AN ABSTRACT OF THE THESIS OF
Miles Orchinik for the degree of Doctor of Philosophy
in Zoology presented on March 13. 1992.
Title: Biochemical and Behavioral Characterization of Steroid Receptors in
Neuronal Membranes
Redacted for Privacy
Abstract approved:
Frank L. Moore
It is well established that steroid hormones modulate brain function
behaviors through intracellular receptors that act as ligand-dependent transcription
factors, regulating specific genes. Recent evidence indicates that steroids can also
elicit rapid neuronal responses independently of gene expression, presumably
through steroid receptors in neuronal membranes. However prior to this thesis,
there was little direct evidence for steroid receptors in neuronal membranes.
This thesis investigates the receptor mechanisms mediating the rapid
suppression of male reproductive behavior following stress or corticosteroid
administration in an amphibian, Taricha granulosa. The rapidity of this
behavioral response is more consistent with a membrane-bound corticosteroid
receptor than with intracellular receptor-mediated events.
Radioligand binding studies, using [3H]corticosterone, reveal that a
genuine, high-affinity receptor for corticosterone exists in neuronal membranes.
These binding sites are most enriched in synaptic membrane fractions and have a
pharmacology distinct from intracellular receptors. Receptor autoradiography
indicates the highest densities of receptors are found in the neuropil, rather than
over cell bodies, in limbic brain regions.
Behavioral studies demonstrate that these membrane-bound receptors most
likely mediate the stress-induced suppression of behavior; the potencies of
steroids to bind to these receptors are highly correlated with their potencies to
inhibit male behavior.
Additional radioligand binding studies demonstrate that this corticosteroid
receptor is distinct from the GABAA receptor complex. Select steroids, but not
corticosterone, are potent modulators of GABAA receptors in Taricha brains and
mammalian brains, suggesting that this mechanism of steroid action has been
highly conserved and is physiologically important.
The signal transduction mechanism associated with the corticosteroid
receptor, like many neurotransmitter receptors, appears to involve G proteins.
CHICorticosterone binding was negatively modulated by guanyl nucleotides in a
manner consistent with a G protein-coupled transmembrane receptor.
Together, these studies provide the first evidence for a G protein-coupled
steroid receptor in neuronal membranes and the best evidence that membrane-
bound steroid receptors are behaviorally relevant. These findings also support the
conclusion that steroids regulate brain function through multiple mechanisms of
action, genomic and nongenomic.
Biochemical and Behavioral Characterization of
Steroid Receptors in Neuronal Membranes
by
Miles Orchinik
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed March 13, 1992
Commencement June 1992
APPROVED:
Redacted for Privacy
Professor of Zoology in charge of major
Redacted for Privacy
Chair of Department of Zoology
Redacted for Privacy
Dean of G
,1
ate Schoo
Date thesis is presented
Typed by
13 March 1992
Miles Orchinik
ACKNOWLEDGEMENTS
Graduate school has been a highly rewarding intellectual adventure
primarily because of the interactions I've had with my mentors, Frank Moore and
Tom Murray in the College of Pharmacy. Frank has been the perfect graduate
advisor. He has tried to instill in me some fundamental sense of how to think
like a biologist, how to go about asking meaningful biological questions. Tom
has tried to provide me with the theoretical foundation and technical tools
required to answer basic questions in neurobiology. Tom also imposed
uncompromisingly high standards for scientific research. I will always be
grateful to Frank and Tom for these lessons and would like to thank them for
their wisdom, patience and enthusiasm.
I would also like to thank my committee members Carl Schreck, Joanne
Leong and Philip Brownell for constructive criticisms of grant proposals,
manuscripts and presentations over the years. I owe a great deal to Paul Franklin
who has taken the time on countless occasions to try to help me figure out some
apparently anomalous data. Pierre Deviche, Sunny Boyd, Cathy Propper,
Georgeen Gaza and Sandy Potter were very helpful when I was first getting
started in the lab. I'd also like to thank my fellow graduate students, particularly
those in Frank's and Phil's labs, who have been sounding boards, critics, and
great intellectual resources.
A number of people assisted in the lab and in the field and this work
would have been much more difficult without their help. I would like to thank
Yasi Aidinejad, David Booth, Valerie Caldwell, Dana Desonie, Brian Ford, Deb
Hildebrand, Chris Lowry, Lori McKinney, Jim Morgan, Michele Schroeder,
Mitch Smith, Scott Stonum, Josh Tacchini and Lisa Young for their help. The
Zoology Department staff, particularly Sedonia Washington and Shirley Baker,
provided stress management by helping me to get papers and grant proposals in
the mail.
I would also like to acknowledge the National Science Foundation for a
NSF Graduate Fellowship which paid my salary for three years and for an
Improvement of Doctoral Dissertation grant which funded much of this research.
I thank my parents for not laughing when they heard I was going back to
school. Finally, Dana Desonie deserves special thanks for good cheer, endless
patience, love and support, for knowing when it was time to take a day off, and
most of all, for doing my laundry before the Fidia Symposium.
TABLE OF CONTENTS
Chapter
I. GENERAL INTRODUCTION
Genomic versus Non-genomic Actions of Steroids in the
Central Nervous System
The Problem
Hypotheses and Objectives
Page
1
1
5
7
II. A CORTICOSTEROID RECEPTOR IN NEURONAL
MEMBRANES
Abstract
Text
Acknowledgements
III. GUANYL NUCLEOTIDES MODULATE BINDING TO
STEROID RECEPTORS IN NEURONAL MEMBRANES
Abstract
Introduction
Methods
Results
Discussion
Acknowledgements
IV. STEROID MODULATION OF GABAA RECEPTORS
IN AN AMPHIBIAN BRAIN
Abstract
Introduction
Materials and Methods
Results
Discussion
V. CONCLUSIONS
12
13
14
33
34
35
36
39
42
45
64
65
66
68
71
76
79
109
Summary
Speculation
109
113
BIBLIOGRAPHY
118
LIST OF FIGURES
Page
Figure
I.1
11.1
Model for the regulation of male Taricha
reproductive behavior.
10
Binding of [3H]CORT to crude synaptosomal membranes.
24
A. Equilibrium saturation binding.
B. Association of [3H]CORT specific binding to
brain membranes.
C. Dissociation of [3H]CORT from neuronal membranes.
11.2
11.3
Distribution of putative, membrane-bound [3H]CORT
binding sites in the preoptic area (POA).
28
Inhibition of male sexual behavior.
30
A. Latency of response to CORT.
B. Linear relationship between potency of steroids to
inhibit [3H]CORT binding and potency to inhibit
sexual behavior.
111.1
111.3
111.4
111.5
11I.6
The effect of MgC12 on the specific binding of
[3H]CORT to neuronal membranes.
52
Equilibrium saturation binding of [3H]CORT to neuronal
membranes in the presence or absence of guanyl nucleotide
54
Guanyl nucleotides inhibit the specific binding of
[3H]CORT in Taricha brain membranes.
56
Effect of MgC12 on the inhibition of[3H]CORT
binding by guanyl nucleotides.
58
Dissociation of specifically bound [311]CORT
from Taricha neuronal membranes.
60
Effect of disulfide bond reducing agent on the sensitivity
of [3H]CORT binding to negative modulation by GTP -yS.
62
LIST OF FIGURES
Page
Figure
IV.1
Equilibrium saturation binding of [35S]TBPS to
neuronal membranes.
91
A. Saturation isotherm.
B. Scatchard-Rosenthal replot of saturation data.
D/.2
Inhibition of 135STTBPS binding by GABA.
93
IV.3
Steroid modulation of [35S]TBPS binding.
95
IV.4
Equilibrium saturation binding of VITiflunitrazepam
to Taricha neuronal membranes.
97
A. Saturation isotherm.
B. Scatchard-Rosenthal replot of saturation data.
IV.5
Effect of GABA and pentobarbital on [3H]flunitrazepam
specific binding.
W.6
Modulation of 1311]flunitrazepam binding by steroids.
IV.7
GABA-stimulated
synaptoneurosomes.
99
101
uptake into Taricha
103
W.8
Effect of CORT on 36a- uptake.
105
W.9
Representative receptor autoradiograms showing
[35S]TBPS binding sites in Taricha midbrain.
107
LIST OF TABLES
Page
Table
II.1
IV.1
IV.2
Potency of steroids as inhibitors of the specific binding
of [31.1]CORT to crude synaptosomal membranes.
22
Potencies of steroids as inhibitors of [35S]TBPS
binding.
87
Distribution of r5S]TBPS and [31-1]flunitrazepam
specific binding sites.
89
PREFACE
This thesis is composed of manuscripts which have been published, are
in press, or are submitted for publication. Chapter II was published as Orchinik,
M., Murray, T.F., and Moore, F.L. (1991) A corticosteroid receptor in neuronal
membranes. Science 252: 1848-1851. Chapter III is in press, Orchinik, M.,
Murray, T.F., Franklin, P.H., and Moore, F.L. (1992) Guanyl nucleotides
modulate binding to steroid receptors in neuronal membranes. Proc. Natl. Acad.
Sci. USA. Chapter IV is prepared for submission, Orchinik, M., Murray, T.F.,
and Moore, F.L. (1992) Steroid modulation of GABAA receptors in an amphibian
brain.
Portions of the work have also appeared in review papers: 1) Orchinik,
M., Murray, T.F., Moore, F.L. (1991) Corticosteroid receptor in neuronal
membranes associated with rapid suppression of sexual behavior. In: Costa, E.
and Paul, S.M., Eds. Neurosteroids and Brain Function. Fidia Research
Foundation Symposia Series, vol 8. Thieme Medical Publishers, New York. pp.
125-132. 2) Moore, F.L., Orchinik, M. (1991) Multiple molecular actions for
steroids in the regulation of reproductive behaviors. Sem. Neurosci. 3:489-496.
Portions of the work have appeared in published abstracts: 1) Orchinik,
M. and Moore, F.L. (1988) Distribution of GABAA receptors in an amphibian
brain. Amer. Zool. 28:118A. 2) Orchinik, M. and Moore, F.L. (1989) Steroid
modulation of GABAA receptors: Novel mechanism for regulation of sexual
behavior. Soc. Neurosci. Abstr. 15:759. 3) Orchinik, M., Murray, T.F., and
Moore, F.L. (1990) Novel steroid-binding site on synaptic membranes may
mediate stress-induced inhibition of sexual behavior. Soc. Neurosci. Abstr.
16:765. 4) Orchinik, M., Murray, T.F., and Moore, F.L. (1991) Membranebound corticosteroid receptor is coupled to a G-protein. Soc. for Neurosci.
Abstr. 17:1408. 5) Rose, J.D., Moore, F.L., and Orchinik, M. (1991) Rapid
neurophysiological actions of corticosterone related to stress-induced inhibition of
sexual behavior in an amphibian. Soc. Neurosci. Abstr. 17:1057.
Frank Moore and Thomas Murray have contributed to every aspect of
this work. Both have made constructive suggestions concerning relevant
experiments, interpretation of data, and manuscript revisions. Dr. Paul Franklin
helped refine and interpret the G protein experiments in Chapter
the manuscript and made valuable critical suggestions.
he also read
BIOCHEMICAL AND BEHAVIORAL CHARACTERIZATION OF
STEROID RECEPTORS IN NEURONAL MEMBRANES
CHAPTER I: GENERAL INTRODUCTION
Genomic versus Non-genomic Actions of Steroids
in the Central Nervous System
One significant breakthrough in modern cellular biology has been the
discovery of the molecular mechanism that mediates many of the cellular
responses to steroid hormones--the intracellular steroid receptor. The
intracellular steroid receptors belong to a family of receptors for gonadal and
adrenal steroids, thyroid hormones, vitamin D metabolites, retinoids, as well as a
number of as yet undetermined ligands in vertebrate and invertebrate cells
(Evans, 1988; Fuller, 1991). In the now classical model of steroid hormone
action, steroids diffuse across plasma membranes and bind to cytosolic or nuclear
receptors in target cells. The binding of ligand to receptor facilitates the binding
of the activated receptor complex to consensus sequences on DNA molecules, the
hormone response elements, thereby altering gene transcription (Yamamoto,
1985; Green and Chambon, 1988; Carson-Jurica, 1990; Beato, 1991). Thus, the
classical steroid receptors act as ligand-dependent transcription factors, and the
responses of cells to steroids results from steroid-directed changes in protein
synthesis.
Steroids have profound influences on the brain, ranging from the life-long
organizational effects of gonadal steroids on sexually dimorphic brain regions that
2
occur during development (Phoenix et al., 1959; Arnold and Gorski, 1984) to
cyclic, transient effects of gonadal and adrenal steroids on neuronal activity and
behavior (Weeks, 1991; Silver et al., 1992; Pfaff, 1989; Mc Ewen et al., 1991).
These actions of steroids can be ascribed to the positive and negative regulation
of gene expression, including genes for neurotransmitter receptors, neuropeptides,
G proteins, and enzymes for neurochemical metabolism (reviewed in Mc Ewen et
al., 1986; Mc Ewen, 1991; Costa and Paul, 1991). However, in addition to the
well-characterized, classical steroid effects on the regulation of gene expression,
there is a subclass of effects produced by gonadal and adrenal steroids that cannot
easily be accounted for by the classical model of steroid action.
Fifty years ago, Seyle discovered that steroids can have potent anesthetic
and anti-convulsant effects (Seyle 1941, 1942). Since that time there have been
numerous observations of steroid-induced changes in neuronal excitability (Kelly
et al., 1977; Teyler et al., 1980; Nabekura et al., 1986; Majewska et al., 1986;
Havens and Rose, 1988; Hua and Chen, 1989; Wong and Moss, 1991;
Dubrovsky et al., 1990; Ffrench-Mullen and Spence, 1991), or neurosecretion
(Keller-Wood and Dallman, 1984; Dluzen and Ramirez, 1989; Borski et al.,
1991) that occur with a latency of seconds to very few minutes. The rapidity of
these actions would seem to preclude a genomic mechanism of action. However,
latency alone may not be sufficient to distinguish between genomic and
nongenomic actions of steroids, especially when responses occur with a latency of
minutes, rather than seconds or hours (Mc Ewen, 1991). Recognizing this, many
3
rapid steroid effects have been demonstrated to occur when access to intracellular
steroid receptors is blocked (Godeau et al., 1978; Hua and Chen, 1989; Dluzen
and Ramirez, 1989), in brain regions or brain preparations lacking intracellular
receptors (Edwardson and Bennett, 1974; Drouva et al., 1985; Smith et al., 1987;
Su et al., 1988; Lan et al., 1990; Wilson et al., 1989; Inoue and Kuriyama,
1989; Sokolovsky et al., 1981), or in the presence of protein synthesis inhibitors
(Nabekura et al., 1986; Schumacher et al., 1990).
