as sympathomimetic 

17 cj 176 , 177 , 77 . 

effects, ganglionic blockade (but see also ) 

172a 

and adrenergic blocking action. 

Despite the embarassing wealth of findings, the most 
reasonable explanation of the mood-elevating effect of the 
hydrazines, is the inhibition of MAO and blockade of amine - 
release, leading to the accumulation of brain 5 -ELT and 
possibly other amines. The main evidence in favor of this 
choice is that many compounds which inhibit MAO seem to have 
an anti-depressant effect irrespective of their structure, 
although their is no correlation between in vitro and 
in vivo studies. (Usdin aid Usdin examined an exhaustive 
series of psychotropic compounds for MAO inhibition in vitro 
They found some but not all hallucinogens, tranquilizers and 
anti-depressant s were MAOI in vitro and concluded: "No 
obvious relationship between nsvchotropic action and MAO 

*1 nO 

inhibition can be discerned.” [:> a As previously mentioned, 
however, studies in vitro are of limited value in this 
field because of a host of factors, such as metabolism of 
the agent in vivo and regional specialization of the brain 
with regard to such properties as biochemistry and perme- 

N 

ability barriers). Additional evidence for the relationship 
between elevated 5-HT levels and anti-depression is that 
other modes of elevating the brain amines such as administer 









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ing the precursors of the catheeholaraines, dihydroxypheny¬ 
lalanine, or the precursor of 5-HT, 5 hydroxytryptophan, 
are associated with c entral stimulati on, Bonny - 

castle ejt al. have shown that a variety of central depressant 
drugs, including sedatives, hypnotics, analgesics, fixed 
and volatile anesthetics, produced a significant elevation 
of rat brain 5-HT, His data suggested that the 5-HT ele¬ 
vation was secondary to the central nervous system de- 
pression, not the cause of it). Shore x a noted a 
temporal relationship between the pharmacological effects 
of iproniazid and an increase in the levels of brain 5>-HT 
and NE. The many studies relating amines to behavioral 
changes induced by other drugs such as reserpine are also 
in favor of this hypothesis. Indeed, the know ledge that 
iproniazid, an antidepressant, raised £-HT levels in the 
brain, while reserpine, a tranquilizer, lowered 5-HT in 
brain,stimulated great interest in the study of brain 
biochemistry and pharmacology. 

To compound the interest in the MAOI, studies show 

1 ? 8 , 179 

their efficacy in relieving angina pectoris their 

hypotensive effect,'*’^ their anti-convulsent effect, 

l82 i81_L 

their anti-inflammatory effect, * an analgesic effect, 

18 £ 

and even an anti-fertility action. They are clearly a 
fascinating group of compounds. 

In most cases, only the hydrazines have been studied in 
these experiments. Whether or not the many other classes 
of compounds which inhibit MAO and which are not hydrazines 








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105 


possess these properties as well, remains to be investi¬ 
gated. Among the other classes of inhibitors of MAO which 

have been reported, are agents which combine with sulfhydryl 

186 ...... n , , 1^7 , _ . 188 

groups, aliphatic alcohols, chlorpromazme , the 

189 190 

harmala alkaloids, p-tolyl ether of choline, trans-2- 

191 

phenylcyclopropylamine (tranylcypromine), ^-methyl-and 

192 

q'-ethyltryptamine (etryptamine ), N-benzyl-N-methyl-2propyny- 
1 amine (pargyline), N-methyl-^ -methyltryptamine,and 

others. (The work to be described here on quaternary 
nitrogen compounds as inhibitors of MAO was begun when only 
the hydrazines were established as MA0I and enthusiasm for 
MA0I as anti -depressant s was at its height}. 

Certain properties of t he hydrazines as MA0I need to be 
pointed out to place in proper context some of the author's 
studies of the quaternary nitrogen compounds. The hydrazines 
and tranylcypromine require preincubaticn with MAO in order 
to exert their maximum inhibitory effect, whereas the harmala 
alkaloids do not require preincubation. The hydrazines and 
tranylcypromine are irreversible inhibitors once they have had 
prolonged contact with MAO, manifested by the fact that ex¬ 
tensive dialysis does not diminish the extent of inhibition. 
The harmala alkaloids are reversible, competitive inhibitors. 
Both of these classes of compounds will raise brain 5-HT and 

catecholamine levels in various organs (e.g. in brain and 

. 189, 195, 196 

heart) of many species, including the rat. 

Studies of these inhibitors in vitro have invariably been 

done on particulate MAO because it has never been entirely 



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106 


freed from its mitochondrial matrix* In one study, MAO 

solubilized with the detergent isoctylphenoxypolyethoxyethanol 
197 

was used. In the presence of this solubilizing agent, MAO 
activity will not sediment despite centrifugation conditions 
sufficient to bring down microsomes. (Recently MAO has been 
obtained from homoginatea subjected to sonic oscillations 
in a form which did not sediment after centrifugation at 

19 6 

35,000g for 30 minutes without the presence of a detergent). 

While investigating new inhibitors of MAO, the author dis¬ 
covered that various confounds which contained a quaternary 
nitrogen atom, usually in the form of a quaternary pyridinium 
moiety, were effective MAOI. At first, it seemed that the 
possibility of this class of compounds being of clinical 
or even of investigative use, was slight because of the 

evidence that highly ionic molecules cannot penetrate the 

199 

blood-brain barrier. However, it seemed likely that a 

long-chain N-alkyl group would aihance the lipid solubility 

of the quaternary pyridinium molecule and thus allow it to 

penetrate the blood-brain barrier, which is known to be most 

200 

permeable to compounds of high lipid solubility. With 
this in mind, pyridine aldoxime dodecyliodide (PAD), which 
has a quaternaxy pyridinium nucleus with a long chain N-alkyl 
radicle, was investigated as a possible inhibitor of MAO. 






