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Studies on agents which modify mast cell stimulation-secretion coupling

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Title:
Studies on agents which modify mast cell stimulation-secretion coupling
Creator:
Heiman, Ann S
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English
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vii, 98 leaves. : ill. ; 29 cm.

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Subjects / Keywords:
Calcium ( jstor )
Glucocorticoids ( jstor )
Histamine release ( jstor )
Histamines ( jstor )
Mast cells ( jstor )
Memory interference ( jstor )
Methylation ( jstor )
Phospholipids ( jstor )
Rats ( jstor )
Secretion ( jstor )
Anti-Inflammatory Agents -- immunology ( mesh )
Dissertations, Academic -- Pharmacology and Therapeutics -- UF ( mesh )
Hypersensitivity, Immediate -- drug therapy ( mesh )
Mast Cells -- drug effects ( mesh )
Pharmacology and Therapeutics thesis Ph.D ( mesh )
Phorbols -- immunology ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida.
Bibliography:
Bibliograpy: leaves 87-97.
General Note:
Photocopy of typescript.
General Note:
Vita.
Statement of Responsibility:
by Ann S. Heiman.

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University of Florida
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11123158 ( OCLC )
ACG7303 ( NOTIS )
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Full Text











Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy



















STUDIES ON AGENTS WHICH MODIFY
MAST CELL STIMULATION-SECRETION COUPLING




By


ANN S. HEIMAN


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1984




Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
STUDIES ON AGENTS WHICH MODIFY
MAST CELL STIMULATION-SECRETION COUPLING
By
ANN S. HEIMAN
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1984


This dissertation is dedicated to Tom who is both my best friend
and my husband. Together with him I celebrate personal growth and
development.


ACKNOWLEDGEMENTS
I express sincere thanks and gratitude to my mentor, Fulton Crews,
and to the other members of my advisory committee, Allen Neims, Stephen
Baker, Stephen Russell and William Kern, for their invaluable assistance
in my development as a pharmacologist. Special acknowledgement goes to
Mrs. Judy Adams whose typing and editing skills are surpassed only by
her patience and pleasant manner. And, I am thankful to all my fellow
lab workers and students for creating a pleasant working atmosphere.


PREFACE
"Of all intellectual activity, science alone has flourished in the
last centuries, science alone has turned out to have the kind of
universality among men which the times require."
J. Robert Oppenheimer
This dissertation is composed of an introductory chapter, two
chapters written in standard manuscript style and a final conclusion
and significance chapter. Due to this format, some material may appear
redundant. Please accept any repeated material as important to the
overall contribution of this research to further understandings of mast
cell stimulation-secretion coupling.
iv


TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS ; 111
PREFACE iv
ABSTRACT vi
CHAPTER ONE INTRODUCTION 1
General Characterization of Mast Cells
and Histamine Release 1
Classes of Agents which Release Histamine
from Mast Cells 4
Other Mediators Released by Mast Cells 7
Role of Calcium in Mast Cell Exocytosis 9
Phospholipid Metabolism During Mast Cell
Activation 10
Conceptual Model for Mast Cell Exocytosis .... 23
CHAPTER TWO INHIBITION OF IMMUNOGLOBULIN, BUT NOT
POLYPEPTIDE-BASE, STIMULATED RELEASE OF
HISTAMINE AND ARACHIDONIC ACID BY
ANTI-INFLAMMATORY STEROIDS 28
Introduction 28
Materials and Methods 29
Results 33
Discussion 45
CHAPTER THREE CHARACTERIZATION OF THE EFFECTS OF PHORBOL
ESTERS ON RAT MAST CELL SECRETION 53
Introduction 53
Materials and Methods 55
Results 59
Discussion 75
CHAPTER FOUR CONCLUSIONS AND SIGNIFICANCE 81
REFERENCES 87
BIOGRAPHICAL SKETCH 98
v


Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
STUDIES ON AGENTS WHICH MODIFY
MAST CELL STIMULATION-SECRETION COUPLING
By
Ann S. Heiman
August, 1984
Chairman: Fulton T. Crews
Major Department: Pharmacology and Therapeutics
Mast cells are long-lived cells which reside in connective tissue
and tissues surrounding blood vessels, meninges, sheaths of peripheral
nerves and submucosa of the small intestine and respiratory system.
They respond to a variety of peptides and participate in concert with
bound IgE and antigen to release mediators of immediate
hypersensitivity.
The present, detailed investigations were conducted with purified,
normal mast cells and the results presented in terms of 1) selective
effects of anti-inflammatory steroids and 2) characterization of
actions of tumor-promoting phorbol esters upon mast cells stimulated
with IgE-dependent secretagogues, peptide-basic agents or the calcium
ionophore A23187.
With respect to the selective effect of anti-inflammatory steroids,
the interesting findings were 1) long-term (18-21 hr) pretreatment
vi


was required for manifestation of inhibitory effects, 2) the inhibitory
effect was limited to anti-inflammatory types of steroids, 3) a
specific glucocorticoid receptor mechanism was involved, 4) inhibition
of release of histamine and arachidonic acid resulted when cells were
stimulated with IgE-dependent secretagogues, 5) no inhibition was noted
when cells were stimulated with polypeptide-basic agents or ionophore
A23187, and 6) IgE-dependent secretagogue stimulated calcium flux was
decreased.
Characterization of the effects of phorbol esters on rat mast cell
secretion revealed that 1) phorbol esters tremendously potentiated
ionophore A23187 stimulated release of histamine, 2) extracellular
calcium was required, 3) histamine release by IgE-like secretagogues
was potentiated by the presence of the phospholipid phosphatidyl serine
and phorbol ester, 4) structure-activity relationships suggested a
single mechanism of action, perhaps mediated by a single receptor, 5)
phorbol esters either alone or with ionophore A23187 increased
phosphorylation of mast cell proteins, and 6) mast cells have low
levels of calcium-phospholipid-dependent protein kinase to which
phorbol esters bind.


CHAPTER ONE
INTRODUCTION
General Characterization of Mast Cells and Histamine Release
Within the Gel! and Coombs (1968) scheme for classifying
immunological responses, histamine release from mast cells (and
basophils) comprises the type I sensitivity commonly called immediate,
anaphylactic or reagin-dependent hypersensitivity. This reaction
occurs upon re-exposure to a specific antigen which interacts with its
immunoglobulin E (IgE) antibody bound to the surface of mast cells and
basophils and triggers a sequence of activation-secretion coupling
events which culminate in the release of several inflammatory mediators
(Altman, 1981). Minimum requirements for an immediate hypersensitive
event are allergen and mast cell or basophil with specific IgE bound to
the cell surface Fc receptor. Initial exposure to an allergen requires
collaboration between it, macrophages and T lymphocytes which stimulate
B lymphocytes to differentiate to plasma cells and synthesize and
release IgE-class antibodies. Investigations into the regulation of
the IgE antibody formation response have led to the hypothesis that
certain IgE-class specific subpopulations of both helper and
suppressive T cells work together with B cells to modulate the antibody
response (Tadamitsu, 1982). Normally, suppressive mechanisms
predominate to keep IgE levels low; therefore, allergic diseases may
represent a "breakthrough" phenomenon (Katz, 1978a, 1978b).
1


2
Mast cells to which IgE binds> reside predominantly in connective
tissue and skin surrounding blood vessels, meninges, sheaths of
peripheral nerves and submucosa of the small intestine and respiratory
system. These are long-lived cells, capable of regranulation in
contrast to their circulating counterparts, the basophils, which are
short-lived, incapable of regranulation and can move into tissues in
response to chemo-attractants (Altman, 1981). Both cell types store
certain mediators of hypersensitivity reactions, such as histamine
which is bound to a water-insoluble complex of protein and heparin
within cytoplasmic granules. Rat peritoneal mast cells reportedly
contain 250-500 granules per cell, and this may comprise up to 70% of
the cellular dry weight (Bergendorff and Uvnas, 1973).
Release of histamine and other mediators in an immediate
hypersensitivity episode is the outcome of a complex biochemical series
of events beginning with activation of the cell surface IgE receptor.
Several investigative groups have reported that normal rat mast cells
have approximately 2 x 105 IgE receptors/cell (Conrad et aj_., 1975;
Ishizaka et a]_., 1975; Mendoza and Metzger, 1976). Cross-linking of
receptor-bound IgE by multi-valent antigen, anti-IgE or direct
cross-linking of IgE receptors by anti-receptor IgE triggers the
sequence of activation-secretion events (Ishizaka and Ishizaka, 1968;
Ishizaka et al_., 1971; Ishizaka and Ishizaka, 1978). Activation of a
serine esterase closely associated with IgE receptors has been
postulated as a very early event in stimulus activated mast cells and
may play a general role in secretory cell activation (Taylor and
Sheldon, 1974). According to the mobile receptor hypothesis of


3
Cuatrecasas (1974), membrane-associated receptors and enzymes acquire
affinities to form complexes upon receptor occupation and may thus
"uncover" active sites of these enzymes of mediator release. Other
enzymes which fit such a hypothesis include phospholipase &2>
phospholipase C, protein kinases such as the calcium and phospholipid
dependent protein kinase (Ca/PL-PK) and the phospholipid
methyl transferases. While different classes of secretagogues may
utilize different enzymes in the initial transmembrane signalling
sequences, it is thought they employ a common sequence during the
secretion phase. This latter phase involves microtubule assembly
(Lagunoff and Chi, 1976) which facilitates intracytoplasmic granule
movement and culminates in very poorly understood granule-plasma
membrane fusion and granule content extrusion.
Degranulation of mast cells and basophils is sensitive to both pH,
with optimum release at pH 7, and temperature, secretion is inhibited
at 0 or 45*C (Moran £t 1_., 1962; Baxter and Adamik, 1975). There is
an absolute requirement for calcium, and in the case of IgE-mediated
degranulation, there must be extracellular calcium ions available
(Foreman and Monger, 1972; Baxter and Adamik, 1978). Availability of
metabolic energy in the form of ATP is a basic prerequisite for
histamine release and can be derived either from oxidative
phosphorylation or glycolysis. Reports indicate that cellular ATP
levels decrease by 30-35% during the release process (Kazimierczak and
Diamant, 1978).
From this general description of the mast cell and the complex
sequence of events involved in degranulation, we can see that it


4
entails an active secretory process and has been considered a
prototypic secretory cell (Douglas, 1968) having the following distinct
components: 1) activation by specific stimuli, 2) extrusion of
granular material, and 3) cellular recovery and restoration of granule
contents (Kirshner and Viveros, 1972).
Classes of Agents which Release Histamine from Mast Cells
Immediate type hypersensitivity reactions by definition involve
antigen and antibody; however other diverse chemical as well as
physical stimuli are capable of inducing granule extrusion (Foreman,
1981; Lagunoff et al_., 1983; Kazimierczak and Diamant, 1978; Ho et al.,
1979). Rather than bridge receptor-bound IgE molecules, these other
chemical stimuli interact with specific mast cell receptors to initiate
stimulation and secretion. Types of agents capable of eliciting
non-cytotoxic mediator release from mast cells and basophils are quite
diverse. An abbreviated categorization of these agents central to
these investigations includes immunologic agents, lectins, polycationic
amines and polypeptides and ionophores.
Within the immunologic agent category fall IgE antibody, IgE
generated toward another species IgE (i.e. anti-IgE), anti-receptor IgE
and the anaphylatoxins C3a and C5a. Release elicited by the first
three agents proceeds via activation of the IgE Fc cell surface
receptor, as previously described. Anaphylatoxins C3a and C5a,
generated from complement components, are believed to induce
degranulation by occupation of their own receptors, independent of the
IgE Fc receptor (Siraganian and Hook, 1976; Hartman and Glovsky, 1981),
but the requirements for release are identical to the IgE-dependent


5
mechanism. Both require extracellular calcium, an energy source, are
temperature dependent and are potentiated by exogenous
phosphatidyl serine (PS).
Lectins are hemagglutinins which possess saccharide-specific
binding sites and include Jack bean concanavalin A (con A), as well as
wheat germ, castor bean and lentil agglutinins. While all these agents
reportedly elicit release, most is known about con A induced histamine
release. Critical determinants of the extent of con A induced release
may vary dependent upon the strain of rat used, presence or absence of
extracellular calcium, the degree of cell sensitization and requirement
for exogenous PS (Lagunoff et ajL, 1983). However, there are data
which convincingly suggest that con A, tetravalent at physiological pH,
cross-links bound IgE by interaction with saccharide moieties located
near the Fc region of the IgE molecule (Siraganian and Siraganian,
1975; Fewtrell £t al_., 1979). Though normal rat mast cells may be
quite resistant to con A, cells collected from animals infected with
Nippostrongylus brasiliensis, known to increase IgE synthesis, become
more sensitive to con A (Keller, 1973). Lastly, monomeric antibody
directed against the Fc portion of IgE inhibits con A induced histamine
release, indicating that this lectin binds to the Fc region of IgE
bound to the mast cell surface (Magro and Bennich, 1977). In
Sprague-Dawley rats, the strain used in these investigations, the
requirements for con A induced mast cell degranulation are identical to
those for IgE-induced release.
The prototypic polycationic amine releasing agent is compd 48/80, a
polymerization product of equimolar p-methoxy-N-methyl phenethylamine


6
and formaldehyde. Polypeptides in this group include somatostatin,
substance P, bradykinin, ACTH and neurotensin (see table compiled by
Lagunoff et al_., 1983). These agents share a common feature, the
presence of at least two basic residues; it has been suggested that any
molecule possessing two basic groups separated by an aliphatic chain of
about five carbons or an aromatic skeleton of corresponding length is
likely to be a mast cell histamine liberator (Patn, 1958). Recent
evidence suggests that compd 48/80 and somatostatin induce histamine
secretion in mast cells by interacting with the same cell surface
receptor (Theoharides et £l_., 1981). Release induced by agents of this
class differ from the above classes in that there is no enhancement by
exogenous PS, no requirement for extracellular calcium, and no
stimulation of phospholipid methylation (Hirata et al_., 1979). This
suggests that initial events of the activation-secretion response of
mast cells may be different for polycationic amines and basic
polypeptides than IgE-like secretagogues.
Ionophores, compounds which facilitate transport of ions through a
lipid barrier separating two aqueous environments, serve as carriers
for various ions. The most completely described is known simply as
A23187, a monocarboxylic antibiotic isolated from Streptomyces
chartreusensis; 1t is capable of eliciting secretion from a wide
variety of cells, including mast cells. In the secretory process, the
ionophore A23187 functions as a calcium carrier, is dependent upon
extracellular calcium and is capable of bypassing the regulatory effect
of cAMP upon mast cell secretion (Foreman et 1976). It is
likewise indifferent to the presence of exogenous PS.


7
For these investigations, we have chosen at least one agent from
each of these four groups: sheep anti-rat IgE (anti-IgE); the antigen
ovalbumin, con A; compd 48/80 or somatostatin and the calcium ionophore
A23187.
Other Mediators Released by Mast Cells
Histamine is but one of many pharmacological principles released by
mast cells. All mediators can be divided into two broad but useful
categories; those like histamine, which are stored and those which are
generated as a consequence of specific activation of the target cell
(Kazimierczak and Diamant, 1978). Pharmacological action,
physiochemical characteristics and metabolism of both preformed and
generated mediators have been reviewed in detail by Ho et al_. (1979).
Preformed mediators are stored within basophil and mast cell membrane
bound granules composed largely of proteoglycan and protein and include
histamine, serotonin (in some species, Yurt and Austen, 1977),
eosinophil chemotactic factor of anaphylaxis (ECF-A), neutrophil
chemotactic factor (NCF) and heparin. Included with stored,
granule-associated mediators are enzymes also released following cell
activation. Chymase, a chymotrypsin-like enzyme, is presumed to be
stored with serotonin at its active site in rat mast cell granules
(Lagunoff and Pritzl, 1976). N-Acetyl-g-D-glucosaminidase and
arylsulfatase A are two other stored enzymes released during
degranulation and are thus presumed to reside in secretory granules
(MacDonald-Lynch et al_., 1978).
Mediators generated subsequent to mast cell activation consist
predominantly of prostaglandins (PGs) and leukotrienes (LTs), the


8
oxidative products of arachidonic acid (AA). They are generated by the
release of AA from phospholipid membrane stores following
secretagogue-induced perturbation which is believed to allow expression
of phospholipases (Flower and Blackwell, 1976).
Metabolism of AA occurs by two independent pathways: the
cyclooxygenase and lipoxygenase pathways. In the rat and human mast
cell, the former pathway initially generates the unstable cyclic
endoperoxides PGG2 and PGH2 which are converted predominantly to PGD2
by the action of a specific synthetase (Lewis and Austen, 1981).
Metabolism via the lipoxygenase pathway generates the intermediate
5-hydroperoxyeicosatetraenoic acid (5-HPETE) which is converted to the
closely related mono-hydroxyeicosatetraenoic acid (5-HETE) or the
unstable intermediate 5S-oxido-trans-7,9-trans,ll,14-cis-
eicosatetraenoic acid (LTA4). From LTA4, two enzymatic pathways have
been described, one leading to the potent chemotactic factor LTB^
(5S,12R,dihydroxy,6,14,cis,8,10-trans-eicosatetraenoic acid) (Borgeat
and Samuelsson, 1979) and the other leading to 5S-hydroxy,6R-S-
glutathionyl-7,9-trans,ll,14-cis-eicosatetraenoic acid (LTC4) by the
addition of glutathione. LTC4 is then metabolized to the corresponding
cysteinylglycine derivative LTD4 then the cysteine derivative LTE4
(Lewis &t 1_., 1982). In stimulated human neutrophils, the
5-lipoxygenase pathway is preferentially activated to generate 5-HETE
and LTB4. In contrast, rat and human mast cells elaborate PGD2 during
coupled activation-secretion (Lewis et al_., 1982) when stimulated with
anti-IgE. LTC4 has been identified following stimulation of a murine
mastocytoma cell line with the calcium ionophore A23187 (Samuelsson et


9
al., 1980). It has been suggested that in human and rat mast cells
challenged with IgE-dependent stimuli, only PGD2 is generated but the
secondary cell types recruited may be the source of the other oxidative
AA metabolites active in immediate hypersensitivity (Lewis and Austen,
1981).
For the series of experiments described in these chapters, we have
chosen to study histamine as the granule-contained secretion marker and
AA, in the form of [14C]-AA incorporated in vitro, as the marker
generated during mast cell stimulation.
Role of Calcium in Mast Cell Exocytosis
According to Gomperts (1983), only one early event in the mast cell
stimulus-response sequence has been unequivocally identified and that
is the elevation of cytosolic calcium into the micromolar range. It
has been stated that this is the sole necessary precursor of secretion.
"Stimulus coupling," first proposed by Douglas and Rubin (1961), has
been introduced to emphasize the central role of calcium as a second
messenger in secretory processes (Pearce et aK, 1983). Experimental
results with calcium ionophores such as A23187, ionomycin and
chlortetracycline which form lipid-soluble complexes with the cation
and directly transfer it across the cell membrane bypassing the normal
messenger-receptor interaction, have served as evidence that calcium is
the second messenger which couples activation with the granular release
(Cochrane and Douglas, 1974; Lichtenstein, 1975; Bennett et al_., 1979;
Pearce £t al_., 1983). Though most investigators agree that ionophores
transport extracellular calcium to the intracellular spaces,


10
ionophore-induced histamine release in rat mast cells in the absence of
extracellular calcium (thus presumably by mobilization of internal
calcium stores) has been reported (Johansen, 1980). A second line of
evidence that calcium is the second messenger has centered around mast
cell exocytosis following microinjection of calcium ions into the cells
while microinjections of magnesium or potassium were without effect
(Kanno et al_., 1973).
The source of calcium differs dependent upon the type of
secretagogue. Antigen-induced, as well as other IgE-dependent
exocytosis, requires extracellular calcium, whereas ligands such as
compd 48/80, peptide 401 and polylysine induce secretion in the absence
of extracellular calcium (Foreman, 1981). Lanthanides, which compete
with calcium for extracel1ular binding sites thought to be the calcium
channel, thus prevent calcium transport across membranes and inhibit
mast cell histamine secretion induced by IgE-like secretagogues
dependent upon extracellular calcium. They do not affect release by
compd 48/80, peptide 401 and the like (Foreman and Monger, 1973; Pearce
and White, 1981; Amellal and Landry, 1983).
Relationships between histamine secretion and 4^Ca^+ uptake by mast
cells have been thoroughly investigated by Foreman et al_. (1977).
Stimulation with antigen-antibody, dextran or con A induced an uptake
of ^Ca^+ which correlated well with the magnitude of histamine
release. PS enhanced both ^Ca^+ uptake and histamine release. When
cells were stimulated with antigen in the presence of non-labeled
calcium, then exposed to tracer ^Ca^+ over a period of 1-5 min, a
64% drop in calcium uptake was noted within 1 min following stimulation


11
indicating a very transient membrane permeability to the cation.
Raised intracellular levels of cAMP produced with theophylline or
dibutyryl cAMP inhibited antigen-induced ^5Ca^+ uptake and histamine
release, but had no effect when the ionophore A23187 was used as
stimulant. It appears, then, that cAMP may inhibit secretion by
reducing membrane permeability to calcium and may be involved in
limiting calcium entry after cell stimulation.
Evidence for mobilization of membrane or intracellular calcium
stores to provide the needed rise of cytosolic calcium ions is given by
results of mast cell stimulation in the absence of extracellular
calcium following short-term (10 min) pretreatments with chelating
agents. Peptide-basic secreting agents including compd 48/80,
bradykinin, somatostatin, polylysine and polymyxin B were capable of
robust release responses in the absence of calcium (Ennis et aJL 1980;
Baxter and Adamik, 1978; Cochrane et al_., 1982). Based upon such
investigation, three pools of calcium have been postulated: 1) calcium
very loosely bound to the outer cell membrane which may migrate to the
cellular cytosol, 2) a superficial, membrane associated store
(removable by brief EGTA exposure) and 3) deeply sequestered calcium
stores (removable by prolonged exposure to EGTA (Ennis et al_., 1980).
While the first store is utilized by both IgE-like and IgE-independent
secretagogues as well as ionophores, the second store is mobilized by
polypeptide-base type secretagogues. Deeply buried depot translocation
may be modulated by occupancy of the two more superficial pools.
Membrane events implicated in mast cell calcium translocation
include turnover of phosphatidylinositol (PI) (Gomperts et al_., 1980;


12
Cockcroft and Gomperts, 1979; Kennerly et al_., 1979b,c). In rat
peritoneal mast cells stimulated with antigen, anti-IgE, con A,
chymotrypsin and compd 48/80, turnover of PI and incorporation of
radiolabel into PI took place regardless of the presence or omission of
extracellular calcium. Concomitant histamine release was either
abolished or reduced depending upon calcium requirements of the ligand.
This was taken as evidence for involvement of the PI response in the
regulation of calcium channels and mobilization of sequestered calcium
in the mast cell (Pearce, 1982; Cockcroft and Gomperts, 1979).
Another membrane event implicated in calcium translocation was
phospholipid methylation where two membrane-bound enzymes
(methytransferases I and II) act sequentially to convert endogenous PE
to PC. Methylation has been demonstrated in rat leukemic basophils and
peritoneal mast cells during stimulation with antigen, con A and
anti-IgE (Crews et al., 1980; Hirata et al_., 1979; Ishizaka et al.,
1980). Because the methyl transferase inhibitor, 3-deaza-adenosine,
blocked phospholipid methylation, calcium influx and histamine release
in a dose-dependent manner, it was suggested that phospholipid
methylation may be a primary and obligatory event in calcium
translocation and histamine release by IgE-dependent mast cell
activation.
From available data, it is still not possible to determine the
relative contributions of these two pathways of lipid metabolism to the
sequence of mast cell activation-secretion coupling events. They may
represent events of differing initial activation sequences, events of a
complex but unified sequence, or consequential events of cell


13
activation. Both the PI response and phospholipid methylation will be
discussed in more detail in the next section.
The mechanism whereby calcium induces the mast cell exocytotic
response is not known. In many systems, the cation produces its
effects by interaction with specific binding proteins exemplified by
the ubiquitous polypeptide calmodulin, proposed as a universal
intracellular receptor for calcium (Means and Dedman, 1980; Cheung,
1980). Calmodulin mediates activity of a number of important enzymes
including cyclic nucleotide phosphodiesterase, brain adenylate cyclase,
calcium dependent ATPase, phospholipase A, myosin light chain kinase,
as well as other specific kinases involved in phosphorylation of
membrane and cytosolic proteins (Pearce, 1982).
In smooth muscle cells, neuroleptic drugs reportedly inhibit
contraction induced by various stimuli by inhibiting calmodulin
(Kerrick et 1_., 1981). Seven such agents including five phenothiazine
derivatives, imipramine and pimozide have recently been studied for
their effects upon mast cell secretion evoked by specific antigen,
compd 48/80 or ionophore A23187 (Douglas and Nemeth, 1982). An
inhibitory potency series generated with the agents for compd 48/80 and
A23187 induced release correlated closely with potencies which
inhibited phosphodiesterase activation by Ca-calmodulin and also
reflected their affinity for binding to calmodulin (Weiss et al.,
1980). Specific antigen-evoked secretion was more sensitive to
inhibition and gave a different rank order of the neuroleptic agents,
an effect shown not to be related to the magnitude of antigen-induced
secretion. Since A23187 elicited ^Ca^+ uptake was not reduced by the


14
neuroleptics, it was suggested by Douglas and Nemeth that since
non-specific membrane effects had been ruled out, their action was
distal to the intracellular rise of calcium ions either on the calcium
receptor or the processes it activates to effect exocytosis. These
results suggested that mast cells mc(y have a calcium-binding protein(s)
which resembles calmodulin and that it is involved in
receptor-activated exocytosis. Its function may be related to
calcium-dependent protein phosphorylation which has been correlated
with mast cell degranulation (Sieghart et £l_., 1978).
Exogenously added calmodulin inhibited mast cell histamine release
elicited by compd 48/80, polymyxin B and A23187 (ID^q about 2 uM) but
not con A (McClain et al_., 1983). Calmodulin itself did not elicit
release, did not appear to enter the cells and did not compete with
compd 48/80 or polymyxin B for cell surface binding sites. EGTA
washing of mast cells reduced subsequent calmodulin-induced inhibition.
So, release may involve externally-bound calcium but the relationship
of this exogenous calmodulin effect to the actual in vivo mechanism
remains unknown.
Care must be used in interpreting these results since a very recent
report on human platelet work stated that these same agents, namely
trifluoperazine, chlorpromazine and W-7 were powerful inhibitors of the
calmodulin independent Ca/PL-PK also known as C-kinase (Sanchez et al.,
1983). In quin 2 loaded platelets, the secretory response stimulated
by phorbol ester, exogenous diacyl glycerol (DAG) or collagen was
suppressed by the neuroleptics at 20-60 uM at basal levels of
cytoplasmic free calcium. Inhibition could be overcome by treatment of


15
platelets with A23187 (40 nM) which elevated cytoplasmic calcium to
700 nM (sub-threshold for calcium alone to evoke secretion). In
contrast, the response to thrombin which was accompanied by elevation
in levels of cytosolic free calcium was barely affected. These data
suggested that the most prominent effect of phenothiazines, at least on
platelets, could be interference with Ca/PL-PK rather than
calmodulin-dependent processes.
Though there is sufficient evidence to support the view that
elevation of cytosolic calcium is the second messenger in mast cell
secretion, subsequent events which calcium initiates or influences
remain largely unknown. Likely events, by analogy with other secretory
cells or from the results discussed above include involvement of
calmodulin, interaction with cyclic nucleotides and cytoskeletal
elements such as microfilaments and microtubules, and activation of
phospholipases and Ca/PL-PK while methyl transferase activation and PI
metabolism may modulate calcium movement.
Phospholipid Metabolism During Mast Cell Activation
Phospholipid methylation. As mentioned in the previous section,
the two predominant phospholipid metabolizing systems are the PI cycle
and phospholipid methylation. Some evidence for the increase of both
these events during mast cell and/or basophil stimulation has been
reported (Crews, 1982).
Phosphatidylcholine (PC) is a major constituent of biomembrane
phospholipids including those of mast cells and is synthesized by at
least two described pathways, the CDP-choline pathway and successive
N-methylations of phosphatidylethanolamine (PE) via methyl transferase
activity (Kennedy and Weiss, 1956; Hirata et al_., 1979).