These rapid, non-genomic events are presumed to be mediated through
steroid recognition sites on neuronal membranes. However, despite the evidence
that steroids can act at the level of neuronal membranes, the existence of
physiologically relevant steroid receptors has remained largely inferred (Mc Ewen
et al., 1988), and the receptor and transduction mechanisms remained unknown.
At the beginning of the studies described herein, there was only one paper
demonstrating saturable binding sites for steroids to brain membranes (Towle and
Sze, 1983). They found that gonadal and adrenal steroids saturably bound to
partially purified synaptic membranes, but not with high affinity (Kd = 120 nM
for corticosteroids), such that the physiological relevance of these binding sites
was doubted (Schumacher, 1990). Two other radioligand binding studies have
been recently published, describing steroid binding to neuronal membranes. Ke
and Ramirez (1990) demonstrated that progesterone, when conjugated to iodinated
bovine serum albumin, bound with low nanomolar affinity to neuronal
membranes, but the results could not be replicated with progesterone alone.
4
Majewska et al. (1990) found that a "neurosteroid" (see below), dehydroepiandro-
sterone sulfate, bound with micromolar affinity to neuronal membranes. The
methodology employed in that study was questionable; the use of a filtration
assay to quantify low-affinity ligand binding to membranes is suspect.
Binding sites for steroids on cell surfaces of non-neuronal tissue have also
been described (Suyemitsu and Terayama, 1975; Koch et al., 1978; Pietras and
Szego, 1979; Blondeau and Baulieu, 1984; Bression et al., 1986; Trueba et al.,
1987; Gametchu, 1987; Patino and Thomas, 1990; Ping et al., 1991; Ibarrola et
al., 1991) that might account for some of the rapid actions of steroids that occur
outside of the central nervous system (reviewed in Duval et al., 1983;
Schumacher, 1990). As with the rapid effects of steroids on neuronal activity,
the non-genomic steroid effects in peripheral tissue were also largely ignored as
the details of the transcriptional model of steroid action were elaborated.
Interest in the rapid effects of steroids on brain function was aroused when a
potential molecular mechanism was discovered. Investigations into the actions of
anesthetic steroids revealed that steroids can allosterically modulate receptors for
7-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the brain
(Harrison and Simmonds, 1984). Subsequently it was discovered that
physiological concentrations of endogenous steroid metabolites are also potent
modulators of GABAA receptors (Majewska et al., 1986). Finally, a biosynthetic
pathway for steroid compounds was discovered in the brain (reviewed in Baulieu,
1991), including a pathway for the synthesis of "neurosteroids" that are active at
5
the GABAA receptor. A number of labs are currently attempting to establish a
physiological role for endogenous GABAA receptor-active steroids (for review,
Paul and Purdy, 1992). However, as demonstrated in Chapters 3 and 4, not only
can select steroids modulate the GABAA/benzodiazepine receptor chloride
ionophore complex, but other neuroactive steroids appear to use cell-surface
receptors besides sites on the GABAA receptor.
The Problem
Reproductive behavior in all vertebrates is regulated through interactions
among external cues and a suite of steroids, neurotransmitters, neuropeptides and
their receptors (Fig. I.1). In addition, the same brain regions--the preoptic area,
amygdala, hypothalamus, for example--appear to have key roles in regulation of
reproductive behavior in all vertebrates. Therefore, the basic regulatory
mechanisms that govern reproductive behaviors appear to have been highly
conserved among vertebrates (Moore, 1990).
Because of the relative simplicity
of behavioral patterns and brain organization in non-mammalian vertebrates, and
their robust behavioral responses to neurochemical manipulation, "lower"
vertebrates may constitute powerful model systems for understanding the
mechanisms that regulate reproductive behaviors.
In most vertebrates stress and reproduction are reciprocally related
(Greenberg and Wingfield, 1987; Sapolsky, 1987). This is true for the
amphibian Taricha granulosa (rough-skinned newt). For example, in Taricha,
6
stress suppresses the circulating levels of testosterone; this response may be due
to the suppression of GnRH release by the adrenal (interrenal) steroid
corticosterone (Moore and Zoeller, 1985). Acute stress also inhibits the
reproductive behavior of male Taricha, the amplectic clasping of females;
corticosterone is a causal factor in this behavioral response (Moore and Miller,
1984). The behavioral response to corticosterone appears to involve GABA,
inasmuch as the stress- or corticosterone-induced suppression of clasping behavior
is blocked when males are pretreated with a GABA synthesis inhibitor or treated
concurrently with a GABAA receptor antagonist (Boyd and Moore, 1990; Moore
and Orchinik, 1991).
These behavioral and endocrinological studies using Taricha invoke
questions of the molecular mechanisms of steroid action because the onset of the
response to stress or corticosterone is rapid; male clasping behavior is suppressed
within 30 minutes of injection (Moore and Miller, 1984). Further examination of
the response latency revealed that male sexual behavior is significantly
suppressed, relative to saline-injected animals, within 8 minutes of intraperitoneal
injection of corticosterone (Orchinik et al., 1991a). More recent work indicates
that the excitability of Taricha hindbrain neurons involved in clasping behavior is
altered within 2 minutes of topical application of corticosterone (Rose et al.,
1991; Orchinik et al., 1991b). Behavioral and electrophysiological responses that
are this rapid are inconsistent with the classical model of steroid-directed changes
in protein synthesis.
7
Hypotheses and Objectives
The insufficiency of the classical model of steroid hormone action to
account for the rapid behavioral and neuronal responses to corticosterone in
Taricha provided the motivational basis for the studies undertaken in this thesis.
The major objective of the studies described herein was to identify and
characterize receptor and transduction mechanisms that could account for the
rapid behavioral responses to corticosteroids.
The first hypothesis, the subject of Chapter 2, was that a physiologically
relevant, high-affinity receptor for corticosteroids exists in neuronal membranes.
The rapidity of the response to corticosterone dictated that the receptor be
localized on the cell surface. Although there was no precedent for a high-affinity
receptor in neuronal membranes for any naturally-occurring steroid, the exquisite
sensitivity of Taricha males to corticosterone injection (significant inhibition of
behavior by intraperitoneal injection of 1 yg) necessitated a high-affinity
recognition site. Chapter 2 describes the pharmacological studies performed to
satisfy basic criteria in validating a genuine, neurochemical receptor system.
A related hypothesis was that the corticosteroid recognition site on
neuronal membranes played a role in the suppression of reproductive behavior.
This hypothesis was tested in Chapter 2 in a series of behavioral experiments
designed to determine if the affinity of steroids for the receptor is correlated with
their potencies to suppress clasping behavior. This question was also addressed
indirectly in receptor autoradiographic studies--functional receptors should be
8
localized in brain regions involved in the regulation of male sexual behavior.
The remaining hypotheses related to the transduction mechanisms
associated with non-genomic steroid effects. The first of these hypotheses tested
was that the potent steroidal ligands for mammalian GABAA receptors modulate
GABAA receptors in Taricha brains in a similar manner. If so, this would
provide strong evidence that this mechanism of steroid action has been conserved
through vertebrate evolution, and therefore is likely to be important
physiologically. Experimental tests of this hypothesis are described in Chapter 4.
As a corollary to this hypothesis, I tested the hypothesis that the
corticosteroid receptor in neuronal membranes is a steroid recognition site on the
GABAA receptor. This was a reasonable hypothesis because the behavioral
response to corticosterone involves an interaction with the GABAA receptor
system, and we found that other steroids can modulate GABAA receptor function
in Taricha brain membranes. This hypothesis, which we eventually rejected, is
addressed in a cursory manner in Chapter 2, but in depth in Chapter 4.
After rejecting the hypothesis that the rapid responses to corticosterone are
mediated through a recognition site on the GABAA receptor, we examined an
alternative hypothesis--that the corticosterone receptor utilizes guanyl nucleotide
binding proteins (G proteins) in neuronal membranes in signal transduction.
More than a hundred receptors for hormones and neurotransmitters have been
identified that couple to G proteins (Simon et al., 1991), although none have been
receptors for steroids. We performed radioligand binding studies to determine if
9
ligand binding to the corticosteroid receptor is modulated in a manner consistent
with a G protein-coupled receptor. These studies are detailed in Chapter 3.
Together the studies in this thesis provide strong evidence that the stressinduced suppression of reproductive behavior in Taricha males is mediated by
corticosteroid receptors that are coupled to G proteins in neuronal membranes.
These receptors are not associated with the GABAA receptor complex, but
GABAA receptors in Taricha brains are modulated by steroids very much like
mammalian GABAA receptors. This second non-genomic mechanism of steroid
action appears to have been highly conserved and therefore is probably an
important molecular mechanism. These studies indicate that, in addition to
classical intracellular receptor-mediated transcriptional events, steroids regulate
vertebrate brain function and behavior through the use of multiple receptor and
transduction mechanisms associated with neuronal membranes.
10
Fig. I.1. Model for the regulation of male Taricha reproductive behavior.
Clasping behavior is regulated by the integration of external cues with multiple
excitatory and inhibitory neuromodulators. As in the mammalian brain,
reproductive behavior is facilitated by actions of gonadal steroids. Steroids
facilitate behavior, in part, through the regulation of the activity of
neurotransmitter and neuropeptides, and their receptors. In response to inhibitory
stimuli, such as stress, the hypothalamic-pituitary-adrenal axis is activated.
Corticosterone, released from the adrenals, antagonizes the excitatory pathways
regulated by arginine vasotocin (the amphibian homologue to arginine
vasopressin) and gonadotropin releasing hormone (GnRH). The behavioral
effects of corticosterone also involve the inhibitory neurotransmitter GABA.
These neurochemical signals and regulatory mechanisms have been highly
conserved in vertebrate evolution.
Inhibitory Stimuli
Excitatory Stimuli
I
Testosterone
y
CORT
+
7
Vasotocin
GnRH
+
--Ipp., Behavioral Response
+
GABA
12
CHAPTER H. A CORTICOSTEROID RECEPTOR IN
NEURONAL MEMBRANES
Miles Orchinik., Thomas F. Murray, Frank L. Moore
M. Orchinik and F. Moore, Department of Zoology, Oregon State University,
Corvallis, OR 97331.
T. Murray, College of Pharmacy, Oregon State University, Corvallis, OR 97331.
13
Abstract
Steroids may rapidly alter neuronal function and behavior through poorly
characterized, direct actions on neuronal membranes. The membrane-bound
receptors mediating these behavioral responses have not been identified.
[3H]Corticosterone labels a population of specific, high-affinity recognition sites
(ICd= 0.51 nM) in synaptic membranes from an amphibian brain. These binding
sites were localized by receptor autoradiography in the neuropil, outside the
regions of perikarya. The affinities of corticoids for this [3II]corticosterone
binding site were linearly related to their potencies in rapidly suppressing male
reproductive behavior. Thus, it appears that brain membranes contain a
corticosteroid receptor that could participate in the regulation of behavior.
14
Text
In the classic model of steroid hormone action, steroids bind to
intracellular receptors, which act as ligand-dependent transcription factors that
regulate gene expression (Yamamoto, 1985; Justafsson et al., 1987; Carson-Jurica
et al., 1990). In addition to these well-known actions of steroid receptors,
steroids may alter brain function through nongenomic mechanisms (Schumacher,
1990; Majewska, 1987; Duval et al., 1983). For example, in rats, short-term
exposure to the gonadal steroid progesterone is associated with rapid changes in
behavior, and this effect occurs in the absence of new protein synthesis
(Schumacher et al., 1990). Both gonadal and adrenal steroid hormones can alter
neuronal firing activity within milliseconds to minutes of administration (Kelly et
al., 1977; Nabekura et al., 1986; Riker et al., 1982; Saphier and Feldman,
1988), and these responses can occur in brain regions lacking classic steroid
receptors (Smith et al., 1987) or if steroid access to intracellular receptors is
blocked (Hua and Chen, 1989). These events appear to be mediated by direct
steroid action on neuronal membranes, but there is little information concerning
steroid binding to membrane-bound recognition sites in the brain (Towle and Sze,
1983; Ke and Ramirez, 1990; Majewska et al., 1990).
To investigate the possibility that glucocorticoid receptors occur on
neuronal membranes, radioligand binding studies were performed on synaptic
(P2) membranes from brains of an amphibian, Taricha granulosa, known to have
15
rapid behavioral responses to corticosterone (CORT) (Moore and Miller, 1984;
Boyd and Moore, 1990; Moore, 1990). Equilibrium saturation binding
experiments', 2 indicated that [3H]CORT binding to brain membranes was
specific, saturable, and of high affinity (Kd, 0.51 nM) (Fig. II. 1A). Kinetic
experiments indicated that specific binding of [3H]CORT was relatively rapid and
reversible (Figs. II. 1, B and C). The Kd value calculated from kinetic data (0.16
Whole brains from males were homogenized in cold
0.32 M sucrose containing 5 mM Hepes (pH=7.45),
centrifuged at 1,000g (15 min) and the resulting
supernatant centrifuged at 30,000g (40 min).
The P2
pellet was frozen, then resuspended in 150 volumes
1
(weight to volume) cold buffer (25 mM Hepes, 1 mM EDTA
free acid, 60 µg /ml bacitracin, pH=7.45) for 3 hours to
dissociate endogenous ligand. After centrifugation at
30,000g (30 min), the final pellet was frozen,
resuspended in assay buffer (25 mM Hepes, 0.5 mM EDTA
salt, 200 mM NaC1, pH=7.45) to a protein concentration of
450 to 550 µg /ml.
Protein concentration was determined
by Bio-Rad microassay with a bovine serum albumin
standard.
Incubations (45-55 Ag protein in 300 Al) were
terminated after 2 hours by vacuum filtration and a 9 ml
wash with buffer over Whatman GF-C filters.
2
Radioactivity bound to filters was measured by liquid
scintillation spectroscopy. Nonspecific binding was
determined by addition of 10 AM unlabeled CORT.
Specific
binding was typically 75% to 80% of total binding.
All
binding experiments were performed in triplicate and were
repeated at least twice, with similar results.
16
nM) was close to the Kd estimated from equilibrium saturation data. The affinity
of this site differs from the low affinity (120 nM) of the [3H]CORT binding site
described in the synaptosomal fraction from rats (Towle and Sze, 1983).
The specific binding of [3H]CORT was temperature sensitive--greatest at
30°C, but eliminated when assays were performed at 60°C.3
Specific binding
was also inhibited in a concentration-dependent manner by treatment of the
membranes with the protease trypsin (0.001
1.0 mg/ml). Together, these data
suggest that [3H]CORT binds to a proteinaceous recognition site in the P2
fraction.'
The binding site was highly specific for CORT (Table IL1) and did not
have a pharmacological signature that resembled that of mammalian (Mc Ewen et
al., 1986; De Kloet and Reul, 1987; Funder and Sheppard, 1987) or amphibians
(Medhi et al., 1984; Lange and Hanke, 1988) intracellular corticoid receptors.