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PAD was conceived and synthesized by Wilson and Gins burg 

to obtain a more lipid-soluble analog of pyridine aldoxime 

202 203 
methiodide (PAM) which was designed and shown by Wilson" 

• * 

to be 



an extraordinarily effective reactivator of cholinesterase 

inhibited by organophosphorous compounds such as diisopropyl- 

fluorophosphate* Wilson reasoned that because of the R-alkyl 

radicle PAD would be more likely than PAM to penetrate the 

blood-brain barrier and to reactivate organophosphorous- 

inhibited brain cholinesterase. PAM itself has been shown 

to enter the brain although no similar direct evidence 

is available that PAD can pass the blood-brain barrier. 

(The studies presented in this paper strongly suggest that 

PAD and related compounds gain entrance into rat brain). 

PAD (in conjunction with atropine) has been shown to be an 

effective antidote in poisoning, due to organophosphorous 

205 

cholinesterase inhibitors. In addition to reactivating 

cholinesterase, PAD can also inhibit cholinesterase, block 

conduction at the node of Ranvier, increase the permeability 

20 & 

of nerve fibers to sodium ions and depolarize resting 
207 

nerve fibers. Some of these effects have been attributed 

to the reaction of PAD with the postulated acetylcholine 

2o6 

receptor protein along the nerve axon.” "" The work presented 
here shows that, in addition to these effects on acetylcholine 




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108 


metabolism and on the proteins associated with it, PAD 

exerts two important effects on the metabolism of 5-HT: 

(i) inhibition in v itro of MAO, the enzyme that is mainly 

responsible for the catabolism of 5-HT; and (ii) release 

of 5-HT from brain and mast cells in which 5-HT may be 

bound to particulate components. Most of these studies 

209, 210 

with PAD have been published. 

The studies with PAD indicated it would be very in¬ 
teresting to investigate dodecyl iproniazid iodide, the 
long-chain N-alkyl derivative of iproniazid* This 



Dodecyl Iproniazid iodide 

compound would have two functional groups that could in¬ 
hibit MO, at least in vitro , as well as being able to re¬ 
lease 5-HT. Synthesis of this compound was accomplished 
in the Lederle Laboratories and a small amount was graciously 
supplied to the author* 

E arly in the study of the effect of quaternary nitrogen 
compounds on MO, the author felt that it might be profitable 
to investigate the role of thiamine in MO becau.se thiamine 
has a quaternary nitrogen in its thiazole moiety* 



Thiamine Hydrochloride 
















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Freliminaiy studies with thiamine - deficient animals 

revealed brain MAO to be elevated, which could come 

about if thiamine or a metabolite of it were natural 

endogenous MAOI. The elevation of brain MAO in thiamine- 

deficient rats was independently confirmed and published 

211 

by Gal and Drewes who, whoever did not speculate on 
any role thiamine might play as a MAOI. A few experi¬ 
ments and an intensive literature search which unearthed 
a number of relevant findings enabled the author to 

publish some speculations about the effect of thiamine on 
212 

MAO. These will be detailed in the Discussion section 


of this thesis 


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MATERIALS AND METHODS 

5-HT creatinine sulfate and d1-5-hydroxytryptophan 
(5-KTP) were obtained from the California Corporation 
for Biochemical Research, Kynuramine was obtained from 
the Regis Chemical Coupaiy; decamethonium, hexamethonium, 

PAM, and cetyl pyridinium iodide were purchased from t he 
K & K Chemical Company. Stigmonene bromide was a gift of 
the Warner-Chilcott Company. Iproniazid phosphate was a 
gift from Hoffman-LaEoche, Inc. Catrcn (JB-516 was a gift 
from Lakeside Laboratories. Haimaline was a gift of Pro¬ 
fessor Nicholas G-iarman of the Yale Department of Pharma¬ 
cology. Thiamine and thiamine pyrophosphate were purchased 
from Schwartz Biochemicals, as was diphosphopyridine nucleo¬ 
tide. Succinylcholine, d-tubocurarine, neostigmine and 
N-methyl nicotinamine were obtained from various commerical 

sources. PAD was synthesized by the method of Wilson and 
60 

Ginsburg. Long-chain alkyl pyridinium compounds were syn- 

213 

thesized by the method of Knight and Shaw. Dodecyl 
iproniazid iodide was synthesized by the Lederle Laboratories; 
the author is very grateful to Dr. Paul Bell, Director of 
Biochemical Research, Lederle Laboratories, for supplying 
this compound* 

5-HT was determined spectrophotome trie ally by the method 
of Udenfriend et al t ^ Numerous experiments were performed 
to show that PAD does not interfere with the determination 
of 5-HT by this method. Catecholamines were determined by 
a spectrofluorometric techniques according to the method 


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110 


MAO activity was assayed spectro- 

photometrie ally All ancubations were for 10 minutes 

within the housing of a Beckman DU spectrophotometer* 

The results obtained were occasionaly checked by another 

method, involving the liberation o f ammonia* 5-HTF 

decarboxylase was determined by the technique of G-addum 
215 

and Giarrnan, Mitochondria were prepared from rat liver 

216 

according to the method of Hogeboom, and frozen at 

-20°C until needed. Each milliliter of the mitochondrial 

suspension used contained the mitochondria from 1 g of liver 

(wet weight). MAO from such mitochondrial preparations was 

"solubilized'' by the method of Zeller jet al. All compounds 

were administered to animals intraperitoneally. The rats used 

were males of approximately l£0 -g weight (Sprague-Dawley 

strain). The mice were DBA/2 males weighing about 20g, The 

mast cells used were the Dunn-Potter murine mastocytoma, 

Q6 

ascites form.' PAD was dissolved in a solution containing 
NaHCO- 1 mg/ml. In all experiments involving PAD, the 

-> 9 

control animals were given the NaHCO^ solution. 







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EXPERIMENTAL RESULTS 


(1) Effect of PAD on the MAO activity of homogenates 
of liver and brain of various species . 

Table 1 demonstrates that PAD can inhibit the MAO of 
both liver and brain of a number of species. Because of 
the crudeness of these preparations, it is not possible 
to say whether MAO of brain or liver is more susceptible 
to inhibition by PAD. 