16
In normal mast cells, Hirata et al_. (1979) have reported a
transient rise in membrane phospholipid methylation which precedes
histamine release during the early stages of con A-induced
activation-secretion coupling, as assessed by the incorporation of
[^H]-methyl groups into the mast cell lipid fraction. In the presence
of calcium, labeled PC was further metabolized, presumably by
phospholipase to ^orm lysophosphatidylcholine. Both phospholipid
methylation and histamine release were inhibited by a-methylmannoside
which prevents binding of con A to its cell surface receptors.
Similarly, the methyl transferase inhibitors 5'-deoxyisobutylthio-3-
deazaadenosine or 3-deazaadenosine (3DZA) plus homocysteine thiolactone
inhibited both phospholipid methylation and histamine release. In the
absence of calcium, methylation but no subsequent histamine release
occurred. When compd 48/80 and ionophore A23187 were employed as
degranulating agents, no stimulation of phospholipid methylation was
observed. These results were taken to imply that while phospholipid
methylation was involved in the activation-secretion signal
transduction by con A, it was bypassed by both compd 48/80 and A23187
perhaps indicating different mechanisms of histamine release (Morita
and Siraganian, 1981).
Results similar to those described for con A stimulated normal
cells were then reported for 2H3 rat basophilic leukemia (RBL) cells
sensitized with specific IgE than stimulated with the corresponding
antigen and extended to include concomitant inhibition of histamine and
[^C]-AA in 3DZA treated cells. More recent studies with RBL cells by
Crews et al_. (1981) have shown that there was a parallel increase


17
between [^H]-methyl incorporation into phospholipids and ^5Ca^+ influx
which preceded release of histamine and AA release. Thus it was
suggested that phospholipid methylation may play a role in calcium
flux.
With a slightly different approach, by generating rabbit antibodies
to the IgE receptor for RBL cells (anti-RBL) which acted as a divalent
anti-receptor binding agent in normal rat mast cells, it was shown that
anti-RBL challenge Increased phospholipid methylation which reached a
maximum at 15 sec then rapidly declined and was followed by an increase
of 4Ca^+ uptake, then histamine release (Ishizaka, 1982). Monomeric
antibodies failed to induce any of these changes. With the use of
inhibitors described above, corroborative results were reported, again
suggesting that phospholipid methylation may be intrinsic for ^Ca^+
influx and histamine release induced by IgE-receptor stimulation.
Additionally, a 3-fold rise of mast cell cAMP, superimposed upon
[3n]-methyl incorporation was measured, with a maximum at 15 sec,
followed by a sharp decline, then a gradual rise to double basal level
again at 3 min following addition of anti-RBL. Methylation inhibitors
such as S-isobutyryl-3-deazaadenosine (3-deaza-SIBA) also partially
inhibited the initial rise in cAMP suggesting that cAMP may in turn
regulate phospholipid methylation. In a dose dependent manner,
theophylline, at millimolar levels increased cAMP, inhibited
methylation, 45Ca2+ influx and subsequent histamine release.
More recently, Daeron et al_. (1982) have reported that pretreatment
of mouse mast cells overnight with 10" ^ to 10" M dexamethasone did not
itself alter mast cell cAMP levels, but when the cells were stimulated


18
with specific antigen, phospholipid methyl at ion, 45Ca^+ influx and
histamine release were all inhibited by at least 75%. However, the
critical experiments, measurement of cAMP during stimulation were not
reported. Thus, while it is still not known how dexamethasone prevents
activation of methyltransferases, it does not appear to be by
alteration of resting cAMP levels.
Cumulatively, the data can be interpreted to indicate that
phospholipid methyltransferases: 1) which effect synthesis of PC to PE
are involved in mast cell receptor-mediated activation, 2) selectively
increase phospholipid methylation associated with IgE-receptor mediated
activation, 3) act prior to an influx of calcium and subsequent
histamine release, and 4) when inhibited, likewise inhibit calcium
influx as well as histamine and AA release. Therefore, phospholipid
methylation may play a critical role in the early events of
IgE-mediated mast cell activation-secretion coupling.
PI metabolism. While there is much information on phosphoinositide
metabolism and hormone and neurotransmitter action as well as the role
of PI hydrolysis as a transducing mechanism in many diverse tissues and
cell types (Berridge, 1981; Farese, 1983a; Farese, 1983b), there is
very little information on the role of PI in mast cells or basophils.
Yet, since the inositol phospholipids have been assigned an important
role in mediating actions which generate intracellular calcium signals,
as do mast cells, PI involvement in mast cell activation-secretion
coupling might be expected.
According to the postulate put forward by Michel 1 and colleagues,
PI hydrolysis is coupled to receptor activation and is responsible for


19
calcium influx or mobilization of intracellular calcium stores. This
is supported by observations that the PI cycle (measured directly as
hydrolysis or indirectly by increased labeling) occurs with many
stimulants that operate via receptors and employ calcium as "second
messenger" (Farese, 1983a). In this pathway, PI, Ptdlns-4,5P2 and/or
Ptdlns-4P are hydrolyzed by a phosphodiesterase such as phospholipase C
to yield DAG and inositol phosphate(s). DAG can serve as a substrate
for diacyl glycerol lipase to yield AA, the predominant fatty acid at
the 2-position, or diacyl glycerol kinase to yield PA which can be
picked up by the appropriate CTP transferase and combined with inositol
phosphate to reform PI. Relevance of this pathway arises from the
following: PA is known to have calcium ionophoric activity, DAG is a
known fusogen, and, in concert with PS and calcium, activates Ca/PL-PK
while Ptdlns-4,5P2 and Ptdlns-4P avidly bind calcium and could modulate
membranous calcium stores and/or alter activities of membrane
associated enzymes or transport proteins.
Cockcroft and Gomperts (1979) have published evidence for a role of
PI turnover in stimulus-secretion coupling in mast cells of rats
sensitized to either ovalbumin or the parasitic helminth
Nippostrongylus brasiliensis. Purified mast cells were preincubated
with either [^H]-inositol or 32_p. for 30 min, and then stimulated with
specific antigen, con A or the non-IgE directed secretagogue compd
48/80 for 15 min. Stimulated PI labeling was independent of
extracellular calcium though IgE-dependent histamine release was highly
calcium dependent. These results were taken as an indication that the
mast cell can be placed on the list of tissues and cell types which


20
exhibit calcium-independent PI responses and thus mcy represent
receptor mediated events proximal to calcium signals. Stimulated PI
labeling is believed to be a secondary consequence of the breakdown of
PI initiated by either the IgE-directed specific angigen, con A or
compd 48/80. At first glance, it seems puzzling that a secretagogue
which is independent of extracellular calcium would be involved in the
turnover of a phospholipid involved in calcium gating; however compd
48/80 (10 pgm/ml) stimulated PI synthesis was reflected as 2400 dpm
^P-PI/1-2 x 105 cells while con A induced PI synthesis was 10-fold
greater. Though not stated, these results also suggest that
calcium-gating may not be the sole function of PI metabolism in the
mast cell.
In a subsequent report (Gomperts et al_., 1980), an extensive table
of tissues which can be stimulated by the ionophore A23187 and also
exhibit a PI response was used as evidence to suggest that the PI
response indicates a receptor mediated biochemical step in the
activation of calcium channels as postulated by Michel! (1975).
Activation of metabolism of the predominant mast cell phospholipids
was investigated by incorporation of ^2p. into individual classes of
phospholipids during stimulation by anti-IgE, con A, compd 48/80 or
calcium ionophore A23187 (Kenner!y et al_., 1979a). Increased
incorporation into PA, PI and PC (4- to 10-fold increases) occurred
within 15 min while no significant incorporation into PS, PE or
sphingomyelin (SM) was discerned. Kinetic analyses of phospholipid
metabolism during anti-IgE stimulation showed that PA labeling
increased most rapidly from 15 sec to 2 min after stimulation while PI


21
and PC labeling increased more slowly and reached a maximum after 6 min
of stimulation. Major changes in PA labeling occurred before mediator
release, PI and PC changes were concomitant with histamine release.
These increases of ^P.. incorporation were presumed to indicate that
mast cell receptor-induced stimulation generated DAG which is then
converted to PA, PI and PC. These results support the hypothesis that
selective mast cell phospholipid metabolism may play a critical role in
the biochemical events which control mediator release.
Since DAG is a precursor for each of the phospholipids, experiments
were designed to determine levels of DAG during mediator release from
mast cells stimulated with compd 48/80 (Kenner! y et al_., 1979b). In
[3H]-AA prelabeled cells, a small but significant absolute label
accumulation in DAG (250-300 cpm/3 x 103 cells) was noted within 60 sec
of 48/80 addition. Unfortunately, this increase was too small to
demonstrate an absolute loss from any of the labeled presursor lipids
PI, PC, PE and triglyceride. In broken cell preparations, 2-[l-^C]-
arachidonoyl-DAG was rapidly converted to free AA, monoacyl glycerol and
triglyceride. Substrate preference of 2-[l-14C]-arachidonyl-DAG over
l-El-^Cj-arachidonyl-PC suggested that degradation was mediated by a
DAG lipase, demonstrated in mast cells by Lewis etal_. (1979). Since
the time course of 3^P-PA accumulation in the previously described
investigations was more rapid than for DAG, it was suggested that mast
cell DAG kinase may initially remove newly formed DAG until stimulated
DAG production exceeds the enzyme's capacity, then the abrupt rise in
DAG levels is measured. Postulated roles for DAG and its metabolites
in mast cell secretion included facilitation of membrane fusion and


22
substrate for AA-derived mediator formation. These same observations
were extended to anti-IgE stimulated mast cells (Kennerly et al_.,
1979c).
Results of experiments conducted with [^H]-glycerol loaded mast
cells stimulated with either ionophore A23187 or compd 48/80 and
designed to study PI breakdown in activated mast cells have been
published by Ishizuka and co-workers (1983). Following stimulation of
cells by A23187, radioactivity in both PA and PI dropped slightly at 10
sec then went on to accumulate at 1.5 times the starting level at 60
sec. In contrast, radioactivity in DAG progressively increased to a
3-fold increase by 5 min. To investigate the possibility that
A23187-induced activation stimulated de novo PI and PA synthesis,
uptake of [^H]-glycerol was investigated during stimulation. Enhanced
uptake of [^H]-glycerol into PI and PA by 7- and 4-fold, respectively,
was measured. Though the percent total histamine released was
comparable, compd 48/80 was much less effective in accelerating PI
metabolism than A23187. In another report, Ishizuka and Nozawa (1983)
have discussed results of the same experimental design but with
specific antigen as the stimulant. In presence of 0.5 mM magnesium,
antigen stimulation (Ascaris suum extracts coupled to
2,4-dinitrophenyl) induced a loss of 30% PI from mast cells preloaded
with [^H]-glycerol followed by a PI resynthesis to 1.5-fold increase
over basal levels at 5 min into stimulation.
Concomitant with the decrease in PI radioactivity, a corresponding
increase was measured for DAG which exhibited a much greater initial
rise than did labeled PA, suggesting antigen stimulated receptor


23
mediated turnover of PI. Thus, while compd 48/80 and ionophore
stimulated de novo PI synthesis, antigen stimulation induced a
turnover. Unfortunately, the former investigations were carried out
with Sprague-Dawley rat mast cells while the antigen-stimulated mast
cells were derived from Wistar rats. Barring strain differences, it
appears that mast cell PI metabolism may differ for different classes
of secretagogues.
To date, this is the evidence that the PI response may play a role
in mast cell activation-secretion. The PI response and the
phospholipid methylation pathway are alike since they 1) are minor
membrane lipids which undergo rapid turnover, 2) are associated with
receptor stimulated activation, 3) may be involved in calcium
mobilization, and 4) can serve as sources of AA. Their exact routes of
metabolism and their physiological roles in mast cell degranulation
require further experimentation.
Conceptual Model for Mast Cell Exocytosis
Delineation of events involved in mast cell release has evolved
primarily from the impact of pharmacologic tools upon
activation-secretion. For the most part, these tools have been enzyme
inhibitors and antagonists and thus block events at different points
along the presumed step-wise pathway of activation-degranulation.
In development of this model, we will focus upon the previously
defined IgE dependent secretagogues including con A, specific antigen,
and anti-IgE as well as polypeptide-basic types which include compd
48/80 and somatostatin. Though the conditions required to elicit mast
cell release may be different for these two classes of secretagogues,


24
they all have one basic feature in commonthey are all polyvalent.
Sequentially, then, the first step in mast cell activation is ligand
receptor binding and aggregation by the polyvalent secretagogue.
Use of the serine esterase inhibitors phenyl methylsulfonylfluoride
(PMSF) or diisopropylfluorophosphate (DFP) to demonstrate blockade of
histamine release as well as PC degradation suggests that activation of
this enzyme may be an early event. When DFP is present during antigen
challenge, no histamine release results. If it is removed prior to
stimulation, release does occur, thus suggesting activation of this
esterase by receptor aggregation. Since the inhibitors prevent PC
degradation, serine esterase activation presumably occurs prior to
activation of phospholipase ^.
Phospholipid methylation which is stimulated selectively by IgE
dependent secretagogues, but not polypeptide-base types, is inhibited
by 3-deaza-SIBA or 3-deaza-adenosine. This results in the inhibition
of ^Ca^+ uptake as well as histamine release. In the absence of
extracellular calcium, methylation, but not IgE-dependent release
occurs, suggesting that phospholipid methylation may precede and/or
modulate calcium influx.
A transient rise in cAMP has been measured during mast cell
stimulation, but when the intracellular level is increased by
theophylline or dibutyryl cAMP, antigen-induced 45Ca^+ uptake and
histamine release are inhibited but no effect was noted with A23187 as
secretagogue. This suggests that cAMP may also be involved in calcium
mobilization but its relationship to phospholipid methylation remains
unclear. Fluctuations in cAMP levels may parallel these other events


25
but may be primarily involved in microtubule assembly-disassembly in
preparation for cellular degranulation.
All these mentioned events precede both histamine and AA release.
Activation of the calcium-dependent enzyme phospholipase A2 may then
occur. This enzyme is reportedly inhibited by mepacrine by some
unknown mechanism of action, or by a-parabromoacetophenone (PBP) which
modifies a histidine residue essential for its activity. Such
inhibition blocks anti-IgE induced histamine release. Other evidence
which suggests a role of phospholipase A2 in mast cell secretion
includes exogenous application of the enzyme which induces non-cytotoxic
release and release of histamine from stimulated cells which is
accompanied by release of [^C]-AA from prelabeled phospholipids.
Oxidative metabolism of released AA via the cyclooxygenase pathway
is exquisitely sensitive to inhibition by indomethacin and other
non-steroidal anti-inflammatory agents while the lipoxygenase pathway
remains undisturbed (Vane, 1971). However, indomethacin, at 10 uM, a
concentration which completely suppresses oxidative conversion of AA,
has no effect on the time course and amount of histamine released by
A23187, con A or anti-IgE suggesting that AA metabolism is not
necessary for or does not precede histamine release.
The inhibitors which block later events are the phenothiazines,
believed to inhibit calmodulin and/or Ca/Pl-PK. These agents block
degranulation by both IgE-dependent agents, polypeptide-base agents, as
well as ionophoretic compounds. Since they block A23187 induced
release of histamine without altering ^5Ca2+ uptake, it is assumed that
the drugs act on processes which effect exocytosis.


26
Very little is known about protein phosphorylation-
dephosphorylation, assembly-disassembly of cytoskeletal elements and
generation of granule-plasma membrane fusogen events which are believed
to comprise a final common pathway in mast cell release.
The goal of the present studies is to investigate the actions of
two additional pharmacologic probes, hydrocortisone and 12-0-tetra-
decanoyl-phorbol acetate on events of mast cell activation-secretion
coupling. This is the first characterization of either of these agents
on purified, normal rat mast cells. Relevance of the hydrocortisone
investigations arises particularly from the successful treatment of
moderate to severe allergic asthma with anti-inflammatory steroids.
The mechanisms by which anti-inflammatory steroids ameliorate symptoms
remain unclear. Since this is a type I immediate hypersensitive
reaction which involves mast cells, results of these present
investigations with purified populations of mast cells exposed to
hydrocortisone could lend valuable information concerning the
mechanisms of action of anti-inflammatory steroids in cells which play
a central role in allergic reactions.
In contrast, 12-0-tetradecanoyl-phorbol acetate, a prototypic
active tumor promoting agent, is known to activate inflammatory type
responses in several types of leukocytes. Its mechanism of action in
certain types of these cells is becoming more clearly delineated.
Thus, results of these investigations in mast cells could lend a new
perspective on events of activation-secretion coupling.
Far-reaching goals of these investigations are to provide new
approaches to pharmacologic modulation of components of the immune


27
system involved in allergic reactions and to gain further insight into
basic exocytotic mechanisms since the mast cell is considered the model
secretory cell.


CHAPTER TWO
INHIBITION OF IMMUNOGLOBULIN, BUT NOT POLYPEPTIDE-BASE,
STIMULATED RELEASE OF HISTAMINE AND ARACHIDONIC ACID
BY ANTI-INFLAMMATORY STEROIDS
Introduction
It is well established that glucocorticoids are potent
anti-inflammatory agents effective in ameliorating the symptoms of
immediate hypersensitivity and other allergic reactions (Lewis and
Austen, 1981). Mechanisms of anti-inflammatory glucocorticoid action
are not well delineated despite studies involving many varied cell
types (Lewis and Piper, 1976; Gryglewski, 1976; Hong and Levine, 1976).
Collectively, the data suggest that corticosteroids decrease the
release of arachidonic acid from membrane phospholipids and thereby
decrease prostaglandin generation without directly altering the enzymes
involved in prostaglandin synthesis. Glucocorticoid induced
inhibition of mast cell release may be related to the effectiveness of
these agents in the treatment of asthma and other allergic reactions.
The effects of glucocorticoids on mast cell release of both histamine
and arachidonic acid have not been clearly delineated.
To clarify biochemical mechanisms of anti-inflammatory steroid
J
action upon events in mast cell activation-secretion coupling, we
conducted investigations on the effects of hydrocortisone and other
glucocorticoids on the secretion of histamine and arachidonic acid and
its metabolites. We report here that anti-inflammatory glucocorticoids
28


29
selectively inhibit release of histamine and [l-14C]-arachidonic acid
(AA) and its metabolites. Only IgE-like secretagogue induced release
is steroid sensitive. Stimulation of both [1-^C]-AA and histamine
release by IgE independent secretagogues such as compd 48/80,
somatostatin, and the calcium ionophore, A23187, is not altered by
steroid pretreatment.
Materials and Methods
Ovalbumin Sensitization
Male or female Sprague-Dawley rats, 300-400g (random-bred colony,
University of Florida, Gainesville, FL) were injected i.m. with 0.5 ml
of a saline suspension containing 10 yg ovalbumin (Miles Research
Products, Elkhart, IN) and 20 mg of Al(OH3) (Amphogel, Wyeth,
Philadelphia, PA). Eight to ten days later a booster of the same dose
was administered i.m. Animals were decapitated and the sensitized mast
cells collected four days after the booster injection.
Mast Cell Preparation
Rat mast cells were obtained by lavage similar to the method of
Sul 1 i van et al_., 1975. Briefly, animals were decapitated and injected
intraperitoneally and intrathoracically with freshly prepared buffer
containing 25 mM PIPES, 0.4 mM MgCl2, 5 mM KC1, 10 mM NaCl, 0.1% (w/v)
BSA or gelatin, 5.6 mM glucose^ and 10 U/ml heparin in distilled water,
pH 7.4. Trunks of the animals were massaged 100 times, the abdominal
and thoracic cavities opened, the cell suspensions recovered and
delivered to 50 ml polypropylene centrifuge tubes. Cells were washed
and resuspended in the above buffer at one animal equivalent of cells
per ml. Each ml of cells was carefully layered over 2 ml of buffer


30
containing 23% metrizamide (Accurate Chemical and Scientific Corp.,
Westbury, NY). Mast cells were purified by passage through the
metrizamide layer during centrifugation at 180xg for 8 min as described
by Yurt et al_., 1977 and Coutts et al_., 1980. Average yields were
1-1.5 x 10 mast cells/rat. Trypan blue exclusion (Sullivan et al.,
1975) was used to assess cell viability (average >97%) and toluidine
blue staining was used as a purity criterion (average approximately
90%).
Passive Sensitization
Purified mast cells were suspended in RPMI 1640 culture medium
(5 x 10 cells/ml) (Flow Laboratory McLean, VA) containing 150 U/ml
penicillin and 150 pg/ml streptomycin, 4 mM glutamine, 5% fetal calf
serum, 200 ng/ml monoclonal mouse IgE anti-DNP antibody (Miles Research
Laboratories, Elkhart, IN) and the cells incubated for 90 min.
Pretreatment of Mast Cells with Steroids
Hydrocortisone, as well as the other steroids tested, were
dissolved in ethanol at a concentration of 1 mM then diluted to the
appropriate final concentration in the above RPMI 1640 medium. Cells
were suspended in the steroid-RPMI 1640 culture medium and incubated at
37C with 5% CO2 for 1 hr for acute steroid effects and 18-21 hr
(overnight) for long-term effects. Cells were washed in fresh culture
medium prior to [1-^C]-AA incorporation or in the PIPES buffered salt
solution prior to histamine release experiments.
Histamine Release and Measurement
Cells were suspended in the PIPES buffered salt solution containing
1 mM CaCl2 to give 1 x 10 cells/0.45 ml. Aliquots of 0.45 ml were


31
dispersed to polypropylene tubes and histamine release initiated by
addition of 50 pi of secretagogue. Histamine release by IgE dependent
agents was carried out in the presence of 50 yg/ml bovine brain extract
enriched in phosphatidyl serine. Incubations were carried out for
indicated times at 37C with gentle agitation and release terminated by
addition of 1 ml cold buffer then centrifugation at 5000xg for 10 min
at 4C. In each assay, a separate group of tubes was used for the
determination of total cellular histamine by adding 0.4N HC104 prior to
incubation. Supernatant histamine was determined by the
o-phthaldialdehyde spectrophotofluorometric procedure (Shore et al,
1959) as modified by Anton and Sayre (1969) and Siraganian (1976).
Percent histamine released was calculated as follows: ng supernatant
histamine/ng total cellular histamine x 100.
Incorporation and Release of [!-* ^C]-AA and Its Metabolites
Purified mast cells were suspended in the RPMI 1640 culture medium
(described above) with [1-^C]-AA (0.3 yCi/ml) then incubated for 60
min at 37C. Cells were washed twice with RPMI 1640 medium and once
with PIPES buffered salt solution containing 0.1% BSA as described
previously (Crews et al_., 1981). Cells were resuspended in PIPES
buffered salt solution and allowed to equilibrate at 37C for 30 min.
Release of [1-^C]-AA and its metabolites was carried out as described
above for histamine. Total release was quantitated by liquid
scintillation spectroscopy.
Measurement of ^5Ca Uptake
The method of Foreman et al. (1977) was used for the measurement of
^5Ca2+ flux. Briefly, purified mast cells were suspended in Tyrode's


32
solution, 2.5 x 105 cells/50 yl. One hundred microliters of versilube
F50 silicone oil (General Electric Corp.) were placed in the bottom of
a 400 yl microfuge tube and 40 yl Tyrode's solution containing ^Ca^+
(5.5 yCi/ml) followed by 10 yl secretagogue in Tyrode's solution were
layered on the oil and warmed to 37C. Five minute incubations were
initiated by addition of 50 yl mast cells and incubations halted by
centrifugation in excess of 10,000xg for 30 sec in a Beckman B
microfuge. Cell pellets were recovered by slicing off the microfuge
tube tips, and vigorously shaking in vials with an aqueous solution of
Triton X-100 (1%, v/v). ^5Ca^+ was assessed by addition of Liquiscint
(National Diagnostics) to the solubilized pellets and counting in a
liquid scintillation spectrometer.
Chemicals and Reagents
All steroids used were purchased from Sigma (St. Louis, M0) as were
compd 48/80, con A, and somatostatin. The calcium ionophore, A23187,
was purchased from Cal biochemical (LaJolla, CA). All other chemicals
used were reagent grade. Radioligands were purchased from Amersham,
[1-^C]-AA, 60 mCi/mmole, and ^5Ca^+, 40 mCi/mg calcium.
Statistical Analysis
Data are expressed as the mean S.E.M. The Student's t-test was
used to evaluate the differences between two means for significance.
The criterion for significance was p<0.05. The EC^g (effective
concentration for 50%) was calculated by probit analysis (Goldstein,
1964).


33
Results
To characterize the effects of glucocorticoids upon release of
histamine and [1-^C]-AA and its metabolites by rat mast cells,
purified mast cells were incubated with hydrocortisone (3 x Iff6 M)
overnight and then stimulated with known secretagogues. Hydrocortisone
pretreatment markedly inhibited both histamine and [1-^C]-AA release
stimulated by concanavalin A (con A), the antigen ovalbumin (OA) and
anti-immuglobulin E antibody (anti-IgE) (fig. 1). Histamine and
[1-^C]-AA release were inhibited to a similar extent. For example,
hydrocortisone treatment inhibited anti-IgE stimulated histamine and
[1-14C]-AA by 83.7 4.1% and 76.6 1.5%, respectively. In contrast
to IgE-like secretogogues, hydrocortisone pretreatment did not alter
the release of either histamine or [1-^C]-AA stimulated by the
polypeptide somatostatin, compd 48/80 or the calcium ionophore, A23187
(fig. 2). Hydrocortisone pretreatment does not significantly alter the
total amounts of mast cell histamine or incorporation of [l-^Cl-AA
(Heiman and Crews, 1984). Since AA and its metabolites have been
implicated in the mechanism of histamine release, we examined the
effects of exogenous AA on hydrocortisone's inhibition of con A
stimulated histamine release. Exogenous AA did not reverse
hydrocortisone's inhibition of con A stimulated release. Con A
(10 pg/ml) released 28.5 0.8% and 28.2 1.0% total cellular
histamine in control cells with and without AA (1 pM), respectively.
Cells treated for 18 hr with hydrocortisone released 13.4 0.8% and
11.8 0.2% total cellular histamine with and without exogenous AA
(1 pM), respectively. These results indicate that hydrocortisone


34
Figure 1. Effect of hydrocortisone pretreatment on the release of
histamine and [1-1^C]-AA and its metabolites during
stimulation with IgE-like secretagogues. Cells were
stimulated for 30 min by addition of indicated secretagogue.
Final stimulant concentrations were: 0A, 100 ug/ml; con A,
3 ug/ml; anti-IgE, 1:1000 dilution. Spontaneous histamine
release ranged from 8-12.9% and spontaneous [1-1^C]-AA
release from 219 to 431 dpms/2.5 x 10^ cells; these have
been subtracted from values depicted. Open bars indicate
untreated cells, striped bars represent hydrocortisone
pretreated cells. Each bar represents a typical mean
S.E.M. of triplicates repeated on at least two other
occasions. All hydrocortisone treated groups were
significantly different from controls at p<0.05 as assessed
by Student's t-test.


35
2000
O
LU
CO
<
ID
CE £
co 1800
o
o 2
m
< *
i-
UJ
2 ^ 1000
OJ
O E
Z a
< -a
<
<
O
800
UJ
co
u 60
GC
UJ
1 40
h-
co
z
a
UJ
< 20
3
2
co
*
AA RELEASE
JLt
48/80
HISTAMINE
1
SOMAT
RELEASE
X
i
A23187
48/80
SOMAT
A23187
Figure 2. Effect of hydrocortisone pretreatment on histamine and
[1-14c]_aa release during stimulation wlth IgE-independent
secretagogues. Cells were stimulated for 15 min with compd
48/80, 1 ug/ml; somatostatin, 100 yg/ml; A23187, 3 ym/ml.
Spontaneous histamine release ranged from 3.8 to 9.0% and
spontaneous [1-14C]-AA release ranged from 128-210
dpms/2.5 x 10^ cells; these have been subtracted from data
shown. Open bars represent untreated cells and striped bars
the hydrocortisone pretreated cells. Each bar represents a
typical mean S.E.M. of triplicates repeated on at least
two other occasions.


36
inhibits IgE-like release, i .e. anti-IgE, antigens and con A, but not
polypeptide-base or ionophore stimulated release.
To further characterize the actions of glucocorticoids on mast cell
release, time courses and concentration response curves were determined
for IgE-like and IgE-independent secretagogues. Treatment with
hydrocortisone (3 x 10"6 M) overnight slowed both the initial rate of
histamine release and the total amount of histamine released by con A,
but did not affect the rate or amount of histamine released by compd
48/80 (fig. 3). Results very similar to those for con A were obtained
with anti-IgE as the stimulant (data not shown). To determine if the
inhibition of stimulated release was due to a decrease in sensitivity
or a loss of responsiveness, complete concentration responses curves
were performed. Response curves for ovalbumin and compd 48/80
indicated that hydrocortisone pretreatment markedly decreased release
at all ovalbumin concentrations tested, but did not alter compd 48/80
induced release (fig. 4). The ovalbumin ED^g for histamine release
(untreated 1.14 0.3 yg/ml; hydrocortisone, 1.19 0.2 yg/ml) was not
shifted by hydrocortisone pretreatment. For [1-^C]-AA release, the
untreated EDgg for ovalbumin was 0.70 0.1 yg/ml, that for
hydrocortisone treated cells was 0.43 0.2 yg/ml. These data suggest
that hydrocortisone treatment selectively decreases maximal release of
histamine and [1-^4C]-AA initiated by an IgE-like secretagogue
stimulation, but not by IgE-independent types of stimulation. These
results, also suggest a close correspondence between release of
histamine and [1-^C]-AA and its metabolites.