Neither the mineralocorticoid aldosterone, the glucocorticoid dexamethasone, or
Specific binding was low at 2°C, moderate at 15°C
and 45°C, negligible at 60°C. Binding studies were
3
routinely performed at 15°C, in the physiological
temperature range of Taricha.
4
Experiments in which perfused and nonperfused
brains were compared indicated that it was unlikely that
[3H]CORT binding activity was due to interaction with
plasma corticosteroid binding proteins.
Amphibian intracellular corticoid receptors have
high affinity (Kd < 10 nM) for aldosterone and/or
dexamethasone as well as CORT.
5
17
any other Type I or Type II corticoid receptor ligands we tested displayed high
affinity for this membrane-associated binding site. Furthermore, the [3H]CORT
binding site did not appear to be associated with the gamma-aminobutyric acid
(GABAA) receptor. The steroid metabolites, 5a-pregnan-3a,21-dio1-20-one (5aTHDOC) and 5a-pregnane-3a-o1-20-one (3a-OH-DHP), are potent modulators of
mammalian (Majewska et al., 1986; Gee, 1988; Puia et al., 1990) and Taricha
(Orchinik and Moore, 1989) GABAA receptors, but 5a-THDOC inhibited
[3H]CORT binding with only modest affinity, while 3a-OH-DHP was devoid of
activity (Table II.1). In addition, nonsteroidal GABAA receptor ligands did not
displace [3H]CORT binding to membranes. The inability of GABAA receptor
ligands to alter [311] -CORT binding is consistent with the inability of CORT to
alter GABAA receptor ligand binding or GABA-stimulated chloride flux in rodent
(Gee et al., 1987; Harrison et al., 1987; Turner et al., 1989; Morrow et al.,
1990) and Taricha brains (Orchinik and Moore, 1989).
Because the P2 fraction contains mitochondria as well as pre- and postsynaptic membranes (Whittaker, 1969), and [3H]CORT binds to adrenal and liver
(Satre and Vignais, 1974; Cozza et al., 1990; Leschenko and Sergeyev, 1987)
mitochondria, we examined [3H]CORT binding to synaptosomal and
mitochondrial fractions prepared by discontinuous sucrose gradient
centrifugation.6 As a marker for synaptic membranes, we measured the binding
Synaptosomes, mitochondria, nuclei were prepared
from fresh brains as described in Whittaker (1969).
The
6
18
of the muscarinic cholinergic antagonist, quinuclidinyl benzilate, [3H]QNB.7 As
a mitochondrial marker, we measured succinate cytochrome C reductase activity
(Feyereisen et al.,
1985).
The succinate cytochrome c reductase assay indicated
that mitochondria were well separated from synaptic membranes (homogenate,
1.2;
mitochondrial fraction,
10.6;
synaptosomal fraction,
2.8
units/minutemg
protein).
Our data indicate that [3H]CORT binds to synaptic membranes. The
specific binding of [314]CORT was most enriched, more than 11-fold, in the
synaptosomal fraction (ratio of [3H]CORT specific binding activity relative to
binding activity in homogenate: crude cytosolic,
mitochondrial, 7.04; synaptosomal,
11.50).
0.06;
nuclear,
0.59;
Furthermore, [3H]CORT binding
activity paralleled the enrichment of muscarinic receptor specific binding in the
synaptosomal fraction (ratio of [3H]QNB specific binding activity relative to
[3H]QNB binding activity in homogenate: crude cytosolic,
mitochondrial,
3.06;
0.06;
nuclear,
0.68;
synaptosomal, 11.20).8 The minimal amount of [3H]CORT
"crude cytosolic" fraction resulted from a 48,000g (45
min) centrifugation of the initial homogenate. All
fractions were resuspended in assay buffer to a protein
concentration of 600 µg /ml.
P2 fraction resuspensions were incubated with 0.5
nM [3H]QNB + 10 uM scopalamine (to determine nonspecific
binding).
8
The greater enrichment of [3H]CORT binding in the
mitochondrial fraction relative to [3H]QNB suggests there
19
binding in the nuclear and crude cytosolic fractions demonstrated that under these
conditions [3H]CORT binding to intracellular receptors is negligible, thus
providing further evidence that [3H]CORT binding activity in the synaptosomal
fraction is not attributable to intracellular receptor contamination.
To further substantiate the presence of CORT binding sites in synaptic
membranes we performed in vitro receptor autoradiography. Sections of Taricha
brain were incubated with [3HICORT in the presence of unlabeled ligands specific
for intracellular receptors.' Under these conditions, [3H]CORT specific binding
sites were detectable, with the greatest density in distinct regions of neuropil in
areas including the amygdala, preoptic area, and hypothalamus (Fig. 11.2). These
[3H]CORT binding sites were located almost exclusively outside regions of
may be binding sites for CORT in neuronal mitochondria,
as well as in synaptic membranes.
Thaw mounted 25-gm brain sections were incubated
for 30 min at 22°C with assay buffer containing 200 nM
9
dexamethasone and ZK91587, then incubated for 2 hours
with 2 nM [3H]CORT in buffer containing 200 nM
dexamethasone and ZK91587.
Nonspecific binding was
determined in alternate sections by the addition of 10 gM
CORT. The reaction was terminated by two 3-min washes in
ice cold buffer. Sections were dried under cool air,
apposed to 3H-sensitive film for 2 months, and stained
with toluidine blue for histology. The autoradiogram was
analyzed by computer-assisted densitometry (DUMAS).
Specific binding was determined by subtraction of
nonspecific binding from the aligned total binding
sections.
20
perikarya. The localization of binding sites in neuropil, areas rich in synaptic
terminals, provides corroborating evidence for CORT binding sites on synaptic
membranes.
A series of behavior experiments were designed to determine if [3H]-
CORT binding sites could be behaviorally relevant receptors.' Stress can
suppress the sexual behavior of male Taricha; this rapid response is dependent
upon the adrenal (interrenal) steroid CORT and is mimicked by CORT injection
(Moore and Miller, 1984; Boyd and Moore, 1990; Moore, 1990). Male sexual
behavior was suppressed within 8 min of intraperitoneal injection, compared to
vehicle-injected controls (Fig. II.3A). A behavioral response this rapid is
consistent with the presence of CORT receptors on synaptic membranes. Sexual
behavior was inhibited by a low dose of CORT (2.5 nmol; Fisher Exact Test, P
< 0.005), consistent with a high-affinity recognition site for CORT. We
established dose responses for a series of steroids as inhibitors of male sexual
m
Adult male Taricha in breeding condition were
collected locally and experiments conducted in the field.
Males (mean weight, 22 g) received intraperitoneal
injections (0.1 ml) of steroid or vehicle (amphibian
Ringers with 8% EtOH /2% dimethyl sulfoxide). Testing was
initiated 5 min after injection, when stimulus females
were added to tanks holding males. Males displaying
sexual behavior (dorsal amplectic clasping of female)
were removed from tanks. Females received 500 Ag
progesterone by injection 24 hours before tests to
enhance attractivity (Moore, 1978).
21
behavior (Fig. II.3B). The potencies of corticoids to rapidly inhibit sexual
behavior were linearly related to their potencies to inhibit [3H]CORT binding
(slope ± SE= 0.58 ± 0.06; P < 0.003). These data suggest that this binding
site, or pharmacologically similar receptors in the spinal cord or peripheral tissue,
could be involved in stress-induced suppression of reproductive behavior.
In conclusion, our binding studies reveal the presence of a specific, highaffinity ligand-receptor interaction (Limbird, 1986; Weiland and Molinoff, 1981)
in synaptic membranes. Autoradiographic studies provide further evidence for
the localization of CORT binding sites in synaptic membranes (in brain regions
known to regulate sexual behaviors). The linear relationship between the
potencies of compounds to interact with this CORT receptor and to inhibit behavior suggests that this receptor may mediate CORT regulation of sexual
behavior.'
u
CORT induces sexual behavior in female rats
within 5 min of intravenous injection through unknown
mechanisms (Kubli-Garfias, 1990).
22
Table 11.1. Potency of steroids as inhibitors of the specific binding of rlIJCORT
to crude synaptosomal membranes.
values and slopes determined by nonlinear
regression analysis (LIGAND); data are mean ± SEM values. Inhibition
reported as percent inhibition of basal [311]CORT specific binding at a
concentration of 1µM competitor (Sigma, Steraloids, New England Nuclear; RU
38486 courtesy of Roussel-UCLAF, France); 0.5 nM [3H]CORT. The highest
concentration of solvent used (0.3% Et0H/0.1% dimethyl sulfoxide) was added
to control tubes. Potent modulators of GABAA receptors designated by *.
Compound
Ki
Inhibition
(nM)
(%)
Slope
=
Corticosterone
0.11 + .006
100
0.96 + 0.03
Cortisol
3.75 + .56
100
0.92 + 0.08
Aldosterone
293
+ 13
69
1.06 + 0.06
5a-THDOC*
297
± 14
70
0.96 + 0.05
RU 28362
569
+ 40
36
1.14 + 0.24
Progesterone
759
+ 113
37
0.99 + 0.10
Testosterone
1,138
± 63
32
0.96 + 0.03
ZK91587
>5,000
13
Dexamethasone
>5,000
11
RU 38486
>5,000
8
3a-OH-DHP*
>5,000
5
Table II. 1
24
Fig. 11.1. Binding of [3H]CORT to crude synaptosomal membranes. (A)
Equilibrium saturation binding. P2 fraction resuspensions were incubated with
increasing concentrations of [3H]CORT (101.6 Ci/mmol; NEN, Boston, MA.).
Specific binding (filled circles) equals total binding (not shown) minus nonspecific
binding (open circles). Estimates of Ici= 0.51 +.04 nM and B.,,= 146 ± 4.4
fmol/mg protein obtained by non-linear regression analysis (Lundon 1). The data
were best fit by a one-site model. Inset: Scatchard replot of data was linear; Hill
coefficient= 0.98 (LIGAND). (B) Association of [3H]CORT specific binding to
brain membranes. P2 fraction resuspensions were incubated with 0.5 nM
[3H]CORT for increasing intervals. Data were fit by a one site model
(LIGAND); kb, = 0.054 ± 0.005 min-1. (C) Dissociation of [3H]CORT from
neuronal membranes. P2 fraction resuspensions were incubated for 2 hours with
0.5 nM CHJCORT prior to addition of 100 AM cold CORT. Data best described
with a one site model; k1 = 0.013 ± 0.001 min-1.
A
0,0
0.5
1.0
3
1.5
Free [ H]CORT (nM)
Fig. II.1
2.0
B
70
kobs= 0.054 min
0
30
60
90
Time (minutes)
Fig. II.1
-1
120
150
C
90
0
60
120
Time (minutes)
Fig. 11.1
180
240
28
Fig. 11.2. Distribution of putative, membrane-bound [3H]CORT binding sites in
the preoptic area (POA). Section (left) showing darkly stained perikarya of POA
neurons. Autoradiogram of identical section (right) shows localization of
CHICORT specific binding sites in the neuropil surrounding perikarya of POA
neurons.
Fig. 11.2
30
Fig. 11.3. Inhibition of male sexual behavior. (A) Latency of response to CORT.
Males were injected with 32 nmol (11 go CORT or vehicle, n= 14. Arrow
indicates time of addition of females to tanks. Data reported as cumulative
percentage of claspers at 1 min intervals. CORT injected males were
significantly inhibited (*) within 3 min of testing (Fisher Exact Test, P = 0.025).
(B) Linear relationship between potency of steroids to inhibit [WORT binding
and potency to inhibit sexual behavior. Males were injected with one of 5 - 7
doses of steroid, or of vehicle (n= 24 for each dose of each steroid; except RU
28362, n=14). Data recorded as number of claspers in 20 minute tests (except
cortisol; 60 minute tests, performed late in the breeding season). ED50 values to
inhibit male clasping behavior versus controls were obtained by probit analysis
(Tallarida and Jacob, 1979).
100
L.,
0
>
0
A
80
*mom
..c
a)
m
60
o
m
x
a)
0
40
a)
6
20
0
0
5
10
15
tMinutes After Injection
Fig. 11.3
20
25
B
L.
0
>
0
a)
CD
o
n
x
a)
0
Lf-
O
"---
o
E
c
c5
2
U)
0
LLJ
1
0
2
1
3
3
Inhibition of [ H]CORT Binding
(log <i, nv1)
Fig. 11.3
4
33
Acknowledgements
We thank A. Blaustein, D. Crews, P. Deviche, K. Moore, C. Propper,
and K. Yamamoto for helpful criticisms of the manuscript. For assistance in the
field, we thank C. Lowry, L. McKinney, J. Morgan, M. Schroeder, J. Tacchini,
B. Ford, and D. Desonie. This work supported by NSF grants BNS-8901500
(F.L.M. and M.O.) and BNS-8909173 (F.L.M.). M.O. was supported by an
NSF Graduate Fellowship.
34
CHAPTER TEL GUANYL NUCLEOTIDES MODULATE BINDING
TO STEROID RECEPTORS IN NEURONAL MEMBRANES
(corticosteroid receptors/G proteins/stress/sexual behavior)
Miles Orchinik *t, Thomas F. Murray*, Paul H. Franklin*, Frank L. Moore*
`Department of Zoology, Oregon State University, Corvallis, OR 97331-2914,
*College of Pharmacy, Oregon State University, Corvallis, OR 97331
Communicated by George Somero
t To whom reprint requests should be addressed
35
Abstract
The recently characterized corticosteroid receptor on amphibian neuronal
membranes appears to mediate rapid, stress-induced changes in male reproductive
behaviors. Because the transduction mechanisms associated with this receptor are
unknown, we performed radioligand binding studies to determine whether this
steroid receptor is negatively modulated by guanyl nucleotides. The binding of
[3H]corticosterone to neuronal membranes was inhibited by nonhydrolyzable
guanyl nucleotides in both equilibrium saturation binding and competition studies.
The addition of guanyl nucleotide plus unlabeled corticosterone induced a rapid
phase of [3H]corticosterone dissociation from membranes that was not induced by
the addition of unlabeled ligand alone. Furthermore, the equilibrium binding of
[3H]corticosterone and the sensitivity of the receptor to modulation by guanyl
nucleotides were both enhanced by Mg2+. These results are consistent with the
formation of a ternary complex of steroid, receptor, and guanine nucleotide-
binding protein that is subject to regulation by guanyl nucleotides. Therefore
rapid signal transduction through corticosteroid receptors on neuronal membranes
appears to be mediated by guanine nucleotide-binding proteins.