TABLE 1. EFFECT of PAD on the MAO activity of LIVER and BRAIN 

HOMOGENATES of RAT t RABBIT, GUINEA PIG, and MOUSE 

% Inhibition of MAO 

, Liver . Brain ^ 

Species 10“%* * 10 M 10“% lO^M 


Rat 

90 

25 

41 

16 

Rabbit 

69 

20 

60 

20 

Guinea Pig 

100 

20 

40 

12 

Mouse 

66 

18 

45 

15 


The brains and livers of adult animals killed by 
decapitation were excised, homogenized in 4 vol of cold 
distilled water, and filtered through cheesecloth; 0.2 
mi of brain homogenate and 0.1 mi of liver homogenate 
were used for each assay of MAO activity. All assays 
were done in duplicate with less than 5$ variation. 

* Concentrations of PAD. 









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(2) Effect of PAD on MAO activity of washed rat liver mlto 
chondria and compariscn of PAD with other inhibitors of MAO , 

Table 2 shows that the inhibition of MAO in vitro , obtain¬ 
able with PAD, is substantial. As can be seen in Table 3> PAD 
is less potent than is JB-516 (Catron, one of the most potent 
hydrazines used clinically) as an inhibitor of MAO in vitro , 
but is more potent than iproniazid. Table 3 also indicates 
that a number of long-chain N-alkyl quaternary pyridine com¬ 
pounds can inhibit MAO in vitro . 


TABLE 2. EFFECT of PAD on MAO activity of Washed BAT LIVER 

MITOCHONDRIA in vitro 


Coneentration of PAD % Inhibition of MAO activity 


10"^M 100 

5 x 10"?M 75 

10"?M I|X) 

4 x 10"°M 20 


Preincubation of enzyme and PAD was not performed. 

Four deteiminations at each concentration of inhibitor 
were performed with less than 5$ variation. 


TABLE 3. COMPARISON of PAD and Related Compounds with Pheny- 

lisopropyl-hydrazine and iproniazid as MAO inhibitors 
in v it no 

Inhibitor (10~^M) % Inhibition 


JB-516 

100 

PAD 

40 

Isonicotinic acid dodecyliodide 

40 

Dodecyl pyridine iodide 

25 

Tetradecl pyridine iodide 

20 

Cetyl pyridine iodide 

20 

Iproniazid 

5 


JB-516 and iproniazid were preincubated with the 
washed rat liver mitochondria for 15 min. before 
kynuramine was added. Four assays with each in¬ 
hibitor were performed with less than %% variation. 















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114 


(3) Effect of pre incub at ion on the Inhibition of 
rat liver mitochondrial MAO by PAD . 

PAD, unlike hydrazine-derived inhibitors of MAO, does 
not require preincubation to exert its maximal inhibitory 
action on MAO. Table 4 gives the results of an experiment 
showing that pre Incubation of MAO with PAD produced no 
effect on the inhibition obtained. 


TAB IE k • EFFECT of PREINCUBATION on the INHIBITION of 


MITOCHONDRIA by PAD 


Concentration of PAD 

MAO activity 

(^O.D./IO min) 


No preinc. 

Preinc. 

4 x 10"%! 

0.172 

0.170 

0.100 

0.100 


The substrate, kynuramine, was added to the re¬ 
action mixture prior to the addition of MAO, or 30 
min. after MAO. In both series, reactions were run 
in duplicate with and without PAD. 







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115 


(I}.) Effect of dialysis on the inhibition of rsit liver 
Mitochondrial MAO by PAD , 

The hydrazine inhibitors irreversibly inhibit MAO, while 
harmaline is a reversible inhibitor. 7 The inhibition due 
to PAD is not reversible by extensive dialysis, despite the 
fact that no preincubation is necessary. The results given 
in Table 5 show that, in an experiment comparing harmaline 
and PAD, only the harmaline inhibition of MAO was reversible. 

TABLE 5* Effect of Dialysis on PAD and Harmaline Inhibition 

Mitochondrial Mao 


A0.D./10 min/l ml of mixture 
Inhibitor » No dialysis Dialysis 

“ ——— "~--' j o7i 3 o r T — 0.110 

PAD (1.5 x 10"-%) , 0.0 0.0 

Harmaline (1.5 x 10"%l) Q.0-* 0.60 


Of the mitochondrial suspension, 0.4 ml was added 
to 7*6 ml of a solution of NaHCOo, 1 mg/ml. The same 
amount of mitochondria also was added to 7*6 ml of the 
NaHCO^ solution which contained PAD at a concentration 
of 1*5 x 10- 7 M. Each mixture was divided in half. 
One-half was dialyzed for 24 hr. against 3 changes of 
4 I., of d istilled water at 4° C; the other half was 
left in a test tube at 4° G * After 24 hr* 1-0 ml. of 
each mixture was test for MAO activity. The same 
experiment was performed simultaneously with harmaline 
at the same concentration (1.5 x 10"’ M). These ex¬ 
periments were performed twice with less than %% 
variation. 

■&MA0 activity could not be determined because 
haimaline absorbed too strongly at 360 mu at this 
concentration; however, harmaline has been reported 
to inhibit MAO completely at concent rations much 
lower than this.l^ 









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(5) Effect of PAD on solubilized MAO , 

It might be argued that, because of its detergent-like 
structure, PAD exerts a nonspecific solubilizing action on 
MAO. This was tested by observing the effect of PAD on MAO 
solubilized with isooctylphenoxypolyethoxyethanol. At three 
different concentrations of PAD, the percentage inhibition of 
the solubilized enzyme and of that in the mitochondrial sus¬ 
pension were the same (see Table 6) although the total 
activity of the solubilized enzyme was only one-fifth as 
great. This is in contrast with the findings of Cotzias 
et al.-*-97 who noted an increase in enzyme activity after 
solubilization• 


TABLE 6. EFFECT of PAD on "Solubilized" MAO, as compared 


with the Mitochondrial MAO 



% Inhibition 

% Inhibition 

Concentration 

of mitochondrial 

of solubilized 

of PAD 

MAO 

MAO 

2.5 x 10“^ 

5.0 x io“£ 

100 

100 

74 

74 

1.0 x 10"> 

ko 

4o 


Cutscum was added to the mitochondrial suspension 
to make a *->% solution. It was shaken intermittently 
in the cold for 4 hr and then centrifuged at 100,000 
x g for 1 hr. The supernatant fraction was decanted 
and tested for MAO activity and inhibition by PAD. 
This was compared with the activity and inhibition 
obtained with another aliquot of the mitochondrial 
MAO. All assays were done in duplicate with less 
than 5$ variation. 