37
Figure 3. Time course of con A and compd 48/80 induced histamine
release. Cells were preincubated with and without
hydrocortisone (3 x 106 M) for 21 hr and then stimulated to
release histamine by the addition of either con A (10 ygm)
in presence of 50 pgm/ml bovine brain extract or compd 48/80
(3 ugm/ml). Histamine release is calculated as a percent of
total releasable mast cell histamine. Each point represents
the mean for triplicate determinations from one of two
experiments of similar design.


38
Figure 4. Dose dependent [1-14C]-AA and histamine release by antigen
ovalbumin (OA) and compd 48/80. Cells were stimulated to
release histamine for 30 min by the addition of OA and 15
min for compd 48/80. Each point represents the mean
S.E.M. of triplicate determinations from one experiment.
Results are typical of those from at least two other
experiments of similar design.


39
To determine the duration of hydrocortisone pretreatment required
for inhibition of release initiated by IgE-like secretagogues, mast
cells were incubated for 24 hr with various times of exposure to
hydrocortisone (3 x 106 M). No inhibition of histamine release was
found after 1, 3 or 9 hr of treatment, but following 12 hr of exposure
to hydrocortisone, there was a significant decrease in histamine
release (fig. 5). This time dependent inhibition slowly increased to
approximately 95% inhibition after 24 hr of pretreatment. A similar
time dependent inhibition was noted for IgE-like release of [1-^C]-AA
and its metabolites. Such a delay in the onset of inhibition is
suggestive that induction of a protein may be involved in the mechanism
of the glucocorticoid action on mast cell secretion.
To explore the possibility that steroid-induced protein synthesis
is involved in the inhibition of IgE-mediated histamine release, we
incubated cells with various concentrations of cycloheximide and
actinomycin D, protein synthesis inhibitors with different sites of
action. These agents alone inhibited histamine release (data not
shown); therefore, we could not demonstrate reversal of the
glucocorticoid inhibitory effect with either cycloheximide or
actinomycin D. In rat thymus cells, glucocorticoid-receptor complex
translocation to the cell nucleus is temperature dependent, i.e. this
migration is halted at 4C (Mosher et al_., 1971; Wira and Munck, 1974).
Thus, we studied the effects of temperature on the actions of
hydrocortisone in our system. Preincubation of mast cells for 21 hr
at 4C or 37C did not alter subsequent con A induced release at 37C
(fig. 6). As shown previously, 21 hr of preincubation with


40
30--

o
V
a>
01 20--

c
6
o

X
10- -
wO O.
o*
*A
\
\
O
O
.o.
Control OO
Con. A A-A
A i
'O'
o
NA
O
3 6 9 12 15 18
Treatment with Hydrocortisone ( hr.)
21
24
Figure 5. Time dependent inhibition of histamine release by
hydrocortisone. Shown is an experiment typical of two other
experiments. All cells were incubated for 24 hr, but
drug-treated cells were exposed to hydrocortisone (3 x
10 M) for the number of hours indicated. Open circles
indicate spontaneous histamine release and closed triangles
indicate con A (10 ygm/ml) stimulated release of histamine
from treated cells. Con A stimulated release from control
cells ranged from 28-30% throughout the 24 hr time course.
Points represent the mean of two determinations. Duplicates
varied by less than 10%.


41
Preincubation Temp.
Hydrocortisone
Con A
4 C
- +
4 C
+ +
- +
37 C
- +
37 C
+ +
- +
Figure 6. Temperature dependent inhibition of histamine release by
hydrocortisone. Mast cells were preincubated with and
without hydrocortisone (3 x 10~6 M) at 4C or 37C for 21
hr. Cells were washed, slowly warmed to 37C and challenged
to release histamine for 30 min by addition of con A
(10 pg/ml). Bars represent the mean percent of total mast
cell histamine S.E.M. for three separate determinations.


42
hydrocortisone at 37C markedly inhibited con A induced histamine
release. However, 21 hr of preincubation at 4C with hydrocortisone
did not alter subsequent con A induced histamine release at 37C
(fig. 6).
To delineate the specificity of the glucocorticoid induced
inhibition of release, we incubated mast cells with several different
types of steroids. We found that fluocinolone, dexamethasone, and
hydrocortisone inhibited con A stimulated histamine release in a
dose-dependent manner following a 21 hr preincubation (fig. 7).
Steroid ICgg values were 1.4 x 10" M, 3.4 x 10" M, and 3.6 x 10"^ M,
respectively. This order of potency parallels both their in vivo
anti-inflammatory potencies (Gilman et al_., 1980) and their affinities
for the glucocorticoid cytosolic receptor (Dausse et al_., 1977). Cells
were also treated overnight with high concentrations (10"5 M) of the
non-glucocorticoid steroids, estradiol, and testosterone. Stimulated
histamine release by con A from untreated cells was 17.0 0.7% while
cells treated with testosterone and estradiol released 15.4 0.8% and
17.4 0.4% total histamine, respectively. Using fixed concentrations
of anti-inflammatory steroids, inhibition was also obtained for release
of [1-^C]-AA and its metabolites (table 1). Thus, only
anti-inflammatory steroids inhibit mast cell release by IgE-like
secretagogues.
The importance of calcium in exocytotic mechanisms has been well
documented for IgE-like mast cell secretagogues (Foreman et al_., 1977;
Baxter and Adamik, 1978). To further investigate the site of action of
glucocorticoids, we treated cells with hydrocortisone for 21 hr and


43
Figure 7. Inhibition of mast cell histamine release by
anti-inflammatory steroids. Steroids were dissolved in
ethanol (which comprised 0.1% of the pretreatment incubation
volume) and cells incubated with indicated concentrations
for 21 hr. Cells were washed, resuspended and challenged
to release histamine for 30 min by addition of con A
(10 yg/ml). Points represent the mean percent of total
histamine released S.E.M. for three separate
determinations.


44
TABLE 1
INHIBITION OF [1-14C]-AA RELEASE BY ANTI-INFLAMMATORY STEROIDS
Treatment
dpms [1-14C]-AA released/lO cells
Histamine
(% Released)
None
14,817
43
Fluocinolone
(108 M)
447
9.7
Dexamethasone
(10-8 M)
819
10
Hydrocortisone
(10-6 M)
875
12.3
Cells were pretreated for 18 hr with indicated concentrations of
steroids, washed, resuspended in PIPES buffered salt solution
containing 1 mM CaCl2 and 1% BSA, 0.45 ml aliquots containing 2.5 x
106 cells dispensed and release initiated by addition of con A
(10 ygm/ml) in presence of 50 ugm/ml BBE. Incubations at 37C were
continued for 30 min. Subtracted background [1-14C]-AA releases
averaged 2686 dpm/lO cells, and subtracted background histamine
releases averaged 5.7%. Values represent the mean of duplicates from
an experiment replicated with similar outcomes on two other
occasions.


45
then stimulated histamine and [1-^C]-AA and metabolite release with
the calcium ionophore, A23187, which artificially induces calcium flux.
Ionophore induced release was not significantly inhibited by
hydrocortisone treatment (fig. 8), suggesting that the secretory and AA
release processes following the influx of cal dun are not altered by
glucocorticoid treatment. Since release stimulated by IgE-like
secretogogues is known to depend upon extracellular calcium (Baxter and
Adamik, 1978), we studied the effect of hydrocortisone treatment on the
influx of ^Ca^+. Stimulation of rat mast cells with antigen caused a
rapid influx of calcium which was completed 5 min after addition of the
secretagogue. Pre-treatment with hydrocortisone for 18 hr markedly
reduced calcium influx Induced by con A, antigen, and anti-IgE (fig.
9). These results suggest that glucocorticoids inhibit plasma membrane
calcium flux and thereby selectively inhibit secretagogues dependent on
extracellular calcium, i.e. IgE-like secretagogues.
Discussion
Antigen and anti-IgE antibodies are thought to stimulate release
from rat mast cells by cross-linking IgE-Fc receptor complexes
(Ishizaka and Ishizaka, 1978). Con A, 1n a manner similar to antigen,
appears to act by cross-linking IgE bound to the cell surface
(Slraganian and Siraganian, 1975). These IgE-like secretagogues have
several properties which distinguish them from the polypeptide-base
secretagogues, e.g. somatostatin and compd 48/80. IgE-l1ke
secretagogues are dependent upon extracellular calcium, potentiated by
phosphatidyl serine (Baxter and Adamik, 1978), stimulate phospholipid


46
Figure 8. Effects of hydrocortisone treatment on A23187-induced
release of histamine and [l-14c]-AA and its metabolites.
Cells were treated as described in the legend of figure 4,
then resuspended and challenged to release histamine by
addition for 15 min of indicated concentrations of the
calcium ionophore, A23187. Points represent the mean
percent of total histamine released S.E.M. for three
determinations.


47
Figure 9. Effect of hydrocortisone pretreatment on IgE-dependent
4Ca2+ uptake. Mast cells were preincubated with and
without hydrocortisone (3 x 10 M) then challenged for
5 min with the following secretagogues in the presence of
5.5 pCi/ml 45Ca2+: con A 10 ug/ml, DNP-BSA 0.1 jig/ml,
anti-IgE 1:100 dilution. Bars represent the mean stimulated
uptake of 4Ca2+ cpm/2 x 10 cells S.E.M. for three
separate determinations. Background 4Ca2+ values were 300
17 cpm/2 x 10 cells. All hydrocortisone treated groups
were significantly different from controls at p<0.05 as
assessed by Student's t-test.


48
methylation (Hirata et. al_., 1979) and are inhibited by methylation
inhibitors (Crews et al_., 1981). Somatostatin and compd 48/80 do not
have any of these requirements or effects. These polypeptide-base
secretagogues appear to act by releasing internal stores of calcium
(fig. 10). Somatostatin and compd 48/80 have been suggested to act on
the same membrane receptor (Theoharides et al_., 1981). They are both
strong secretagogues which typically release more than 40-50% of the
total cellular histamine as compared to IgE-like secretagogues which
have a maximal response of about 20-30% total cellular histamine. The
strength of stimulation may play a role in the glucocorticoid affect.
However, submaximal concentrations of compd 48/80 which release
approximately 30% of total cellular histamine are not altered by
glucocorticoid treatment (fig. 4). These findings, along with the
differential effects of the glucocorticoids, suggest that release of
histamine and arachidonic acid and its metabolites induced by the
IgE-Fc receptor complex occurs by triggering mechanisms different from
those of somatostatin-48/80 induced release and that glucocorticoids
act by selectively uncoupling IgE-mediated release (fig. 10).
Glucocorticoids can act by membrane stabilization and/or induction
of the synthesis of specific proteins (fig. 10). The latter 1s thought
to involve the binding of glucocorticoids to a cytosolic receptor, and
formation of a complex which translocates to the nucleus and increases
synthesis of specific mRNA (Grody et al_., 1982). Cytosolic
glucocorticoid receptors have been demonstrated in mast cells (Daeron
et al_., 1982). Other studies have indicated that the translocation of
the glucocorticoid-receptor complex is temperature dependent (Mosher et


49
Figure 10. Schematic diagram of possible sites of cortisol action on
mast cell exocytosis. Cortisol could act through a
cytoplasmic receptor which when activated migrates to the
nucleus and induces the synthesis of specific proteins. A
membrane effect is also possible. The specific proteins
synthesized appear to interfere with the coupling of the
IgE receptors to the influx of calcium. Phospholipid
methylation (i.e. PE>PC) and/or phosphatidylinositol
turnover (i.e. PI>DAG>PA) have been implicated in the
coupling of IgE receptors to calcium influx and the release
of arachidonic acid (AA). Phospholipid methylation may be
inhibited by steroid treatment (Daeron et al_., 1982).
Somatostatin and compd 48/80 appear to release internal
stores of calcium. Therefore, these secretagogues are not
inhibited by glucocorticoids. Abbreviations: IgE,
immunoglobulin E; Ag, antigen; con A, concanavalin A;
somat., somatostatin; 48/80, compd 48/80; Fc, cell surface
receptor to which IgE binds; ATP, adenosine triphosphate;
RNA, ribonucleic acid.


50
al., 1971; Wira and Munck, 1974). The following data suggest that
glucocorticoids inhibit rat mast cell histamine release by inducing the
synthesis of specific proteins. Membrane stabilization occurs acutely,
whereas we have demonstrated that the inhibition of histamine and
arachidonic acid release by glucocorticoids requires several hours of
treatment. Inhibition of con A (IgE-1ike) induced histamine release by
glucocorticoids is temperature dependent, as is glucocorticoid-receptor
complex translocation. Various glucocorticoid inhibitory potencies
reported here parallel their in vivo anti-inflammatory potencies
(Gilman et al_., 1980) and their affinities for the cytosolic receptor
(Dausse et aU, 1977). Thus, it is likely that induction of a specific
protein is an important component of the glucocorticoid action on rat
mast cells.
Studies on rat mast cells (Hirata et al_., 1979) and rat basophilic
leukemia cells (Crews et al_., 1981; McGivney et al_., 1981) have
suggested that the stimulus-secretion coupling sequence is as follows:
antigen cross-linking of IgE, stimulation of phospholipid methylation,
influx of calcium, activation of phospholipase initiating fusion of
granule and plasma membranes (fig. 10). Our findings indicate that
glucocorticoids selectively inhibit IgE-mediated release by reducing
IgE stimulated calcium flux. Studies in mouse mast cells have
suggested that glucocorticoids alter anti-IgE stimulated histamine
release by inhibiting phospholipid methylation (Daeron et al_., 1982).
These studies indicated that steroid treatment does not change IgE-Fc
receptor number or affinity. Other investigators studying
anti-inflammatory steroid action have found that steroids induce the


51
synthesis of a phospholipase inhibitory protein in rat leukocytes
(macrocortin) (Blackwell et al_., 1980), rabbit neutrophils
(lipomodulin) (Hirata et al_., 1980), and mouse mast cells (Daeron et
al., 1982). Our finding that the glucocorticoid inhibitory effect upon
arachidonic acid release occurs only for IgE-like secretagogues and is
not overcome by exogenous AA is inconsistent with a general inhibition
of phospholipase. Several subtypes of phospholipase may exist, with
only the IgE-receptor associated form of the enzyme exhibiting
susceptibility to anti-inflammatory pretreatment. It has now been
proposed that the majority of lipomodulin secreted from neutrophils is
phosphorylated (therefore inactive) then split into several smaller
molecules the smallest of which is a 16,000 dalton protein (the size of
macrocortin) also generated by lymphocytes and called IgE-suppressive
factor or glycosylation inhibiting factor (Vede et al_., 1983). This
suggests, then, that a fragment of lipomodulin is involved in the
selective formation of IgE-suppressive factors which modulate T cell
subset responses to immunologic stimuli. A similar protein, generated
by glucocorticoid treated mast cells could contribute to the
selectivity of the inhibition induced. Alternatively, glucocorticoids
may also synthesize a phospholipid methyl transferase inhibitory
protein. Additional studies are necessary to delineate the mechanisms
of glucocorticoid specificity.
Whatever the mechanism of glucocorticoid action, our findings
indicate that glucocorticoids selectively inhibit IgE-mediated release,
i.e. anti-IgE, antigen and con A, but do not alter IgE-independent
stimulation of histamine and arachidonic acid release by somatostatin,


52
compd 48/80 or the calcium ionophore, A23187. The precise mechanisms
of steroid action in asthma and other allergic reactions are unknown.
Responsiveness differs for various types of allergic reactions
(Patterson, 1979). Our finding suggests that steroid treatment may
reduce allergic reactions mediated by antigen-IgE, but not reactions
stimulated by peptides and toxins which act through other receptors.


CHAPTER THREE
CHARACTERIZATION OF THE EFFECTS OF
PHORBOL ESTERS ON RAT MAST CELL SECRETION
Introduction
Phorbol esters (PEs), the most potent tumor promoting agents in the
mouse skin bioassay, also cause a variety of biological responses in
diverse cell types (Blumberg, 1980). These include lymphocyte
mitogenesis (Touraine et aK, 1977; Abb et al_., 1979), platelet
aggregation and serotonin release (Muf son et al_., 1979; Zucker et al.,
1974), PMN superoxide anion production (Lehrer and Cohen, 1981),
interleukin 2 production by mouse EL4 thymoma cells (Kraft et al.,
1982), as well as histamine release from a mixed population of human
leukocytes (Schleimer et al_., 1980).
We became interested in studying the effects of PEs alone and in
conjunction with various secretagogues for several important reasons.
First, it is well recognized that when applied to the skin, PEs elicit
signs of acute inflammation, mediators of which are released by mast
cells (Boutwell, 1974). Second, skin has been shown to contain
specific binding sites for PEs (Delclos et al_., 1980), and skin
contains a large number of mast cells. Recent studies have suggested
that PEs may specifically activate a calcium/phospholipid dependent
protein kinase (Ca/PL-PK) (Yamanishi et al_., 1983; Castagna et al.,
1982; Ashendel et al_., 1983). An investigation of the action of PEs on
mast cell secretion could provide a valuable tool for delineating the
53


54
role of this protein kinase in mediator release since experimental
results with stimulated mast cells have shown that during secretion
there is a rapid calcium-dependent phosphorylation of certain protein
bands (Sieghart et al_., 1978; Theoharides et al_., 1980; Wells and Mann,
1982).
In the purified mast cell system, receptor-mediated stimulation can
occur via^ two major classes of secretagogues which we call IgE-like and
polypeptide-base as well as other types of secretagogues (Crews and
Heiman, 1984; Lagunoff et al_., 1983). We define IgE-like as those
secretagogues which appear to activate mast cells by interacting with
bound immunoglobulin E. We call somatostatin and compd 48/80
polypeptide-base secretagogues since these agents appear to release
histamine through a cell surface receptor which is not IgE. Properties
which distinguish the two classes of secretagogues which we investigate
are: IgE-like agents (antigen, anti-IgE, concanavalin A) require
extracellular calcium, are potentiated by phosphatidyl serine (Baxter
and Adamik, 1978), stimulate phospholipid methylation, and are
sensitive to glucocorticoid pretreatment when used to stimulate mast
cell secretion (Crews and Heiman, 1984). Polypeptide-base
secretagogues (somatostatin, compd 48/80) do not require extracellular
calcium and do not respond to the conditions described for IgE-like
secretagogues.
Our interest in the Ca/PL-PK centers in the similarities for its
activation and mast cell secretion, namely calcium mobilization and
enhancement of release by PS. Abundant occurrence of Ca/PL-PK and its
endogenous substrate proteins in the particulate fraction of human


55
neutrophils suggests that protein phosphorylations by this enzyme may
be involved in membrane associated neutrophil functions. This
hypothesis was further strengthened by the demonstration, in these same
studies, that phosphorylation was inhibited by trifluoperazine, an
agent which inhibits neutrophil chemotaxis, aggregation and
degranulation (Helfman et al_., 1982). Trifluoperazine is also known to
inhibit rat mast cell secretion elicited by antigen, compd 48/80 and by
the calcium ionophore A23187 (Douglas and Nemeth, 1982).
We report here that 1) PE tremendously potentiates A23187
stimulated release of histamine and arachidonic acid, 2) extracellular
calcium is required for release in the presence of PE and A23187, 3)
histamine release by IgE-like secretagogues is potentiated by PS and
TPA, 4) structure activity relationships for the effects of various PE
analogs on mast cell histamine release suggest a single mechanism of
action, perhaps mediated by a single receptor, 5) TPA either alone or
with A23187 significantly increased phosphorylation of mast cell
protein(s), and 6) mast cells have low levels of Ca/PL-PK.
Materials and Methods
Mast cell preparation. Rat mast cells were obtained by lavage
similar to the method of Sullivan et al_. (1975). Briefly, animals were
decapitated and injected intraperitoneally and intrathoracically with
freshly prepared buffer containing 25 mM PIPES, 0.4 mM MgC^, 5 mM KC1,
10 mM NaCl, 0.1% (w/v) BSA or gelatin, 5.6 mM glucose, and 10 U/ml
heparin in distilled water, pH 7.4. Trunks of the animals were
massaged, the ventral wall reflected, the cell suspensions withdrawn
and delivered to 50 ml polypropylene centrifuge tubes. Cells were


56
washed and resuspended in the above buffer at one animal equivalent of
cells per ml. Each ml of cells was carefully layered over 2 ml of 23%
metrizamide (Accurate Chemical and Scientific Corp., Westbury, NY) also
prepared in the above buffer without heparin. Mast cells were purified
by passage through the metrizamide layer during centrifugation at 180xg
for 8 min as described by Yurt et al_. (1977) and Coutts et al_. (1980).
Average yields were 1-1.5 x 106 mast cells/rat. Trypan blue exclusion
was used to assess cell viability (average >97%) and toluidine blue
staining was used as a purity criterion (average approximately 90%).
Preparation of secretagogues. Ten times the final concentrations
of all secretagogues were prepared in the PIPES buffered salt solution
described above. In cases where IgE-like secretagogues were used,
500 ygm/ml phosphatidyl serine (PS) was added to the buffer and
sonicated for 2 min prior to addition of concanavalin A (con A) or
sheep anti-rat IgE (anti-IgE).
PEs were dissolved in DMSO as stock solutions of 1 mg/ml and were
diluted in buffer. During secretagogue induced release from mast
cells, DMSO comprised .001% of the incubated volume and in vehicle
treated control cells did not alter mediator release.
Histamine release and measurement. Cells were suspended in the
PIPES buffered salt solution containing 1 mM CaC^ to give 1 x 10^
cells/0.45 ml. Aliquots of 0.45 ml were dispensed to polypropylene
tubes and histamine release initiated by addition of 50 pi of
secretagogue. Histamine release by IgE dependent agents was carried
out in the presence of 50 pgm/ml phosphatidyl serine from bovine brain.
Incubations were carried out for indicated times at 37C with gentle


57
agitation and release terminated by addition of 1 ml cold buffer then
centrifugation at 4000xg for 10 min at 4C. In each assay, a separate
group of tubes was used for the determination of total cellular
histamine by adding 0.4N HCIO^ prior to incubation. Supernatant
histamine was determined by the o-phthaldialdehyde spectrophoto-
fluorometric procedure (Shore et al_., 1959) as modified by Anton and
Sayre (1969) and Siraganian (1976). Percent histamine released was
calculated as follows: ng supernatant histamine/ng total cellular
histamine x 100. Spontaneous histamine release, specified in each
figure legend, has not been subtracted.
Phosphorylation of mast cell proteins. In a method similar to that
employed by Wells and Mann (1982), cells were suspended in the PIPES
buffered salt solution containing 1 mM CaC^ at 2 x 10 mast cells/ml
and incubated at 37C for 30 min with [^P]orthophosphate, carrier
free, at a concentration of 1 mCi/ml. Cells were then washed twice in
5 ml warmed buffer and the pelleted cells resuspended to a final cell
density of 10 cells/50 pi. Fifty micro-liters of resuspended cells
were delivered to warmed glass tubes and stimulation initiated by
addition of 5 pi of secretagogue. Incubations were 60 sec and
terminated by the addition of 50 pi of gel sample buffer containing
20% w/v glycine, 4% w/v SDS, 0.008% bromophenol blue and 10 pi
mercaptoethanol/ tube. Tubes were placed in a boiling water bath for
3 min, cooled, sealed and stored overnight at -20C prior to SDS-PAGE.
SDS-PAGE. Electrophoresis was carried out in a Bio-Rad dual slab
cell. The resolving gel was 8% acrylamide (w/v) with an
acrylamide:bis-acrylamide ratio of 29:1. Samples (50 pi) were applied


58
to wells cast in the stacking gel with a 15-slot comb and gels
calibrated by applying the following MW standards from a commercial
standards kit (Sigma): carbonic anhydrase, 29 kDa; egg albumin,
45 kDa; BSA, 66 kDA; g-galactosidase, 116 kDA. Slabs were stained with
0.2% Coomassie brilliant blue R, dried and autoradiographed using SAR-6
x-ray film (Kodak) at -70C. To quantitate phosphorylation,
autoradiograms were scanned with a densitometer (E-C Apparatus Corp.)
and peak heights used to represent relative units of radioactivity
(Ueda et al_., 1973).
Measurement of Ca/PL-PK. Total, solubilized fractions of cerebral
cortex, resident peritoneal cells (RPC), RPC-devoid of mast cells
(RPC mast cells; cells which form a band at the buffer-matrizamide
interface) and mast cells were prepared according to the method
described by Hel fman et al_. (1982). Washed RPCs were resuspended in 2
ml Hank's balanced salt solution (HBSS) and treated for 5 min at 4C
with 5 mM DFP. Cells were washed twice in cold HBSS, mast cell and
RPC mast cell fractions prepared as described above, pelleted and
resuspended in 0.5 ml homogenization buffer (50 mM Tris-HCl, pH 7.5;
1% v/v g-mercaptoethanol; 2 mM EGTA; 0.1% v/v Triton X-100; 1 mM
phenyl methylsulfonyl fluoride). Dissected cerebral cortex tissue was
delivered into 5 volumes of homogenization buffer then homogenized by
hand in a glass-teflon tissue homogenizer. Cortical homogenate served
as positive Ca/PL-PK control in all experiments. All tissue
preparations were then sonicated at 50 watts for 20 sec (Sonifier Cell
Disruptor, Ultrasonics, Inc., Plainview, NY), stirred gently for 1 hr
at 4C, then centrifuged at 100,000xg for 60 min. The resulting


59
supernatants were used as the source of enzyme. Ca/PL-PK enzyme assay
conditions were as follows. Total incubation volumes were 200 yl and
contained 25 mM PIPES (pH 6.5), 10 mM MgCl2, 250 yM EGTA, 300-500 yM
CaCl2, 1 nmol (containing 0.9 to 1.4 x 106 cpms of [Y-32P]ATP), 40 ygm
histone 1, 3-4 ygm supernatant protein in the presence or absence of
50 ygm phosphatidyl serine. Reactions were carried out for 4 min at
30C and halted by the addition of 4 ml trichloroacetic acid (TCA)
containing 0.25% w/v sodium tungstate. Forty microliters of 1.625% BSA
were added and tubes centrifuged at 2000 rpm (500xg) for 1 min.
Supernatants were aspirated, pellets redissolved in 0.1 ml 0.5N NaOH
then precipitated again with TCA-tungstate solution. Precipitates were
washed in this manner three times, redispersed in 0.1 ml 0.5N NaOH and
an aliquot counted after addition of 5 ml Liquiscint (National
Diagnostics, Somerville, NJ).
Chemicals and reagents. All phorbol esters used were purchased
from Sigma (St. Louis, M0) as were compd 48/80, con A, somatostatin,
phosphatidyl serine, and ATP. The calcium ionophore, A23187, was
purchased from Cal biochemical (LaJolla, CA) and anti-IgE from Miles.
Culture medium was purchased from Flow Laboratories and [1-^C]-AA and
[^P]orthophosphate from Amersham. All other chemicals used were
reagent grade.
Results
Time Course and Calcium Requirement for TPA Induced Histamine Release
The time course of TPA induced release of mast cell histamine in
the presence and absence of subthreshold concentration of the calcium
ionophore, A23187, is shown in figure 11. While A23187 (0.05 yg/ml) or


60
Figure 11. Time course for TPA induced histamine release. Purified
rat mast cells were incubated with TPA, 10 ng/ml in the
presence and absence of A23187, 0.05 ygm/ml for the
indicated times at 37C. Spontaneous histamine release was
4 0.5% and has not been subtracted. A23187 alone
released 6.6 0.4%. Each point represents the mean
percent histamine released SEM of triplicates from an
experiment replicated with similar outcomes on two other
occasions.


61
TPA (10 yg/ml) alone released less than 10% of total cellular histamine
over the 3 hr time course, these agents acted synergistically to
produce a significant release after 5 min. Histamine was linearly
released for about 20 min, reaching a plateau of approximately 60%
total histamine released at 30 min. After this initial release period,
there was a slow gradual increase in histamine release reaching
approximately 70-80% after 3 hr and up to 90-95% after 5 hr. To
determine the role of extracellular calcium, cells were preincubated
for 90 min in PIPES-buffered salt solution containing either 10"^ M
EDTA or 2 mM calcium chloride then stimulated with TPA plus ionophore
A23187 in the same medium. Cells preincubated then stimulated in
presence of EGTA released only 6% histamine over spontaneous release
(5%), while cells preincubated and stimulated in presence of calcium
released 60% total cellular histamine.
Effect of TPA on Concentration Response of the Calcium Ionophore
A23187
The calcium ionophore, A23187, is thought to stimulate histamine
release by directly elevating free intracellular calcium levels.
Concentration response curves for A23187 with and without TPA are
depicted in figure 12. The histamine release elicited by ionophore
with TPA was potentiated (shifted to the left) when compared with
A23187 alone. The ECgg for the ionophore was approximately 832 ng/ml
and was reduced to 56.3 ng/ml when TPA was present as a co-stimulant.
When the extracellular calcium was removed, neither the co-stimulants
TPA-A23187 nor A23187 alone stimulated release.