36
Introduction
Steroid hormones modulate behavior and neuroendocrine function through
the regulation of neuronal protein synthesis, an action mediated by intracellular
steroid receptors (Evans, 1988; Carson-Jurica, 1990). However, not all effects of
steroid hormones on brain function involve these genomic mechanisms
(Schumacher, 1990). Evidence for nongenomically-mediated actions of steroids
includes changes in neuronal excitability or neurosecretion that occur within
seconds of steroid administration, when de novo protein synthesis is inhibited,
when access to intracellular receptors is blocked, or in regions of the brain where
intracellular steroid receptors have not been identified (Kelly et al., 1977;
Nabekura et al., 1986; Smith et al., 1987; Hua and Chen, 1989; Dubrovsky et
al., 1990). These actions of steroids that occur independently of classical
intracellular receptors may have important roles in the regulation of behavior.
For example, progesterone induces reproductive behavior in female rats, in part,
through the modulation of hypothalamic oxytocin receptors; this rapid steroid
effect is independent of de novo protein synthesis (Schumacher et al., 1990).
The binding sites for steroids on the membranes of neurons (Towle and
Sze, 1983; Ke and Ramirez, 1990), as well as other cells (Suyemitsu and
Terayama, 1975; Pietras and Szego, 1979; Koch et al., 1978; Blondeau and
Baulieu, 1984; Gametchu, 1987; Patino and Thomas, 1990), might mediate some
rapid cellular responses to steroids. However, little is known about the
37
transduction mechanisms associated with these recognition sites. Only a few
general types of transduction mechanisms account for the multitude of cellular
responses initiated by cell surface receptors: a receptor may be an integral part of
a ligand-gated ion channel, a transmembrane-regulated enzyme, or a
transmembrane protein that couples to guanine nucleotide-binding regulatory
proteins (G proteins) (Hollenberg, 1991). It is now well established that some
steroids can directly modulate the functioning of a ligand-gated ion channel, the
GABAA receptor (Majewska et al., 1986; Lan et al., 1990). It appears that
steroids may utilize alternate mechanisms as well. We recently characterized a
high-affinity corticosteroid receptor in neuronal membranes from an amphibian
brain (Orchinik et al., 1991a) which appears to be physiologically relevant in
mediating the stress-induced suppression of male reproductive behavior (Moore
and Miller, 1984; Boyd and Moore, 1990; Moore and Orchinik, 1991). This
receptor is not associated with the GABAA receptor chloride channel complex
(Orchinik et al., 1991a). Therefore, we have conducted studies to determine
whether the transduction mechanism utilized by this steroid receptor involves G
proteins.
Members of the superfamily of G protein-coupled transmembrane
receptors exhibit common structural and regulatory motifs (Raymond et al.,
1990). One characteristic of these G protein-coupled receptors is that the binding
of hormone or neurotransmitter to the receptor is subject to heterotropic negative
modulation by guanine nucleotides (Birnbaumer et al., 1990; Gilman, 1987).
38
Therefore to address this question with respect to the membrane-bound
corticosteroid receptor, we performed a series of radioligand binding studies to
determine if [3H]corticosterone binding is sensitive to regulation by guanyl
nucleotides. The data support the conclusion that signal transduction through this
corticosteroid receptor in neuronal membranes is mediated by G proteins.
39
Methods
Animals.
Adult male rough-skinned newts (Taricha granulosa) were
collected locally (Benton Co., OR). Animals (mean weight 20 g) were
maintained in the lab in large tanks under a lighting and temperature regimen that
approximated natural conditions. Animals were maintained and sacrificed in
accordance with I. A. C. U. C. guidelines.
Membrane preparation. Brains were rapidly removed and homogenized
in 40 volumes (original weight to volume) of cold 0.32M sucrose containing 5
mM Hepes (pH=7.45). The whole brain homogenate was centrifuged at 1,000 x
g (15 min), and the resulting supernatant was centrifuged at 30,000 x g (40 min,
4°C). The P2 pellet was frozen and thawed, then resuspended in 150 volumes
(original weight to volume) of cold buffer (25 mM Hepes, 10 mM EDTA free
acid, 60 /.4g/m1 bacitracin, pH=7.45) for 2-3 hrs at 4°C to dissociate endogenous
ligands and remove endogenous cations. The suspension was centrifuged at
30,000 x g (30 min). The resulting pellet was washed in 150 volumes of 25 mM
Hepes buffer (pH=7.45) and centrifuged again at 30,000 x g for 30 minutes.
The final pellet was resuspended in approximately 900 Al assay buffer/brain to a
protein concentration of 250-350 µg /ml. Assay buffer contained 25 mM Hepes
and 10 mM MgC12 (unless specified otherwise), pH=7.45. Protein concentration
was determined by the Bradford method using Pierce Coomassie protein assay
reagent (Rockford, Ill.), and BSA standard.
40
Radioligand binding assays. The binding of radiolabeled corticosterone
(CORT) to crude synaptic membranes was initiated by the addition of 100 fil
[3H]CORT (0.75 nM final concentration) to 100 d of the membrane preparation
and 100 til of inhibiting compound or buffer. For equilibrium saturation binding
experiments a range of [3H]CORT concentrations, from 0.03 - 5.0 nM, were
used. The assays were incubated for 2 h at 30°C (21), unless specified
otherwise, and the reactions terminated by rapid filtration and 9 ml rinse with
cold buffer (25 mM Tris, pH =7.45) over Whatman GF-C filters, using a Brandel
harvester (M-24R). Radioactivity bound to the filters was quantified by standard
liquid scintillation spectroscopy. Non-specific binding was defined as that
occurring in the presence of 1 or 10 gM unlabelled CORT. At the concentration
of [3H]CORT used, specific binding was typically 80% of total binding. The data
from saturation and competition experiments were analyzed by nonlinear
regression analysis, using LUNDON I Saturation Analysis Software (Lundon
Software, Cleveland, OH) and EBDA (Elsevier-Biosoft, Cambridge, UK),
respectively.
Kinetic experiments were performed to study the guanine nucleotideinduced dissociation of [3H]CORT from membrane receptors. [3H]CORT (0.75
nM) was allowed to equilibrate with Taricha brain membranes for 2 h as above.
Dissociation was initiated by the addition of 25 IA of 25 /1M unlabelled CORT or
CORT plus 1 mM guanosine 5'-0-(3-thiotriphosphate) (GTP-TS). The estimates
of the kinetic parameters were obtained using a non-linear least squares curve
41
fitting program KINETIC (Elsevier-Biosoft).
All binding experiments were performed in triplicate and were repeated at
least twice with similar results.
Materials. ril]Corticosterone, [1,2,6,7-[3H(N)]-, specific activity 85-88
Ci/mmol, was purchased from New England Nuclear. Unlabeled CORT and
guanine nucleotides were obtained from Sigma.
42
Results
The specific binding of [3H]CORT to Taricha neuronal membranes was
enhanced in a concentration-dependent manner by the addition of MgC12 (Fig.
111.1). The EC50 of Mg' for stimulation of [3H]CORT binding was 0.51 ± .17
mM. This enhancement of binding appeared to be cation specific; NaC1 and
CaC12 did not appreciably alter [3H]CORT specific binding (data not shown). In
subsequent assays, 10 mM MgC12 was included in the assay buffer. Equilibrium
saturation experiments (Fig. 111.2) indicated that [3H]CORT bound to a single
population of high affinity recognition sites with a Kd of 0.14 ± 0.01 nM and a
B. of 183 ± 4 fmol/mg protein. The specific binding of [3H]CORT was
reduced by GTP--yS, a nonhydrolyzable analog of GTP (Fig. 111.2). In the
presence of 100 AM GTP--yS, the saturation isotherm was best fit by a one-site
model yielding estimates of Kd = 0.21 ± 0.02 nM and B. = 140 ± 3 fmol/mg
protein; the increase in Kd was not significant (T = 3.13; P = 0.089).
In titration experiments (Fig. 111.3), the nonhydrolyzable guanyl
nucleotides GTP-7S and guanyl-5'-yl-imidodiphosphate [Gpp(NH)p], inhibited up
to 88% of the specific binding of [3H]CORT in a concentration-dependent
manner; IC50 = 43.2 ± 11 AM and 104 ± 65 AM, respectively. The inhibition
of [3H]CORT binding by GTP-7S was temperature sensitive; GTP--yS was less
potent at 15°C (IC50 = 234 AM), than at 30°C. The rank order potency of
nucleotides to inhibit [31-1] CORT binding was GTP-7S > Gpp(NH)p > > GDP>
43
GMP > > ATP. The potency of GTP (data not shown) was variable between
experiments, but was generally similar to GDP. The limited potency of the
endogenous nucleotide is presumably due to the high level of GTPase activity in
neuronal membranes (Birnbaumer et al., 1990; Leid et al., 1988; Keen et al.,
1991).
As shown in Fig. III.4, the ability of guanine nucleotides to inhibit
[3H]CORT binding was enhanced by MgC12. The efficacies of GTP-7S and
Gpp(NH)p as modulators of [3H]CORT binding were markedly decreased when
MgC12 was excluded from the assay buffer.
To determine the effects of guanyl nucleotides on dissociation kinetics, we
compared the dissociation of [3H1CORT initiated by unlabeled CORT alone with
that initiated by GTP -yS plus CORT (Fig. III.5). Dissociation of [311]CORT
initiated by CORT alone was monophasic, yielding a dissociation rate constant (k
1) of 0.014 ± .0006 mie. In contrast, dissociation of [3H]CORT initiated by the
simultaneous addition of CORT plus GTP-7S was biphasic (Fig. II1.5). The
initial rapid phase of dissociation was described by a k, = 0.515 ± .134 min-1,
in which 22% of [3H]CORT dissociated. The subsequent slow dissociation was
described by a rate constant (1(.1 = 0.015 ± .0004 min-1) similar to the k,
obtained by the addition of CORT alone. The addition of GTP-7S alone induced
a rapid dissociation of approximately 20% of [3H]CORT binding within 2 minutes
(data not shown).
Considering that coupling of membrane receptors to G proteins may be
44
dependent upon the oxidation state of cysteine residues (Fraser, 1989; Boege et
al., 1991), we performed GTP--yS titration experiments in the presence or
absence of 100 iLM dithiothreitol, a disulfide bond reducing agent. The
equilibrium binding of [3H]CORT was slightly decreased, but the potency of
GTP-7S to inhibit [3H]CORT binding was dramatically reduced, when membranes
were homogenized and assayed in buffers containing dithiothreitol (Fig. 111.6).
45
Discussion
Our results provide evidence for a novel molecular mechanism for the
rapid modulation of brain function and behavior by steroid hormones. The data
support the conclusion that signal transduction through the recently described
corticosteroid receptor in neuronal membranes is mediated by guanyl nucleotide-
binding proteins. Of the more than one hundred different G protein-coupled
receptors that have been identified (Simon et al., 1991), none have been steroid
hormone receptors. However, the current findings that ligand binding to the
CORT receptor was negatively modulated by guanyl nucleotides in equilibrium
saturation, titration and kinetic experiments, provides strong evidence that this
receptor is allosterically regulated by heterotrimeric G proteins. Given these
findings, it appears that there are multiple receptor and transduction mechanisms
that steroid hormones may utilize to modulate neuronal activity, including the
regulation of transcription via soluble intracellular receptors, the direct
modulation of ligand-gated ion channels, and the regulation of cellular effector
mechanisms via G protein-coupled receptors.
Since the discovery that GTP regulates the binding of glucagon (Rodbell et
al., 1971), it has become clear that the negative modulation of agonist binding by
guanyl nucleotides is a generalized phenomenon among G protein-coupled
receptors (De Lean et al., 1980; Birnbaumer, 1990). This regulation is believed
to be due to a heterotropic interaction between nucleotide binding to G protein
46
and agonist binding to receptor. In the absence of receptor-bound ligand, G
proteins exist as heterotrimers with GDP bound. The binding of agonist to the
receptor facilitates the formation of an agonist-receptor-G protein complex and
the dissociation of GDP. GTP is exchanged for GDP; this promotes dissociation
of the G protein into a and By subunits which may activate effector molecules,
and dissociation of the receptor from the G protein-receptor complex. When
dissociated from G proteins, the free receptor displays low affinity for agonists
(Birnbaumer et al., 1990; Gilman, 1987). Dissociation of the ligand from the
receptor, and the hydrolysis of bound GTP, allows for continued activation of the
cycle in response to hormone or transmitter.
The results from each of the experiments presented herein are consistent
with the formation of a receptor-G protein complex which is subject to allosteric
regulation by guanyl nucleotides. The binding of [3H]CORT to Taricha brain
membranes was inhibited in a concentration-dependent manner by guanyl
nucleotides. As in most G protein-coupled receptor systems, the nonhydrolyzable
nucleotides had greater efficacy in inhibiting ligand binding than the hydrolyzable
compounds, and the adenosine nucleotide was without effect. Also, GTP-7S
induced a rapid phase of [3H]CORT dissociation from membrane receptors that
was not induced by CORT alone, consistent with a nucleotide-induced shift in the
affinity state of the CORT receptor. We also found that MgCl2 enhanced the
specific binding of [3H]CORT, an effect not induced by CaC12 or NaCl. In many
receptor systems, Mg2+ facilitates formation of the high affinity state of the
47
receptor, presumably by binding to the G protein (Birnbaumer et al., 1990;
Gilman, 1987). Therefore, the increase in binding elicited by Mg' likely
reflects a conversion from the low- to the high- affinity state of the receptor for
CORT. The EC50 value (0.51 mM) for this Mg' effect is similar to the K. of
Mg' for hormone receptor-mediated activation of G, (Iyengar and Birnbaumer,
1982).
The inhibition of ['WORT binding by GTP--yS in saturation isotherms
was described by an apparent decrease in the number of binding sites, rather than
a decrease in affinity. The increase in Kd (from 140 to 210 pM) was modest,
similar to the affinity shift induced in GABAB receptors by GTP (Hill et al.,
1984), for example. However, the rapid dissociation of [3H]CORT binding
induced by GTP -yS suggests a more pronounced affinity shift, at least in a
subpopulation of CORT receptors (Fig. I11.5). In many systems, guanyl
nucleotides induce a 50- to 100-fold decrease in receptor affinity, usually
measured as a decreased ability of agonists to displace radiolabeled antagonist
binding. No antagonists to the CORT receptor are currently available (Orchinik
et al., 1991b), and the highest concentration of [3H]CORT used was 5 nM, which
effected a 97% occupancy of high-affinity recognition sites. Therefore, a 50- to
100-fold shift in affinity of a subpopulation of receptors would not be detectable
under the assay conditions used, but rather would be manifest as a reduction in
the apparent B. Similar decreases in apparent B,,, have been reported in
saturation isotherms studying guanyl nucleotide regulation of [3H]agonist binding
48
to G protein-coupled receptors, such as a- (Rouot et al., 1980) and B-adrenergic
(Williams and Lefkowitz, 1977) receptors.