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(6) Ability of PAD to inhibit MAO in vivo . 

PAD was administered intraperitoneally in various 
regimens to rats which were then sacrificed at varying 
times after the last dose* The range of dosages was from 
10 to 16 mg/kg for 1-7 days, and the elapsed tin® after 
the last administration of PAD was 1-5 hr. Brains and 
livers were treated as in (1). Little difference could 
be detected by this method in the activity of the homo¬ 
genates on MAO of control snd treated animals, whereas 
animals given comparable dosages of the hydrazine in¬ 
hibitors showed extensive or complete inhibition of MAO. 

(7) Effect of PAD on the levels of 5-HT in rat brain . 

A sensitive indicator of MAO inhibition, Mi vivo, is 
an increase in t he brain level of 5-HT. It was the re foie 
of interest to determine whether PAD could raise this level. 
The results, shown in Table 7, indicate that PAD lowered 
rat brain levels of 5-HT when these were determined Ij. hours 
after the last administration of PAD; 2l| hours after a 
dose that produced extemsive lowering of 5-KT in brain, 
the level bad returned to normal. 












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118 


TABLE 7* Effect of PAD on 5-HT levels in Brain 


PAD No. of days Change 

(mg/kg) administered (% +) 


1. 

30 

1 

-15 + 2 

2. 

15 

7 

-36 + 4 

3. 

20 

2 

-40 + 4 

4. 

50 

2 

- 61 + + 5 

5. 

65 

1 

No change 24 hr 




after injection 


Drags were given intraperitoneally. fiach 

t roup consisted of 6 male rats. In the first 
experiments the treated animals and controls 
were killed by exsanguination 4 hr after PAD 
and their brains homogenized in 5 ml of 0.1 N 
EC1. In the fifth experiment the animals were 
killed 24 hr after administration of PAD. 





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119 


(8) Effect of PAD on the catecholamine levels of the 
rat brain . 

No effect on rat brain catecholamines was observed when 
high doses of PAD were given in intraperitoneally for either 
short or long periods. 

(9) Effect of PAD on 5-hydroxytryptophan decarboxylase 
of rat kidney in vitro . 

PAD, 10 ^M, pro dined no significant inhibition of rat 
kidney f?-hydroxy tryptophan decarboxylase. 

(10) Effect of PAD on 5-HT of mast cells . 

The ascites mast cells of the Dum-Potter mouse masto¬ 
cytoma contains 5-ET which behaves as though it were bound 

218 

in granules in pharmacologic experiments. These cells 
are more suitable for studies on amine release than are 
the non-nucleated platelets that often are used. Table 8 
shows that PAD and other long-chain quaternary pyridinium 
compounds, but not PAM, released 5-HT from mast cells. 

JB-516 also caused a similar release. 








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TABIE 8. 


Release of 5-HT from Mast Cells 


Drug (0.5 mg/ml) % Release 


" -———~ 

PAD 100 
Tetradecyl pyridine iodide 100 
Cetyl pyridine iodide 100 
PAM 20 
JB-516 60 


The mast cells were removed from 
DBA/2 mice by aspiration of the peri¬ 
toneal contents, and suspended in Hank’s 
medium. 1 ' Duplicates were incubated in 
20-ml beakers at 37° for 2 hr in a 
Dubnoff shaker, with and without in¬ 
hibitors dissolved in Hank’s medium, g 
Each beaker contained approximately 10° 
cells. After incubation the cells were 
centrifuged and the supernatant fractions 
decanted. The cells were washed with more 
Hank’s medium, centrifuged, and the 2 super¬ 
natant fractions combined. The 5-HT content 
of both the cells and the supernatant frac¬ 
tions were measured spectrophotofluoro- 
metrically. There was less than 5% 
variation in the duplicates. 









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121 


(H) Effect of PAD and JB-516 on levels of 5-HT in 
rats after administration of reserpine . 

It is conceivable that the release of 5-HT by PAD in 
the rat brain masked some inhibition of MAO that otherwise 
would have been detected through increased levels of 5-HT* 

It was thought that the administration of PAD, after maximal 
release of 5-HT by reserpine, might then peimit the detection 
of MAO inhibition through the accumulation of newly synthe¬ 
sized 5-HT. Table 9 shows that inhibition of MAO could not 
be demonstrated in vivo even by this method whereas that 
caused by JB-516 is readily apparent. 


TABLE 9. 5-HT Levels after Reserpine followed by either 

PAD or JB-516 


Compound administered 

5-ht 

levels (jug) 

Range 

Reserpine 


0.09 

0.08-0.10 

Reserpine + PAD 


0.09 

0.08-0.10 

Reserpine + JB-516 


O.i.f.6 

O.U3-0.49 


Eighteen rats were given ii mg of reserpine 
per kg; 6 of these were given 15 mg of PAD 
per kg. and 6 were given 10 mg of JB-516 
per kg. 5 Hr after the reserpine. The 5-HT 
levels at 1 hr after the last injection were 
determined. 








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122 


(12) Effect on rats of pretreatment with PAD followed 
by reserpine, and pretreatment with reserpine followed by 

PAD . 

Pretreatment cf rodents with an inhibitor of MAO can 

17 ^ 

reverse the sedative effects of reserpine. Since PAD 
possesses some of the properties of each of these compounds, 
it was of interest to observe the performance of PAD in both 
these roles. As can be seen in Table 10, the behavior of 
PAD was not typical of an inhibitor of MAO, since it did not 
reverse the sedative effects of reserpine when given as 
pretreatment; rather, it delayed them. On the other hand, 

PAD did not act like reserpine, since it did not produce 
hyperactivity in rats pretreated with JB-£l 6 . 