% HISTAMINE RELEASED
62
A23187 (NG/ML) LOG SCALE
Figure 12. Effect of TRA on the dose-response of the calcium ionophore
A23187. Purified rat mast cells were incubated at 37C for
30 min with various concentrations of A23187 in the
presence and absence of TPA, 10 ng/ml. Spontaneous
histamine release which has not been subtracted was 4.1
0.5%. Each point represents the mean SEM of triplicate
determinations from one experiment. Results are typical of
those from at least two other experiments of similar
design.


63
Structure-activity Relationship for the Effect of Various PE Analogs on
Mast Cell Histamine Release
Specificity of the PE effect was explored by examining the
structure activity relationships for a series of five PEs. These
studies indicated an order of potency: TPA > 4g-PDD > POE (Fig.13).
Approximate EC^g values for the active PEs were: 5.4 ng/ml, 83.1 ng/ml,
and 807 ng/ml, respectively. Agents inactive as tumor promotors, e.g.
4a-PDD and 4a-phorbol were inactive as releasers of histamine.
Active-inactive isomeric pairs are suggestive of receptor specificity.
In experiments of a similar design cells were loaded with [1-^C]-AA
prior to stimulation with TPA and subthreshold A23187. The maximal
response of both histamine and [1-^C]-AA release occurred at 3 ng/ml
TPA with 32.1 0.8% and 4421 510 cpms/10^ cells, respectively.
Basal histamine release in presence of subthreshold A23187 was 4.7
0.4% while [1-^C]-AA release was 1528 46 cpms/10^ cells. Thus,
co-stimulation with TPA-A23187 appears to activate concomitant
histamine and AA release. Neither TPA alone, nor the inactive tumor
promoter 4a-phorbol induced release of these mediators.
Effect of TPA on IgE-like Secretagogue Induced Histamine Release
To characterize the effect of TPA on mast cell histamine release,
concentration response curves were assessed for anti-IgE (Fig. 14, top)
and con A (Fig. 14, bottom). At all concentrations of anti-IgE and con
A tested, inclusion of TPA increased histamine release. Although TPA
increased con A stimulated histamine release, it did not significantly
change the ECgg of con A for release, being 0.68 pgm/ml and 0.66 ygm/ml


64
Figure 13. Specificity of the PE effect on mast cell histamine
release. Purified rat mast cells were incubated at 37C
for 30 min with the indicated concentrations of PEs in the
presence of 0.05 ymg A23187. Abbreviations are: TPA,
12-0-tetradecanoylphorbol-13-acetate; 4bPD0, 48-phorbol
12&,13a-didecanoate; POE, phorbol 12-myristate 13-acetate
4-0-methyl ether; PE, 4a-phorbol. Background release in the
presence of A23187 was 17 to 22% and has not been
subtracted. Points represent the mean percent of total
histamine released SEM for triplicate determinations from
one experiment. Similar results were obtained in one other
experiment of identical design.


65
Figure 14. Effect of TPA on IgE-like secretagogue induced histamine
release. Indicated concentrations of anti-IgE (top panel)
and con A (bottom panel) in the presence of exogenous PS
(50 ygm/ml) and in the presence and absence of TPA
(10 ng/ml) were used to stimulate histamine release from
purified rat mast cells. Spontaneous histamine release,
not subtracted, was 7-7.6% for Anti-IgE experiments and
4-5.5% for con A experiments. Incubations were carried out
at 37C for 45 min. Each point represents the mean percent
histamine released SEM of triplicates from one
experiment. Results are typical of those from several
other experiments of a similar design.


66
in the presence and absence of TPA, respectively. Thus, TPA exhibited
a synergistic relationship with both con A and anti-IgE.
Enhancement of Stimulated Histamine Release by Phosphatidyl serine and
TPA
Since IgE-like secretagogues are known to be enhanced by exogenous
PS, the interaction of TPA with PS was investigated. Con A alone
elicited a very slight release response, which was greatly potentiated
in the presence of PS (Table 2). A similar situation exists for
anti-IgE. There was no significant dependence of peptide-base
secretagogue induced release upon exogenous PS (Grosman and Diamant,
1975). When TPA was present with IgE-like secretagogues, but without
PS, small increases in release were observed. This release was
synergistically enhanced when both PS and TPA were present. The
responses were more than additive even though maximal concentrations of
PS and TPA were used.
Effect of TPA on Polypeptide-Base Secretagogue Induced Histamine
Release
To determine the actions of TPA on compd 48/80 and somatostatin
induced mast cell release, complete concentration response curves were
done in the presence and absence of TPA (Fig. 15). In contrast to the
IgE-like secretagogues, TPA and compd 48/80 co-stimulation was barely
more than additive at 0.1 ygm/ml compd 48/80, and less than additive at
all other concentrations. Release induced by TPA and/or somatostatin
were less than additive at low concentrations and virtually identical
at higher concentrations. Neither potentiation nor synergism was
evident.


67
TABLE 1
EFFECT OF PS ON HISTAMINE RELEASE BY IgE-LIKE SECRETAGOGUES
Releasing
Agent
Histamine Release
(% Total Cellular Histamine)
A1 one
PS
(50 ygm/ml)
TPA
(10 ng/ml)
PS + TPA
None
1.1 0.1
3.1 0.4
5.9 0.4
7.6 0.2
con A
(10 pgm/ml)
3.8 0.3
20.5 0.9
13.0 1.0
44.7 1.5
anti-IgE
(1:1000 dil.)
2.3 0.7
14.0 1.2
7.7 0.3
33.0 2.4
Purified mast cells were washed, equilibrated for 30 min, pelleted
then resuspended in PIPES buffered salt solution containing 1 mM CaClo.
0.45 ml cells were aliquoted and stimulated by addition of 50 pi of
indicated releasing agents. Incubations were carried out for 45 min at
37C with secretagogue alone, and with PS and/or TPA. Values represent
the mean percents of triplicate S.E.M. of total mast cell histamine.
Similar results were obtained, in experiments of similar design, on two
other occasions.


68
Figure 15. Effect of TPA on polypeptide-base secretagogue induced
histamine release. Purified rat mast cells were incubated
with the indicated concentrations of compd 48/80 (top panel)
and somatostatin (bottom panel) in the presence and absence
of TPA, 10 ng/ml, for 30 min at 37C. Spontaneous release
which has not been subtracted, was 4% for compd 48/80 alone,
5% with TPA (top panel) and 8% for somatostatin alone, 14%
with TPA (bottom panel). Each point represents the mean
percent histamine released S.E.M. of triplicates from one
experiment. Results are typical of those from two other
experiments of a similar design.


69
Effect of Secretagogues on Mast Cell Protein Phosphorylation
It is becoming increasingly evident that activation-release events
of secretory cells are regulated, at least in part by protein
phosphorylation-dephosphorylation. Recent studies suggest that PEs
bind to and activate the Ca/PL-PK. Therefore, we investigated protein
phosphorylation in mast cells. Autoradiographs of protein
phosphorylations during 60 sec stimulation with con A, 48/80, anti-IgE,
and TPA alone, suboptimal A23187 alone and TPA-suboptimal A23187
together are depicted in figure 16. Although a variety of proteins are
phosphorylated, only a few are markedly changed during stimulation. We
found significant changes in the phosphorylation of four major bands
with apparent molecular weights of 78, 59, 55 and 48 kDa. The most
consistent and largest changes occurred in the 48 kDa band (Fig. 16).
Stimulation of mast cells with con A, anti-IgE, compd 48/80, TPA and
TPA-A23187 at least doubled the phosphorylation of the proteins
migrating with an apparent molecular weight of 48 kDa. Con A, compd
48/80, and TPA-A23187 stimulated histamine release corresponded with
the increase in phosphorylation of the 48 kDa band. In contrast,
anti-IgE and TPA alone stimulated much larger increases in
phosphorylation of the 48 kDa band than histamine release. Although
low dose ionophore (0.05 ygm/ml) had little effect on 48 kDa
phosphorylation or histamine release (Figs. 16 and 17)
secretion-inducing doses of A23187 (1 ygm/ml) stimulated
phosphorylation of the 48 kDa band and other bands similar to con A,
anti-IgE and compd 48/80. Thus, TPA alone and TPA-A23187 stimulate


70
100-
80-
a b c d
e f g h
- P 78
- P 60
-P 55
-P 48
Figure 16. Autoradiographs of SDS-PAGE gels showing secretagogue
induced phosphorylation of mast cell proteins. Incubation
periods were for 60 sec and release of histamine from
identically handled but unlabeled cells incubated with
secretagogue for conventional 15 or 30 min are given in
table 3. Secretagogues (concentrations shown in legend to
figure 17) were: a) PS, b) con A + PS, c) anti-IgE + PS,
d) none, e) compd 48/80, f) TPA, g) A23187, and h) TPA +
A23187. Apparent molecular weights were determined by
calibrating the gel with standard proteins of known
molecular weight. Phosphorylated proteins are shown on the
right.


INCREASED PROT. PHOS. 71
PHOSPHORYLATION
* '1
^ HISTAMINE RELEASE
CON A A-IGE 48/80 TPA A23 A23+TPA
Figure 17. Phosphorylation of a 48 kOa protein during mast ceil
stimulation. Phosphorylation experiments were incubated 60
sec, histamine release in identically handled, unlabeled
cells, for 30 min (also shown in table 3). Subtracted
phosphorylation peak heights were: buffer 46 4.4 units;
PS 53.2 4.2 units. Subtracted spontaneous histamine
release was 6.8 1% for buffer and 9.7 + 2.3% for PS
controls. Secretagogue concentrations are shown in table
3. Values represent the mean S.E.M. of arbitrary unit
densitometric peak heights of two identically conducted
experiments each performed in triplicate. One asterisk
indicates those values which differ significantly from
controls at p<0.05, two asterisks indicate those differing
from controls at p<0.01, one-way ANOVA.


72
phosphorylation of the 48 kDa proteins similar to the more classical
secretagogues.
The densitometric peak heights of three other major bands (i.e. 78,
59 and 55 kDa) are presented in table 3. Interestingly,
phosphorylation of the 78 kDa band was significantly elevated only by
compd 48/80. For the 59 kDa proteins, there was a trend toward
increased phosphorylation for compd 48/80, con A and anti-IgE with 13%,
12% and 41% increases in peak height when compared with appropriate
controls. An exact opposite trend is noted for TPA alone or in
conjunction with A23187 where peak heights dropped 17% and 24%,
respectively. In the 55 kDa protein band phosphorylations, mean
increases in peak heights were: 21%, 48/80; 33%, con A; and 43%,
anti-IgE, TPA, A23187 and TPA-A23187 had much smaller effects on the
phosphorylation of these proteins. A lack of correlation between
phosphorylation and histamine release is not surprising since each
secretagogue has a somewhat different time course for release.
Phosphorylation changes occurred very fast and were measured after
1 min of stimulation, whereas release was determined 30 min after
stimulation. These experiments do indicate that TPA and TPA-A23187 can
modify mast cell protein phosphorylation.
Assessment of Mast Cell Ca/PL-PK Activity
Since TPA has been shown to bind and activate the Ca/PL-PK, we
compared the activity of this kinase in cerebral cortex, which is known
to have a high activity, to that in resident peritoneal cells and
purified mast cells. Table 4 depicts results of two experiments
showing kinase activity using histone 1 as substrate. By comparison,


TABLE 3
PHOSPHORYLATION OF 78 kDa, 59 kDa, AND 55 kDa PROTEINS DURING MAST CELL STIMULATION
Densitometric Peak Heights (Arbitrary Units)
Apparent Protein Molecular Weights (kDa)
Secretagogue % Total
(cone) 78 59 55 Histamine Release
Buffer
68.6

5.6
65.3

4.0
90

3.8
6.8

1.0
48/80 (3 ygm/ml)
112

16.4*
73.2
+
12.6
109
+
5.8*
79.7
+
2.7**
TPA (10 ng/ml)
77.4

9.2
52.8

10.2*
97.2

10.4
25.1

2.0**
A23187 (.05 ugm/ml)
64

6.0
120.4

6.4
98.6

8.4
17.4

5.0
TPA + A23187
55.4
+
7.4
100
+
12.4*
91
+
5.0
56.5

4.7**
PS (50 ngm/ml)
73.4

7.6
110

5.4
80.4
+
9.2
9.7

2.3
con A + PS (10 pgm/ml)
71

7.4
123.6

9.2
106.4

12.0
39.4
+
4.9**
anti-IgE + PS (1:1000 dil)
76.4

9.4
155.6

15.6*
114.6

3.8*
29.2

2.1**
Experiments were carried as described in Methods and legend of figure 7. One asterisk indicates
those values which differed significantly from controls at p<0.05 and two asterisks indicate those
differing from controls at p<0.01, one-way ANOVA. Shown is the mean S.E.M. for 6 separate
determinations.


TABLE 4
IN VITRO Ca/PL-PK ACTIVITY IN CEREBRAL CORTEX, MAST CELLS AND RESIDENT PERITONEAL CELLS
Tissue
cpm 32P incorporated
+PS -PS
Ca/PL-PK activity
pmol P/ygm protein/4 min
Experiment 1
Cerebral cortex
74,829
72,143
4753
6027
16.0
RPC mast cel Is
34,120
35,434
5516
5329
5.2
Mast cells
5341
4401
2512
2727
'
0.4
Experiment 2
Cerebral cortex
34,305 1521
1229
84
8.9
RPC mast cells
11,488 1102
1240
716
2.8
Mast cells
1542 394
586
3
0.26
RPC
1736 89
948
14
0.21
Enzyme assays on
total solubilized
fractions
were
conducted as described in methods
with 4 pgm protein per assay tube. Abbreviations used: RPC, resident peritoneal cells
were those cells collected by lavage; RPC mast cells, RPC devoid of mast cells
represent a mixed population of white cells which band during metrizamide purification of
mast cells. Data presented for experiment 1 are duplicates while those from experiment 2
are mean S.E.M. of triplicates.


75
mast cells had 10-fold and 40-fold less activity than resident
peritoneal cells without mast cells (RPC mast cells) and cortex,
respectively. These relationships were not altered when endogenous
proteins were used as substrate (data not shown). In RPC fractions
which contained approximately 6% mast cells, Ca/PL-PK activity was
reduced to the level of purified mast cell homogenate activity. Thus,
the presence of mast cells markedly reduced the measured Ca/PL-PK
activity in RPC cells suggesting an endogenous inhibitor. Exogenous
histamine and heparin did not inhibit Ca/PL-PK activity and other
experiments to clearly establish an inhibitor have proved difficult
suggesting that any inhibitor that is present is very labile. Since
these assays were carried out with optimal conditions for Ca/PL-PK
activity, addition of PEs did not significantly increase activity.
Unfortunately, the enzyme activity is very low in mast cells' making it
difficult to demonstrate enhancement of suboptimal activity with either
DAG or PEs.
Discussion
We have studied the interaction of PEs with purified rat mast
cells. Although PEs alone cause only a small release of histamine,
they tremendously potentiate A23187 induced release. Concentrations of
ionophore which produce negligible release alone, release approximately
50% of mast cell histamine in the presence of TPA. PEs also
synergistically enhance release stimulated by anti-IgE and con A, two
secretagogues which release histamine through an interaction with IgE.
Polypeptide-base secretagogues such as compd 48/80 and somatostatin,
are not potentiated by PEs. In contrast to stimulation involving IgE,


76
PEs are only additive or less than additive with polypeptide-base
secretagogues. Anti-IgE, con A and the calcium ionophore, A23187, all
require extracellular calcium to stimulate histamine release, whereas,
somatostatin and compd 48/80 do not. Thus, stimulation of histamine
release by secretagogues which require extracellular calcium is
enhanced by PEs while release by agents which do not require
extracellular calcium was not enhanced (Fig. 18).
PEs have been shown to stimulate and potentiate A23187 induced
stimulation in a variety of cell types. Studies on human leukocytes
have shown that PEs enhance anti-IgE and A23187 stimulated histamine
release similar to our findings with purified rat peritoneal mast cells
(Schleimer et al_., 1982). In addition, the EC^g values and overall
potency series for the various PEs (e.g., TPA > 4g-PDD > POE 4g-PDD
and PE) are similar for rat peritoneal mast cells, human basophils
(Schleimer et al_., 1980), the mouse ear inflammation assay (Driedger
and Blumberg, 1979) and platelet serotonin release (Yamanishi et al.,
1983). The potency of TPA and the presence of active-inactive isomeric
pairs suggests that PEs may be acting at a specific site. The ability
of PEs to enhance the response to the calcium ionophore and/or other
stimulants known to cause calcium flux suggests that PEs may act by
enhancing the sensitivity of cells to calcium activation. Thus, if the
concentration of ionophore is proportional to the amount of calcium
entering the cell, mast cells are approximately 15-fold more sensitive
to calcium (i.e. the ECgg for A23187 is reduced 15-fold, see Fig. 13).
PEs have been shown to increase the affinity of the Ca/PL-PK for
calcium (Kishimoto et al_., 1980). Taken together, these findings


77
Ag
Con A
anti- IgE
va./
48/80
Somat.
phosphorylation
Figure 18. Hypothetical model of the role of Ca/PL-PK in mast cell
activation-secretion coupling. Multiple mechanisms may
exist for activation of Ca/PL-PK. IgE-like secretagogues
may activate the enzyme in the presence of PS primarily
through elevation of cytosolic calcium levels by
phospholipid methylation. Polypeptide-base secretagogues,
which do not significantly alter cytosolic calcium levels,
may primarily stimulate the turnovers of phosphatidylinositol
forming diacyl glycerol which lowers the calcium requirement
for the enzyme. Abbreviations used: AA, arachidonic acid;
Ag, antigen; con A, concanavalin A; DAG, diacyl glycerol;
48/80, compd 48/80; PC, phosphatidylcholine; PE,
phosphatidylethanolamine; PI, phosphatidylinositol; PNE,
phosphatidyl-N-methylethanol amine; PS, phosphatidyl serine;
somat, somatostatin; TPA, 12-0-tetradecanoyl-phorbol-13-
acetate.


78
suggest that the Ca/PL-PK may be involved in A23187 stimulated
histamine release.
PEs bind to a high affinity receptor site in a variety of tissues.
Recent studies have found that the PE binding site co-purifies with the
Ca/PL-PK. This protein kinase is activated by calcium and phospholipid
with phosphatidyl serine being the preferred lipid. We have shown that
mast cells have Ca/PL-PK activity although not as much as brain tissue
or other peritoneal cells. The high activity of rat peritoneal cells
with the mast cells removed is likely to be due to neutrophils which
are known to have large amounts of Ca/PL-PK (Helfman et al_., 1982) and
are readily activated by PEs. PEs enhance the sensitivity of Ca/PL-PK
to calcium and PS. The PEs apparently mimmic the action of
diacyglycerol, a lipid which is formed during mast cell stimulation
(Kennerly et al_., 1979), and which also enhances the affinity of
Ca/PL-PK for calcium and PS. Our finding that exogenous PS and TPA
enhance anti-IgE and con A stimulated histamine release in a
synergistic manner suggests that the stimulation of secretion by
anti-IgE and con A may involve activation of Ca/PL-PK.
Protein kinases and protein phosphorylation-dephosphorylation have
been implicated in stimulation secretion coupling in a number of
tissues. Previous studies on mast cells have reported changes in the
phosphorylation of four protein bands having molecular weights of
approximately 42-46, 56-59, 67-68 and 78 kDa during stimulation with
compd 48/80 (Sieghart et al_., 1978) and anti-IgE (Wells and Mann,
1982). We found that certain secretagogues increased ^P incorporation
into proteins having apparent molecular weights of 48, 55, 59 and


79
78 kDa. Considering differences in requirements for extracellular
calcium, PS, and the time course of histamine release, it is not
surprising that we found differences in protein phosphorylation 1 min
after stimulation. We found our most consistent increases in
phosphorylation in the 48 kDa band. Anti-IgE, con A, compd 48/80, TPA
alone and TPA-A23187 all markedly increased phosphorylation of this
band. PEs and PE-A23187 have been shown to stimulate secretion from
neutrophils and platelets, respectively, and to cause prominent
increases in the phosphorylation of proteins having apparent molecular
weights of approximately 47 kDa and 43 kDa, respectively. Both of
these proteins have been shown to be substrates for the Ca/PL-PK. It
is possible that these proteins with apparent molecular weights of
42-48 kDa represent closely related proteins which play a role in
secretion from a number of cells.
The studies discussed above suggest that the Ca/PL-PK is involved
in stimulation-secretion coupling in neutrophils, platelets and other
tissues. We have found Ca/PL-PK activity in extracts from rat
peritoneal mast cells. Furthermore, our finding that the PEs, which
are known to bind to and stimulate the Ca/PL-PK, enhance histamine
release by agents which mobilize extracellular calcium and that PEs
stimulate protein phosphorylation suggests that activation of the
Ca/PL-PK may be involved in histamine release. PEs did not enhance
release stimulated by somatostatin or compd 48/80. Compd 48/80
stimulated the largest increases in protein phosphorylation, including
the 48 kDa band, and the greatest release of histamine. It is possible
that compd 48/80 stimulates secretion through a completely separate


80
mechanism or that it maximally activates the Ca/PL-PK such that PEs do
not further enhance activation. In any case, our results suggest that
the Ca/PL-PK may play a role in stimulation-secretion coupling in mast
cel Is.


CHAPTER FOUR
CONCLUSIONS AND SIGNIFICANCE
Experimental results presented in this dissertation demonstrate for
the first time that the pharmacological probes hydrocortisone, a
prototypic anti-inflammatory steroid, and TPA, a prototypic tumor
promoter, exert significant and selective effects upon mast cell
stimulated release.
In the case of anti-inflammatory steroid action, we have
demonstrated that in cells treated in vitro, long-term (more than 9 hr)
pretreatment is required for manifestation of inhibitory effects.
Exertion of the inhibitory influences appear to be due to induction of
the synthesis of specific proteins. Inhibition of ^5Ca^+ uptake,
histamine and [1-^C]-AA release in anti-inflammatory steroid treated
cells was limited to secretion by IgE-like secretagogues. Addition of
exogenous AA did not overcome the inhibitory effect. No significant
inhibition was noted in cells stimulated with polypeptide-base
secretagogues or the receptor-independent stimulating agent ionophore
A23187.
Other investigators have shown in mouse mast cells, glucocorticoid
pretreatment did not alter IgE-Fc receptor number, affinity of IgE for
its receptor or basal cAMP levels. Pretreatment did, however, inhibit
IgE-induced phospholipid methylation, 45Ca^+ uptake as well as
histamine release. Phospholipase inhibitory protein (lipomodulin)
levels were increased in anti-inflammatory steroid pretreated cells.
81


82
This evidence suggests that in mast cells, anti-inflammatory
steroids exert inhibitory influences early in the biochemical sequence
of activation-secretion events. However, several possibilities for
glucocorticoid action exist and include: 1) uncoupling cellular
activation from influx of extracellular calcium, 2) inhibition of
phospholipid methyl transferases, 3) synthesis of phospholipase
inhibitory protein which interacts selectively with a subtype of
phospholipase associated with Fc receptors, and 4) in a manner
analogous to lymphocytes, generation of IgE-suppressive factors, which
modulate T cell subset responses to immunologic stimuli may also act
selectively in mast cells.
Time courses of glucocorticoid induced inhibition of IgE-like
release argue against competition for extracellular calcium binding
sites as do the lanthanides, agents which also selectively inhibit
IgE-like but not polypeptide-base induced release. Anti-inflammatory
steroid induced inhibition is reminiscent of methyl transferase
inhibitor action as well as theophylline and dibutyryl cAMP induced
inhibition of IgE-antigen elicited methylation, 45Ca2+ uptake and
histamine release. Ionophore A23187 treatment can overcome inhibition
by all of these agents, presumably since it functions as a calcium
carrier which bypasses receptor-mediated effects which may include both
transient cAMP and methyl transferase activation.
As documented earlier, phospholipid methylation has been shown to
act prior to calcium influx and glucocorticoids prior to methylation.
Thus, it appears that one mechanism of glucocorticoid action in mast
cells is by methyl transferase inhibition. This is also the case in


83
other white cells as Honma et al_. (1981) have shown a decrease in
phospholipid methylation in dexamethasone treated mouse myeloid
leukemia cells during differentiation. In chronic leukemia cells in
culture, 6 hr pretreatment with anti-inflammatory steroids did not
influence the activity of phospholipid methyl transferase I in forming
PME from PE, however, there was a decrease in the conversion of PME to
PC by phospholipid methyl transferase II. A similar effect in mast
cells could explain glucocorticoid induced inhibition patterns.
Our data does not rule out the generation of phospholipase
inhibitory proteins such as lipomodulin as another mechanism of
anti-inflammatory steroid action. However, results supportive of a
generalized generation of lipomodulin in glucocorticoid pretreated mast
cells included only IgE-mediated versus A23187 mediated release. The
model is complicated in view of the results of the present
investigations where another class of receptor-stimulating agents,
polypeptide-base secretagogues were included. Selectivity of the
phospholipase inhibitory proteins to IgE-like coupled responses must
now be evoked. The fact that exogenously added AA, if it resembles
endogenous release, is not sufficient to overcome the presumed
phospholipase inhibitory effects of glucocorticoids suggests that other
mechanisms may be involved and/or other products of phospholipase
activation may be important.
In contrast to the inhibitory effects of glucocorticoids, we have
demonstrated that the pharmacologic probe, TPA is capable of increasing
mast cell release. In our characterizations of the effects of PEs upon
mast cell release, we have shown that, in the presence of extracellular


84
calcium, PEs tremendously potentiate ionophore A23187 stimulated
release of both histamine and AA. PEs act synergistically with
IgE-like secretagogues in the presence of PS while interactions with
peptide-base types of secretagogues are minimal. Structure-activity
relationships for various PE analogues on mast cell histamine release
suggest a single mechanism of action, perhaps mediated by a single
receptor. In many tissues, the PE receptor is now thought to be a
Ca/PL-PK; we have demonstrated low levels of this enzyme in mast cells.
Activation of Ca/PL-PK by PEs occurs in the presence of PS by
substituting for DAG to increase the affinity of the enzyme for
calcium. Our findings that exogenous PS and TPA enhance IgE-like
stimulated histamine release in a synergistic manner suggest
involvement of the enzyme Ca/PL-PK in mast cell stimulation-secretion
coupling. In contrast, peptide-base secretagogues may effect secretion
through a completely separate mechanism or through maximum activation
of Ca/PL-PK thereby abolishing enhancement by TPA.
In platelets, signal induced PI breakdown is linked to Ca/PL-PK
activation by generation of DAG. The synthetic analogue of DAG,
1-oleoyl-2-acetylglycerol (OAG) which gains access to intact platelets
with no sign of membrane damage, activated Ca/PL-PK without inducing PI
turnover or AA generation. In a similar manner, synergistic effects of
OAG and ionophore A23187 have been noted in mast cells. When
stimulated with OAG alone, mast cells released histamine to some
extent, but in the presence of low concentrations of ionophore A23187,
release was dramatically enhanced (Nishizuka, 1984). The suggestion
that protein phosphorylation by Ca/PL-PK and the mobilization of


85
calcium are indispensible and synergistically effective for causing
full physiological responses is supported by the following. Turnover
of PI and incorporation of radiolabel into PI take place in the mast
cell stimulated with antigen, anti-IgE, and con A or compd 48/80
regardless of the presence or omission of extracellular calcium. Thus,
PI metabolism may concomitantly participate in activation of Ca/PL-PK,
the regulation of calcium channels or mobilization of sequestered
calcium, as well as the generation of AA following receptor activation
of the mast cell.
McPhail et al_. (1984) have recently stated that unsaturated fatty
acids, including AA, directly activate Ca/PL-PK in the presence of PS
by increasing affinity of the enzyme for calcium. However, at higher
concentrations, a loss of enzyme activity which could not be reversed
by PS was noted. Like DAG, AA may also have an ability to directly
activate and regulate Ca/PL-PK thus giving it a new role in modulation
of cellular responses. We have noted, in data not shown, that mast
cells can be stimulated to secrete histamine by exogenous AA in the
presence of extracellular calcium. That stimulation may be via
activation of Ca/PL-PK.
We have also investigated glucocorticoid-TPA interactions and have
found that, in the presence of suboptimal concentrations of ionophore
A23187, addition of various concentrations of TPA to glucocorticoid
pretreated cells did not overcome the inhibition of release of either
histamine of [1-*4C]-AA. Glucocorticoid pretreatment of mast cells may
induce synthesis of a Ca/PL-PK inhibitory protein such as the phorbol
ester binding inhibitory factor (PEBIF) described by Hamel et al.
(1984).


86
Relevance of this work toward progress in basic medical research of
human allergy is fortified by the similarity of initial triggering
events of IgE-mediated release from rat mast cells and purified human
lung mast cells.


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Full Text
UNIVERSITY OF FLORIDA
3 1262 08554 3683


Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
STUDIES ON AGENTS WHICH MODIFY
MAST CELL STIMULATION-SECRETION COUPLING
By
ANN S. HEIMAN
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1984

This dissertation is dedicated to Tom who is both my best friend
and my husband. Together with him I celebrate personal growth and
development.