Many of our observations are consistent with findings in the B-adrenergic
receptor system, where the receptor-G protein interactions have been extensively
characterized (Raymond et al., 1990). The efficacies of guanyl nucleotides to
regulate agonist binding to EDTA-washed membranes were enhanced by MgC12
in both the CORT (Fig. 111.4) and B-adrenergic (O'Donnell et al., 1984) receptor
systems. This phenomenon may be related to the ability of Mg2+ to promote the
formation of the ternary complex (agonist-receptor-G protein), which increases
the guanyl nucleotide sensitivity of [3H]CORT binding. The modulation of
[3H]CORT binding by guanine nucleotides was also temperature-sensitive, in a
manner similar to the B-adrenergic system (O'Donnell et al., 1984). In addition,
treatment of the membranes with 100 /,M dithiothreitol resulted in a slight
reduction of the equilibrium binding of [3H]CORT, but a much greater reduction
in the efficacy of GTP--yS as an inhibitor of [3H]CORT binding. Similarly, in the
B-adrenergic system, the oxidation state of cysteine residues appears to be
important not only for ligand binding, but also in maintaining the receptor in a
conformation required for activation of G proteins (Fraser, 1989; Boege et al.,
1991). It is noteworthy that a 10- to 50-fold higher concentration of dithiothreitol
is routinely included in assays of intracellular adrenal steroid receptors,
suggesting that the steroid recognition sites on the membrane-bound and
intracellular receptors may differ significantly.
49
Little is known about the interaction of small, hydrophobic ligands, such
as steroids, with membrane-bound receptors. It has been suggested that these
compounds would be unlikely to bind to receptor proteins in the lipid milieu of
plasma membranes. However, agonist binding has been characterized (Devane et
al., 1988), an effector mechanism determined (Howlett et al., 1986), and a
receptor cloned and expressed (Matsuda et al., 1990) for at least one other class
of lipophilic compounds, the cannabinoids. Although cannabinoids do not inhibit
j3H-JCORT binding (data not shown), there are similarities in the ligand-receptor
interactions between the CORT and cannabinoid receptors systems. Equilibrium
saturation studies in both receptor systems indicate that ligands bind with high
specificity to a single population of recognition sites with subnanomolar affinity
(Orchinik et al., 1991a; Devane et al., 1988). Ligand binding to both the CORT
and cannabinoid receptors was enhanced by MgC12, while guanyl nucleotides
inhibited binding and induced a rapid dissociation of ligand from the receptors.
The maximal inhibition of cannabinoid binding by guanyl nucleotides reported
was also less than 100% (Devane et al., 1988), but the deduced molecular
structure of the receptor indicates clearly that it belongs to the family of
membrane-spanning receptors that couple to G proteins (Matsuda et al., 1990).
The sensitivity of agonist binding to inhibition by nucleotides in different receptor
systems may depend on the tightness of coupling between the receptor and G
protein, the membrane preparation, cation and ligand concentration, and the
relative abilities of various agonists to discriminate different affinity states of the
50
receptor.
There is precedent for the hypothesis that a membrane-bound steroid
receptor may be coupled to G proteins. Progesterone induces maturation of
Xenopus oocytes through mechanisms that are independent of classical
intracellular receptors (Godeau et al., 1978), and progesterone binding sites on
oocyte membranes have been described (Blondeau and Baulieu, 1984; Sadler and
Mailer, 1982). While progesterone inhibits cholera toxin-stimulated adenylate
cyclase activity in oocyte membranes (Finidori-Lepicard et al., 1981), the
inhibition of adenylate cyclase activity is not mediated by the pertussis toxin-
sensitive G, (Sadler et al., 1984). Therefore, the mechanism of progesterone
action in oocyte membranes cannot easily be explained by the conventional model
of receptor-G protein interaction (Smith, 1989). However, the demonstration that
the CORT receptor in Taricha neuronal membranes is sensitive to negative
modulation by guanyl nucleotides is entirely consistent with the coupling of a
steroid receptor to heterotrimeric G proteins in the brain.
The latency of electrophysiological responses to CORT is consistent with
the time course of a receptor-G protein-effector mechanism of action. The
excitability of Taricha hindbrain neurons is altered within 2 min of topical CORT
application, and the response to CORT is reversed within 2-5 minutes following
removal of CORT (Rose et al., 1991). Some neurons in the rat brain respond to
corticosteroid administration with the same latency (7), which also would be
consistent with a steroid receptor-G protein interaction. It is reasonable to
51
speculate that a membrane receptor for estradiol also couples to G proteins,
inasmuch as estradiol depolarizes some neurons in the rat hypothalamus within 35 min of application, through a decrease in IC+ conductance that is apparently
mediated by cAMP (Nabekura et al., 1986; Minami et al., 1990). Therefore, in
the mammalian brain as well, it is likely that some steroid hormones may effect
rapid alterations in brain function through the use of receptor and transduction
mechanisms similar those described in Taricha brains.
52
Fig. BI.1. The effect of MgC12 on the specific binding of [3H]CORT to neuronal
membranes. The membrane preparation was incubated with 0.75 nM [3H]CORT
and increasing concentrations of MgC12. The addition of MgC12 enhanced
E3H-JCORT binding with ECso = 0.51 ± 0.17 mM. The hyperbolic fit shown
was derived using least squares regression analysis with Graph Pad-In Plot (San
Diego, CA).
50
40
30
4-1
c
a)
u
20
L._
10
0
0
5
10
15
20
25
MgCl2 Concentration (mM)
Fig. 111.1
30
35
54
Fig. 111.2. Equilibrium saturation binding of [3H]CORT to neuronal membranes
in the presence or absence of guanyl nucleotide. Neuronal membranes were
incubated with increasing concentrations of [3H]CORT. Nonspecific binding
(open squares) was defined as that occurring in the presence of 1 or 10 AM
unlabeled CORT. Specific binding in the absence of guanyl nucleotide (open
circles) was described by Kd = 0.14 ± 0.01 nM and B,n,o, = 183 ± 4 fmol/mg
protein (Hill coefficient = 1.08). In the presence of 100 AM GTP--yS (filled
circles), the saturation isotherm was described by Kd = 0.21 ± 0.02 nM and B.
= 140 ± 3 fmol/mg protein (Hill coefficient = 1.12). In both cases, the data
were best fit by a one-site model using Lundon I software and LIGAND software
(Elsevier-Biosoft, Cambridge, UK). Inset, linear Scatchard-Rosenthal replot of
saturation data.
1,0 -
0,8
0.6
30
90
60
120
160
1.3
0,4
1,0
0.8
0.5
0.2
0.3
0.0
Bound (fmol/mg protein)
0,0
0
3
2
1
3
[ H]CORT Concentration (nM)
Fig. 111.2
I
I
4
5
180
56
Fig. B11.3. Guanyl nucleotides inhibit the specific binding of [3H]CORT in
Taricha brain membranes. Membranes were incubated with 0.75 nM CHICORT
and increasing concentrations of the nucleotides shown. The titration curves were
replicated three times with similar results. IC50 estimates ± standard error were
obtained for GTP-yS (43.2 ± 11 tkM) and Gpp(NH)p (104 ± 65 AM) using
EBDA software. The inhibition of [31T]CORT binding by GTP (not shown)
varied between experiments, with maximal inhibition ranging from 20% to 35%.
i
i
I
1
100
Q)
C
80
1-6
C
En
U
FE..3
a)
ca.
,--,
0
L.
c
0
C)
0
4-)
}--
4)
0
C.)
60
C
(-)
0
40
ATP
7 GDP
(1)
a...,
GMP
t,)rZ
0 Gpp(NH)p
0 GTPyS
0
/
1
7
1
1
6
5
1
4
log Nucleotide Concentration (Molar)
Fig. 111.3
1
3
58
Fig. 111.4. Effect of MgC12 on the inhibition of [3H]CORT binding by guanyl
nucleotides. Membranes were incubated with 0.75 nM rHiCORT and increasing
concentrations of nucleotide, either without MgC12 added, or with 10 mM MgC12
added. The efficacy of both GTP--yS (left panel) and Gpp(NH)p (right panel) as
inhibitors of [3WORT binding was greatly enhanced by MgC12.
100
100
cr)
if)
80
80
F co 60
a) 0
a
60
C0.-
o
-,
(i)
0
-4-ac
40
40
C_)
r1-1
L-J
Gpp(NH)p without MgCl2
Gpp(NH)p with MgCl2
0 GTP-yS without MgCl2
20
GTP-yS with MgCl2
0
0
//
20
1
-6
-5
-4
-3
-6
-5
log Nucleotide Concentration (Molar)
Fig. 111.4
-4
-3
0
60
Fig. 111.5. Dissociation of specifically bound [3H]CORT from Taricha neuronal
membranes. [3H]CORT (0.75 nM) was allowed to equilibrate with membranes
for 2 hrs, at which time dissociation was initiated by the addition of 25 Al of
unlabelled CORT or CORT plus 1 mM GTP -1S. For CORT alone, k1 was
determined to be 0.014 ± .0006 min-1. The addition of GTP--yS plus CORT
resulted in biphasic dissociation rates, with 22% of [H]CORT dissociating with
k.1 = 0.515 ± .134 min-1, and 78% dissociating with k1 = 0.015 ± .0004 min-1,
as determined by KINETIC software.
1,2
E
a
1.0
-o
0,8
(.)
14-::
0.6
a)
a
O
0,4
V)
0.2
0
10
20
30
40
50
Time (minutes)
Fig. 111.5
60
70
80
62
Fig. 111.6. Effect of disulfide bond reducing agent on the sensitivity of
[3H]CORT binding to negative modulation by GTP-7S. Brains were
homogenized, and membranes prepared and assayed as in Fig. 111.3., or with 100
/AM dithiothreitol added to each buffer. In the presence of dithiothreitol, the
equilibrium binding of [3H]CORT was reduced by 13%. The highest
concentration of GTP--yS inhibited the specific binding of [3H]CORT by 88% in
control membranes, while GTP-7S inhibited only 32% of rITICORT specific
binding to membranes in the presence of dithiothreitol.
200
i
.
I
01
C
'-6
c
150
E5
0
F0
a)
a
cn
100
H
0U
E
=1
re=
L--1
O Control
e
50
'
0
//
+100p,M Dithiothreitol
L
,
-6
1
-5
-4
log GTPyS Concentration (Molar)
Fig. 111.6
1
64
Acknowledgements
The authors are grateful to James D. Rose for allowing us to cite
unpublished data. Research was supported by grants from the National Science
Foundation, BNS-8901500 (F.L.M. and M.O.) and BNS-8909173 (F.L.M.).
M.O. was supported, in part, by a National Science Foundation Graduate
Fellowship.
CHAPTER W. STEROID MODULATION OF GABAA
RECEPTORS IN AN AMPHIBIAN BRAIN
Miles Orchinik, Thomas F. Murray' and Frank L. Moore
Department of Zoology and 'College of Pharmacy, Oregon State University.
Corvallis, OR 97331
65
66
Abstract
Compelling evidence indicates that certain steroids are potent modulators
of the GABAA receptor /Cl-channel complex in the rat brain. The 3ahydroxylated ring A reduced metabolites of progesterone and deoxycorticosterone
are released in response to stress and alter ligand binding to GABAA receptors
and potentiate GABA-stimulated Ct flux. However, the physiological relevance
of this nongenomically-mediated mechanism of steroid action is still unclear. We
performed radioligand binding studies, using an amphibian, Taricha granulosa, to
determine if steroid modulation of GABAA receptors has been highly conserved
during vertebrate evolution. The binding parameters for the convulsant t-butyl
bicyclophosphorothionate ([35S]TBPS) and the benzodiazepine [3H]flunitrazepam
were similar in Taricha and mammalian brains. The allosteric regulation of
[35S]TBPS
and [3H]flunitrazepam binding by GABA were also similar between
species. We also found that the rank order and absolute potencies of steroids to
inhibit [35S]TBPS binding and enhance [3H]flunitrazepam binding to GABAA
receptors were similar in Taricha and rat brains. In autoradiographic studies,
[3H]flunitrazepam binding sites were more uniformly distributed than [35SJTBPS
binding sites, but [35S]TBPS binding sites in all brain regions were inhibited by
3a-OH-5a-pregnan-20-one. As in mammalian studies, corticosterone was far less
potent in inhibiting [35S]TBPS binding or enhancing [3H]flunitrazepam binding.
Corticosterone also had no effect on GABA-stimulated Cl- uptake into Taricha
67
synaptoneurosomes. Therefore, the recently described corticosteroid receptor in
Taricha neuronal membranes does not appear to be associated with the GABAA
receptor. However, since GABAA receptors are modulated by steroids in a very
similar manner in the brains of mammals and a "primitive" vertebrate, this
mechanism appears to have been highly conserved and therefore is likely to serve
important roles in regulating vertebrate brain function.
68
Introduction
Seyle discovered 50 years ago that certain steroids have anti-convulsant
and anesthetic properties (Seyle, 1941, 1942) in addition to their classical
endocrine actions. More recent evidence indicates that a number of endogenous,
neuroactive steroids can alter neuronal excitability, neurosecretion, neurochemical
receptors, or behavior within seconds to minutes of administration (reviews:
Duval, 1983; Schumacher, 1990; Mc Ewen, 1991; Chadwick and Widdows, 1990;
Costa and Paul, 1991; Moore and Orchinik, 1991; Paul and Purdy, 1992). In
contrast to the classical actions of steroid hormones that are mediated through
intracellular receptor-directed changes in protein synthesis (Yamamoto, 1985;
Evans, 1988; Carson-Jurica et al., 1990), the actions of neuroactive steroids are
presumed to occur through interaction with specific recognition sites on neuronal
membranes.
The best characterized neuroactive steroids are the 3a-hydroxylated ring A
reduced metabolites of progesterone and deoxycorticosterone, 3a-OH-5a-pregnan-
20-one (3a-DHP) and 3a,21-0H-5a-pregnan-20-one (5a-THDOC), respectively
(reviews: Lambert et al., 1987; Gee, 1988; Schumacher and Mc Ewen, 1989;
Harrison et al., 1989; Lan et al., 1991; Paul and Purdy, 1992). Compelling
evidence indicates that these steroids alter neuronal excitability through direct
modulation of the GABAA (gamma-aminobutyric acid)/benzodiazepine receptor-
chloride ionophore complex (Harrison and Simmonds, 1987; Majewska et al.,
69
1986; Callachan, 1987; Morrow et al., 1987; Gee et al., 1988; Peters et al.,
1988; Turner et al., 1989; Lan et al., 1990; Im et al., 1990). These naturally
occurring steroids are among the most potent GABAA receptor ligands known,
capable of enhancing GABA-activated C1 conductance in nanomolar
concentrations. Sensitivity to steroids in transiently expressed GABAA receptor
subunits (Puia et al., 1990; Lan et al., 1990; Shingai et at , 1991; Hill - Yenning
et al., 1991; Woodward et al., 1992) strongly suggests that steroid recognition
sites reside on GABAA receptors, however these steroid recognition sites have not
been characterized in radioligand binding studies.