TABLE 10* Effect on Rats of Pretreatment with PAD followed 

by reserpine, and of pre treatment with 
JB-516 followed by PAD 

Drug administered Behavioral observations 

Reserpine Sedation in 20 min 

PAD followed by reserpine Sedation in 60 min 

JB-516 followed by reserpine Enhanced alertness and activity 
JB-516 followed by PAD No change from untreated animals 

JB-516 No change frcm untreated animals 


Each group consisted of 6 animals; these were kept in 
individual cages. Observations were continued for 1+ hr. 
All doses (mg/kg) were: reserpine 5; PAD, 50; JB-£l 6 , 20. 
PAD was given for 2 days, the last dose 4 . days prior to 
reserpine or 30 min after JB-516. JB-51& was given 30 

min before PAD or reserpine. 











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123 


(13 ) Effects of various quaternary ammonium derivatives 
on MAO in vitro . 

Of many readily available compounds tested, decamethonium 

(8£% inhibition at 10“^M), hexamethonium (25>% at 10“^M), and 

-P 

thiamine ( 70 % at 10 M) exhibited weak inhibitory actions. 
Choline, acetylcholine, PAM, diphosphopyridine nucleotide, 
N-methyl nicotinamide, suecinylcholine, d-tubocurarine, 
stigmonene bromide, and neostigmine were inactive. Several 
short-chain N-alkyl derivatives of heterocyclic nitrogen 
compounds were also good inhibitors in vitro . 

(Ill) In vivo and in V itro Studies with Thiamine and 
MAO . 

As mentioned previously, studies by the author and 
211 

Gal and Drewes demonstrated that in thiamine-defic¬ 
ient rats the MAO-activity of brain and intestine is 
increased. However, rats given thiamine, 70mg/kg subcutane¬ 
ously, and sacrificed after 2 hours, x^hen thiamine pyrophos- 

220 

phate levels in liver are elevated, showed no inhibition 
of the MAO-activity of liver, brain, and intestine, as com¬ 
pared with untreated controls. Studies in vitro showed that 

-2 

thiamine at a concentration of 10 M inhibits the MAO of 
rat liver mitochondria 70%, whereas thiamine pyrophosphate 
is slightly less effective. 














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124 


(l£) Effect of Dodecyl Iproniazid Iodide on MAO 
In vitro . 

Table 11 demonstrates that dodecyl iproniazid iodide 
is an effective inhibitor of rat liver mitochondrial MAO 
without any preincubation, but that is an appreciably 
more effective inhibitor after preincubation. 

Table 11. Effect of Dodecyl Iproniazid Iodide on MAO 

Activity of Washed Rat Liver Mitochondria With 
and Without Preincubation 


Concentration of 

% Inhibition 

of MAO 

Dodecyl Iproniazid 
Iodide 

No Preinc. 

Preinc. 

1 x lO^l 


95 % 


The Substrate, kynuramine, was added to the 
reaction mixture which contained dodecyl ipron¬ 
iazid iodide prior to the addition of mito¬ 


chondrial MAO, or 30 minutes after the MAO. In 
both series, reactions were run in duplicate 
with and without dodecyl iproniazid iodide. 











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125 


(16) Ability of Dodecyl Iproniazid Iodide to Inhibit 
MAO in vitro, 

Dodecyl iproniazid iodide, 20 mg/kg, was given intro- 
peri toneally to rats which were sacrificed four hours 
later. Liver specimens were treated as in (1), Dodecyl 
iproniazid iodide produced a 70$ inhibition of rat liver 
MAO under these conditions, 

(17) Effect of Dodecyl Iproniazid Iodide on the Levels 
of 5-HT in Rat Brain , 

Dodecyl iproniazid iodide, 20 mg/kg, was given intra- 
peri toneally to 6 rats which were sacrificed four hours 
later. Brains were excised and treated as in (7), Dodecyl 
iproniazid iodide increase 5-HT levels by 50$ under these 


conditions 








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DISCUSSION 


FAD as an Inhibitor of Monoamine Oxidase 

These studies demonstrate that the long-chain N-alkyl 
quaternary pyridinium compounds have a unique series of 
effects on brain amines and on MAO* PAD was studied ex¬ 
tensively as a prototype of this class, but some of the 
experiments performed demonstrate that these properties 
are probably possessed by most or all of the whole class 
of compounds. 

PAD ranks among the most potent MAO inhibitors in vitro 
when related to published comparative data from studies 
with iproniazid,^-53 ^<3 the a ufchor’s studies with PAD, 
iproniazid, and other MAOI. PAD irreversibly inhibited 
MAO in vitro , without preincubation. This sets it apart 
from the "hydrazines," which required pre incubation, and 
from the harmala alkaloids, which are reversible in¬ 
hibitors. Dodecyl iproniazid iodide, which was designed 
as a combination of a hydrazine MAOI and a long-chain 
N-alkyl pyridinium compound, was found to be an effective 
inhibitor without preincubation, but to be an even more 
effective inhibitor after pre incubation. This could be 
due to two separate modes of MAO inhibition: that attribu¬ 
table to the N-alkyl pyridinium moiety, not requiring any 
preincubation and accounting for most of the i\$% inhibition 
due to the hydrazine grouping requiring preincubation and 
accounting for the additional 50 % inhibition noted after 
30 minutes pre incubation. As mentioned above, the potency 






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127 


of PAD as an in vitro Inhibitor is considerable, and it 
is not unreasonable to predict that more potent inhibitors 
of MO, containing a quaternary pyridinium nitrogen, can 
be synthesized. 

Every attempt to demonstrate inhibition of MAO in vivo 
with these compounds was unsuccessful: (i) There was no 
decrease in the MAO activity of liver and brain homo¬ 
genates of PAD-treated animals; (ii) 5-HT levels in brain 
decreased rather than increased after treatment with PAD; 
(iii) PAD produced no increase in the 5-HT levels of rats 
pretreated with reserpine to release nearly all of the 5-HT 
initially present, thus eliminating the possibility that 
release of 5-HT by PAD was obscuring a simultaneous accumu¬ 
lation du© to inhibition of MAO. Rats given JB-516, a 
potent MAOI, readily accumulated 5-HT under the same con¬ 
ditions indicating that after the initial release by re¬ 
serpine and degradation by MAO, newly formed 5-HT would 
accumulate, though its binding might be impaired by the 
reserpine. In the PAD-treated animals no accumulation 
was noted, probably because MAO was not inhibited.(iv) 
Finally, there was no reversal of the reserpine-induced 
depression in rats pretreated with PAD. 