ACKNOWLEDGEMENTS
I express sincere thanks and gratitude to my mentor, Fulton Crews,
and to the other members of my advisory committee, Allen Neims, Stephen
Baker, Stephen Russell and William Kern, for their invaluable assistance
in my development as a pharmacologist. Special acknowledgement goes to
Mrs. Judy Adams whose typing and editing skills are surpassed only by
her patience and pleasant manner. And, I am thankful to all my fellow
lab workers and students for creating a pleasant working atmosphere.

PREFACE
"Of all intellectual activity, science alone has flourished in the
last centuries, science alone has turned out to have the kind of
universality among men which the times require."
J. Robert Oppenheimer
This dissertation is composed of an introductory chapter, two
chapters written in standard manuscript style and a final conclusion
and significance chapter. Due to this format, some material may appear
redundant. Please accept any repeated material as important to the
overall contribution of this research to further understandings of mast
cell stimulation-secretion coupling.
iv

TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS ; ill
PREFACE 1 v
ABSTRACT vi
CHAPTER ONE INTRODUCTION 1
General Characterization of Mast Cells
and Histamine Release 1
Classes of Agents which Release Histamine
from Mast Cells 4
Other Mediators Released by Mast Cells 7
Role of Calcium in Mast Cell Exocytosis 9
Phospholipid Metabolism During Mast Cell
Activation 10
Conceptual Model for Mast Cell Exocytosis .... 23
CHAPTER TWO INHIBITION OF IMMUNOGLOBULIN, BUT NOT
POLYPEPTIDE-BASE, STIMULATED RELEASE OF
HISTAMINE AND ARACHIDONIC ACID BY
ANTI-INFLAMMATORY STEROIDS 28
Introduction 28
Materials and Methods 29
Results 33
Discussion 45
CHAPTER THREE CHARACTERIZATION OF THE EFFECTS OF PHORBOL
ESTERS ON RAT MAST CELL SECRETION 53
Introduction 53
Materials and Methods 55
Results 59
Discussion 75
CHAPTER FOUR CONCLUSIONS AND SIGNIFICANCE 81
REFERENCES 87
BIOGRAPHICAL SKETCH 98
v

Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
STUDIES ON AGENTS WHICH MODIFY
MAST CELL STIMULATION-SECRETION COUPLING
By
Ann S. Heiman
August, 1984
Chairman: Fulton T. Crews
Major Department: Pharmacology and Therapeutics
Mast cells are long-lived cells which reside in connective tissue
and tissues surrounding blood vessels, meninges, sheaths of peripheral
nerves and submucosa of the small intestine and respiratory system.
They respond to a variety of peptides and participate in concert with
bound IgE and antigen to release mediators of immediate
hypersensitivity.
The present, detailed investigations were conducted with purified,
normal mast cells and the results presented in terms of 1) selective
effects of anti-inflammatory steroids and 2) characterization of
actions of tumor-promoting phorbol esters upon mast cells stimulated
with IgE-dependent secretagogues, peptide-basic agents or the calcium
ionophore A23187.
With respect to the selective effect of anti-inflammatory steroids,
the interesting findings were 1) long-term (18-21 hr) pretreatment
vi

was required for manifestation of inhibitory effects, 2) the inhibitory
effect was limited to anti-inflammatory types of steroids, 3) a
specific glucocorticoid receptor mechanism was involved, 4) inhibition
of release of histamine and arachidonic acid resulted when cells were
stimulated with IgE-dependent secretagogues, 5) no inhibition was noted
when cells were stimulated with polypeptide-basic agents or ionophore
A23187, and 6) IgE-dependent secretagogue stimulated calcium flux was
decreased.
Characterization of the effects of phorbol esters on rat mast cell
secretion revealed that 1) phorbol esters tremendously potentiated
ionophore A23187 stimulated release of histamine, 2) extracellular
calcium was required, 3) histamine release by IgE-like secretagogues
was potentiated by the presence of the phospholipid phosphatidyl serine
and phorbol ester, 4) structure-activity relationships suggested a
single mechanism of action, perhaps mediated by a single receptor, 5)
phorbol esters either alone or with ionophore A23187 increased
phosphorylation of mast cell proteins, and 6) mast cells have low
levels of calcium-phospholipid-dependent protein kinase to which
phorbol esters bind.

CHAPTER ONE
INTRODUCTION
General Characterization of Mast Cells and Histamine Release
Within the Gel! and Coombs (1968) scheme for classifying
immunological responses, histamine release from mast cells (and
basophils) comprises the type I sensitivity commonly called immediate,
anaphylactic or reagin-dependent hypersensitivity. This reaction
occurs upon re-exposure to a specific antigen which interacts with its
immunoglobulin E (IgE) antibody bound to the surface of mast cells and
basophils and triggers a sequence of activation-secretion coupling
events which culminate in the release of several inflammatory mediators
(Altman, 1981). Minimum requirements for an immediate hypersensitive
event are allergen and mast cell or basophil with specific IgE bound to
the cell surface Fc receptor. Initial exposure to an allergen requires
collaboration between it, macrophages and T lymphocytes which stimulate
B lymphocytes to differentiate to plasma cells and synthesize and
release IgE-class antibodies. Investigations into the regulation of
the IgE antibody formation response have led to the hypothesis that
certain IgE-class specific subpopulations of both helper and
suppressive T cells work together with B cells to modulate the antibody
response (Tadamitsu, 1982). Normally, suppressive mechanisms
predominate to keep IgE levels low; therefore, allergic diseases may
represent a "breakthrough" phenomenon (Katz, 1978a, 1978b).
1

2
Mast cells to which IgE binds> reside predominantly in connective
tissue and skin surrounding blood vessels, meninges, sheaths of
peripheral nerves and submucosa of the small intestine and respiratory
system. These are long-lived cells, capable of regranulation in
contrast to their circulating counterparts, the basophils, which are
short-lived, incapable of regranulation and can move into tissues in
response to chemo-attractants (Altman, 1981). Both cell types store
certain mediators of hypersensitivity reactions, such as histamine
which is bound to a water-insoluble complex of protein and heparin
within cytoplasmic granules. Rat peritoneal mast cells reportedly
contain 250-500 granules per cell, and this may comprise up to 70% of
the cellular dry weight (Bergendorff and Uvnas, 1973).
Release of histamine and other mediators in an immediate
hypersensitivity episode is the outcome of a complex biochemical series
of events beginning with activation of the cell surface IgE receptor.
Several investigative groups have reported that normal rat mast cells
have approximately 2 x 105 IgE receptors/cell (Conrad et aj_., 1975;
Ishizaka et a]_., 1975; Mendoza and Metzger, 1976). Cross-linking of
receptor-bound IgE by multi-valent antigen, anti-IgE or direct
cross-linking of IgE receptors by anti-receptor IgE triggers the
sequence of activation-secretion events (Ishizaka and Ishizaka, 1968;
Ishizaka et al_., 1971; Ishizaka and Ishizaka, 1978). Activation of a
serine esterase closely associated with IgE receptors has been
postulated as a very early event in stimulus activated mast cells and
may play a general role in secretory cell activation (Taylor and
Sheldon, 1974). According to the mobile receptor hypothesis of

3
Cuatrecasas (1974), membrane-associated receptors and enzymes acquire
affinities to form complexes upon receptor occupation and may thus
"uncover" active sites of these enzymes of mediator release. Other
enzymes which fit such a hypothesis include phospholipase Ag,
phospholipase C, protein kinases such as the calcium and phospholipid
dependent protein kinase (Ca/PL-PK) and the phospholipid
methyl transferases. While different classes of secretagogues may
utilize different enzymes in the initial transmembrane signalling
sequences, it is thought they employ a common sequence during the
secretion phase. This latter phase involves microtubule assembly
(Lagunoff and Chi, 1976) which facilitates intracytoplasmic granule
movement and culminates in very poorly understood granule-plasma
membrane fusion and granule content extrusion.
Degranulation of mast cells and basophils is sensitive to both pH,
with optimum release at pH 7, and temperature, secretion is inhibited
at 0° or 45*C (Moran £t ¿1_., 1962; Baxter and Adamik, 1975). There is
an absolute requirement for calcium, and in the case of IgE-mediated
degranulation, there must be extracellular calcium ions available
(Foreman and Monger, 1972; Baxter and Adamik, 1978). Availability of
metabolic energy in the form of ATP is a basic prerequisite for
histamine release and can be derived either from oxidative
phosphorylation or glycolysis. Reports indicate that cellular ATP
levels decrease by 30-35% during the release process (Kazimierczak and
Diamant, 1978).
From this general description of the mast cell and the complex
sequence of events involved in degranulation, we can see that it

4
entails an active secretory process and has been considered a
prototypic secretory cell (Douglas, 1968) having the following distinct
components: 1) activation by specific stimuli, 2) extrusion of
granular material, and 3) cellular recovery and restoration of granule
contents (Kirshner and Viveros, 1972).
Classes of Agents which Release Histamine from Mast Cells
Immediate type hypersensitivity reactions by definition involve
antigen and antibody; however other diverse chemical as well as
physical stimuli are capable of inducing granule extrusion (Foreman,
1981; Lagunoff et al_., 1983; Kazimierczak and Diamant, 1978; Ho et al.,
1979). Rather than bridge receptor-bound IgE molecules, these other
chemical stimuli interact with specific mast cell receptors to initiate
stimulation and secretion. Types of agents capable of eliciting
non-cytotoxic mediator release from mast cells and basophils are quite
diverse. An abbreviated categorization of these agents central to
these investigations includes immunologic agents, lectins, polycationic
amines and polypeptides and ionophores.
Within the immunologic agent category fall IgE antibody, IgE
generated toward another species IgE (i.e. anti-IgE), anti-receptor IgE
and the anaphylatoxins C3a and C5a. Release elicited by the first
three agents proceeds via activation of the IgE Fc cell surface
receptor, as previously described. Anaphylatoxins C3a and C5a,
generated from complement components, are believed to induce
degranulation by occupation of their own receptors, independent of the
IgE Fc receptor (Siraganian and Hook, 1976; Hartman and Glovsky, 1981),
but the requirements for release are identical to the IgE-dependent

5
mechanism. Both require extracellular calcium, an energy source, are
temperature dependent and are potentiated by exogenous
phosphatidyl serine (PS).
Lectins are hemagglutinins which possess saccharide-specific
binding sites and include Jack bean concanavalin A (con A), as well as
wheat germ, castor bean and lentil agglutinins. While all these agents
reportedly elicit release, most is known about con A induced histamine
release. Critical determinants of the extent of con A induced release
may vary dependent upon the strain of rat used, presence or absence of
extracellular calcium, the degree of cell sensitization and requirement
for exogenous PS (Lagunoff et al_., 1983). However, there are data
which convincingly suggest that con A, tetravalent at physiological pH,
cross-links bound IgE by interaction with saccharide moieties located
near the Fc region of the IgE molecule (Siraganian and Siraganian,
1975; Fewtrell £t al_., 1979). Though normal rat mast cells may be
quite resistant to con A, cells collected from animals infected with
Nippostrongylus brasiliensis, known to increase IgE synthesis, become
more sensitive to con A (Keller, 1973). Lastly, monomeric antibody
directed against the Fc portion of IgE inhibits con A induced histamine
release, indicating that this lectin binds to the Fc region of IgE
bound to the mast cell surface (Magro and Bennich, 1977). In
Sprague-Dawley rats, the strain used in these investigations, the
requirements for con A induced mast cell degranulation are identical to
those for IgE-induced release.
The prototypic polycationic amine releasing agent is compd 48/80, a
polymerization product of equimolar p-methoxy-N-methyl phenethylamine

6
and formaldehyde. Polypeptides in this group include somatostatin,
substance P, bradykinin, ACTH and neurotensin (see table compiled by
Lagunoff et al_., 1983). These agents share a common feature, the
presence of at least two basic residues; it has been suggested that any
molecule possessing two basic groups separated by an aliphatic chain of
about five carbons or an aromatic skeleton of corresponding length is
likely to be a mast cell histamine liberator (Patón, 1958). Recent
evidence suggests that compd 48/80 and somatostatin induce histamine
secretion in mast cells by interacting with the same cell surface
receptor (Theoharides et £l_., 1981). Release induced by agents of this
class differ from the above classes in that there is no enhancement by
exogenous PS, no requirement for extracellular calcium, and no
stimulation of phospholipid methylation (Hirata et al_., 1979). This
suggests that initial events of the activation-secretion response of
mast cells may be different for polycationic amines and basic
polypeptides than IgE-like secretagogues.
Ionophores, compounds which facilitate transport of ions through a
lipid barrier separating two aqueous environments, serve as carriers
for various ions. The most completely described is known simply as
A23187, a monocarboxylic antibiotic isolated from Streptomyces
chartreusensis; 1t is capable of eliciting secretion from a wide
variety of cells, Including mast cells. In the secretory process, the
ionophore A23187 functions as a calcium carrier, is dependent upon
extracellular calcium and is capable of bypassing the regulatory effect
of cAMP upon mast cell secretion (Foreman et , 1976). It is
likewise indifferent to the presence of exogenous PS.

7
For these investigations, we have chosen at least one agent from
each of these four groups: sheep anti-rat IgE (anti-IgE); the antigen
ovalbumin, con A; compd 48/80 or somatostatin and the calcium ionophore
A23187.
Other Mediators Released by Mast Cells
Histamine is but one of many pharmacological principles released by
mast cells. All mediators can be divided into two broad but useful
categories; those like histamine, which are stored and those which are
generated as a consequence of specific activation of the target cell
(Kazimierczak and Diamant, 1978). Pharmacological action,
physiochemical characteristics and metabolism of both preformed and
generated mediators have been reviewed in detail by Ho et al_. (1979).
Preformed mediators are stored within basophil and mast cell membrane
bound granules composed largely of proteoglycan and protein and include
histamine, serotonin (in some species, Yurt and Austen, 1977),
eosinophil chemotactic factor of anaphylaxis (ECF-A), neutrophil
chemotactic factor (NCF) and heparin. Included with stored,
granule-associated mediators are enzymes also released following cell
activation. Chymase, a chymotrypsin-like enzyme, is presumed to be
stored with serotonin at its active site in rat mast cell granules
(Lagunoff and Pritzl, 1976). N-Acetyl-g-D-glucosaminidase and
arylsulfatase A are two other stored enzymes released during
degranulation and are thus presumed to reside in secretory granules
(MacDonald-Lynch et al_., 1978).
Mediators generated subsequent to mast cell activation consist
predominantly of prostaglandins (PGs) and leukotrienes (LTs), the

8
oxidative products of arachidonic acid (AA). They are generated by the
release of AA from phospholipid membrane stores following
secretagogue-induced perturbation which is believed to allow expression
of phospholipases (Flower and Blackwell, 1976).
Metabolism of AA occurs by two independent pathways: the
cyclooxygenase and lipoxygenase pathways. In the rat and human mast
cell, the former pathway initially generates the unstable cyclic
endoperoxides PGG2 and PGH2 which are converted predominantly to PGD2
by the action of a specific synthetase (Lewis and Austen, 1981).
Metabolism via the lipoxygenase pathway generates the intermediate
5-hydroperoxyeicosatetraenoic acid (5-HPETE) which is converted to the
closely related mono-hydroxyeicosatetraenoic acid (5-HETE) or the
unstable intermediate 5S-oxido-trans-7,9-trans,ll,14-cis-
eicosatetraenoic acid (LTA4). From LTA4, two enzymatic pathways have
been described, one leading to the potent chemotactic factor LTB4
(5S,12R,dihydroxy,6,14,cis,8,10-trans-eicosatetraenoic acid) (Borgeat
and Samuelsson, 1979) and the other leading to 5S-hydroxy,6R-S-
glutathionyl-7,9-trans,ll,14-cis-eicosatetraenoic acid (LTC4) by the
addition of glutathione. LTC4 is then metabolized to the corresponding
cysteinylglycine derivative LTD4 then the cysteine derivative LTE4
(Lewis &t ¿]_., 1982). In stimulated human neutrophils, the
5-lipoxygenase pathway is preferentially activated to generate 5-HETE
and LTB4. In contrast, rat and human mast cells elaborate PGD2 during
coupled activation-secretion (Lewis et al_., 1982) when stimulated with
anti-IgE. LTC4 has been identified following stimulation of a murine
mastocytoma cell line with the calcium ionophore A23187 (Samuelsson et

9
al., 1980). It has been suggested that in human and rat mast cells
challenged with IgE-dependent stimuli, only PGD2 is generated but the
secondary cell types recruited may be the source of the other oxidative
AA metabolites active in immediate hypersensitivity (Lewis and Austen,
1981).
For the series of experiments described in these chapters, we have
chosen to study histamine as the granule-contained secretion marker and
AA, in the form of [14C]-AA incorporated in vitro, as the marker
generated during mast cell stimulation.
Role of Calcium in Mast Cell Exocytosis
According to Gomperts (1983), only one early event in the mast cell
stimulus-response sequence has been unequivocally identified and that
is the elevation of cytosolic calcium into the micromolar range. It
has been stated that this is the sole necessary precursor of secretion.
"Stimulus coupling," first proposed by Douglas and Rubin (1961), has
been introduced to emphasize the central role of calcium as a second
messenger in secretory processes (Pearce et aK, 1983). Experimental
results with calcium ionophores such as A23187, ionomycin and
chlortetracycline which form lipid-soluble complexes with the cation
and directly transfer it across the cell membrane bypassing the normal
messenger-receptor interaction, have served as evidence that calcium is
the second messenger which couples activation with the granular release
(Cochrane and Douglas, 1974; Lichtenstein, 1975; Bennett et al_., 1979;
Pearce £t al_., 1983). Though most investigators agree that ionophores
transport extracellular calcium to the intracellular spaces,

10
ionophore-induced histamine release in rat mast cells in the absence of
extracellular calcium (thus presumably by mobilization of internal
calcium stores) has been reported (Johansen, 1980). A second line of
evidence that calcium is the second messenger has centered around mast
cell exocytosis following microinjection of calcium ions into the cells
while microinjections of magnesium or potassium were without effect
(Kanno et al_., 1973).
The source of calcium differs dependent upon the type of
secretagogue. Antigen-induced, as well as other IgE-dependent
exocytosis, requires extracellular calcium, whereas ligands such as
compd 48/80, peptide 401 and polylysine induce secretion in the absence
of extracellular calcium (Foreman, 1981). Lanthanides, which compete
with calcium for extracel1ular binding sites thought to be the calcium
channel, thus prevent calcium transport across membranes and inhibit
mast cell histamine secretion induced by IgE-like secretagogues
dependent upon extracellular calcium. They do not affect release by
compd 48/80, peptide 401 and the like (Foreman and Monger, 1973; Pearce
and White, 1981; Amellal and Landry, 1983).
Relationships between histamine secretion and 4^Ca^+ uptake by mast
cells have been thoroughly investigated by Foreman et al_. (1977).
Stimulation with antigen-antibody, dextran or con A induced an uptake
of ^Ca^+ which correlated well with the magnitude of histamine
release. PS enhanced both ^Ca^+ uptake and histamine release. When
cells were stimulated with antigen in the presence of non-labeled
calcium, then exposed to tracer ^Ca^+ over a period of 1-5 min, a
64% drop in calcium uptake was noted within 1 min following stimulation

11
indicating a very transient membrane permeability to the cation.
Raised intracellular levels of cAMP produced with theophylline or
dibutyryl cAMP inhibited antigen-induced ^5Ca^+ uptake and histamine
release, but had no effect when the ionophore A23187 was used as
stimulant. It appears, then, that cAMP may inhibit secretion by
reducing membrane permeability to calcium and may be involved in
limiting calcium entry after cell stimulation.
Evidence for mobilization of membrane or intracellular calcium
stores to provide the needed rise of cytosolic calcium ions is given by
results of mast cell stimulation in the absence of extracellular
calcium following short-term (10 min) pretreatments with chelating
agents. Peptide-basic secreting agents including compd 48/80,
bradykinin, somatostatin, polylysine and polymyxin B were capable of
robust release responses in the absence of calcium (Ennis et aJL , 1980;
Baxter and Adamik, 1978; Cochrane et al_., 1982). Based upon such
investigation, three pools of calcium have been postulated: 1) calcium
very loosely bound to the outer cell membrane which may migrate to the
cellular cytosol, 2) a superficial, membrane associated store
(removable by brief EGTA exposure) and 3) deeply sequestered calcium
stores (removable by prolonged exposure to EGTA (Ennis et al_., 1980).
While the first store is utilized by both IgE-like and IgE-independent
secretagogues as well as ionophores, the second store is mobilized by
polypeptide-base type secretagogues. Deeply buried depot translocation
may be modulated by occupancy of the two more superficial pools.
Membrane events implicated in mast cell calcium translocation
include turnover of phosphatidylinositol (PI) (Gomperts et al_., 1980;

12
Cockcroft and Gomperts, 1979; Kennerly et al_., 1979b,c). In rat
peritoneal mast cells stimulated with antigen, anti-IgE, con A,
chymotrypsin and compd 48/80, turnover of PI and incorporation of
radiolabel into PI took place regardless of the presence or omission of
extracellular calcium. Concomitant histamine release was either
abolished or reduced depending upon calcium requirements of the ligand.
This was taken as evidence for involvement of the PI response in the
regulation of calcium channels and mobilization of sequestered calcium
in the mast cell (Pearce, 1982; Cockcroft and Gomperts, 1979).
Another membrane event implicated in calcium translocation was
phospholipid methylation where two membrane-bound enzymes
(methytransferases I and II) act sequentially to convert endogenous PE
to PC. Methylation has been demonstrated in rat leukemic basophils and
peritoneal mast cells during stimulation with antigen, con A and
anti-IgE (Crews et al., 1980; Hirata et al_., 1979; Ishizaka et al.,
1980). Because the methyl transferase inhibitor, 3-deaza-adenosine,
blocked phospholipid methylation, calcium influx and histamine release
in a dose-dependent manner, it was suggested that phospholipid
methylation may be a primary and obligatory event in calcium
translocation and histamine release by IgE-dependent mast cell
activation.
From available data, it is still not possible to determine the
relative contributions of these two pathways of lipid metabolism to the
sequence of mast cell activation-secretion coupling events. They may
represent events of differing initial activation sequences, events of a
complex but unified sequence, or consequential events of cell

13
activation. Both the PI response and phospholipid methylation will be
discussed in more detail in the next section.
The mechanism whereby calcium induces the mast cell exocytotic
response is not known. In many systems, the cation produces its
effects by interaction with specific binding proteins exemplified by
the ubiquitous polypeptide calmodulin, proposed as a universal
intracellular receptor for calcium (Means and Dedman, 1980; Cheung,
1980). Calmodulin mediates activity of a number of important enzymes
including cyclic nucleotide phosphodiesterase, brain adenylate cyclase,
calcium dependent ATPase, phospholipase A, myosin light chain kinase,
as well as other specific kinases involved in phosphorylation of
membrane and cytosolic proteins (Pearce, 1982).
In smooth muscle cells, neuroleptic drugs reportedly inhibit
contraction induced by various stimuli by inhibiting calmodulin
(Kerrick et ¿1_., 1981). Seven such agents including five phenothiazine
derivatives, imipramine and pimozide have recently been studied for
their effects upon mast cell secretion evoked by specific antigen,
compd 48/80 or ionophore A23187 (Douglas and Nemeth, 1982). An
inhibitory potency series generated with the agents for compd 48/80 and
A23187 induced release correlated closely with potencies which
inhibited phosphodiesterase activation by Ca-calmodulin and also
reflected their affinity for binding to calmodulin (Weiss et al.,
1980). Specific antigen-evoked secretion was more sensitive to
inhibition and gave a different rank order of the neuroleptic agents,
an effect shown not to be related to the magnitude of antigen-induced
secretion. Since A23187 elicited ^Ca^+ uptake was not reduced by the

14
neuroleptics, it was suggested by Douglas and Nemeth that since
non-specific membrane effects had been ruled out, their action was
distal to the intracellular rise of calcium ions either on the calcium
receptor or the processes it activates to effect exocytosis. These
results suggested that mast cells mc(y have a calcium-binding protein(s)
which resembles calmodulin and that it is involved in
receptor-activated exocytosis. Its function may be related to
calcium-dependent protein phosphorylation which has been correlated
with mast cell degranulation (Sieghart et £l_., 1978).
Exogenously added calmodulin inhibited mast cell histamine release
elicited by compd 48/80, polymyxin B and A23187 (ID^q about 2 uM) but
not con A (McClain et al_., 1983). Calmodulin itself did not elicit
release, did not appear to enter the cells and did not compete with
compd 48/80 or polymyxin B for cell surface binding sites. EGTA
washing of mast cells reduced subsequent calmodulin-induced inhibition.
So, release may involve externally-bound calcium but the relationship
of this exogenous calmodulin effect to the actual in vivo mechanism
remains unknown.
Care must be used in interpreting these results since a very recent
report on human platelet work stated that these same agents, namely
trifluoperazine, chlorpromazine and W-7 were powerful inhibitors of the
calmodulin independent Ca/PL-PK also known as C-kinase (Sanchez et al.,
1983). In quin 2 loaded platelets, the secretory response stimulated
by phorbol ester, exogenous diacyl glycerol (DAG) or collagen was
suppressed by the neuroleptics at 20-60 uM at basal levels of
cytoplasmic free calcium. Inhibition could be overcome by treatment of

15
platelets with A23187 (40 nM) which elevated cytoplasmic calcium to
700 nM (sub-threshold for calcium alone to evoke secretion). In
contrast, the response to thrombin which was accompanied by elevation
in levels of cytosolic free calcium was barely affected. These data
suggested that the most prominent effect of phenothiazines, at least on
platelets, could be interference with Ca/PL-PK rather than
calmodulin-dependent processes.
Though there is sufficient evidence to support the view that
elevation of cytosolic calcium is the second messenger in mast cell
secretion, subsequent events which calcium initiates or influences
remain largely unknown. Likely events, by analogy with other secretory
cells or from the results discussed above include involvement of
calmodulin, interaction with cyclic nucleotides and cytoskeletal
elements such as microfilaments and microtubules, and activation of
phospholipases and Ca/PL-PK while methyl transferase activation and PI
metabolism may modulate calcium movement.
Phospholipid Metabolism During Mast Cell Activation
Phospholipid methylation. As mentioned in the previous section,
the two predominant phospholipid metabolizing systems are the PI cycle
and phospholipid methylation. Some evidence for the increase of both
these events during mast cell and/or basophil stimulation has been
reported (Crews, 1982).
Phosphatidylcholine (PC) is a major constituent of biomembrane
phospholipids including those of mast cells and is synthesized by at
least two described pathways, the CDP-choline pathway and successive
N-methylations of phosphatidylethanolamine (PE) via methyl transferase
activity (Kennedy and Weiss, 1956; Hirata et al_., 1979).