In the rat, concentrations of 3a-DHP and 5a-THDOC in the plasma and
brain increase significantly in response to stress (Purdy et al., 1991). When
administered, these steroids possess hypnotic and anxiolytic properties similar to
the benzodiazepines and barbiturates (Crawley et al., 1986; Kavaliers and Wiebe,
1987; Mendelson et al., 1987; Bitran et al., 1991; Wieland et al., 1991). This
has led researchers to speculate that these steroids may be endogenous ligands for
GABAA receptors, mediating anxiolytic or sedative processes in response to stress
(Paul and Purdy, 1992). But thus far, a physiological role for endogenous
GABAA-active steroids has not been established (Delville et al., 1991; Costa and
Paul, 1991). If steroid modulation of GABAA receptors has been highly
conserved through vertebrate evolution, it would strengthen the conclusion that
this mechanism of action for neuroactive steroids is likely to subserve important
physiological roles. Therefore, we investigated the possibility that GABAA
70
receptors are modulated by 3a-hydroxylated, ring A reduced pregnane steroids in
a "primitive", non-mammalian vertebrate brain.
As a second point of interest, male reproductive behavior is suppressed by
stress or injection of corticosterone in an amphibian, Taricha granulosa (Moore
and Miller, 1984; Moore and Orchinik, 1991). The response to corticosterone is
rapid; behavioral changes are apparent within 8 minutes of intraperitoneal
injection (Orchinik et al., 1991a), and the excitability of hindbrain neurons is
altered within 2 minutes of corticosterone application (Rose et al., 1991). Since
the behavioral response to corticosterone (CORT) is blocked by the GABAA
receptor antagonist bicuculline (Boyd and Moore, 1990), this response might be
mediated through a steroid recognition site on GABAA receptors. We have
previously characterized a high-affinity corticosteroid receptor in neuronal
membranes (Orchinik et al., 1991a; Orchinik et al., 1992); in this current study
we examine the hypothesis that the membrane-bound corticosteroid recognition
site is associated with the GABAA receptor complex.
Our results indicate that steroids modulate GABAA receptors in a primitive
vertebrate brain in a manner very similar to mammalian brains. However, the
pharmacological profile of the steroid recognition sites on GABAA receptors is
distinct from the biochemical and behavioral profile of the corticosterone
receptors in neuronal membranes.
71
Materials and Methods
Animals. Adult male Taricha granulosa (rough-skinned newts), mean
weight 20 g, were collected from local ponds (Benton County, OR) and
maintained in a laboratory tank in dechlorinated water at 17°C with
approximately natural photoperiod. Animals were maintained and sacrificed in
accordance with I.A.C.U.C. guidelines.
Radioligand Binding Assays. Neuronal (P2) membranes were prepared
from whole Taricha brains (Orchinik et al., 1991a). Briefly, P2 pellets were
subjected to 2 freeze/thaw cycles and a 4 hour preincubation in a large volume of
cold, hypoosmotic buffer to remove endogenous GABA and steroids. The well
washed P2 pellet was resuspended in assay buffer (25 mM HEPES, 0.5 mM
EDTA salt, 200 mM NaC1, pH=7.45) to a protein concentration of 400-500
µg /ml. The binding of radiolabeled t-butylbicyclophosphorothionate (TBPS) or
the central benzodiazepine ligand, flunitrazepam, to neuronal membranes was
initiated by the addition of 100 Al [3H]flunitrazepam or [35S]TBPS to 100 Al of the
P2 membrane preparation and 100 Al of steroid, GABA, competing compound or
buffer. Steroids were added to the membranes 15 minutes prior to the addition of
radiolabeled compound. The membranes were incubated for 2 hours at 15°C
(both radioligands reach equilibrium within 2 hours) and the reactions were
terminated by rapid filtration and 9 ml rinse with ice cold buffer (25 mM Tris,
200 mM NaCl, pH=7.45) over Whatman GF-C filters, using a Brandel harvester
72
(M-24R). Filters were soaked in 0.5% polyethylenimine prior to use in [35S]TBPS binding studies. Radioactivity bound to the filters was quantified by
standard liquid scintillation spectroscopy using 7 ml of Ecolume (ICN
Biomedicals Inc) as scintillant. Non-specific binding was defined as that
occurring in the presence of 100 /AM picrotoxin ([35S]TBPS) or 10 gM diazepam
([3H]flunitrazepam). Protein concentration was determined by the Bio-Rad
protein microassay method (Bio-Rad, Richmond CA), using bovine serum
albumin as standard. For each experiment, the binding curves were performed in
triplicate and the experiments were repeated at least twice, with similar results.
Estimates of Kd and B. were obtained by non-linear regression analysis
using LUNDON I Saturation Analysis Software (Lundon Software, Cleveland,
OH) or LIGAND (Elsevier-Biosoft, Cambridge, UK). EC50 and IC50 estimates
were obtained by least squares regression analysis using EBDA (Elsevier-Biosoft,
Cambridge, UK) or Graph Pad-In Plot (San Diego, CA). Hyperbolic and
sigmoidal curves were fit using Graph Pad-In Plot.
Measurement of 36c1 Uptake. Synaptoneurosomes were prepared from
whole Taricha brains as described (Schwartz et al., 1985) with minor
modifications. Freshly removed brains were homogenized in 10 volumes (v/w)
cold buffer (20 mM HEPES, 145 mM NaCl, 5 mM KC1, 1 mM MgC12, 1 mM
CaC12, 10 mM D-glucose; pH 7.4) using a glass Dounce (8 strokes) and filtered
through 2 layers of nylon mesh (pore size: 160 gm). The filtrate was washed
with 30 volumes of buffer and centrifuged (1000 x g, 15 min, 4°C) twice. The
73
final pellet was resuspended in 2.5 volumes (vol/original weight) buffer to a
protein concentration of approximately 5 mg/ml.
36a- uptake was determined as described by Allan and Harris (1986) with
minor modifications. Aliquots of the membranes (150 gl) were pre-incubated at
22°C for 10 min. Uptake was initiated by the addition of 0.1 gCi 36a- (150 gl)
with or without GABA, and gently vortexing. Bicuculline methiodide,
'
picrotoxin, or steroids were added 10 min prior to the addition of Cl. Influx
was terminated by the addition of 3 ml cold buffer containing 100 gM picrotoxin,
followed by rapid filtration under partial vacuum over glass-fiber filters on a
single manifold harvester, and additional wash with 6 ml cold buffer. Whatman
GF-C filters were presoaked in 0.1% bovine serum albumin. The radioactivity
bound to filters was quantified by standard liquid scintillation spectrometry. The
amount of 36a- bound to filters in the absence of tissue was subtracted from the
total binding in of each tubes. GABA-stimulated 36C1- influx was determined to
be maximal between 5 - 15 seconds at 22°C, so incubations were carried out for
12 seconds. All determinations were performed in triplicate.
Receptor Autoradiography. Thaw mounted 25-gm brain sections, cut in
the transverse plane, were dried in a desiccator overnight under partial vacuum at
-5°C and stored at -80°C until use. To determine the distribution of r5SUBPS
binding sites, slides were pre-incubated for 30 min in cold assay buffer (25 mM
HEPES, 200 mM NaC1, 0.5 mM EDTA; pH = 7.45), then incubated for 2 hours
with 15 nM [35S]TBPS at 15°C. Nonspecific binding was determined in alternate
74
sections by the addition of 200 izM picrotoxin. To assess potential regional
differences in sensitivity of [35S]TBPS binding to modulation by steroids, sections
were incubated with 15 nM [35S]TBPS and 10 AM 3a-DHP or 100 AM CORT.
To visualize benzodiazepine binding sites, sections were incubated with 5 nM
[3H]flunitrazepam (2 hours, 15°C), in the presence or absence of 20 /.4M
diazepam to determine nonspecific binding. For both radioligands, the slides
were rinsed twice (2 x 1-min) in ice cold buffer, dipped in ice cold dH2O, and
dried under cool air. [35S]TBPS treated slides were apposed to XAR-2 X-OMAT
film (Kodak) and [3H]flunitrazepam treated slides apposed to Ultrafilm VHF
sensitive film (Reichert-Jung) for 3 wks. Sections were then stained for
histology. The autoradiograms were analyzed by computer-assisted densitometry
(DUMAS). Specific binding was determined by subtracting nonspecific binding
from the total binding sections. Due to the difficulties in making comparisons
between GABAA receptor ligands with differing optimal binding conditions, the
results are presented in a semi-quantitative form reflecting the relative distribution
of binding sites for each of the ligands. The mean optical density values for
specific binding in each region were grouped into one of 4 ranks: + Detectable;
+ + Low density; + + + Moderate density; + + + + High density.
Materials. [311]Flunitrazepam (103.1 Ci/mmol) and [35S]TBPS (87.5
111.5 Ci/mmol) and 36C1- (14.97 mCi/g) were obtained from New England
Nuclear (Boston, MA). Steroids were obtained from Steraloids (Wilton, NH) or
Sigma (St. Louis, MO). Other chemicals were obtained from Sigma or Research
75
Biochemicals Incorporated (Natick, MA).
76
Results
The convulsant [35S]TBPS, bound to a single population of recognition
sites in Taricha neuronal membranes (Fig. IV. lA and B) with a Kd = 17.1 ± 1.3
nM and B. = 901 ± 32 fmol/mg protein (Hill coefficient = 0.998). The
binding of [35S]TBPS was inhibited in a concentration-dependent manner by
GABA (IC50 = 1.41 ± 0.12 ilM; Fig. D/.2). In most experiments, bicuculline
methiodide enhanced [35S]TBPS binding by approximately 10%, relative to
control tubes (data not shown), presumably due to residual endogenous GABA
despite the extensive washing in tissue preparation.
The binding of 2.5 nM [35S]TBPS was potently inhibited by the steroids
3a-DHP and 5a-THDOC in titration experiments (Fig. IV.3; Table IV.1). When
GABA was excluded from the incubation buffer, the potency of 3a-DHP to
inhibit [35S]TBPS binding was decreased more than 3-fold (IC50 = 47.2 ± 9.8
nM). In contrast, 100 AM CORT inhibited only a fraction of [35S]TBPS specific
binding, in the presence or absence of GABA. CORT was devoid of inhibitory
activity in the presence of bicuculline.
The equilibrium saturation binding of [3H]flunitrazepam to Taricha
neuronal membranes (Fig. IV.4) was described by a one site model with Kd
4.03 ± 0.55 nM and B. = 2830 ± 83 fmol/mg protein (Hill coefficient =
0.965). The binding of [3H]flunitrazepam was enhanced by GABA (EC50 = 1.95
± 0.15 gM) and by the barbiturate sodium pentobarbital (EC50 = 228 ± 35 ihM;
77
Fig. IV.5).
The steroids 3a-DHP and 5a -THDOC enhanced [3H]flunitrazepam binding
(Fig. IV.6) with EC50 values = 116 ± 50 nM and 225 ± 96 nM, respectively.
In contrast, CORT had little or no effect on basal (Fig. IV.6) or barbituratestimulated [3H]flunitrazepam binding (data not shown). The addition of 3µM
GABA did not enhance the potencies of either 3a-DHP or CORT as modulators
of [3H]flunitrazepam binding.
GABA stimulated 36C1 flux into Taricha brain synaptoneurosomes (Fig.
IV.7), with an estimated EC50 = 5.8 ± 2.5 AM, similar to the EC50 for GABAmediated inhibition of [35S]TBPS binding. The basal level of 36a- uptake was
62.9 nmol/mg protein/12 seconds. Of this, 10.9 nmol/mg protein/12 seconds was
blocked by the addition of 200 AM picrotoxin or bicuculline methiodide, GABAA
receptor antagonists. The addition of CORT (1 nM
1 AM) produced no
discernable effect on either GABA-stimulated or GABA-independent 36C1- influx
(Fig. IV.8). Because of the large quantity of tissue required for 36a- flux
studies, dose response curves for other steroids were not performed.
To determine if neuroanatomical differences in distribution might underlie
the differences in the densities of GABAA receptors defined with [35S]TBPS
versus [3H]flunitrazepam in membrane preparations, and to detect potential
differences in the regional sensitivity to steroids, we performed receptor
autoradiography (Fig. IV.9). [35S]TBPS and [3H]flunitrazepam binding sites were
found in all brain regions, but [3H]flunitrazepam binding sites were more
78
uniformly distributed than the [35S]TBPS binding sites (Table IV.2). Convulsant
sites were most enriched in the midbrain and dorsal thalamus, while moderate to
high densities of benzodiazepine binding sites were found throughout the
telencephalon, midbrain and hindbrain. The greatest discrepancy between TBPS
and benzodiazepine binding sites was found in the telencephalon, where
[35S]TBPS binding sites were sparse, but [311]flunitrazepam sites were abundant.
Receptor autoradiography indicated that [35S]TBPS binding was inhibited
in every brain region by 10 AM 3a-DHP. There were no marked regional
differences in the sensitivity of [35S]TBPS binding to steroid; 3a-DHP inhibited
between 80 92% of [35S]TBPS specific binding in each of the 13 brain regions
examined. There were no brain regions with any apparent sensitivity of
[35S]TBPS
binding to modulation by CORT. However, in contrast to 3a-DHP,
100 AM CORT inhibited less than 10% of [35S]TBPS specific binding in every
brain region examined.
79
Discussion
GABAA receptors in Taricha brains are allosterically modulated by
ligands, including steroids, in a manner very similar to that described for the rat
brain. The convulsant [35S]TBPS bound with an affinity (Kd = 17 nM) similar to
the Kd reported for dialyzed mammalian brain membranes (Squires et al., 1983).
As in the rat brain, TBPS binding was inhibited by low micromolar
concentrations of GABA. The benzodiazepine flunitrazepam also bound to
Taricha brain membranes with an affinity (Kd = 4 nM) similar to the rat brain
(Stephenson, 1988; Knapp et al., 1990), and as in mammals, the binding of
[3H]flunitrazepam was enhanced by both GABA and barbiturates. Significantly,
both [35S]TBPS and [3H]flunitrazepam binding were potently modulated by 3a-
hydroxylated 5a-reduced steroids, as in the rat brain (Majewska et al., 1986) and
the potency of steroids to inhibit [35S]TBPS binding was enhanced by GABA (Gee
et al., 1987). The rank order and absolute potencies of a series of steroids to
modulate [35S]TBPS binding were in similar in Taricha and rat brains (Table
IV.1). These studies suggest that the recognition sites for steroids on GABAA
receptors have been highly conserved through vertebrate evolution and thus
portend physiologically important functions.