There are several questions to be considered in seeking 
an explanation for the absence of in vi vo -lnhib it ion of 
MAO. Can PAD penetrate the rat blood-brain barrier and 
reach the cells of the central nervous system? Can PAD 







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128 


enter the various types of cells of the rat liver? If 
penetration does occur, was the amount of PAD administered 
adequate to produce detectable inhibition? 

The evidence for the ability of PAD end other quaternary 
pyridiniums to penetrate the rat blood-brain barrier is the 
decrease in the 5-HT levels which PAD produces, and the in¬ 
crease in brain levels of 5-HT which dodecyl iproniazid 
iodide produces* The release of 5-HT from mast cells in 
vitro by PAD supports the hypothesis that the release is 
mediated directly by this agent rather than in some in¬ 
direct manner* There is the possibility that the releasing 
effect is produced by less PAD than is required for the in¬ 
hibitory effect on MAO, so that sufficient PAD enters the 
brain to produce the former but not the latter effect. 

The exclusion of PAD from the rat liver is another possi¬ 
bility that cannot yet be dismissed, although it is not a 
very attractive one. The demonstration of the inhibition 
of liver MAO by dodecyl iproniazid iodide makes this very 
unlikely* 

The question of adequate dosage is less troublesome, as 

can be shown by comparing PAD with iproniazid* After rats 

were given a single dose of iproniazid of 5 x 10“^ moles/kg 

body weight, MAO activity determined subsequently in vitro 

221 

did not return to normal for £ days* In the most vigorous 
attempt to demonstrate MAO inhibition by PAD, l*[j. x 10"^ 
moles/kg of PAD was given to each of the six rats daily for 




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129 


seven days, and no Inhibition of MO of brain or liver 
was detected in any rat, despite the evidence presented 
herein that PAD, like iproniazid, is an irreservible in¬ 
hibitor, and that the in vitro -assay used in these ex- 
110 

perimenfcs is more sensitive than is the one formerly 
221 

in use. Ancfc her possible explanation is that PAD is 

rapidly detoxified in vivo but not in vitro by the mito¬ 
chondria* Thus, in vivo , PAD which reached amine-contain¬ 
ing granules before reaching mitochondria could exert its 
releasing action, whereas the mitochondrial detlxification 
would prevent PAD from inhibiting MAO, a mitochondrial 
enzyme. If so, analogs of PAD that are not rapidly in¬ 
activated in vivo might be synthesized, and a useful MOI 
might result* However, recent studies of the metabolism 

222 ppo 

of PAM in man and in lower animals J revealed that the 
drug is excreted largely unchanged, although small amounts 
of a number of other metabolites were noted. Whether the 
same holds true for PAD remains to be established. 

Perhaps the best explanation for the difference found 

22k 

in vitro and in vivo is the recent work by Aebi. ' Aebi 
examined mitochondrial MAO in various states of preservation 
of the structure of mitochondria and showed that the enzyme 
behaved differently under each set of circumstances. The 
activity of MOI was also influenced by the morphologic 
state of the mitochondria. The effectiveness of the MOI 
studied>varied with the intactness of the mitochondria 









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leading Aebi to conclude that it was very difficult to 
predict what results would be obtained in the intact 
state (in vivo ) from studies on mitochondria that were 

223 

to some extent disrupted (in v itro ). Pletscher and Gey 

have noted many other compounds which inhibit MAO in v itro 

226 

but not in vivo * Werke et_ al. have recently reported 
that N-benzyl -N* - i sop ropy Ihydrazine inhibits MO in vivo 
but not in vitro and speculate that many effective inhibitors 
have probably been missed because most screening methods for 
new MAO I are of an in vitro nature. 

The weak ability of hexamethonium to inhibit MAO is of 
some interest in t he light of the recent demonstration 

176 

that inhibitors of MAO can produce ganglionic blockade. 

This is not to suggest that the effects of hexamethonium 

are due to MAO inhibition; rather, it is meant to point up 

the reciprocity between these two activities that might lead 

to an exploration of other similarities. It is interesting, 

moreover, that hexamethonium potentiated the effects of 5-HT 

227 

in some experiments with the isolated guinea pig ileum. 

Could this have been due to MO inhibition? 

In the same light, the actions of PAD on both acetyl¬ 
cholinesterase and acetylcholine receptors, and on MAO may 
be reiterated. It would be on interest to determine whether 
the influences of PAD on the release of 5-HT and on the 
activity of MAO are of any importance for understanding the 
observations of Dettbaxn and Wilson already alluded to.^°^ , ^°^ 















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220 

It is worthy of note that chlorpromazine' and tolazollne 
also inhibit both MAO and cholinesterase. 

PAD and Amine Release 

In experiments to determine whether PAD could increase 
5-HT levels as would be expected of a MAOI, it was noted 
that instead of increasing 5-HT levels, PAD actually 
produced a decrease in t he anount of the amine. The 
extent of lowering was found to be dependent upon the 
dosage of PAD. That 5-HT release accounted for the de¬ 
crease in 5-HT levels noted in Table 7 is indicated by 
the inability of PAD to inhibit 5-HTP decarboxylase and 
the ability of PAD to produce similar changes in the 5-HT 
levels of mast cells (Table 8). 

In the experiments with mast cells, the 5-KT which 
sedimented with the cells and which was present in the 
supernatant fluid was determined with and without treat¬ 
ment of the cells id.th PAD, other quaternary pyridinium 
ions, etc. All the 5-ET was recovered in either the super 
natant fluid or the cell mass. The quaternary pyridiniums 
including PAD, effected a transfer of the 5-HT from the 
cells to the supernatant fluid. This strongly suggests 
release of the amine. 