16
In normal mast cells, Hirata et al_. (1979) have reported a
transient rise in membrane phospholipid methylation which precedes
histamine release during the early stages of con A-induced
activation-secretion coupling, as assessed by the incorporation of
[^H]-methyl groups into the mast cell lipid fraction. In the presence
of calcium, labeled PC was further metabolized, presumably by
phospholipase to ^orm lysophosphatidylcholine. Both phospholipid
methylation and histamine release were inhibited by a-methylmannoside
which prevents binding of con A to its cell surface receptors.
Similarly, the methyl transferase inhibitors 5'-deoxyisobutylthio-3-
deazaadenosine or 3-deazaadenosine (3DZA) plus homocysteine thiolactone
inhibited both phospholipid methylation and histamine release. In the
absence of calcium, methylation but no subsequent histamine release
occurred. When compd 48/80 and ionophore A23187 were employed as
degranulating agents, no stimulation of phospholipid methylation was
observed. These results were taken to imply that while phospholipid
methylation was involved in the activation-secretion signal
transduction by con A, it was bypassed by both compd 48/80 and A23187
perhaps indicating different mechanisms of histamine release (Morita
and Siraganian, 1981).
Results similar to those described for con A stimulated normal
cells were then reported for 2H3 rat basophilic leukemia (RBL) cells
sensitized with specific IgE than stimulated with the corresponding
antigen and extended to include concomitant inhibition of histamine and
[^C]-AA in 3DZA treated cells. More recent studies with RBL cells by
Crews et al_. (1981) have shown that there was a parallel increase

17
between [^H]-methyl incorporation into phospholipids and ^5Ca^+ influx
which preceded release of histamine and AA release. Thus it was
suggested that phospholipid methylation may play a role in calcium
flux.
With a slightly different approach, by generating rabbit antibodies
to the IgE receptor for RBL cells (anti-RBL) which acted as a divalent
anti-receptor binding agent in normal rat mast cells, it was shown that
anti-RBL challenge Increased phospholipid methylation which reached a
maximum at 15 sec then rapidly declined and was followed by an increase
of ^®Ca^+ uptake, then histamine release (Ishizaka, 1982). Monomeric
antibodies failed to induce any of these changes. With the use of
inhibitors described above, corroborative results were reported, again
suggesting that phospholipid methylation may be intrinsic for ^®Ca^+
influx and histamine release induced by IgE-receptor stimulation.
Additionally, a 3-fold rise of mast cell cAMP, superimposed upon
[3n]-methyl incorporation was measured, with a maximum at 15 sec,
followed by a sharp decline, then a gradual rise to double basal level
again at 3 min following addition of anti-RBL. Methylation inhibitors
such as S-isobutyryl-3-deazaadenosine (3-deaza-SIBA) also partially
inhibited the initial rise in cAMP suggesting that cAMP may in turn
regulate phospholipid methylation. In a dose dependent manner,
theophylline, at millimolar levels increased cAMP, inhibited
methylation, 45Ca2+ influx and subsequent histamine release.
More recently, Daeron et al_. (1982) have reported that pretreatment
of mouse mast cells overnight with 10" ^ to 10"® M dexamethasone did not
itself alter mast cell cAMP levels, but when the cells were stimulated

18
with specific antigen, phospholipid methylation, 45Ca^+ influx and
histamine release were all inhibited by at least 75%. However, the
critical experiments, measurement of cAMP during stimulation were not
reported. Thus, while it is still not known how dexamethasone prevents
activation of methyltransferases, it does not appear to be by
alteration of resting cAMP levels.
Cumulatively, the data can be interpreted to indicate that
phospholipid methyltransferases: 1) which effect synthesis of PC to PE
are involved in mast cell receptor-mediated activation, 2) selectively
increase phospholipid methylation associated with IgE-receptor mediated
activation, 3) act prior to an influx of calcium and subsequent
histamine release, and 4) when inhibited, likewise inhibit calcium
influx as well as histamine and AA release. Therefore, phospholipid
methylation may play a critical role in the early events of
IgE-mediated mast cell activation-secretion coupling.
PI metabolism. While there is much information on phosphoinositide
metabolism and hormone and neurotransmitter action as well as the role
of PI hydrolysis as a transducing mechanism in many diverse tissues and
cell types (Berridge, 1981; Farese, 1983a; Farese, 1983b), there is
very little information on the role of PI in mast cells or basophils.
Yet, since the inositol phospholipids have been assigned an important
role in mediating actions which generate intracellular calcium signals,
as do mast cells, PI involvement in mast cell activation-secretion
coupling might be expected.
According to the postulate put forward by Michel 1 and colleagues,
PI hydrolysis is coupled to receptor activation and is responsible for

19
calcium influx or mobilization of intracellular calcium stores. This
is supported by observations that the PI cycle (measured directly as
hydrolysis or indirectly by increased labeling) occurs with many
stimulants that operate via receptors and employ calcium as "second
messenger" (Farese, 1983a). In this pathway, PI, Ptdlns-4,5P2 and/or
Ptdlns-4P are hydrolyzed by a phosphodiesterase such as phospholipase C
to yield DAG and inositol phosphate(s). DAG can serve as a substrate
for diacyl glycerol lipase to yield AA, the predominant fatty acid at
the 2-position, or diacyl glycerol kinase to yield PA which can be
picked up by the appropriate CTP transferase and combined with inositol
phosphate to reform PI. Relevance of this pathway arises from the
following: PA is known to have calcium ionophoric activity, DAG is a
known fusogen, and, in concert with PS and calcium, activates Ca/PL-PK
while Ptdlns-4,5P2 and Ptdlns-4P avidly bind calcium and could modulate
membranous calcium stores and/or alter activities of membrane
associated enzymes or transport proteins.
Cockcroft and Gomperts (1979) have published evidence for a role of
PI turnover in stimulus-secretion coupling in mast cells of rats
sensitized to either ovalbumin or the parasitic helminth
Nippostrongylus brasiliensis. Purified mast cells were preincubated
with either [^H]-inositol or 32_p. for 30 min, and then stimulated with
specific antigen, con A or the non-IgE directed secretagogue compd
48/80 for 15 min. Stimulated PI labeling was independent of
extracellular calcium though IgE-dependent histamine release was highly
calcium dependent. These results were taken as an indication that the
mast cell can be placed on the list of tissues and cell types which

20
exhibit calcium-independent PI responses and thus mcy represent
receptor mediated events proximal to calcium signals. Stimulated PI
labeling is believed to be a secondary consequence of the breakdown of
PI initiated by either the IgE-directed specific angigen, con A or
compd 48/80. At first glance, it seems puzzling that a secretagogue
which is independent of extracellular calcium would be involved in the
turnover of a phospholipid involved in calcium gating; however compd
48/80 (10 pgm/ml) stimulated PI synthesis was reflected as 2400 dpm
•^P-PI/1-2 x 105 cells while con A induced PI synthesis was 10-fold
greater. Though not stated, these results also suggest that
calcium-gating may not be the sole function of PI metabolism in the
mast cell.
In a subsequent report (Gomperts et al_., 1980), an extensive table
of tissues which can be stimulated by the ionophore A23187 and also
exhibit a PI response was used as evidence to suggest that the PI
response indicates a receptor mediated biochemical step in the
activation of calcium channels as postulated by Michel! (1975).
Activation of metabolism of the predominant mast cell phospholipids
was investigated by incorporation of ^2p. into individual classes of
phospholipids during stimulation by anti-IgE, con A, compd 48/80 or
calcium ionophore A23187 (Kenner!y et al_., 1979a). Increased
incorporation into PA, PI and PC (4- to 10-fold increases) occurred
within 15 min while no significant incorporation into PS, PE or
sphingomyelin (SM) was discerned. Kinetic analyses of phospholipid
metabolism during anti-IgE stimulation showed that PA labeling
increased most rapidly from 15 sec to 2 min after stimulation while PI

21
and PC labeling increased more slowly and reached a maximum after 6 min
of stimulation. Major changes in PA labeling occurred before mediator
release, PI and PC changes were concomitant with histamine release.
These increases of ^P.. incorporation were presumed to indicate that
mast cell receptor-induced stimulation generated DAG which is then
converted to PA, PI and PC. These results support the hypothesis that
selective mast cell phospholipid metabolism may play a critical role in
the biochemical events which control mediator release.
Since DAG is a precursor for each of the phospholipids, experiments
were designed to determine levels of DAG during mediator release from
mast cells stimulated with compd 48/80 (Kenner! y et al_., 1979b). In
[3H]-AA prelabeled cells, a small but significant absolute label
accumulation in DAG (250-300 cpm/3 x 103 cells) was noted within 60 sec
of 48/80 addition. Unfortunately, this increase was too small to
demonstrate an absolute loss from any of the labeled presursor lipids
PI, PC, PE and triglyceride. In broken cell preparations, 2-[l-^C]-
arachidonoyl-DAG was rapidly converted to free AA, monoacyl glycerol and
triglyceride. Substrate preference of 2-[l-14C]-arachidonyl-DAG over
l-[l-^C]-arachidonyl-PC suggested that degradation was mediated by a
DAG lipase, demonstrated in mast cells by Lewis etal_. (1979). Since
the time course of 3^P-PA accumulation in the previously described
investigations was more rapid than for DAG, it was suggested that mast
cell DAG kinase may initially remove newly formed DAG until stimulated
DAG production exceeds the enzyme's capacity, then the abrupt rise in
DAG levels is measured. Postulated roles for DAG and its metabolites
in mast cell secretion included facilitation of membrane fusion and

22
substrate for AA-derived mediator formation. These same observations
were extended to anti-IgE stimulated mast cells (Kennerly et al_.,
1979c).
Results of experiments conducted with [^H]-glycerol loaded mast
cells stimulated with either ionophore A23187 or compd 48/80 and
designed to study PI breakdown in activated mast cells have been
published by Ishizuka and co-workers (1983). Following stimulation of
cells by A23187, radioactivity in both PA and PI dropped slightly at 10
sec then went on to accumulate at 1.5 times the starting level at 60
sec. In contrast, radioactivity in DAG progressively increased to a
3-fold increase by 5 min. To investigate the possibility that
A23187-induced activation stimulated de novo PI and PA synthesis,
uptake of [^H]-glycerol was investigated during stimulation. Enhanced
uptake of [^H]-glycerol into PI and PA by 7- and 4-fold, respectively,
was measured. Though the percent total histamine released was
comparable, compd 48/80 was much less effective in accelerating PI
metabolism than A23187. In another report, Ishizuka and Nozawa (1983)
have discussed results of the same experimental design but with
specific antigen as the stimulant. In presence of 0.5 mM magnesium,
antigen stimulation (Ascaris suum extracts coupled to
2,4-dinitrophenyl) induced a loss of 30% PI from mast cells preloaded
with [^H]-glycerol followed by a PI resynthesis to 1.5-fold increase
over basal levels at 5 min into stimulation.
Concomitant with the decrease in PI radioactivity, a corresponding
increase was measured for DAG which exhibited a much greater initial
rise than did labeled PA, suggesting antigen stimulated receptor

23
mediated turnover of PI. Thus, while compd 48/80 and ionophore
stimulated de novo PI synthesis, antigen stimulation induced a
turnover. Unfortunately, the former investigations were carried out
with Sprague-Dawley rat mast cells while the antigen-stimulated mast
cells were derived from Wistar rats. Barring strain differences, it
appears that mast cell PI metabolism may differ for different classes
of secretagogues.
To date, this is the evidence that the PI response may play a role
in mast cell activation-secretion. The PI response and the
phospholipid methylation pathway are alike since they 1) are minor
membrane lipids which undergo rapid turnover, 2) are associated with
receptor stimulated activation, 3) may be involved in calcium
mobilization, and 4) can serve as sources of AA. Their exact routes of
metabolism and their physiological roles in mast cell degranulation
require further experimentation.
Conceptual Model for Mast Cell Exocytosis
Delineation of events involved in mast cell release has evolved
primarily from the impact of pharmacologic tools upon
activation-secretion. For the most part, these tools have been enzyme
inhibitors and antagonists and thus block events at different points
along the presumed step-wise pathway of activation-degranulation.
In development of this model, we will focus upon the previously
defined IgE dependent secretagogues including con A, specific antigen,
and anti-IgE as well as polypeptide-basic types which include compd
48/80 and somatostatin. Though the conditions required to elicit mast
cell release may be different for these two classes of secretagogues,

24
they all have one basic feature in common—they are all polyvalent.
Sequentially, then, the first step in mast cell activation is ligand
receptor binding and aggregation by the polyvalent secretagogue.
Use of the serine esterase inhibitors phenyl methylsulfonylfluoride
(PMSF) or diisopropylfluorophosphate (DFP) to demonstrate blockade of
histamine release as well as PC degradation suggests that activation of
this enzyme may be an early event. When DFP is present during antigen
challenge, no histamine release results. If it is removed prior to
stimulation, release does occur, thus suggesting activation of this
esterase by receptor aggregation. Since the inhibitors prevent PC
degradation, serine esterase activation presumably occurs prior to
activation of phospholipase ^.
Phospholipid methylation which is stimulated selectively by IgE
dependent secretagogues, but not polypeptide-base types, is inhibited
by 3-deaza-SIBA or 3-deaza-adenosine. This results in the inhibition
of ^Ca^+ uptake as well as histamine release. In the absence of
extracellular calcium, methylation, but not IgE-dependent release
occurs, suggesting that phospholipid methylation may precede and/or
modulate calcium influx.
A transient rise in cAMP has been measured during mast cell
stimulation, but when the intracellular level is increased by
theophylline or dibutyryl cAMP, antigen-induced 45Ca^+ uptake and
histamine release are inhibited but no effect was noted with A23187 as
secretagogue. This suggests that cAMP may also be involved in calcium
mobilization but its relationship to phospholipid methylation remains
unclear. Fluctuations in cAMP levels may parallel these other events

25
but may be primarily involved in microtubule assembly-disassembly in
preparation for cellular degranulation.
All these mentioned events precede both histamine and AA release.
Activation of the calcium-dependent enzyme phospholipase A2 may then
occur. This enzyme is reportedly inhibited by mepacrine by some
unknown mechanism of action, or by a-parabromoacetophenone (PBP) which
modifies a histidine residue essential for its activity. Such
inhibition blocks anti-IgE induced histamine release. Other evidence
which suggests a role of phospholipase A2 in mast cell secretion
includes exogenous application of the enzyme which induces non-cytotoxic
release and release of histamine from stimulated cells which is
accompanied by release of [^C]-AA from prelabeled phospholipids.
Oxidative metabolism of released AA via the cyclooxygenase pathway
is exquisitely sensitive to inhibition by indomethacin and other
non-steroidal anti-inflammatory agents while the lipoxygenase pathway
remains undisturbed (Vane, 1971). However, indomethacin, at 10 uM, a
concentration which completely suppresses oxidative conversion of AA,
has no effect on the time course and amount of histamine released by
A23187, con A or anti-IgE suggesting that AA metabolism is not
necessary for or does not precede histamine release.
The inhibitors which block later events are the phenothiazines,
believed to inhibit calmodulin and/or Ca/Pl-PK. These agents block
degranulation by both IgE-dependent agents, polypeptide-base agents, as
well as ionophoretic compounds. Since they block A23187 induced
release of histamine without altering ^5Ca2+ uptake, it is assumed that
the drugs act on processes which effect exocytosis.

26
Very little is known about protein phosphorylation-
dephosphorylation, assembly-disassembly of cytoskeletal elements and
generation of granule-plasma membrane fusogen events which are believed
to comprise a final common pathway in mast cell release.
The goal of the present studies is to investigate the actions of
two additional pharmacologic probes, hydrocortisone and 12-0-tetra-
decanoyl-phorbol acetate on events of mast cell activation-secretion
coupling. This is the first characterization of either of these agents
on purified, normal rat mast cells. Relevance of the hydrocortisone
investigations arises particularly from the successful treatment of
moderate to severe allergic asthma with anti-inflammatory steroids.
The mechanisms by which anti-inflammatory steroids ameliorate symptoms
remain unclear. Since this is a type I immediate hypersensitive
reaction which involves mast cells, results of these present
investigations with purified populations of mast cells exposed to
hydrocortisone could lend valuable information concerning the
mechanisms of action of anti-inflammatory steroids in cells which play
a central role in allergic reactions.
In contrast, 12-0-tetradecanoyl-phorbol acetate, a prototypic
active tumor promoting agent, is known to activate inflammatory type
responses in several types of leukocytes. Its mechanism of action in
certain types of these cells is becoming more clearly delineated.
Thus, results of these investigations in mast cells could lend a new
perspective on events of activation-secretion coupling.
Far-reaching goals of these investigations are to provide new
approaches to pharmacologic modulation of components of the immune

27
system involved in allergic reactions and to gain further insight into
basic exocytotic mechanisms since the mast cell is considered the model
secretory cell.

CHAPTER TWO
INHIBITION OF IMMUNOGLOBULIN, BUT NOT POLYPEPTIDE-BASE,
STIMULATED RELEASE OF HISTAMINE AND ARACHIDONIC ACID
BY ANTI-INFLAMMATORY STEROIDS
Introduction
It is well established that glucocorticoids are potent
anti-inflammatory agents effective in ameliorating the symptoms of
immediate hypersensitivity and other allergic reactions (Lewis and
Austen, 1981). Mechanisms of anti-inflammatory glucocorticoid action
are not well delineated despite studies involving many varied cell
types (Lewis and Piper, 1976; Gryglewski, 1976; Hong and Levine, 1976).
Collectively, the data suggest that corticosteroids decrease the
release of arachidonic acid from membrane phospholipids and thereby
decrease prostaglandin generation without directly altering the enzymes
involved in prostaglandin synthesis. Glucocorticoid induced
inhibition of mast cell release may be related to the effectiveness of
these agents in the treatment of asthma and other allergic reactions.
The effects of glucocorticoids on mast cell release of both histamine
and arachidonic acid have not been clearly delineated.
To clarify biochemical mechanisms of anti-inflammatory steroid
action upon events in mast cell activation-secretion coupling, we
conducted investigations on the effects of hydrocortisone and other
glucocorticoids on the secretion of histamine and arachidonic acid and
its metabolites. We report here that anti-inflammatory glucocorticoids
28

29
selectively inhibit release of histamine and [l-14C]-arachidonic acid
(AA) and its metabolites. Only IgE-like secretagogue induced release
is steroid sensitive. Stimulation of both [1-^C]-AA and histamine
release by IgE independent secretagogues such as compd 48/80,
somatostatin, and the calcium ionophore, A23187, is not altered by
steroid pretreatment.
Materials and Methods
Ovalbumin Sensitization
Male or female Sprague-Dawley rats, 300-400g (random-bred colony,
University of Florida, Gainesville, FL) were injected i.m. with 0.5 ml
of a saline suspension containing 10 yg ovalbumin (Miles Research
Products, Elkhart, IN) and 20 mg of Al(OH3) (Amphogel, Wyeth,
Philadelphia, PA). Eight to ten days later a booster of the same dose
was administered i.m. Animals were decapitated and the sensitized mast
cells collected four days after the booster injection.
Mast Cell Preparation
Rat mast cells were obtained by lavage similar to the method of
Sul 1 i van et al_., 1975. Briefly, animals were decapitated and injected
intraperitoneally and intrathoracically with freshly prepared buffer
containing 25 mM PIPES, 0.4 mM MgCl2, 5 mM KC1, 10 mM NaCl, 0.1% (w/v)
BSA or gelatin, 5.6 mM glucose^ and 10 U/ml heparin in distilled water,
pH 7.4. Trunks of the animals were massaged 100 times, the abdominal
and thoracic cavities opened, the cell suspensions recovered and
delivered to 50 ml polypropylene centrifuge tubes. Cells were washed
and resuspended in the above buffer at one animal equivalent of cells
per ml. Each ml of cells was carefully layered over 2 ml of buffer

30
containing 23% metrizamide (Accurate Chemical and Scientific Corp.,
Westbury, NY). Mast cells were purified by passage through the
metrizamide layer during centrifugation at 180xg for 8 min as described
by Yurt et al_., 1977 and Coutts et al_., 1980. Average yields were
1-1.5 x 10® mast cells/rat. Trypan blue exclusion (Sullivan et al.,
1975) was used to assess cell viability (average >97%) and toluidine
blue staining was used as a purity criterion (average approximately
90%).
Passive Sensitization
Purified mast cells were suspended in RPMI 1640 culture medium
(5 x 10® cells/ml) (Flow Laboratory McLean, VA) containing 150 U/ml
penicillin and 150 pg/ml streptomycin, 4 mM glutamine, 5% fetal calf
serum, 200 ng/ml monoclonal mouse IgE anti-DNP antibody (Miles Research
Laboratories, Elkhart, IN) and the cells incubated for 90 min.
Pretreatment of Mast Cells with Steroids
Hydrocortisone, as well as the other steroids tested, were
dissolved in ethanol at a concentration of 1 mM then diluted to the
appropriate final concentration in the above RPMI 1640 medium. Cells
were suspended in the steroid-RPMI 1640 culture medium and incubated at
37°C with 5% CO2 for 1 hr for acute steroid effects and 18-21 hr
(overnight) for long-term effects. Cells were washed in fresh culture
medium prior to [1-^C]-AA incorporation or in the PIPES buffered salt
solution prior to histamine release experiments.
Histamine Release and Measurement
Cells were suspended in the PIPES buffered salt solution containing
1 mM CaCl2 to give 1 x 10® cells/0.45 ml. Aliquots of 0.45 ml were

31
dispersed to polypropylene tubes and histamine release initiated by
addition of 50 pi of secretagogue. Histamine release by IgE dependent
agents was carried out in the presence of 50 yg/ml bovine brain extract
enriched in phosphatidyl serine. Incubations were carried out for
indicated times at 37°C with gentle agitation and release terminated by
addition of 1 ml cold buffer then centrifugation at 5000xg for 10 min
at 4°C. In each assay, a separate group of tubes was used for the
determination of total cellular histamine by adding 0.4N HC104 prior to
incubation. Supernatant histamine was determined by the
o-phthaldialdehyde spectrophotofluorometric procedure (Shore et al♦,
1959) as modified by Anton and Sayre (1969) and Siraganian (1976).
Percent histamine released was calculated as follows: ng supernatant
histamine/ng total cellular histamine x 100.
Incorporation and Release of [1-^C]-AA and Its Metabolites
Purified mast cells were suspended in the RPMI 1640 culture medium
(described above) with [1-^C]-AA (0.3 pCi/ml) then incubated for 60
min at 37°C. Cells were washed twice with RPMI 1640 medium and once
with PIPES buffered salt solution containing 0.1% BSA as described
previously (Crews et al_., 1981). Cells were resuspended in PIPES
buffered salt solution and allowed to equilibrate at 37°C for 30 min.
Release of [1-^C]-AA and its metabolites was carried out as described
above for histamine. Total release was quantitated by liquid
scintillation spectroscopy.
Measurement of ^5Ca Uptake
The method of Foreman et al. (1977) was used for the measurement of
^5Ca^+ flux. Briefly, purified mast cells were suspended in Tyrode's

32
solution, 2.5 x 105 cells/50 yl. One hundred microliters of versilube
F50 silicone oil (General Electric Corp.) were placed in the bottom of
a 400 yl microfuge tube and 40 yl Tyrode's solution containing ^Ca^+
(5.5 yCi/ml) followed by 10 yl secretagogue in Tyrode's solution were
layered on the oil and warmed to 37°C. Five minute incubations were
initiated by addition of 50 yl mast cells and incubations halted by
centrifugation in excess of 10,000xg for 30 sec in a Beckman B
microfuge. Cell pellets were recovered by slicing off the microfuge
tube tips, and vigorously shaking in vials with an aqueous solution of
Triton X-100 (1%, v/v). ^5Ca^+ was assessed by addition of Liquiscint
(National Diagnostics) to the solubilized pellets and counting in a
liquid scintillation spectrometer.
Chemicals and Reagents
All steroids used were purchased from Sigma (St. Louis, M0) as were
compd 48/80, con A, and somatostatin. The calcium ionophore, A23187,
was purchased from Cal biochemical (LaJolla, CA). All other chemicals
used were reagent grade. Radioligands were purchased from Amersham,
[1-^C]-AA, 60 mCi/mmole, and ^5Ca^+, 40 mCi/mg calcium.
Statistical Analysis
Data are expressed as the mean ± S.E.M. The Student's t-test was
used to evaluate the differences between two means for significance.
The criterion for significance was p<0.05. The EC^g (effective
concentration for 50%) was calculated by probit analysis (Goldstein,
1964).

33
Results
To characterize the effects of glucocorticoids upon release of
histamine and [1-^C]-AA and its metabolites by rat mast cells,
purified mast cells were incubated with hydrocortisone (3 x Iff6 M)
overnight and then stimulated with known secretagogues. Hydrocortisone
pretreatment markedly inhibited both histamine and [1-^C]-AA release
stimulated by concanavalin A (con A), the antigen ovalbumin (OA) and
anti-immuglobulin E antibody (anti-IgE) (fig. 1). Histamine and
[1-^C]-AA release were inhibited to a similar extent. For example,
hydrocortisone treatment inhibited anti-IgE stimulated histamine and
[1-14C]-AA by 83.7 ± 4.1% and 76.6 ± 1.5%, respectively. In contrast
to IgE-like secretogogues, hydrocortisone pretreatment did not alter
the release of either histamine or [1-^C]-AA stimulated by the
polypeptide somatostatin, compd 48/80 or the calcium ionophore, A23187
(fig. 2). Hydrocortisone pretreatment does not significantly alter the
total amounts of mast cell histamine or incorporation of [l-^Cl-AA
(Heiman and Crews, 1984). Since AA and its metabolites have been
implicated in the mechanism of histamine release, we examined the
effects of exogenous AA on hydrocortisone's inhibition of con A
stimulated histamine release. Exogenous AA did not reverse
hydrocortisone's inhibition of con A stimulated release. Con A
(10 pg/ml) released 28.5 ± 0.8% and 28.2 ± 1.0% total cellular
histamine in control cells with and without AA (1 pM), respectively.
Cells treated for 18 hr with hydrocortisone released 13.4 ± 0.8% and
11.8 ± 0.2% total cellular histamine with and without exogenous AA
(1 pM), respectively. These results indicate that hydrocortisone

34
Figure 1. Effect of hydrocortisone pretreatment on the release of
histamine and [1-1^C]-AA and its metabolites during
stimulation with IgE-like secretagogues. Cells were
stimulated for 30 min by addition of indicated secretagogue.
Final stimulant concentrations were: 0A, 100 ug/ml; con A,
3 ug/ml; anti-IgE, 1:1000 dilution. Spontaneous histamine
release ranged from 8-12.9% and spontaneous [1-1^C]-AA
release from 219 to 431 dpms/2.5 x 10^ cells; these have
been subtracted from values depicted. Open bars indicate
untreated cells, striped bars represent hydrocortisone
pretreated cells. Each bar represents a typical mean ±
S.E.M. of triplicates repeated on at least two other
occasions. All hydrocortisone treated groups were
significantly different from controls at p<0.05 as assessed
by Student's t-test.

35
2000
O
UJ
m
<
UJ
co ® 1500 ■
o
o 2
oo
< *
I- »
UJ «
2 ^ 1000
OJ
O E
Z a
< -a
<
<
O
500
UJ
co
5 60
GE
UJ
l 40
h-
m
x
o
UJ
< 20
3
2
co
*
AA RELEASE
JLt
48/80
HISTAMINE
SOMAT
RELEASE
X
i
A23187
48/80
SOMAT
A23187
Figure 2. Effect of hydrocortisone pretreatment on histamine and
[1-14c]-AA release during stimulation with IgE-independent
secretagogues. Cells were stimulated for 15 min with compd
48/80, 1 ug/ml; somatostatin, 100 yg/ml; A23187, 3 ym/ml.
Spontaneous histamine release ranged from 3.8 to 9.0% and
spontaneous [1-14C]-AA release ranged from 128-210
dpms/2.5 x 10^ cells; these have been subtracted from data
shown. Open bars represent untreated cells and striped bars
the hydrocortisone pretreated cells. Each bar represents a
typical mean ± S.E.M. of triplicates repeated on at least
two other occasions.

36
inhibits IgE-like release, i .e. anti-IgE, antigens and con A, but not
polypeptide-base or ionophore stimulated release.
To further characterize the actions of glucocorticoids on mast cell
release, time courses and concentration response curves were determined
for IgE-like and IgE-independent secretagogues. Treatment with
hydrocortisone (3 x 10"6 M) overnight slowed both the initial rate of
histamine release and the total amount of histamine released by con A,
but did not affect the rate or amount of histamine released by compd
48/80 (fig. 3). Results very similar to those for con A were obtained
with anti-IgE as the stimulant (data not shown). To determine if the
inhibition of stimulated release was due to a decrease in sensitivity
or a loss of responsiveness, complete concentration responses curves
were performed. Response curves for ovalbumin and compd 48/80
indicated that hydrocortisone pretreatment markedly decreased release
at all ovalbumin concentrations tested, but did not alter compd 48/80
induced release (fig. 4). The ovalbumin ED^g for histamine release
(untreated 1.14 ± 0.3 yg/ml; hydrocortisone, 1.19 ± 0.2 yg/ml) was not
shifted by hydrocortisone pretreatment. For [1-^C]-AA release, the
untreated EDgg for ovalbumin was 0.70 ± 0.1 yg/ml, that for
hydrocortisone treated cells was 0.43 ± 0.2 ug/ml. These data suggest
that hydrocortisone treatment selectively decreases maximal release of
histamine and [1-^4C]-AA initiated by an IgE-like secretagogue
stimulation, but not by IgE-independent types of stimulation. These
results, also suggest a close correspondence between release of
histamine and [1-^C]-AA and its metabolites.

37
Figure 3. Time course of con A and compd 48/80 induced histamine
release. Cells were preincubated with and without
hydrocortisone (3 x 10“6 M) for 21 hr and then stimulated to
release histamine by the addition of either con A (10 ygm)
in presence of 50 ugm/ml bovine brain extract or compd 48/80
(3 ugm/ml). Histamine release is calculated as a percent of
total releasable mast cell histamine. Each point represents
the mean for triplicate determinations from one of two
experiments of similar design.

38
Figure 4. Dose dependent [1-14C]-AA and histamine release by antigen
ovalbumin (OA) and compd 48/80. Cells were stimulated to
release histamine for 30 min by the addition of OA and 15
min for compd 48/80. Each point represents the mean ±
S.E.M. of triplicate determinations from one experiment.
Results are typical of those from at least two other
experiments of similar design.

