0013-7227/98/$03.00/0
10
Endocrinology
Copyright © 1998 by The Endocrine Society
Muscarinic Regulation of Intracellular Signaling and
Neurosecretion in Gonadotropin-Releasing Hormone
Neurons
LAZAR Z. KRSMANOVIC, NADIA MORES, CARLOS E. NAVARRO,
S. ABDUL SAEED, KRISHAN K. ARORA, AND KEVIN J. CATT
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, Maryland 20892
ABSTRACT
Agonist activation of cholinergic receptors expressed in perifused
hypothalamic and immortalized GnRH-producing (GT1–7) cells in-
duced prominent peaks in GnRH release, each followed by a rapid
decrease, a transient plateau, and a decline to below basal levels. The
complex profile of GnRH release suggested that acetylcholine (ACh)
acts through different cholinergic receptor subtypes to exert stimu-
latory and inhibitory effects on GnRH release. Whereas activation of
nicotinic receptors caused a transient increase in GnRH release, ac-
tivation of muscarinic receptors inhibited basal GnRH release. Nano-
molar concentrations of ACh caused dose-dependent inhibition of
cAMP production that was prevented by pertussis toxin (PTX), con-
sistent with the activation of a plasma-membrane Gi protein. Micro-
molar concentrations of ACh also caused an increase in phosphoino-
T HE DIVERSE actions of acetylcholine (ACh) are medi-
ated by activation of nicotinic (ionotropic) and mus-
carinic (metabotropic) classes of receptors. Nicotinic recep-
tors (nAChRs) are ligand gated receptor channels that are
cation-specific but do not distinguish readily among cations
(1). Of the five subtypes (M1–M5) of muscarinic receptors
(mAChRs), M1, M3, and M5 are coupled to Gq/11 and mediate
activation of phospholipase C (PLC) and InsP3/Ca21 signal-
ing. The M2 and M4 subtypes are coupled to Gi/o, and me-
diate inhibition of cAMP production (2). Both classes of cho-
linergic receptors are expressed throughout the brain and are
abundant in the hypothalamus, where monoaminergic neu-
rons that terminate on GnRH and other peptidergic neurons
are located (3).
The GnRH neurons of the hypothalamus are innervated by
noradrenergic neurons located in the hindbrain, and cat-
echolamines have been implicated in the regulation of GnRH
release. In contrast, there is less evidence for a role of cho-
linergic innervation in the control of GnRH secretion. In early
studies, a rapid atropine-sensitive cholinergic component
was found to be involved in the release of ovulating hormone
in rats and rabbits (4, 5). Subsequently, intraventricular ad-
ministration of atropine was shown to block the release of
LH, FSH, and PRL from the pituitary gland, implicating
cholinergic pathways in the control of gonadotropin secre-
Received April 6, 1998.
Address all correspondence and requests for reprints to: Kevin J. Catt,
M.D., Ph.D., Endocrinology and Reproduction Research Branch, Build-
ing 49, Room 6A-36, NICHD, NIH, Bethesda, Maryland 20892. E-mail:
catt@helix.nih.gov.
4037
Vol. 139, No.
Printed in U.S.A.
sitide hydrolysis that was inhibited by the M1 receptor antagonist,
pirenzepine. In ACh-treated cells, immunoblot analysis revealed that
membrane-associated Gaq/11 immunoreactivity was decreased after 5
min but was restored at later times. In contrast, immunoreactive Gai3
was decreased for up to 120 min after ACh treatment. The agonist-
induced changes in G protein a-subunits liberated during activation
of muscarinic receptors were correlated with regulation of their re-
spective transduction pathways. These results indicate that ACh
modulates GnRH release from hypothalamic neurons through both
M1 and M2 muscarinic receptors. These receptor subtypes are coupled
to Gq and Gi proteins that respectively influence the activities of PLC
and adenylyl cyclase/ion channels, with consequent effects on neu-
rosecretion. (Endocrinology 139: 4037– 4043, 1998)
tion (6). Direct evidence for actions of ACh at the hypotha-
lamic level was provided by studies on the release of LH and
FSH from hypothalamic and pituitary tissue (7–9). More
recently, muscarinic and nicotinic receptors were found to be
involved in the stimulatory and inhibitory actions of ACh on
GnRH and LH release (10 –15). ACh has also been shown to
have a significant role in the regulation of lordosis by acting
on muscarinic receptors in the ventromedial nucleus (16).