The amine-releasing action of PAD was an unexpected 
finding and added a new dimension of interest to the study 
of this compound. Firstly, because the mechanism of many 
of the clinically and experimentally valuable actions of 



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132 


the rauwolfia alkaloids, particularly reserpine has been 

230-232 

thought to be related to amine-release* It is 

likely that study of other amine-releasing agents can 
help illuminate this matter. PAD is particularly useful 
in this regard, because it is a matter of heated dispute 
whether it is through 5-HT or catecholamine depletion 
that reserpine brings about Its actions. This problem 

233 

has been approached by using drugs such as tetrabenazine, 

23 II 

O-noethyl-meta-tyrosine, ‘ conditions such as reserpine 

035 

plus hypothermia, or a diet deficient In 5-hydroxy- 
236 

tryptophan to deplete selectively either the catechol¬ 
amines or 5-ET and to determine whether any correlation 
between behavior and cerebral change in one or the other 
amines could be obtained* At this point, there is good 
evidence in both camps, (perhaps an indication that neither 
amine is involved). Nevertheless, the studies reported 
here indicate that PAD can be of use in this problem. No 
other agent has been reported to the author's knowledge, 
which depletes bring 5-HT but not the catecholamines, 
although it has many other effects, as previously noted. 

This selective action of PAD could be exploited to elucidate 
the physiologic iitport of 5-HT depletion. Qualitatively, 
animals treated with doses of PAD which depleted 65$ of 
their 5-HT seemed no less active and alert than controls 
who had been treated identically, except for not receiving 
PAD. The studies detailed in Table 10 indicated that PAD 
pretreatment delayed, not hastened the onset of sedation 


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133 


in rats given small doses of reserpine. Also rats pre¬ 
treated with phenylisopropylhydrazine (JB-516), an 
effective MAOI, were alerted and activated by reserpine, 
whereas a high dose of PAD had no effect on animals pre¬ 
treated with phenylisopropylhydrazine. These differences 
in the effects of PAD and reserpine, although admittedly 
subjective and in need of quantitative verification, 
indicate that if amine-release is the means by which re¬ 
serpine produces behavioral effects, it is mediated via 
change in catecholamines, rather than 5-HT. (It is of 
course possible, that it is another amine, or no amine 
at all, through which reserpine operates). The second 
reason for being interested in the amine-depleting action 
of PAD is that it is the only compound which has been shown 
to both release brain amines and inhibit MAO, Tranylcypromine, 
one cf the MAOI mentioned previously, has been shown to re- 

237 

lease norepinephrine from the hearts of both rats and cats, 
but it has not been observed to release brain amines. Ipron** 
iazid can diminish the monoa Heines in the cat hypothalamus 
but this was thought to be a non-specific effect.It is 
of interest that PAD’S effect on 5-HT binding is not long- 
lasting* Twenty-four hours after a large dose of PAD, 5-HT 
levels return to normal. 

If a quaternary pyridinium molecule were synthesized 
which could inhibit brain MAO in vivo as well as release 
brain 5-HT, it might turn out to have unique behavioral 
and possibly therapeutic effects since it is by no means 




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certain that it is through catecholamine release rather 
than through 5-HT release by which reserpine acts, or 
that 5-HT depletion ha s no behavioral effect. Studies 
on the treatment of depression in man with a combination 
of reserpine and iproniazid, i.e., with an amine re¬ 
leaser and a MAOI, have indicated that this combination 

239 

has particular value. 

Dodecyl Iproniazid Iodide 

The studies with dodecyl iproniazid iodide showed that 
it possessed the properties that were predicted by the 
author. The inhibition of MAO in vitro without preincu¬ 
bation was probably due to the N-alkyl group bonded to 
the pyridine nitrogen atom, whereas the supplemental 
inhibitory potency noted after preincubation was probably 
due to the hydrazine moiety. Studies in vivo demonstrated 
that it is a potent inhibitor of liver MAO (70^ inhibition 
at 20 mg/kg) and that it raises brain 5-HT levels. This 
implies that it inhibits brain MAO also, or prevents the 
release of brain 5-HT. It could also release brain 5-HT 
despite the 50 % elevation of 5-HT over control values 
because the simultaneous inhibition of MAO would prevent 
its destruction so it might accumulate in its "released” 
state. There were many interesting experiments to do with 
this compound, but unfortunately only a very small amount 
was available to the author and the method of synthesis 
was not available. 





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135 


The effects of PAD and dodecyl iproniazid iodide on 

rat brain imply that both these agents can pass the 

blood-brain barrier and provide additional evidence for 
201 

Wilson’s idea that long-chain N-alkyl radicals can 
be used to enhance the ability of highly ionized mole¬ 
cules to penetrate this barrier* 

The Effect of Thiamine on Monoamine Oxidase 

211 

In a recent publication. Gal and Drewes demon¬ 
strated that in thiamine-deficient rats, the MAO-activity 
of the brain and intestine is increased, as compared to the 
activity of rats fed ad libitum with a normal laboratory 
diet. These workers suggested that the increase might be 
related to the stress theory of Selye. However, it is 
proposed by the author, that, in vivo * thiamine, which 
has a quaternary nitrogen in its thiazole moiety, is either 
itself an inhibitor of MAO, or more likely, is metabolized 
via a pathway which produces another quaternary nitrogen 
compound which is an inhibitor of MAO. Thus, the increased 
MAO-activity in the thiamine-deficient animals might be 
accounted for by a diminution in the amount of this thiamine¬ 
like inhibitor. The MAO-inhibition caused by this thiamine¬ 
like compound also might explain the hypotension and gang¬ 
lionic blockade which thiamine produces in man and experi¬ 
mental animals.Gertner has shown that MA0I block 

IV 6 

ganglionic transmission in cats and dogs, while hypo¬ 
tension in man has been noted with the administration of 
180 


MA0I 










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136 


Experiments presented here show that thiamine and 

thiamine pyrophosphate in vitro are weak inhibitors of 

MAO: at 10-^M thiamine inhibits the MAO of rat liver 

mitochondria 70 % 9 a result of 1ittle significance with 

respect to the proposed mechanism in vivo . Furthermore, 

rats given 70 mg of thiamine per kg subcutaneously, and 

sacrificed after two hours, when thiamine pyrophosphate 

220 

levels in liver are elevated, showed no inhibits on of 

the MAO-activity of liver, brain and intestine as compared 

with untreated controls. However, this does not constitute 

a refutation of the proposed theory, if the rate of formation 

of the postulated thiamine metabolite proceeds maximally with 

the amount of thiamine provided by a normal diet. 