This conclusion is supported by the findings that 3a-hydroxylated reduced
pregnane steroids produce anesthesia in amphibians (Mok and Krieger, 1990),
and indeed, in all vertebrate groups (Oliver et al., 1991). This steroid-induced
80
anesthesia appears to be mediated by GABAA receptors, inasmuch as picrotoxin
antagonizes the anesthetic effect in fish (Oliver et al., 1991). Interestingly, nonvertebrate chordates and many invertebrate phyla possess GABAA-like receptors
that are insensitive to pregnanolone (3a-OH-5B-pregnane-20-one)-induced
anesthesia, suggesting that the steroid recognition site evolved early in chordate
evolution (Oliver et al., 1991). The benzodiazepine binding site on the GABAA
receptor appears to have evolved later phylogenetically than the steroid site
(Nielsen et al., 1978; Hebebrand et al., 1987).
The physiological and behavioral relevance of steroid modulation of
GABAA receptors in Taricha is as yet unknown. However, significant
concentrations of 3a-DHP accumulate in amphibian brains following injection of
the parent compound progesterone (Mok and Krieger, 1990), indicating that the
required converting enzymes are present in amphibians as well as mammals.
Preliminary studies suggest that, as in mammals, the 3a-hydroxylated ring Areduced steroids may modulate some behavioral responses to stress in Taricha
(Orchinik and Moore, 1989). But in contrast to corticosterone, which suppresses
male behavior during stress, injection of 5a-THDOC can produce a short-latency
facilitation of reproductive behavior in mildly stressed males (Orchinik and
Moore, 1989).
A second major objective was to determine whether there is a high-affinity
interaction of corticosterone with GABAA receptors. Most studies have found
that the 3a-hydroxylated, ring A-reduced structure is required for high-affinity
81
modulation of ligand binding or GABA-evoked
conductance (Harrison et al.,
1987; Gee et al., 1988; Morrow et al., 1990; Purdy et al., 1990; Im et al., 1990;
Lan et al., 1991). However, there have been reports showing a modest
corticosterone-induced enhancement of [35S]TBPS binding in the absence of
GABA (Sutanto et al., 1989), as well as biphasic effects of corticosterone on
[35S]TBPS
binding (Majewska, 1987) and GABA-induced contractions of guinea
pig ileum (Ong et al., 1987). Also, species differences might exist in the
structural requirements for steroid interaction with the GABAA receptor.
Our results indicated that corticosterone had very limited efficacy as a
modulator of GABAA receptor function, as in mammalian brains. In the
concentration range of 0.1 nM - 100 AM, with or without GABA, we detected no
significant effects of corticosterone on [35S]TBPS binding, [3H]flunitrazepam
binding, or GABA-stimulated C1 flux in brain membranes. Corticosterone did
not inhibit [35S]TBPS binding to a regionally specific subpopultion of GABAA
receptors in autoradiograms, including the hindbrain where populations of
corticosterone-sensitive neurons are found (Rose et al., 1991).
Receptor autoradiographic studies also found that the distributions of
[35S]TBPS
or [3H]flunitrazepam binding sites did not correspond to the
distribution of membrane-bound [3H]corticosterone receptors (Orchinik et al.,
1991a). In contrast to the GABAA receptor ligands, [3H]corticosterone binding
sites were found in high density in the neuropil surrounding the preoptic area,
hypothalamus, and amygdala.
82
Further, the behavioral responses to injection of GABAA-active steroids
(intraperitoneal and intracerebroventricular) differed from the responses to
corticosterone. In addition, none of the steroidal and nonsteroidal GABAA
receptor ligands tested were potent competitors for [3H]CORT binding sites
(Orchinik et al., 1991a; Orchinik et al., 1991b). Given the potency of
corticosterone in behavioral and electrophysiological studies, the high-affinity
binding of CHICORT to neuronal membranes (Kd < 0.5 nM; Orchinik et al.,
1991a; Orchinik et al., 1992), and the limited potency of corticosterone to
modulate any of the parameters of GABAA receptor function we assayed, we
conclude that the rapid behavioral responses to corticosterone are not due to
direct modulation of GABAA receptors. This conclusion is further supported by
our recent findings that [3H]corticosterone binding to neuronal membranes is
sensitive to modulation by guanyl nucleotides--strong evidence that the
corticosteroid receptor on neuronal membranes is coupled to G proteins (Orchinik
et al., 1992).
Numerous discrepancies in correspondence between the distribution of
GABAA receptors labeled with the convulsant TBPS, benzodiazepines and ligands
specific for the GABA binding site have been reported (Unnerstall et al., 1981;
McCabe and Wamsley, 1986; Wamsley et al., 1986; Schmitz et al., 1988; Olsen
et al., 1990; Burt and Kamatchi, 1991). In well-washed Taricha brain
membranes, the density of [3H]flunitrazepam binding sites was 3-fold higher than
[35S]TBPS
binding sites (B. = 2830 fmol/mg protein vs. 901 fmol/mg protein,
83
respectively). In autoradiograms, [3H]flunitrazepam binding sites were rather
uniformly distributed in Taricha brains, whereas [35S]TBPS sites had a restricted
distribution, being highly concentrated in the midbrain and barely detectable in
much of the telencephalon.
This lack of correspondence could result, in part, from regional variation
in endogenous GABA levels, inasmuch as GABA enhances benzodiazepine
binding but inhibits TBPS binding. However, there is a widespread distribution
of GABAergic neurons and fibers in the brain of a closely related urodele
amphibian, Triturus cristatus (Franzoni and Morino, 1989). The distribution of
GABA immunoreactivity largely parallels the distribution of [3H]flunitrazepam
binding sites in Taricha brains: intensive GABA immunoreactivity in the olfactory
bulb, dorsal and medial pallium, striatum, dorsal thalamus, tectum, midbrain
tegmentum, cerebellum and the lateral acoustic area -all areas with moderate to
heavy [3H]flunitrazepam binding. Given the widespread distribution of GABA in
the urodele brain, it is difficult to attribute the low density of [35S]TBPS binding
sites in the telencephalon relative to the midbrain to inhibition by GABA alone.
In another primitive vertebrate, the eel, [3H]flunitrazepam and [35S]TBPS binding
sites are also differentially distributed, but [35S]TBPS binding sites are more
abundant and more evenly distributed, while benzodiazepine receptors are
enriched in the midbrain (Corda et al., 1989).
A more likely explanation is that the differences in distribution reflect
GABA, receptor heterogeneity, a phenomenon that has been documented in
84
mammals (reviews: Sieghart, 1989; Seeburg et al., 1990; Burt and Kamatchi,
1991; Olsen and Tobin, 1990) and non-mammalian vertebrates (Schmitz et al.,
1988). [35S]TBPS and [3H]flunitrazepam probably label different subpopulations
of GABAA receptors, although amphibians lack the full complement of GABAA
receptor isoforms found in mammals. A single receptor subunit is photoaffinity
labeled with [3H]flunitrazepam in many fish and amphibians, whereas 2 bands are
labeled in reptiles and birds, and multiple receptor subunits are labeled in some
mammalian species (Hebebrand et al., 1987; Deng et al., 1991). This increasing
diversity of flunitrazepam-binding molecules probably reflects the appearance of
multiple forms of the a subunit (Burt and Kamatchi, 1991).
If steroid sensitivity resides on the a-subunit of GABAA receptors (Lan et
al., 1991), and amphibians possess few a-subunit subtypes, one might expect few
regional differences in the steroid sensitivity of GABAA receptors in Taricha
brains. The relatively uniform inhibition [35S]TBPS binding by 3a-OH-DHP in
autoradiograms suggests that all the TBPS binding sites are functionally coupled
to steroid recognition sites in Taricha brains. Similarly, 3a-DHP inhibited
[35S]TBPS binding in autoradiographic sections of rat brains, although modest
differences in potency existed between brain regions (Gee et al., 1988).
However, a more recent study found striking differences in the potency of 3ahydroxylated ring A-reduced steroids (especially 5B-THDOC) to inhibit [35S]TBPS
binding in the frontal cortex versus the spinal cord of rats, as well as a different
rank order potency of steroids in the two regions (Gee and Lan, 1991). This
85
favors the existence of a heterogeneous population of steroid recognition sites
coupled to GABAA receptors. At present, it is not clear whether this
heterogeneity reflects a multiplicity of steroid recognition sites on a single
GABAA receptor, multiple affinity states of the receptor, or different populations
of GABAA receptors having differing sensitivities to steroids (Morrow et al.,
1990; Lan at al, 1990; Puia et al., 1990; Shingai et al., 1991; Vincini et al.,
1991; Woodward et al., 1992). There were no striking differences in the efficacy
of either 3a-DHP or corticosterone to inhibit [35SUBPS binding from
telencephalon to the caudal hindbrain to suggest the presence of multiple steroid
recognition sites on Taricha GABAA receptors. More detailed studies are
required to determine whether a heterogenous population of steroid recognition
sites resides on GABAA receptors in Taricha brains.
In conclusion, the allosteric modulation of GABAA receptor function by
steroids appears to be an ancient characteristic of vertebrate brains. Our studies
demonstrated that steroid modulation of GABAA receptors in Taricha brains is
remarkably similar to that in mammalian brains. This suggests that the
recognition sites for steroids on GABAA receptors have been highly conserved
during vertebrate evolution, making it likely that this mechanism subserves
important physiological roles. However, the pharmacological profiles for the
GABAA receptor and the high-affinity corticosteroid receptor in neuronal
membranes are inconsistent, suggesting that neuroactive steroids utilize multiple
non-genomic mechanisms to regulate brain function.
86
Thus, it appears that steroids can modulate neuronal activity through
intracellular receptors that regulate long-term transcriptional events, G proteincoupled receptors that may regulate second messenger pathways, and direct
modulation of GABAA receptor function.
87
Table IVA. Potencies of steroids as inhibitors of [35S]TBPS binding. Brain
membranes were incubated with 2.5 nM [35S]TBPS, 1µM GABA, and increasing
concentrations of steroids. Steroids were dissolved in a concentrated stock
solution of ethanol or ethanol:DMSO (2:1) and then diluted in buffer. The
highest concentration of solvent used (maximum 0.3%) was added to control
tubes. IC50 values are reported as means ± SE from pooled data representing 3
curves. Inhibition is the maximal percent inhibition of basal [35S]TBPS specific
binding. Highest concentration of steroid used was 100 AM steroid. Also listed
are IC50 values reported for steroid inhibition of 2 nM [35S]TBPS binding to rat
cerebral cortex. Pregnenolone is 3B-OH-5-pregnen-20-one.
Steroid
Current study of Taricha
Inhibition
IC50 (nM)
Published values for rat
Inhibition
IC50 (nM)
3a-DHP
14.3 + 8.9
100
16.7a
100a
5a-THDOC
86.9 + 26
100
88.0a
100a
Pregnenolone SO4
18,200 + 6,500
74
14,000b
=85b
Corticosterone
37,600 + 12,900
34
>100,000a
21a
33
>100,000a
30a
Pregnenolone
>50,000
a Gee et al. (1987)
b Gee et al. (1989) Percent inhibition estimated from curve.
Table IV.1
89
Table IV.2. Distribution of [35S]TBPS and [3H]flunitrazepam specific binding
sites. Multiple (4-6) densitometric determinations of relative optical density were
made for total and non-specific binding in 13 brain regions for each brain. Nonspecific [3H]flunitrazepam binding was relatively uniform, but non-specific
[35S]TBPS binding varied across brain regions. After subtracting non-specific
binding, the mean values for each region were grouped into one of 4 ranks:
+ Detectable; + + Low density; + + + Moderate density;
+ + + + High density. The rankings were consistent if the brains regions were
ranked for individuals or pooled for all animals (n=4 for [35S]TBPS; n=2 for
[3H]flunitrazepam).
Brain Region
Telencephalon
Olfactory bulb
Striatum
Medial Pallium
Dorsal/lateral Pallium
Basal Forebrain complex
Diencephalon
Preoptic Area
Hypothalamus
Dorsal thalamus
Ventral thalamus
TBPS
++
+++
+
+
+ +a
++++
++++
+++
+++
+++
++++
+++
++
++
+++
++
Midbrain
Tectum
Tegmentum
++++
++++
++++
+++
Hindbrain
Cerebellum
Tegmentum
+++
+++
++++
a
b
++
Flunitrazepam
+
+++ in amygdala pars lateralis
++++ in lateral acoustic area
Table IV.2
+4.b
91
Fig. IVA. Equilibrium saturation binding of [35S]TBPS to neuronal membranes.
(A) Saturation isotherm. Membranes were incubated with one of 8 concentrations
of [35S]TBPS for 2 hrs at 15°C. Nonspecific binding was defined as that
occurring in the presence of 100 AM picrotoxin. Specific binding was described
by Kd = 17.1 ± 1.3 nM and B. = 901 ± 32 fmol/mg protein (Hill coefficient
= 0.998). The data were best fit by a one-site model using Lundon I software
and LIGAND software (Elsevier-Biosoft, Cambridge, UK). (B) ScatchardRosenthal replot of saturation data.
92
5000
E
A
4000
1:J
3000
CO
Lr)
t,)
2000
0
m
1000
0
0
5
10
15
20
30
25
35
40
35
Free [ S]1BPS (nM)
0.6
0.5
-or
0.4
a)
a)
0.3
-o
0.2
0
co
0.
1
0.0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Bound (pmol/mg protein)
Fig. IV. 1
0.8
0.9
93
Fig. W.2. Inhibition of [35S]TBPS binding by GABA. Membranes were
incubated with 2.5 nM [35S]TBPS and increasing concentrations of GABA. The
IC50 (concentration that inhibited 50% of specific [35S]TBPS binding) was
estimated as 1.41 ± 0.12 M. The data shown are pooled results from 3 curves.
0
-7
-6
-5
log GABA Concentration (M)
Fig. IV.2
-4
95
Fig. W.3. Steroid modulation of [35S]TBPS binding. The addition of steroids
inhibited the specific binding of the convulsant [35S]TBPS. Increasing
concentrations of various steroids were added to assay tubes 15 minutes prior to
addition of 2.5 nM [35S] -TBPS.
Results are reported as specific binding, percent
of [35S] -TBPS bound in the absence of steroid and presence of 1µM GABA.
Non-specific binding was determined in the presence of 100 µM picrotoxin. The
data shown are results pooled from 3 three competition curves.
100
rn
80
Ca"
U
;117
60
V)
40
V 3aOHDHP
20
0 5aTHDOC
V PregnenoloneSO4
CORT
0
10
9
8
7
6
5
log Steroid Concentration (Molar)
Fig. IV.3
4
97
Fig. W.4. Equilibrium saturation binding of [3H]flunitrazepam to Taricha
neuronal membranes. (A) Saturation isotherm. Membranes were incubated with
one of 8 concentrations of [3H]flunitrazepam for 2 hrs at 15°C. Non-specific
binding was determined in the presence of 10 AM diazepam. Specific binding
data were fit with a one site model yielding estimates of Kd = 4.03 ± 0.55 nM
and B. = 2830 ± 83 fmol/mg protein (Hill coefficient = 0.965 (Lundon I;
LIGAND). (B) Scatchard-Rosenthal replot of saturation data.