39
To determine the duration of hydrocortisone pretreatment required
for inhibition of release initiated by IgE-like secretagogues, mast
cells were incubated for 24 hr with various times of exposure to
hydrocortisone (3 x 10“6 M). No inhibition of histamine release was
found after 1, 3 or 9 hr of treatment, but following 12 hr of exposure
to hydrocortisone, there was a significant decrease in histamine
release (fig. 5). This time dependent inhibition slowly increased to
approximately 95% inhibition after 24 hr of pretreatment. A similar
time dependent inhibition was noted for IgE-like release of [1-^C]-AA
and its metabolites. Such a delay in the onset of inhibition is
suggestive that induction of a protein may be involved in the mechanism
of the glucocorticoid action on mast cell secretion.
To explore the possibility that steroid-induced protein synthesis
is involved in the inhibition of IgE-mediated histamine release, we
incubated cells with various concentrations of cycloheximide and
actinomycin D, protein synthesis inhibitors with different sites of
action. These agents alone inhibited histamine release (data not
shown); therefore, we could not demonstrate reversal of the
glucocorticoid inhibitory effect with either cycloheximide or
actinomycin D. In rat thymus cells, glucocorticoid-receptor complex
translocation to the cell nucleus is temperature dependent, i.e. this
migration is halted at 4°C (Mosher et al_., 1971; Wira and Munck, 1974).
Thus, we studied the effects of temperature on the actions of
hydrocortisone in our system. Preincubation of mast cells for 21 hr
at 4°C or 37°C did not alter subsequent con A induced release at 37°C
(fig. 6). As shown previously, 21 hr of preincubation with

40
30--

o
v
0)
01 20--
®
c
E
o
OT
X
10- -
wO O.
â– O*
*A
\
\
O
•o
.o.
Control 0—0
Con. A A-A
A i
'O'
â– o*
NA
O
3 6 9 12 15 18
Treatment with Hydrocortisone ( hr.)
21
24
Figure 5. Time dependent inhibition of histamine release by
hydrocortisone. Shown is an experiment typical of two other
experiments. All cells were incubated for 24 hr, but
drug-treated cells were exposed to hydrocortisone (3 x
10”® M) for the number of hours indicated. Open circles
indicate spontaneous histamine release and closed triangles
indicate con A (10 ygm/ml) stimulated release of histamine
from treated cells. Con A stimulated release from control
cells ranged from 28-30% throughout the 24 hr time course.
Points represent the mean of two determinations. Duplicates
varied by less than 10%.

41
Preincubation Temp.
Hydrocortisone
Con A
4° C
- +
4° C
+ +
- +
37° C
- +
37° C
+ +
- +
Figure 6. Temperature dependent inhibition of histamine release by
hydrocortisone. Mast cells were preincubated with and
without hydrocortisone (3 x 10~6 M) at 4°C or 37°C for 21
hr. Cells were washed, slowly warmed to 37°C and challenged
to release histamine for 30 min by addition of con A
(10 pg/ml). Bars represent the mean percent of total mast
cell histamine ± S.E.M. for three separate determinations.

42
hydrocortisone at 37°C markedly inhibited con A induced histamine
release. However, 21 hr of preincubation at 4°C with hydrocortisone
did not alter subsequent con A induced histamine release at 37°C
(fig. 6).
To delineate the specificity of the glucocorticoid induced
inhibition of release, we incubated mast cells with several different
types of steroids. We found that fluocinolone, dexamethasone, and
hydrocortisone inhibited con A stimulated histamine release in a
dose-dependent manner following a 21 hr preincubation (fig. 7).
Steroid ICgg values were 1.4 x 10"® M, 3.4 x 10"® M, and 3.6 x 10"^ M,
respectively. This order of potency parallels both their in vivo
anti-inflammatory potencies (Gilman et al_., 1980) and their affinities
for the glucocorticoid cytosolic receptor (Dausse et al_., 1977). Cells
were also treated overnight with high concentrations (10"5 M) of the
non-glucocorticoid steroids, estradiol, and testosterone. Stimulated
histamine release by con A from untreated cells was 17.0 ± 0.7% while
cells treated with testosterone and estradiol released 15.4 ± 0.8% and
17.4 ± 0.4% total histamine, respectively. Using fixed concentrations
of anti-inflammatory steroids, inhibition was also obtained for release
of [1-^C]-AA and its metabolites (table 1). Thus, only
anti-inflammatory steroids inhibit mast cell release by IgE-like
secretagogues.
The importance of calcium in exocytotic mechanisms has been well
documented for IgE-like mast cell secretagogues (Foreman et al_., 1977;
Baxter and Adamik, 1978). To further investigate the site of action of
glucocorticoids, we treated cells with hydrocortisone for 21 hr and

43
Figure 7. Inhibition of mast cell histamine release by
anti-inflammatory steroids. Steroids were dissolved in
ethanol (which comprised 0.1% of the pretreatment incubation
volume) and cells incubated with indicated concentrations
for 21 hr. Cells were washed, resuspended and challenged
to release histamine for 30 min by addition of con A
(10 yg/ml). Points represent the mean percent of total
histamine released ± S.E.M. for three separate
determinations.

44
TABLE 1
INHIBITION OF [1-14C]-AA RELEASE BY ANTI-INFLAMMATORY STEROIDS
Treatment
dpms [1-14C]-AA released/lO® cells
Histamine
(% Released)
None
14,817
43
Fluocinolone
(10“8 M)
447
9.7
Dexamethasone
(10“8 M)
819
10
Hydrocortisone
(10-6 M)
875
12.3
Cells were pretreated for 18 hr with indicated concentrations of
steroids, washed, resuspended in PIPES buffered salt solution
containing 1 mM CaCl2 and 1% BSA, 0.45 ml aliquots containing 2.5 x
106 cells dispensed and release initiated by addition of con A
(10 ygm/ml) in presence of 50 ugm/ml BBE. Incubations at 37°C were
continued for 30 min. Subtracted background [1-14C]-AA releases
averaged 2686 dpm/lO® cells, and subtracted background histamine
releases averaged 5.7%. Values represent the mean of duplicates from
an experiment replicated with similar outcomes on two other
occasions.

45
then stimulated histamine and [1-^C]-AA and metabolite release with
the calcium ionophore, A23187, which artificially induces calcium flux.
Ionophore induced release was not significantly inhibited by
hydrocortisone treatment (fig. 8), suggesting that the secretory and AA
release processes following the influx of cal dun are not altered by
glucocorticoid treatment. Since release stimulated by IgE-like
secretogogues is known to depend upon extracellular calcium (Baxter and
Adamik, 1978), we studied the effect of hydrocortisone treatment on the
influx of ^Ca^+. Stimulation of rat mast cells with antigen caused a
rapid influx of calcium which was completed 5 min after addition of the
secretagogue. Pre-treatment with hydrocortisone for 18 hr markedly
reduced calcium influx Induced by con A, antigen, and anti-IgE (fig.
9). These results suggest that glucocorticoids inhibit plasma membrane
calcium flux and thereby selectively inhibit secretagogues dependent on
extracellular calcium, i.e. IgE-like secretagogues.
Discussion
Antigen and anti-IgE antibodies are thought to stimulate release
from rat mast cells by cross-linking IgE-Fc receptor complexes
(Ishizaka and Ishizaka, 1978). Con A, 1n a manner similar to antigen,
appears to act by cross-linking IgE bound to the cell surface
(S1ragan1an and Siraganian, 1975). These IgE-like secretagogues have
several properties which distinguish them from the polypeptide-base
secretagogues, e.g. somatostatin and compd 48/80. IgE-l1ke
secretagogues are dependent upon extracellular calcium, potentiated by
phosphatidyl serine (Baxter and Adamik, 1978), stimulate phospholipid

46
Figure 8. Effects of hydrocortisone treatment on A23187-induced
release of histamine and [l-14c]-AA and its metabolites.
Cells were treated as described in the legend of figure 4,
then resuspended and challenged to release histamine by
addition for 15 min of indicated concentrations of the
calcium ionophore, A23187. Points represent the mean
percent of total histamine released ± S.E.M. for three
determinations.

47
Figure 9. Effect of hydrocortisone pretreatment on IgE-dependent
4®Ca2+ uptake. Mast cells were preincubated with and
without hydrocortisone (3 x 10“® M) then challenged for
5 min with the following secretagogues in the presence of
5.5 pCi/ml 45Ca2+: con A 10 yg/ml, DNP-BSA 0.1 jig/ml,
anti-IgE 1:100 dilution. Bars represent the mean stimulated
uptake of 4®Ca2+ cpm/2 x 10® cells ± S.E.M. for three
separate determinations. Background 4®Ca2+ values were 300 ±
17 cpm/2 x 10® cells. All hydrocortisone treated groups
were significantly different from controls at p<0.05 as
assessed by Student's t-test.

48
methylation (Hirata et. al_., 1979) and are inhibited by methylation
inhibitors (Crews et al_., 1981). Somatostatin and compd 48/80 do not
have any of these requirements or effects. These polypeptide-base
secretagogues appear to act by releasing internal stores of calcium
(fig. 10). Somatostatin and compd 48/80 have been suggested to act on
the same membrane receptor (Theoharides et ¿1_., 1981). They are both
strong secretagogues which typically release more than 40-50% of the
total cellular histamine as compared to IgE-like secretagogues which
have a maximal response of about 20-30% total cellular histamine. The
strength of stimulation may play a role in the glucocorticoid affect.
However, submaximal concentrations of compd 48/80 which release
approximately 30% of total cellular histamine are not altered by
glucocorticoid treatment (fig. 4). These findings, along with the
differential effects of the glucocorticoids, suggest that release of
histamine and arachidonic acid and its metabolites induced by the
IgE-Fc receptor complex occurs by triggering mechanisms different from
those of somatostatin-48/80 induced release and that glucocorticoids
act by selectively uncoupling IgE-mediated release (fig. 10).
Glucocorticoids can act by membrane stabilization and/or induction
of the synthesis of specific proteins (fig. 10). The latter 1s thought
to involve the binding of glucocorticoids to a cytosolic receptor, and
formation of a complex which translocates to the nucleus and increases
synthesis of specific mRNA (Grody et al_., 1982). Cytosolic
glucocorticoid receptors have been demonstrated in mast cells (Daeron
et al_., 1982). Other studies have indicated that the translocation of
the glucocorticoid-receptor complex is temperature dependent (Mosher et

49
Figure 10. Schematic diagram of possible sites of cortisol action on
mast cell exocytosis. Cortisol could act through a
cytoplasmic receptor which when activated migrates to the
nucleus and induces the synthesis of specific proteins. A
membrane effect is also possible. The specific proteins
synthesized appear to interfere with the coupling of the
IgE receptors to the influx of calcium. Phospholipid
methylation (i.e. PE—>PC) and/or phosphatidylinositol
turnover (i.e. PI—>DAG—>PA) have been implicated in the
coupling of IgE receptors to calcium influx and the release
of arachidonic acid (AA). Phospholipid methylation may be
inhibited by steroid treatment (Daeron et al_., 1982).
Somatostatin and compd 48/80 appear to release internal
stores of calcium. Therefore, these secretagogues are not
inhibited by glucocorticoids. Abbreviations: IgE,
immunoglobulin E; Ag, antigen; con A, concanavalin A;
somat., somatostatin; 48/80, compd 48/80; Fc, cell surface
receptor to which IgE binds; ATP, adenosine triphosphate;
RNA, ribonucleic acid.

50
al., 1971; Wira and Munck, 1974). The following data suggest that
glucocorticoids inhibit rat mast cell histamine release by inducing the
synthesis of specific proteins. Membrane stabilization occurs acutely,
whereas we have demonstrated that the inhibition of histamine and
arachidonic acid release by glucocorticoids requires several hours of
treatment. Inhibition of con A (IgE-1ike) induced histamine release by
glucocorticoids is temperature dependent, as is glucocorticoid-receptor
complex translocation. Various glucocorticoid inhibitory potencies
reported here parallel their in vivo anti-inflammatory potencies
(Gilman et al_., 1980) and their affinities for the cytosolic receptor
(Dausse et al_., 1977). Thus, it is likely that induction of a specific
protein is an important component of the glucocorticoid action on rat
mast cells.
Studies on rat mast cells (Hirata et al_., 1979) and rat basophilic
leukemia cells (Crews et al_., 1981; McGivney et al_., 1981) have
suggested that the stimulus-secretion coupling sequence is as follows:
antigen cross-linking of IgE, stimulation of phospholipid methylation,
influx of calcium, activation of phospholipase initiating fusion of
granule and plasma membranes (fig. 10). Our findings indicate that
glucocorticoids selectively inhibit IgE-mediated release by reducing
IgE stimulated calcium flux. Studies in mouse mast cells have
suggested that glucocorticoids alter anti-IgE stimulated histamine
release by inhibiting phospholipid methylation (Daeron et al_., 1982).
These studies indicated that steroid treatment does not change IgE-Fc
receptor number or affinity. Other investigators studying
anti-inflammatory steroid action have found that steroids induce the

51
synthesis of a phospholipase inhibitory protein in rat leukocytes
(macrocortin) (Blackwell et al_., 1980), rabbit neutrophils
(lipomodulin) (Hirata et al_., 1980), and mouse mast cells (Daeron et
al., 1982). Our finding that the glucocorticoid inhibitory effect upon
arachidonic acid release occurs only for IgE-like secretagogues and is
not overcome by exogenous AA is inconsistent with a general inhibition
of phospholipase. Several subtypes of phospholipase may exist, with
only the IgE-receptor associated form of the enzyme exhibiting
susceptibility to anti-inflammatory pretreatment. It has now been
proposed that the majority of lipomodulin secreted from neutrophils is
phosphorylated (therefore inactive) then split into several smaller
molecules the smallest of which is a 16,000 dalton protein (the size of
macrocortin) also generated by lymphocytes and called IgE-suppressive
factor or glycosylation inhibiting factor (Vede et al_., 1983). This
suggests, then, that a fragment of lipomodulin is involved in the
selective formation of IgE-suppressive factors which modulate T cell
subset responses to immunologic stimuli. A similar protein, generated
by glucocorticoid treated mast cells could contribute to the
selectivity of the inhibition induced. Alternatively, glucocorticoids
may also synthesize a phospholipid methyl transferase inhibitory
protein. Additional studies are necessary to delineate the mechanisms
of glucocorticoid specificity.
Whatever the mechanism of glucocorticoid action, our findings
indicate that glucocorticoids selectively inhibit IgE-mediated release,
i.e. anti-IgE, antigen and con A, but do not alter IgE-independent
stimulation of histamine and arachidonic acid release by somatostatin,

52
compd 48/80 or the calcium ionophore, A23187. The precise mechanisms
of steroid action in asthma and other allergic reactions are unknown.
Responsiveness differs for various types of allergic reactions
(Patterson, 1979). Our finding suggests that steroid treatment may
reduce allergic reactions mediated by antigen-IgE, but not reactions
stimulated by peptides and toxins which act through other receptors.

CHAPTER THREE
CHARACTERIZATION OF THE EFFECTS OF
PHORBOL ESTERS ON RAT MAST CELL SECRETION
Introduction
Phorbol esters (PEs), the most potent tumor promoting agents in the
mouse skin bioassay, also cause a variety of biological responses in
diverse cell types (Blumberg, 1980). These include lymphocyte
mitogenesis (Touraine et aK, 1977; Abb et al_., 1979), platelet
aggregation and serotonin release (Muf son et al_., 1979; Zucker et al.,
1974), PMN superoxide anion production (Lehrer and Cohen, 1981),
interleukin 2 production by mouse EL4 thymoma cells (Kraft et al.,
1982), as well as histamine release from a mixed population of human
leukocytes (Schleimer et al_., 1980).
We became interested in studying the effects of PEs alone and in
conjunction with various secretagogues for several important reasons.
First, it is well recognized that when applied to the skin, PEs elicit
signs of acute inflammation, mediators of which are released by mast
cells (Boutwell, 1974). Second, skin has been shown to contain
specific binding sites for PEs (Delclos et al_., 1980), and skin
contains a large number of mast cells. Recent studies have suggested
that PEs may specifically activate a calcium/phospholipid dependent
protein kinase (Ca/PL-PK) (Yamanishi et al_., 1983; Castagna et al.,
1982; Ashendel et al_., 1983). An investigation of the action of PEs on
mast cell secretion could provide a valuable tool for delineating the
53

54
role of this protein kinase in mediator release since experimental
results with stimulated mast cells have shown that during secretion
there is a rapid calcium-dependent phosphorylation of certain protein
bands (Sieghart et al_., 1978; Theoharides et al_., 1980; Wells and Mann,
1982).
In the purified mast cell system, receptor-mediated stimulation can
occur via^ two major classes of secretagogues which we call IgE-like and
polypeptide-base as well as other types of secretagogues (Crews and
Heiman, 1984; Lagunoff et al_., 1983). We define IgE-like as those
secretagogues which appear to activate mast cells by interacting with
bound immunoglobulin E. We call somatostatin and compd 48/80
polypeptide-base secretagogues since these agents appear to release
histamine through a cell surface receptor which is not IgE. Properties
which distinguish the two classes of secretagogues which we investigate
are: IgE-like agents (antigen, anti-IgE, concanavalin A) require
extracellular calcium, are potentiated by phosphatidyl serine (Baxter
and Adamik, 1978), stimulate phospholipid methylation, and are
sensitive to glucocorticoid pretreatment when used to stimulate mast
cell secretion (Crews and Heiman, 1984). Polypeptide-base
secretagogues (somatostatin, compd 48/80) do not require extracellular
calcium and do not respond to the conditions described for IgE-like
secretagogues.
Our interest in the Ca/PL-PK centers in the similarities for its
activation and mast cell secretion, namely calcium mobilization and
enhancement of release by PS. Abundant occurrence of Ca/PL-PK and its
endogenous substrate proteins in the particulate fraction of human

55
neutrophils suggests that protein phosphorylations by this enzyme may
be involved in membrane associated neutrophil functions. This
hypothesis was further strengthened by the demonstration, in these same
studies, that phosphorylation was inhibited by trifluoperazine, an
agent which inhibits neutrophil chemotaxis, aggregation and
degranulation (Helfman et al_., 1982). Trifluoperazine is also known to
inhibit rat mast cell secretion elicited by antigen, compd 48/80 and by
the calcium ionophore A23187 (Douglas and Nemeth, 1982).
We report here that 1) PE tremendously potentiates A23187
stimulated release of histamine and arachidonic acid, 2) extracellular
calcium is required for release in the presence of PE and A23187, 3)
histamine release by IgE-like secretagogues is potentiated by PS and
TPA, 4) structure activity relationships for the effects of various PE
analogs on mast cell histamine release suggest a single mechanism of
action, perhaps mediated by a single receptor, 5) TPA either alone or
with A23187 significantly increased phosphorylation of mast cell
protein(s), and 6) mast cells have low levels of Ca/PL-PK.
Materials and Methods
Mast cell preparation. Rat mast cells were obtained by lavage
similar to the method of Sullivan et al_. (1975). Briefly, animals were
decapitated and injected intraperitoneally and intrathoracically with
freshly prepared buffer containing 25 mM PIPES, 0.4 mM MgC^, 5 mM KC1,
10 mM NaCl, 0.1% (w/v) BSA or gelatin, 5.6 mM glucose, and 10 U/ml
heparin in distilled water, pH 7.4. Trunks of the animals were
massaged, the ventral wall reflected, the cell suspensions withdrawn
and delivered to 50 ml polypropylene centrifuge tubes. Cells were

56
washed and resuspended in the above buffer at one animal equivalent of
cells per ml. Each ml of cells was carefully layered over 2 ml of 23%
metrizamide (Accurate Chemical and Scientific Corp., Westbury, NY) also
prepared in the above buffer without heparin. Mast cells were purified
by passage through the metrizamide layer during centrifugation at 180xg
for 8 min as described by Yurt et al_. (1977) and Coutts et al_. (1980).
Average yields were 1-1.5 x 106 mast cells/rat. Trypan blue exclusion
was used to assess cell viability (average >97%) and toluidine blue
staining was used as a purity criterion (average approximately 90%).
Preparation of secretagogues. Ten times the final concentrations
of all secretagogues were prepared in the PIPES buffered salt solution
described above. In cases where IgE-like secretagogues were used,
500 pgm/ml phosphatidyl serine (PS) was added to the buffer and
sonicated for 2 min prior to addition of concanavalin A (con A) or
sheep anti-rat IgE (anti-IgE).
PEs were dissolved in DMSO as stock solutions of 1 mg/ml and were
diluted in buffer. During secretagogue induced release from mast
cells, DMSO comprised .001% of the incubated volume and in vehicle
treated control cells did not alter mediator release.
Histamine release and measurement. Cells were suspended in the
PIPES buffered salt solution containing 1 mM CaC^ to give 1 x 10^
cells/0.45 ml. Aliquots of 0.45 ml were dispensed to polypropylene
tubes and histamine release initiated by addition of 50 pi of
secretagogue. Histamine release by IgE dependent agents was carried
out in the presence of 50 pgm/ml phosphatidyl serine from bovine brain.
Incubations were carried out for indicated times at 37°C with gentle

57
agitation and release terminated by addition of 1 ml cold buffer then
centrifugation at 4000xg for 10 min at 4°C. In each assay, a separate
group of tubes was used for the determination of total cellular
histamine by adding 0.4N HCIO^ prior to incubation. Supernatant
histamine was determined by the o-phthaldialdehyde spectrophoto-
fluorometric procedure (Shore et al_., 1959) as modified by Anton and
Sayre (1969) and Siraganian (1976). Percent histamine released was
calculated as follows: ng supernatant histamine/ng total cellular
histamine x 100. Spontaneous histamine release, specified in each
figure legend, has not been subtracted.
Phosphorylation of mast cell proteins. In a method similar to that
employed by Wells and Mann (1982), cells were suspended in the PIPES
buffered salt solution containing 1 mM CaC^ at 2 x 10® mast cells/ml
and incubated at 37°C for 30 min with [^P]orthophosphate, carrier
free, at a concentration of 1 mCi/ml. Cells were then washed twice in
5 ml warmed buffer and the pelleted cells resuspended to a final cell
density of 10® cells/50 pi. Fifty micro-liters of resuspended cells
were delivered to warmed glass tubes and stimulation initiated by
addition of 5 pi of secretagogue. Incubations were 60 sec and
terminated by the addition of 50 pi of gel sample buffer containing
20% w/v glycine, 4% w/v SDS, 0.008% bromophenol blue and 10 pi
mercaptoethanol/ tube. Tubes were placed in a boiling water bath for
3 min, cooled, sealed and stored overnight at -20°C prior to SDS-PAGE.
SDS-PAGE. Electrophoresis was carried out in a Bio-Rad dual slab
cell. The resolving gel was 8% acrylamide (w/v) with an
acrylamide:bis-acrylamide ratio of 29:1. Samples (50 pi) were applied

58
to wells cast in the stacking gel with a 15-slot comb and gels
calibrated by applying the following MW standards from a commercial
standards kit (Sigma): carbonic anhydrase, 29 kDa; egg albumin,
45 kDa; BSA, 66 kDA; g-galactosidase, 116 kDA. Slabs were stained with
0.2% Coomassie brilliant blue R, dried and autoradiographed using SAR-6
x-ray film (Kodak) at -70°C. To quantitate phosphorylation,
autoradiograms were scanned with a densitometer (E-C Apparatus Corp.)
and peak heights used to represent relative units of radioactivity
(Ueda et al_., 1973).
Measurement of Ca/PL-PK. Total, solubilized fractions of cerebral
cortex, resident peritoneal cells (RPC), RPC-devoid of mast cells
(RPC - mast cells; cells which form a band at the buffer-matrizamide
interface) and mast cells were prepared according to the method
described by Hel fman et al_. (1982). Washed RPCs were resuspended in 2
ml Hank's balanced salt solution (HBSS) and treated for 5 min at 4°C
with 5 mM DFP. Cells were washed twice in cold HBSS, mast cell and
RPC - mast cell fractions prepared as described above, pelleted and
resuspended in 0.5 ml homogenization buffer (50 mM Tris-HCl, pH 7.5;
1% v/v g-mercaptoethanol; 2 mM EGTA; 0.1% v/v Triton X-100; 1 mM
phenyl methylsulfonyl fluoride). Dissected cerebral cortex tissue was
delivered into 5 volumes of homogenization buffer then homogenized by
hand in a glass-teflon tissue homogenizer. Cortical homogenate served
as positive Ca/PL-PK control in all experiments. All tissue
preparations were then sonicated at 50 watts for 20 sec (Sonifier Cell
Disruptor, Ultrasonics, Inc., Plainview, NY), stirred gently for 1 hr
at 4°C, then centrifuged at 100,000xg for 60 min. The resulting

59
supernatants were used as the source of enzyme. Ca/PL-PK enzyme assay
conditions were as follows. Total incubation volumes were 200 yl and
contained 25 mM PIPES (pH 6.5), 10 mM MgCl2, 250 yM EGTA, 300-500 yM
CaCl2, 1 nmol (containing 0.9 to 1.4 x 106 cpms of [Y-32P]ATP), 40 ygm
histone 1, 3-4 ygm supernatant protein in the presence or absence of
50 ygm phosphatidyl serine. Reactions were carried out for 4 min at
30°C and halted by the addition of 4 ml trichloroacetic acid (TCA)
containing 0.25% w/v sodium tungstate. Forty microliters of 1.625% BSA
were added and tubes centrifuged at 2000 rpm (500xg) for 1 min.
Supernatants were aspirated, pellets redissolved in 0.1 ml 0.5N NaOH
then precipitated again with TCA-tungstate solution. Precipitates were
washed in this manner three times, redispersed in 0.1 ml 0.5N NaOH and
an aliquot counted after addition of 5 ml Liquiscint (National
Diagnostics, Somerville, NJ).
Chemicals and reagents. All phorbol esters used were purchased
from Sigma (St. Louis, M0) as were compd 48/80, con A, somatostatin,
phosphatidyl serine, and ATP. The calcium ionophore, A23187, was
purchased from Cal biochemical (LaJolla, CA) and anti-IgE from Miles.
Culture medium was purchased from Flow Laboratories and [1-^C]-AA and
[^P]orthophosphate from Amersham. All other chemicals used were
reagent grade.
Results
Time Course and Calcium Requirement for TPA Induced Histamine Release
The time course of TPA induced release of mast cell histamine in
the presence and absence of subthreshold concentration of the calcium
ionophore, A23187, is shown in figure 11. While A23187 (0.05 yg/ml) or

60
Figure 11. Time course for TPA induced histamine release. Purified
rat mast cells were incubated with TPA, 10 ng/ml in the
presence and absence of A23187, 0.05 ygm/ml for the
indicated times at 37°C. Spontaneous histamine release was
4 ± 0.5% and has not been subtracted. A23187 alone
released 6.6 ± 0.4%. Each point represents the mean
percent histamine released ± SEM of triplicates from an
experiment replicated with similar outcomes on two other
occasions.

61
TPA (10 yg/ml) alone released less than 10% of total cellular histamine
over the 3 hr time course, these agents acted synergistically to
produce a significant release after 5 min. Histamine was linearly
released for about 20 min, reaching a plateau of approximately 60%
total histamine released at 30 min. After this initial release period,
there was a slow gradual increase in histamine release reaching
approximately 70-80% after 3 hr and up to 90-95% after 5 hr. To
determine the role of extracellular calcium, cells were preincubated
for 90 min in PIPES-buffered salt solution containing either 10"^ M
EDTA or 2 mM calcium chloride then stimulated with TPA plus ionophore
A23187 in the same medium. Cells preincubated then stimulated in
presence of EGTA released only 6% histamine over spontaneous release
(5%), while cells preincubated and stimulated in presence of calcium
released 60% total cellular histamine.
Effect of TPA on Concentration Response of the Calcium Ionophore
A23187
The calcium ionophore, A23187, is thought to stimulate histamine
release by directly elevating free intracellular calcium levels.
Concentration response curves for A23187 with and without TPA are
depicted in figure 12. The histamine release elicited by ionophore
with TPA was potentiated (shifted to the left) when compared with
A23187 alone. The ECgg for the ionophore was approximately 832 ng/ml
and was reduced to 56.3 ng/ml when TPA was present as a co-stimulant.
When the extracellular calcium was removed, neither the co-stimulants
TPA-A23187 nor A23187 alone stimulated release.

% HISTAMINE RELEASED
62
A23187 (NG/ML) LOG SCALE
Figure 12. Effect of TPA on the dose-response of the calcium ionophore
A23187. Purified rat mast cells were incubated at 37°C for
30 min with various concentrations of A23187 in the
presence and absence of TPA, 10 ng/ml. Spontaneous
histamine release which has not been subtracted was 4.1 ±
0.5%. Each point represents the mean ± SEM of triplicate
determinations from one experiment. Results are typical of
those from at least two other experiments of similar
design.