Substantial indirect evidence can be provided for the 

theory. Although in higher animals most of t he t hiamine 

which is absorbed is excreted unchanged, the fate of the 

2k2 

thiamine which is degraded, is largely unknown. Molluscs, 

carp, and some other lower species possess an enzyme, thiamin- 
2113 

ase, which catalyses the reaction of thiamine with a variety 
of basic compounds with loss of the li-methyl-^-hydroxyethyl- 
thiazole portion, and the formation of other quaternary com¬ 
pounds derived from the pyrimidine portion. 2 ^ No similar 
enzyme has been found in mammalian tissue, but only small 
amounts may be present. Such a reaction could produce a 
potent quaternary inhibitor of MAO. It is significant 
that Minz 2 ^ and von Muralt using labeled thiamine, 

has shown that, on excitation of a peripheral nerve. 








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137 


metabolites of thiamine appear, which are as yet uniden¬ 
tified. Other studies have shown that excitation of the 

2li7-250 

vagal nerve can release thiamine into the heart. 

Four quaternary ammonium ions, one of them a quaternary 
pyridinium molecule have been found in the giant nerve 

251 

fibers of the squid. One of these might be the pos¬ 

tulated inhibitor; If so, this would provide a mechanism 
for preventing the oxidation of any neurohumor which is 
a substrate for MAO. 

The hypotension and ganglionic blockade produced by 
thiamine and by known inhibitors of MAO is suggestive 
that these compounds are related. This action of thia¬ 
mine is unrelated to its properties as coenzyme.This 
blockade is also different in kind from that produced 

by hexamethonium,“ r as is that caused by the inhibitors 
176 

of MAO. 

A final argument which may be offered is that in 
thiamine-deficiency state in man (beri-beri), the initial 
symptoms are emotional - depression, apathy, and disinterest, 

252,253 

a syndrome a physician might now treat with an in¬ 
hibitor of MAO, which indeed would be only replacement 
therapy, if thiamine administration to thiamine-deficient 
organisms does in fact produce an MAO inhibitor* 

It is interesting that thiamine-deficient rats also 

ll6 

show increased cholinesterase activity ‘ and diamine 

117 

oxidase activity, while thiamine is also a weak in¬ 
hibitor of these enzymes, in vitro . It has been proposed 







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that thiamine inhibits both of these enzymes in vivo . 

Thus, there is both precedent for the theory presented and 
a wider scope for a mechanism which would delay the cata¬ 
bolism of neurohumors such as acetylcholine, histamine, 
serotonin, and the catecholamines. 



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139 


SUMMARY OF PART TWO 

1, Pyridine aldoxime dodecyliodide (PAD) was found to 
inhibit the monoamine oxidase (MAO) of homogenates 

of rat, rabbit, guinea pig, and mouse brain and liver 
in vitro . This is the first demonstration that a 
quaternary nitrogen compound can inhibit MAO, 

Numerous other such compounds were also found to 
inhibit MAO in vitro, 

2 , PAD is a more powerful MAO inhibitor in vitro than 
iproniazid which at one time was widely used in the 
treatment of depression, 

3, No preincubation of enzyme and inhibitor is necessary 
and the inhibition is irreversible. This sets it 
apart from, other MAO inhibitors, 

1|. 0 PAD is also an effective inhibitor of "solubilized” 

MAO. 

5, PAD is not an effective MAO inhibitor in vivo . 

Possible explanations for this discrepancy between 
in vivo and in vitro results are discussed. 

6, PAD, like reserpine, was found to lower the 5-hydroxy- 
tryptaraine (5-HT) levels in rat brain, but has no effect 
on the catecholamine levels. It also releases 5-HT from 
the Dunn-Potter mouse mastocytoma ascites cells. 










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7. PAD does not inhibit rat kidney 5-hydroxytryptophane 
decarboxylase in vitro. 

8. These findings suggest that PAD exerts a selective 
releasing action on bound 5-HT. It is the only com¬ 
pound reported which selectively depletes 5-HT but 
not the catecholamines. 

9. PAD treatment produced no accumulaticn of rat brain 
5-HT after initial depletion of 5-HT with reserpine 
whereas 5-PIT did accumulate after treatment with a 
hydrazine type MAO inhibitor under the same conditions. 
This is a more sensitive indication that no MAO in¬ 
hibition occurs in vivo . 

10. Animals given sufficient PAD to deplete their 5-HT 
stores do not have the appearance of animals treated 
with reserpine. In addition, animals pretreated with 
phenylisopropylhydrazine (PIH), and inhibitor of MAO, 
and then given PAD do not have the appearance of animal 
pretreated with PIH and then given reserpine. 

11. Decamethonium, hexamethonium and thiamine are also 
weak inhibitors of MAO in vitro . 

12. The reported increase in brain MAO activity of 
thiamine-deficient rats is interpreted as an indica¬ 
tion that thiamine or probably one of its metabolites 
is an MAO inhibitor in vivo. Relevant data from the 


literature is offered in support of this idea 








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13 • Dodecyliproniazid iodide wag conceived o.f as a mole¬ 
cule combining the MAO inhibitory properties of a 
hydrazine and the 5-HT releasing properties of a 
quaternary nitrogen compound* It was found to in¬ 
hibit MAO in vitro with both the hydrazine moiety 
and the quaternary nitrogen moiety* It was also 
found to release rat brain 5-HT in vivo * This is 
the first compound to both inhibit MO in vivo and 
release brain 5-HT and might have unique psycho- 
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