98
25
A
10
20
30
40
50
60
70
80
3
Free [ H]Flunitrazepam (nM)
0.8
0.6
M
1
O
0.4
0.2
0.0
0.0
0.5
1.0
1.5
2.0
Bound (pmol/mg protein)
Fig. IV.4
2.5
3.0
99
Fig. IV.5. Effect of GABA and pentobarbital on [3H]flunitrazepam specific
binding. Membranes were incubated with 2 nM [3H]flunitrazepam plus
increasing concentrations of GABA or pentobarbital. Both GABA and
pentobarbital enhanced [3H]flunitrazepam binding with EC50 values of 1.95 ±
0.15 I.LM and 228 ± 35 AM, respectively.
0)
C
180
E5
c
CE
-4-
s....,
D
I
rn
LA...
100
0
6
5
4
log Concentration (Molar)
Fig. IV.5
3
101
Fig. IV.6. Modulation of [3H]flunitrazepam binding by steroids. Tissue was
incubated with 2 nM [31-1]-fiunitrazepam and increasing concentrations of steroid.
Non-specific binding was determined in the presence of 10 AM diazepam.
Results are reported as [3H]flunitrazepam specific binding, means and standard
errors, fmol/mg protein from a representative experiment performed in triplicate.
o.)
170
160
0
(.)
_
CL
o
150 -
0
140
5a-THDOC
CORT
130
a.
N
3a-DHP
a_
120
-4-1
10-
?11)
100
0
-9
-8
-7
log Steroid Concentration (Molar)
Fig. IV.6
103
Fig. W.7. GABA-stimulated 360- uptake into Taricha synaptoneurosomes.
36C1-
uptake was initiated by the simultaneous addition of 0.1 /Xi 36a and GABA.
Bicuculline-sensitive 36C1- uptake was defined as the difference between 36C1-
uptake in the presence of GABA and that in the presence of 200 kt.M bicuculline
methiodide. Data are pooled results, reported as nmol 36C1-/mg protein/12
seconds, means ± SE, for a representative experiment performed in triplicate.
Basal 36C1- uptake in the presence of bicuculline was 51.9 nmol/mg protein/12
seconds.
1/
40
w
_..Y
CI
e--.
D
0
0
-+CL
o
35
30
25
20
15
10
5
'
0
//
1
-5
-4
log Concentration GABA Added (M)
Fig. IV.7
105
Fig. W.B. Effect of CORT on 36a- uptake. Synaptoneurosomes were
preincubated for 10 minutes with the concentration of CORT indicated plus.
36C1-
was added to tubes concurrently with either 3µM GABA or 100 AM picrotoxin.
Uptake was terminated after 12 seconds. The data are pooled results, presented
as the percentage of 36a- uptake in the presence of picrotoxin and absence of
steroid; means ± SE, for one experiment performed in triplicate.
//
130
..---..
0
120
1._
C"
O
U
4-
0
1
10
"4-C
a)
U
L
a)
D_
..........
100
90
I
0
J///
1
9
8
7
log Concentration CORI (Molar)
Fig. IV.8
,
107
Fig. P1.9. Representative receptor autoradiograms showing [35S]TBPS binding
sites in Taricha midbrain. Alternate transverse sections were incubated with 15
nM [35S]TBPS +/- 200 AM picrotoxin. Total binding is shown in (A),
nonspecific binding in (B), and pseudocolor-enhanced specific binding in (C).
Binding sites were enriched in the tectum and midbrain tegmentum, but relatively
sparce in the caudal hypothalmus.
A
c
B
1--,
Fig. IV.9
00
109
CHAPTER V: CONCLUSIONS
Summary
The major findings in this thesis can be briefly summarized. A genuine,
high-affinity receptor for corticosteroids, with a unique pharmacology, is present
in neuronal membranes. These receptors are not allosterically linked to GABAA
receptors, but appear to utilize G proteins for signal transduction. A variety of
studies indicate that these receptors may play a key role in mediating rapid
behavioral responses to stress. Other steroid molecules, also stress-related, may
alter brain function through a second, non-genomic mechanism of action--the
modulation of GABAA receptors.
A number of standard pharmacological criteria are applied in establishing
the existence of genuine, physiologically relevant neuroreceptors. These include
the demonstration of saturability and appropriate binding affinity. The kinetics of
receptor occupancy should be consistent with physiological responses and receptor
binding should be reversible. The kinetically derived dissociation constant should
agree with the Kd derived from equilibrium studies. Binding to the recognition
site should be disrupted under conditions that denature proteins; heating the tissue
and treatment with proteases should eliminate specific binding. The recognition
site should exhibit pharmacological specificity. The distribution of receptors
within the brain, and within the neuron, should make sense. Ultimately, one
110
should demonstrate a physiological or behavioral role for the receptor.
Chapter II addressed these criteria. The biochemical and neuroanatomical
criteria were largely satisfied. Chapter II also provided strong correlational
evidence supporting a behavioral role for this receptor. Recent
electrophysiological data (Rose et al., 1991) further support the conclusion that
[3H]corticosterone binding sites in Taricha brains represent functional,
behaviorally relevant, steroid receptors in neuronal membranes.
The neuronal responses initiated by a vast array of membrane-bound
receptors for neurochemical signals are mediated by one of three fundamental
transduction mechanisms: a receptor may be an integral part of a ligand-gated ion
channel, a transmembrane-regulated enzyme, or a transmembrane protein that
couples to G protein. Based on our current understanding of the cycle of
receptor-G protein-effector interaction, one can make predictions concerning the
modulation of [311]corticosterone binding if the receptor is a G protein-coupled
receptor. Implicit in these predictions is the assumption that corticosterone acts
as an agonist at the receptor.
Chapter III was a test of these predictions. If a receptor interacts with G
proteins, Mg2+ should enhance agonist binding by promoting formation of the
high-affinity ternary complex. Guanyl nucleotides, especially the
nonhydrolyzable ones, should negatively modulate agonist binding by promoting
disruption of the high-affinity ternary complex. The decreased affinity of the
111
receptor for agonist binding following exposure to nonhydrolyzable guanyl
nucleotides should result from an increase in the rate of agonist dissociation from
the receptor. Therefore, the shift in affinity state of the receptor should be
reflected in both equilibrium saturation binding and kinetic studies. In general,
one expects guanyl nucleotide effects on agonist binding to be sensitive to Mg'
concentrations. The results in Chapter III were entirely consistent with these
predictions. We were unable to quantify the low affinity state of the receptor in
saturation isotherms because of a technical problem--we lacked a radiolabeled
receptor antagonist. Therefore, Chapter III strongly suggests that corticosterone
has agonist properties at a receptor in neuronal membranes that utilizes G
proteins for signal transduction.
In Chapter IV, we examined that possibility that these corticosteroid
receptors, or a subpopulation of them, is an integral part of a ligand-gated ion
channel, the GABAA receptor. In the mammalian brain there appear to be
stringent structural requirements for steroid interaction with recognition sites on
the GABAA receptor/ chloride ionophore complex. The 3a-hydroxylated ring Areduced pregnane steroids are potent modulators of GABAA receptor function,
while steroids which lack this structure, such as corticosterone, lack efficacy.
Therefore, the objectives in Chapter N were two-fold. First, to determine
whether GABAA receptors in a primitive vertebrate brain are sensitive to
modulation by 3a-hydroxylated ring A-reduced pregnane steroids in a manner
112
similar to mammalian brains. Second, to test the specific hypothesis that
corticosterone is a potent modulator of GABAA receptors.
One would expect steroids to allosterically alter ligand binding in other
species in a similar manner, if steroid recognition sites on GABAA receptors have
been conserved. In membrane preparations, the binding of ligands to the
convulsant site on or near the chloride channel (such as [35S]TBPS) should be
sensitive to negative modulation by reduced pregnane steroids. The sensitivity of
[35S]TBPS binding to inhibition by steroids should be enhanced by GABA. The
potencies of steroids to non-competitively inhibit [35SJTBPS binding should
correlate with their potencies to enhance GABA-stimulated 360- uptake. GABAA
receptor-active steroids should also enhance the binding of central
benzodiazepines, such as [311]flunitrazepam. The studies in Chapter IV indicate
that GABAA receptors in Taricha brains are allosterically modulated by steroids
in a manner very similar to the rat brain. The 3a-hydroxylated ring A-reduced
pregnane steroids were potent modulators of [35S]TBPS and [3H]flunitrazepam
binding, but corticosterone was devoid of activity at the GABAA receptor.
Therefore, the steroid recognition site on GABAA receptors appears to have been
highly conserved during vertebrate evolution, suggesting two things:
corticosteroid receptors are not associated with GABAA receptors and modulation
of GABAA receptors by pregnane steroids is likely to be important in the
regulation of brain function in many species.
113
These studies are the first characterization of a high-affinity recognition
site on brain membranes for any naturally-occurring steroid, and the first to
satisfy the minimal pharmacological criteria expected for a genuine, membrane-
bound steroid receptor. These studies provide the first direct evidence that
steroid receptors may be coupled to G proteins in neuronal membranes. Also,
these data are the first evidence that steroids can modulate ligand binding to
GABAA receptors in a non-mammalian species. Taken together, these studies
make a strong case that non-genomically-mediated steroid actions in the brain
subserve important physiological and behavioral functions.
Speculation
We have identified a corticosteroid receptor in Xenopus brain membranes
with very similar binding characteristics. Several implications follow from this
finding. Since anuran and urodele amphibians diverged evolutionarily
approximately 350 million years ago, steroid regulation of neuronal function
through membrane-bound G protein-coupled receptors appears to be an ancient
characteristic of vertebrate brains. It also suggests that this mechanism was
found among vertebrates on the main line towards mammalian evolution.
It is likely that similar receptor and transduction mechanisms mediate a
number of rapid changes in neuronal activity induced by corticosteroids, as well
as gonadal steroids, in all vertebrate brains. For example, the excitability of
114
certain hypothalamic neurons in the rat brain is rapidly altered by corticosteroids
(Hua and Chen, 1989; Chen et al., 1991). These steroid-induced changes in
membrane potential exhibit a pharmacology and response latency that is consistent
with the corticosteroid receptor and electrophysiological responses characterized
in Taricha brains. Also, the K+ conductance regulated by estradiol in rat
hypothalamic neurons is dependent upon cAMP (Nabekura et al., 1986; Minami
et al., 1990), suggesting that these effects of estradiol are mediated by a G
protein-coupled receptor. There is also a cell-surface receptor for progesterone
on Xenopus oocytes that regulates intracellular cAMP levels, although the
mechanism is unclear (reviewed in Smith, 1989). In fish, cortisol rapidly
regulates prolactin release, as well as intracellular Ca' and cAMP levels (Borski
et al., 1991). I think it is likely that a conserved family of G protein-coupled
steroid receptors exists in vertebrates.
Since the GABAA receptor belongs to a family of ligand-gated ion
channels that share sequence homology and structural motifs, it is possible that
steroids also modulate excitatory amino acid receptors, nicotinic cholinergic
receptors, or glycine receptors. Recent studies suggest this may be the case
(Smith et al., 1987; Wu et al., 1990; Wu et al., 1991).
The unique pharmacology of the membrane-bound corticosteroid receptors
allow them to be distinguished from intracellular corticosteroid receptors. This is
a tool which should facilitate further studies into the regulation of neuroendocrine
115
activity and behavior by neuroactive corticosteroids. Further, the specificity of
the recognition site could facilitate development of therapeutic drugs with the
potential to treat conditions ranging from emotional disorders, particularly clinical
depression, to seizure susceptibility.
Why then haven't high-affinity steroid receptors been characterized in the
mammalian brain? One possibility, knowing that steroid receptors may be
coupled to G proteins, is that Towle and Sze (1983) described genuine receptors,
but their assay conditions isolated the recognition sites in a very low-affinity
state. Another problem relates to the high non-specific binding of lipophilic
steroids to neuronal membranes. Our studies were successful, in part, because
we achieved 80% specific binding using [3H]corticosterone and Taricha brain
membranes. However, we have had little success working with other steroids,
including tritiated high-affinity GABAA receptor-active steroids, because of
problems with nonspecific binding. The differences between the ligands may be
related to the relative tendencies of the steroids to partition into the lipid bilayer.
3a-DHP and 5a-THDOC are more saturated, being reduced in the 5 position,
and therefore more lipophilic than corticosterone. In addition, corticosterone,
with two hydroxyls, is more polar than 3a-DHP. Characterization of the highaffinity G protein-coupled receptors for the lipophilic cannabinoids was dependent
upon the development of suitable ligands. An alternative to radioligand binding
studies for identification of membrane-bound steroid receptors in mammalian
116
brains may be receptor cloning. This task will be less formidable if the receptors
belong to the superfamily of G protein-coupled receptors, and when the effector
mechanisms utilized by the receptor are identified, enabling expression cloning.
There is a unifying concept amidst the growing diversity of mechanisms
utilized by steroids. Steroids may exemplify the concept that the complexity of
signalling in the brain is achieved through the use of relatively few
neurochemicals, but a diversity of receptor subtypes (Schofield et al., 1990). It
appears to be the rule, rather than the exception, that neurotransmitters utilize
multiple receptor subtypes. Acetylcholine, for example, binds to a ligand-gated
ion channel as well as multiple subtypes of G protein-coupled receptors.
Dopamine binds to at least five G protein-coupled receptor subtypes, but in
addition, may activate receptors that are members of the intracellular steroid
receptor family (Power et al., 1991a, 1991b).
This provides a sense of
symmetry--neurotransmitters may utilize classical steroid receptor mechanisms,
while steroids may utilize classical neurotransmitter receptor mechanisms to
regulate brain function.
Steroids appear to utilize a multitude of receptor and transduction
mechanisms that are segregated temporally. Intracellular receptor-mediated
changes in gene transcription may take hours or days, regulation of second
messenger systems through G protein-coupled receptors occurs within minutes,
steroid modulation of ionic conductance through ligand-gated or voltage-gated
117
(ffrench-Mullen and Spence, 1991) ion channels occurs within seconds. It is
possible that the complex, often biphasic physiological responses to adrenal
steroids during stress may be mediated by the activation of multiple receptor
mechanisms with differing temporal characteristics. These responses might result
from activation of different receptor subtypes in differnent regions, or from the
activation of different receptors in the same cell. As an example, vitamin D
metabolites bind to receptors belonging to the family of intracellular steroid
receptors, but also rapidly activate a Ca2+ conductance with a different rank order
potency (Cancela et al., 1988; Farach-Carson et al., 1991). These genomic and
non-genomic reponses can be elicited in the same cell. Therefore, it is possible
that a given steroid may elicit complex responses in a single neuron through the
use of multiple receptor mechanisms.
118
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