63
Structure-activity Relationship for the Effect of Various PE Analogs on
Mast Cell Histamine Release
Specificity of the PE effect was explored by examining the
structure activity relationships for a series of five PEs. These
studies indicated an order of potency: TPA > 4g-PDD > POE (Fig.13).
Approximate EC^g values for the active PEs were: 5.4 ng/ml, 83.1 ng/ml,
and 807 ng/ml, respectively. Agents inactive as tumor promotors, e.g.
4a-PDD and 4a-phorbol were inactive as releasers of histamine.
Active-inactive isomeric pairs are suggestive of receptor specificity.
In experiments of a similar design cells were loaded with [1-^C]-AA
prior to stimulation with TPA and subthreshold A23187. The maximal
response of both histamine and [1-^C]-AA release occurred at 3 ng/ml
TPA with 32.1 ± 0.8% and 4421 ± 510 cpms/10^ cells, respectively.
Basal histamine release in presence of subthreshold A23187 was 4.7 ±
0.4% while [1-^C]-AA release was 1528 ± 46 cpms/10^ cells. Thus,
co-stimulation with TPA-A23187 appears to activate concomitant
histamine and AA release. Neither TPA alone, nor the inactive tumor
promoter 4a-phorbol induced release of these mediators.
Effect of TPA on IqE-like Secretagogue Induced Histamine Release
To characterize the effect of TPA on mast cell histamine release,
concentration response curves were assessed for anti-IgE (Fig. 14, top)
and con A (Fig. 14, bottom). At all concentrations of anti-IgE and con
A tested, inclusion of TPA increased histamine release. Although TPA
increased con A stimulated histamine release, it did not significantly
change the ECgg of con A for release, being 0.68 pgm/ml and 0.66 ygm/ml

64
Figure 13. Specificity of the PE effect on mast cell histamine
release. Purified rat mast cells were incubated at 37°C
for 30 min with the indicated concentrations of PEs in the
presence of 0.05 ymg A23187. Abbreviations are: TPA,
12-0-tetradecanoylphorbol-13-acetate; 4bPDD, 48-phorbol
12&,13a-didecanoate; POE, phorbol 12-myristate 13-acetate
4-0-methyl ether; PE, 4a-phorbol. Background release in the
presence of A23187 was 17 to 22% and has not been
subtracted. Points represent the mean percent of total
histamine released ± SEM for triplicate determinations from
one experiment. Similar results were obtained in one other
experiment of identical design.

65
Figure 14. Effect of TPA on IgE-like secretagogue induced histamine
release. Indicated concentrations of anti-IgE (top panel)
and con A (bottom panel) in the presence of exogenous PS
(50 ygm/ml) and in the presence and absence of TPA
(10 ng/ml) were used to stimulate histamine release from
purified rat mast cells. Spontaneous histamine release,
not subtracted, was 7-7.6% for Anti-IgE experiments and
4-5.5% for con A experiments. Incubations were carried out
at 37°C for 45 min. Each point represents the mean percent
histamine released ± SEM of triplicates from one
experiment. Results are typical of those from several
other experiments of a similar design.

66
in the presence and absence of TPA, respectively. Thus, TPA exhibited
a synergistic relationship with both con A and anti-IgE.
Enhancement of Stimulated Histamine Release by Phosphatidyl serine and
TPA
Since IgE-like secretagogues are known to be enhanced by exogenous
PS, the interaction of TPA with PS was investigated. Con A alone
elicited a very slight release response, which was greatly potentiated
in the presence of PS (Table 2). A similar situation exists for
anti-IgE. There was no significant dependence of peptide-base
secretagogue induced release upon exogenous PS (Grosman and Diamant,
1975). When TPA was present with IgE-like secretagogues, but without
PS, small increases in release were observed. This release was
synergistically enhanced when both PS and TPA were present. The
responses were more than additive even though maximal concentrations of
PS and TPA were used.
Effect of TPA on Polypeptide-Base Secretagogue Induced Histamine
Release
To determine the actions of TPA on compd 48/80 and somatostatin
induced mast cell release, complete concentration response curves were
done in the presence and absence of TPA (Fig. 15). In contrast to the
IgE-like secretagogues, TPA and compd 48/80 co-stimulation was barely
more than additive at 0.1 ygm/ml compd 48/80, and less than additive at
all other concentrations. Release induced by TPA and/or somatostatin
were less than additive at low concentrations and virtually identical
at higher concentrations. Neither potentiation nor synergism was
evident.

67
TABLE 1
EFFECT OF PS ON HISTAMINE RELEASE BY IgE-LIKE SECRETAGOGUES
Releasing
Agent
Histamine Release
(% Total Cellular Histamine)
Alone
PS
(50 ygm/ml)
TPA
(10 ng/ml)
PS + TPA
None
1.1 ± 0.1
3.1 ± 0.4
5.9 ± 0.4
7.6 ± 0.2
con A
(10 pgm/ml)
3.8 ± 0.3
20.5 ± 0.9
13.0 ± 1.0
44.7 ± 1.5
anti-IgE
(1:1000 dll.)
2.3 ± 0.7
14.0 ± 1.2
7.7 ± 0.3
33.0 ± 2.4
Purified mast cells were washed, equilibrated for 30 min, pelleted
then resuspended in PIPES buffered salt solution containing 1 mM CaClo.
0.45 ml cells were aliquoted and stimulated by addition of 50 yl of
indicated releasing agents. Incubations were carried out for 45 min at
37°C with secretagogue alone, and with PS and/or TPA. Values represent
the mean percents of triplicate ± S.E.M. of total mast cell histamine.
Similar results were obtained, in experiments of similar design, on two
other occasions.

68
Figure 15. Effect of TPA on polypeptide-base secretagogue induced
histamine release. Purified rat mast cells were incubated
with the indicated concentrations of compd 48/80 (top panel)
and somatostatin (bottom panel) in the presence and absence
of TPA, 10 ng/ml, for 30 min at 37°C. Spontaneous release
which has not been subtracted, was 4% for compd 48/80 alone,
5% with TPA (top panel) and 8% for somatostatin alone, 14%
with TPA (bottom panel). Each point represents the mean
percent histamine released ± S.E.M. of triplicates from one
experiment. Results are typical of those from two other
experiments of a similar design.

69
Effect of Secretagogues on Mast Cell Protein Phosphorylation
It is becoming increasingly evident that activation-release events
of secretory cells are regulated, at least in part by protein
phosphorylation-dephosphorylation. Recent studies suggest that PEs
bind to and activate the Ca/PL-PK. Therefore, we investigated protein
phosphorylation in mast cells. Autoradiographs of protein
phosphorylations during 60 sec stimulation with con A, 48/80, anti-IgE,
and TPA alone, suboptimal A23187 alone and TPA-suboptimal A23187
together are depicted in figure 16. Although a variety of proteins are
phosphorylated, only a few are markedly changed during stimulation. We
found significant changes in the phosphorylation of four major bands
with apparent molecular weights of 78, 59, 55 and 48 kDa. The most
consistent and largest changes occurred in the 48 kDa band (Fig. 16).
Stimulation of mast cells with con A, anti-IgE, compd 48/80, TPA and
TPA-A23187 at least doubled the phosphorylation of the proteins
migrating with an apparent molecular weight of 48 kDa. Con A, compd
48/80, and TPA-A23187 stimulated histamine release corresponded with
the increase in phosphorylation of the 48 kDa band. In contrast,
anti-IgE and TPA alone stimulated much larger increases in
phosphorylation of the 48 kDa band than histamine release. Although
low dose ionophore (0.05 ygm/ml) had little effect on 48 kDa
phosphorylation or histamine release (Figs. 16 and 17)
secretion-inducing doses of A23187 (1 ygm/ml) stimulated
phosphorylation of the 48 kDa band and other bands similar to con A,
anti-IgE and compd 48/80. Thus, TPA alone and TPA-A23187 stimulate

70
abed e f
- P 78
- P 60
-P 55
-P 48
Figure 16. Autoradiographs of SDS-PAGE gels showing secretagogue
induced phosphorylation of mast cell proteins. Incubation
periods were for 60 sec and release of histamine from
identically handled but unlabeled cells incubated with
secretagogue for conventional 15 or 30 min are given in
table 3. Secretagogues (concentrations shown in legend to
figure 17) were: a) PS, b) con A + PS, c) anti-IgE + PS,
d) none, e) compd 48/80, f) TPA, g) A23187, and h) TPA +
A23187. Apparent molecular weights were determined by
calibrating the gel with standard proteins of known
molecular weight. Phosphorylated proteins are shown on the
right.

INCREASED PROT. PHOS. 71
â–¡ PHOSPHORYLATION
* * '—‘
^ HISTAMINE RELEASE
CON A A-IGE 48/80 TPA A23 A23+TPA
Figure 17. Phosphorylation of a 48 kOa protein during mast ceil
stimulation. Phosphorylation experiments were incubated 60
sec, histamine release in identically handled, unlabeled
cells, for 30 min (also shown in table 3). Subtracted
phosphorylation peak heights were: buffer 46 ± 4.4 units;
PS 53.2 ± 4.2 units. Subtracted spontaneous histamine
release was 6.8 ± 1% for buffer and 9.7 + 2.3% for PS
controls. Secretagogue concentrations are shown in table
3. Values represent the mean ± S.E.M. of arbitrary unit
densitometric peak heights of two identically conducted
experiments each performed in triplicate. One asterisk
indicates those values which differ significantly from
controls at p<0.05, two asterisks indicate those differing
from controls at p<0.01, one-way ANOVA.

72
phosphorylation of the 48 kDa proteins similar to the more classical
secretagogues.
The densitometric peak heights of three other major bands (i.e. 78,
59 and 55 kDa) are presented in table 3. Interestingly,
phosphorylation of the 78 kDa band was significantly elevated only by
compd 48/80. For the 59 kDa proteins, there was a trend toward
increased phosphorylation for compd 48/80, con A and anti-IgE with 13%,
12% and 41% increases in peak height when compared with appropriate
controls. An exact opposite trend is noted for TPA alone or in
conjunction with A23187 where peak heights dropped 17% and 24%,
respectively. In the 55 kDa protein band phosphorylations, mean
increases in peak heights were: 21%, 48/80; 33%, con A; and 43%,
anti-IgE, TPA, A23187 and TPA-A23187 had much smaller effects on the
phosphorylation of these proteins. A lack of correlation between
phosphorylation and histamine release is not surprising since each
secretagogue has a somewhat different time course for release.
Phosphorylation changes occurred very fast and were measured after
1 min of stimulation, whereas release was determined 30 min after
stimulation. These experiments do indicate that TPA and TPA-A23187 can
modify mast cell protein phosphorylation.
Assessment of Mast Cell Ca/PL-PK Activity
Since TPA has been shown to bind and activate the Ca/PL-PK, we
compared the activity of this kinase in cerebral cortex, which is known
to have a high activity, to that in resident peritoneal cells and
purified mast cells. Table 4 depicts results of two experiments
showing kinase activity using histone 1 as substrate. By comparison,

TABLE 3
PHOSPHORYLATION OF 78 kDa, 59 kDa, AND 55 kDa PROTEINS DURING MAST CELL STIMULATION
Densitometric Peak Heights (Arbitrary Units)
Apparent Protein Molecular Weights (kDa)
Secretagogue % Total
(cone) 78 59 55 Histamine Release
Buffer
68.6
±
5.6
65.3
±
4.0
90
±
3.8
6.8
±
1.0
48/80 (3 pgm/ml)
112
±
16.4*
73.2
+
12.6
109
+
5.8*
79.7
+
2.7**
TPA (10 ng/ml)
77.4
±
9.2
52.8
±
10.2*
97.2
±
10.4
25.1
±
2.0**
A23187 (.05 ugm/ml)
64
±
6.0
120.4
±
6.4
98.6
±
8.4
17.4
±
5.0
TPA + A23187
55.4
+
7.4
100
+
12.4*
91
+
5.0
56.5
±
4.7**
PS (50 ngm/ml)
73.4
±
7.6
110
±
5.4
80.4
+
9.2
9.7
±
2.3
con A + PS (10 pgm/ml)
71
±
7.4
123.6
±
9.2
106.4
±
12.0
39.4
+
4.9**
anti-IgE + PS (1:1000 dil)
76.4
±
9.4
155.6
±
15.6*
114.6
±
3.8*
29.2
±
2.1**
Experiments were carried as described in Methods and legend of figure 7. One asterisk indicates
those values which differed significantly from controls at p<0.05 and two asterisks indicate those
differing from controls at p<0.01, one-way ANOVA. Shown is the mean ± S.E.M. for 6 separate
determinations.

TABLE 4
IN VITRO Ca/PL-PK ACTIVITY IN CEREBRAL CORTEX, MAST CELLS AND RESIDENT PERITONEAL CELLS
Tissue
cpm 32P incorporated
+PS -PS
Ca/PL-PK activity
pmol P/ygm protein/4 min
Experiment 1
Cerebral cortex
74,829
72,143
4753
6027
16.0
RPC - mast cel Is
34,120
35,434
5516
5329
5.2
Mast cells
5341
4401
2512
2727
'
0.4
Experiment 2
Cerebral cortex
34,305 ± 1521
1229 ±
84
8.9
RPC - mast cells
11,488 ± 1102
1240 ±
716
2.8
Mast cells
1542 ± 394
586 ±
3
0.26
RPC
1736 ± 89
948 ±
14
0.21
Enzyme assays on
total solubilized
fractions
were
conducted as described in methods
with 4 pgm protein per assay tube. Abbreviations used: RPC, resident peritoneal cells
were those cells collected by lavage; RPC - mast cells, RPC devoid of mast cells
represent a mixed population of white cells which band during metrizamide purification of
mast cells. Data presented for experiment 1 are duplicates while those from experiment 2
are mean ± S.E.M. of triplicates.

75
mast cells had 10-fold and 40-fold less activity than resident
peritoneal cells without mast cells (RPC - mast cells) and cortex,
respectively. These relationships were not altered when endogenous
proteins were used as substrate (data not shown). In RPC fractions
which contained approximately 6% mast cells, Ca/PL-PK activity was
reduced to the level of purified mast cell homogenate activity. Thus,
the presence of mast cells markedly reduced the measured Ca/PL-PK
activity in RPC cells suggesting an endogenous inhibitor. Exogenous
histamine and heparin did not inhibit Ca/PL-PK activity and other
experiments to clearly establish an inhibitor have proved difficult
suggesting that any inhibitor that is present is very labile. Since
these assays were carried out with optimal conditions for Ca/PL-PK
activity, addition of PEs did not significantly increase activity.
Unfortunately, the enzyme activity is very low in mast cells' making it
difficult to demonstrate enhancement of suboptimal activity with either
DAG or PEs.
Discussion
We have studied the interaction of PEs with purified rat mast
cells. Although PEs alone cause only a small release of histamine,
they tremendously potentiate A23187 induced release. Concentrations of
ionophore which produce negligible release alone, release approximately
50% of mast cell histamine in the presence of TPA. PEs also
synergistically enhance release stimulated by anti-IgE and con A, two
secretagogues which release histamine through an interaction with IgE.
Polypeptide-base secretagogues such as compd 48/80 and somatostatin,
are not potentiated by PEs. In contrast to stimulation involving IgE,

76
PEs are only additive or less than additive with polypeptide-base
secretagogues. Anti-IgE, con A and the calcium ionophore, A23187, all
require extracellular calcium to stimulate histamine release, whereas,
somatostatin and compd 48/80 do not. Thus, stimulation of histamine
release by secretagogues which require extracellular calcium is
enhanced by PEs while release by agents which do not require
extracellular calcium was not enhanced (Fig. 18).
PEs have been shown to stimulate and potentiate A23187 induced
stimulation in a variety of cell types. Studies on human leukocytes
have shown that PEs enhance anti-IgE and A23187 stimulated histamine
release similar to our findings with purified rat peritoneal mast cells
(Schleimer et ^1_., 1982). In addition, the EC^q values and overall
potency series for the various PEs (e.g., TPA > 4b-PDD > POE >> 4&-PDD
and PE) are similar for rat peritoneal mast cells, human basophils
(Schleimer et al_., 1980), the mouse ear inflammation assay (Oriedger
and Blumberg, 1979) and platelet serotonin release (Yamanishi et al.,
1983). The potency of TPA and the presence of active-inactive isomeric
pairs suggests that PEs may be acting at a specific site. The ability
of PEs to enhance the response to the calcium ionophore and/or other
stimulants known to cause calcium flux suggests that PEs may act by
enhancing the sensitivity of cells to calcium activation. Thus, if the
concentration of ionophore is proportional to the amount of calcium
entering the cell, mast cells are approximately 15-fold more sensitive
to calcium (i.e. the EC^q for A23187 is reduced 15-fold, see Fig. 13).
PEs have been shown to increase the affinity of the Ca/PL-PK for
calcium (Kishimoto et al_., 1980). Taken together, these findings

77
Ag
Con A
anti- IgE
va./
Mr
48/80
Somat.
phosphorylation
Figure 18. Hypothetical model of the role of Ca/PL-PK in mast cell
activation-secretion coupling. Multiple mechanisms may
exist for activation of Ca/PL-PK. IgE-like secretagogues
may activate the enzyme in the presence of PS primarily
through elevation of cytosolic calcium levels by
phospholipid methylation. Polypeptide-base secretagogues,
which do not significantly alter cytosolic calcium levels,
may primarily stimulate the turnovers of phosphatidylinositol
forming diacyl glycerol which lowers the calcium requirement
for the enzyme. Abbreviations used: AA, arachidonic acid;
Ag, antigen; con A, concanavalin A; DAG, diacyl glycerol;
48/80, compd 48/80; PC, phosphatidylcholine; PE,
phosphatidylethanolamine; PI, phosphatidylinositol; PNE,
phosphatidyl-N-methylethanol amine; PS, phosphatidyl serine;
somat, somatostatin; TPA, 12-0-tetradecanoyl-phorbol-13-
acetate.

78
suggest that the Ca/PL-PK may be involved in A23187 stimulated
histamine release.
PEs bind to a high affinity receptor site in a variety of tissues.
Recent studies have found that the PE binding site co-purifies with the
Ca/PL-PK. This protein kinase is activated by calcium and phospholipid
with phosphatidyl serine being the preferred lipid. We have shown that
mast cells have Ca/PL-PK activity although not as much as brain tissue
or other peritoneal cells. The high activity of rat peritoneal cells
with the mast cells removed is likely to be due to neutrophils which
are known to have large amounts of Ca/PL-PK (Helfman et al_., 1982) and
are readily activated by PEs. PEs enhance the sensitivity of Ca/PL-PK
to calcium and PS. The PEs apparently mimmic the action of
diacyglycerol, a lipid which is formed during mast cell stimulation
(Kennerly et al_., 1979), and which also enhances the affinity of
Ca/PL-PK for calcium and PS. Our finding that exogenous PS and TPA
enhance anti-IgE and con A stimulated histamine release in a
synergistic manner suggests that the stimulation of secretion by
anti-IgE and con A may involve activation of Ca/PL-PK.
Protein kinases and protein phosphorylation-dephosphorylation have
been implicated in stimulation secretion coupling in a number of
tissues. Previous studies on mast cells have reported changes in the
phosphorylation of four protein bands having molecular weights of
approximately 42-46, 56-59, 67-68 and 78 kDa during stimulation with
compd 48/80 (Sieghart et al_., 1978) and anti-IgE (Wells and Mann,
1982). We found that certain secretagogues increased ^P incorporation
into proteins having apparent molecular weights of 48, 55, 59 and

79
78 kDa. Considering differences in requirements for extracellular
calcium, PS, and the time course of histamine release, it is not
surprising that we found differences in protein phosphorylation 1 min
after stimulation. We found our most consistent increases in
phosphorylation in the 48 kDa band. Anti-IgE, con A, compd 48/80, TPA
alone and TPA-A23187 all markedly increased phosphorylation of this
band. PEs and PE-A23187 have been shown to stimulate secretion from
neutrophils and platelets, respectively, and to cause prominent
increases in the phosphorylation of proteins having apparent molecular
weights of approximately 47 kDa and 43 kDa, respectively. Both of
these proteins have been shown to be substrates for the Ca/PL-PK. It
is possible that these proteins with apparent molecular weights of
42-48 kDa represent closely related proteins which play a role in
secretion from a number of cells.
The studies discussed above suggest that the Ca/PL-PK is involved
in stimulation-secretion coupling in neutrophils, platelets and other
tissues. We have found Ca/PL-PK activity in extracts from rat
peritoneal mast cells. Furthermore, our finding that the PEs, which
are known to bind to and stimulate the Ca/PL-PK, enhance histamine
release by agents which mobilize extracellular calcium and that PEs
stimulate protein phosphorylation suggests that activation of the
Ca/PL-PK may be involved in histamine release. PEs did not enhance
release stimulated by somatostatin or compd 48/80. Compd 48/80
stimulated the largest increases in protein phosphorylation, including
the 48 kDa band, and the greatest release of histamine. It is possible
that compd 48/80 stimulates secretion through a completely separate

80
mechanism or that it maximally activates the Ca/PL-PK such that PEs do
not further enhance activation. In any case, our results suggest that
the Ca/PL-PK may play a role in stimulation-secretion coupling in mast
cel Is.

CHAPTER FOUR
CONCLUSIONS AND SIGNIFICANCE
Experimental results presented in this dissertation demonstrate for
the first time that the pharmacological probes hydrocortisone, a
prototypic anti-inflammatory steroid, and TPA, a prototypic tumor
promoter, exert significant and selective effects upon mast cell
stimulated release.
In the case of anti-inflammatory steroid action, we have
demonstrated that in cells treated in vitro, long-term (more than 9 hr)
pretreatment is required for manifestation of inhibitory effects.
Exertion of the inhibitory influences appear to be due to induction of
the synthesis of specific proteins. Inhibition of ^5Ca^+ uptake,
histamine and [1-^C]-AA release in anti-inflammatory steroid treated
cells was limited to secretion by IgE-like secretagogues. Addition of
exogenous AA did not overcome the inhibitory effect. No significant
inhibition was noted in cells stimulated with polypeptide-base
secretagogues or the receptor-independent stimulating agent ionophore
A23187.
Other investigators have shown in mouse mast cells, glucocorticoid
pretreatment did not alter IgE-Fc receptor number, affinity of IgE for
its receptor or basal cAMP levels. Pretreatment did, however, inhibit
IgE-induced phospholipid methylation, 45Ca^+ uptake as well as
histamine release. Phospholipase inhibitory protein (lipomodulin)
levels were increased in anti-inflammatory steroid pretreated cells.
81

82
This evidence suggests that in mast cells, anti-inflammatory
steroids exert inhibitory influences early in the biochemical sequence
of activation-secretion events. However, several possibilities for
glucocorticoid action exist and include: 1) uncoupling cellular
activation from influx of extracellular calcium, 2) inhibition of
phospholipid methyl transferases, 3) synthesis of phospholipase
inhibitory protein which interacts selectively with a subtype of
phospholipase associated with Fc receptors, and 4) in a manner
analogous to lymphocytes, generation of IgE-suppressive factors, which
modulate T cell subset responses to immunologic stimuli may also act
selectively in mast cells.
Time courses of glucocorticoid induced inhibition of IgE-like
release argue against competition for extracellular calcium binding
sites as do the lanthanides, agents which also selectively inhibit
IgE-like but not polypeptide-base induced release. Anti-inflammatory
steroid induced inhibition is reminiscent of methyl transferase
inhibitor action as well as theophylline and dibutyryl cAMP induced
inhibition of IgE-antigen elicited methylation, 45Ca2+ uptake and
histamine release. Ionophore A23187 treatment can overcome inhibition
by all of these agents, presumably since it functions as a calcium
carrier which bypasses receptor-mediated effects which may include both
transient cAMP and methyl transferase activation.
As documented earlier, phospholipid methylation has been shown to
act prior to calcium influx and glucocorticoids prior to methylation.
Thus, it appears that one mechanism of glucocorticoid action in mast
cells is by methyl transferase inhibition. This is also the case in

83
other white cells as Honma et al_. (1981) have shown a decrease in
phospholipid methylation in dexamethasone treated mouse myeloid
leukemia cells during differentiation. In chronic leukemia cells in
culture, 6 hr pretreatment with anti-inflammatory steroids did not
influence the activity of phospholipid methyl transferase I in forming
PME from PE, however, there was a decrease in the conversion of PME to
PC by phospholipid methyl transferase II. A similar effect in mast
cells could explain glucocorticoid induced inhibition patterns.
Our data does not rule out the generation of phospholipase
inhibitory proteins such as lipomodulin as another mechanism of
anti-inflammatory steroid action. However, results supportive of a
generalized generation of lipomodulin in glucocorticoid pretreated mast
cells included only IgE-mediated versus A23187 mediated release. The
model is complicated in view of the results of the present
investigations where another class of receptor-stimulating agents,
polypeptide-base secretagogues were included. Selectivity of the
phospholipase inhibitory proteins to IgE-like coupled responses must
now be evoked. The fact that exogenously added AA, if it resembles
endogenous release, is not sufficient to overcome the presumed
phospholipase inhibitory effects of glucocorticoids suggests that other
mechanisms may be involved and/or other products of phospholipase
activation may be important.
In contrast to the inhibitory effects of glucocorticoids, we have
demonstrated that the pharmacologic probe, TPA is capable of increasing
mast cell release. In our characterizations of the effects of PEs upon
mast cell release, we have shown that, in the presence of extracellular

84
calcium, PEs tremendously potentiate ionophore A23187 stimulated
release of both histamine and AA. PEs act synergistically with
IgE-like secretagogues in the presence of PS while interactions with
peptide-base types of secretagogues are minimal. Structure-activity
relationships for various PE analogues on mast cell histamine release
suggest a single mechanism of action, perhaps mediated by a single
receptor. In many tissues, the PE receptor is now thought to be a
Ca/PL-PK; we have demonstrated low levels of this enzyme in mast cells.
Activation of Ca/PL-PK by PEs occurs in the presence of PS by
substituting for DAG to increase the affinity of the enzyme for
calcium. Our findings that exogenous PS and TPA enhance IgE-like
stimulated histamine release in a synergistic manner suggest
involvement of the enzyme Ca/PL-PK in mast cell stimulation-secretion
coupling. In contrast, peptide-base secretagogues may effect secretion
through a completely separate mechanism or through maximum activation
of Ca/PL-PK thereby abolishing enhancement by TPA.
In platelets, signal induced PI breakdown is linked to Ca/PL-PK
activation by generation of DAG. The synthetic analogue of DAG,
1-oleoyl-2-acetylglycerol (OAG) which gains access to intact platelets
with no sign of membrane damage, activated Ca/PL-PK without inducing PI
turnover or AA generation. In a similar manner, synergistic effects of
OAG and ionophore A23187 have been noted in mast cells. When
stimulated with OAG alone, mast cells released histamine to some
extent, but in the presence of low concentrations of ionophore A23187,
release was dramatically enhanced (Nishizuka, 1984). The suggestion
that protein phosphorylation by Ca/PL-PK and the mobilization of

85
calcium are indispensible and synergistically effective for causing
full physiological responses is supported by the following. Turnover
of PI and incorporation of radiolabel into PI take place in the mast
cell stimulated with antigen, anti-IgE, and con A or compd 48/80
regardless of the presence or omission of extracellular calcium. Thus,
PI metabolism may concomitantly participate in activation of Ca/PL-PK,
the regulation of calcium channels or mobilization of sequestered
calcium, as well as the generation of AA following receptor activation
of the mast cell.
McPhail et al_. (1984) have recently stated that unsaturated fatty
acids, including AA, directly activate Ca/PL-PK in the presence of PS
by increasing affinity of the enzyme for calcium. However, at higher
concentrations, a loss of enzyme activity which could not be reversed
by PS was noted. Like DAG, AA may also have an ability to directly
activate and regulate Ca/PL-PK thus giving it a new role in modulation
of cellular responses. We have noted, in data not shown, that mast
cells can be stimulated to secrete histamine by exogenous AA in the
presence of extracellular calcium. That stimulation may be via
activation of Ca/PL-PK.
We have also investigated glucocorticoid-TPA interactions and have
found that, in the presence of suboptimal concentrations of ionophore
A23187, addition of various concentrations of TPA to glucocorticoid
pretreated cells did not overcome the inhibition of release of either
histamine of [1-*4C]-AA. Glucocorticoid pretreatment of mast cells may
induce synthesis of a Ca/PL-PK inhibitory protein such as the phorbol
ester binding inhibitory factor (PEBIF) described by Hamel et al.
(1984).

86
Relevance of this work toward progress in basic medical research of
human allergy is fortified by the similarity of initial triggering
events of IgE-mediated release from rat mast cells and purified human
lung mast cells.

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BIOGRAPHICAL SKETCH
I, Ann Schwarzbauer, spent my youthful years in Menasha, Wisconsin,
involved in the work of dairy farming and in developing an appreciation
for plants, animals and space which country life can instill. Adding
to this an excellent education at St. Mary Grade and High School, I was
given the best of basics with which to begin my adult life.
The following four years of my enrollment in the University of
Wisconsin were very eventful and culminated in my Bachelor of Science
in medical microbiology. Very shortly thereafter, and perhaps
unexpectedly for a farmer's daughter, I married, not a travelling
salesman, but Tom, a psychologist.
Our partnership strengthened and developed into personally
fulfilling alternations where one of us is employed while the other is
a student. My present turn as a student is now closing and with it I
have come around full circle from "farmacology" to pharmacology.
98

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Fulton T. Crews, Chairman
Assistant Professor of
Pharmacology and Therapeutics
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Steph^h F. Baker
Associate Professor of
Pharmacology and Therapeutics
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Wil liam R. Kern
Associate Professor of
Pharmacology and Therapeutics

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Professor of Pharmacology
and Therapeutics
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Stephen W. Russell
Professor of Pathology
This dissertation was submitted to the Graduate Faculty of the
College of Medicine and to the Graduate Council, and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Phil osophy.
August 1984
Dean for Graduate Studies
and Research
for

UNIVERSITY OF FLORIDA
3 1262 08554 3683




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