In the present studies, the direct actions of ACh on GnRH
release were analyzed in cultured hypothalamic neurons and
GnRH-producing (GT1–7) cells.
Materials and Methods
Tissue and cell culture
Hypothalamic tissue was removed from fetuses of 17-day pregnant
Sprague-Dawley rats. The borders of the excised hypothalami were
delineated by the anterior margin of the optic chiasm, the posterior
margin of the mamillary bodies, and laterally by the hypothalamic sulci.
After dissection, hypothalami were placed in ice-cold dissociation buffer
containing 137 mm NaCl, 5 mm KCl, 0.7 mm Na2HPO4, 26 mm HEPES,
and 100 mg/liter gentamicin, pH 7.4. The tissues were washed and then
incubated in a sterile flask with dissociation buffer supplemented with
0.2% collagenase, 0.4% BSA, 0.2% glucose, and 0.05% DNase I. After 60
min incubation in a 37 C waterbath with shaking at 60 cycles/min, the
tissue was gently triturated by repeated aspiration into a smooth-tipped
Pasteur pipette. Incubation was continued for another 30 min, after
which the tissue was again dispersed. The cell suspension was passed
through sterile mesh (200 mm) into a 50-ml tube, sedimented by cen-
trifugation for 10 min at 200 3 g, and washed once in dissociation buffer
and once in culture medium consisting of 500 ml DMEM containing
0.584 g/liter l-glutamate and 4.5 g/liter glucose, mixed with 500 ml F-12
medium containing 0.146 g/liter l-glutamine, 1.8 g/liter glucose, 100
mg/ml gentamicin, 2.5 g/liter sodium bicarbonate, and 10% heat-inac-
4038 MUSCARINIC REGULATION OF GnRH SECRETION
tivated FCS. Each dispersed hypothalamus yielded about 1.5 3 106 cells.
Immortalized GnRH neurons (GT1–7 cells) obtained from Dr. R. I.
Weiner (University of California at San Francisco) were cultured under
the same conditions as primary hypothalamic cells.
Perifusion procedure
Cells were incubated in 50-ml tubes containing 1.5 3 107 cells, 0.3 ml
preswollen Cytodex-2 beads, and 30 ml of culture medium for 24h in 5%
CO2/air. The suspension was then transferred into 60-mm dishes and
culture was continued for 14 – 60 days, with replenishment of culture
medium every second day. Before perifusion, the cell-bead mixture was
collected by sedimentation and resuspended in Krebs-Ringer buffer
containing 1 mg/ml BSA, 1 mg/ml glucose, 20 mm bacitracin, pH 7.4.
After gassing for 1 h with 95% O2/5% CO2, the beads were loaded into
a temperature-controlled 0.5 ml chamber. Perifusion was performed at
a flow rate of 10 ml/h at 37 C for at least 1 h to establish a stable baseline
before addition of agents made up in the same medium. Fractions were
collected at either 1- or 5-min intervals and stored at 220 C before RIA
using 125I-GnRH, GnRH standards, and primary antibody donated by
Dr. V. D. Ramirez (University of Illinois, Urbana, IL). The intra and
interassay coefficients of variation at 80% binding in standard samples
(15 pg/ml) were 12–14%, respectively. GnRH pulses were identified and
their parameters were determined by algorithm cluster analysis. Indi-
vidual point sds were calculated using a power function variance model
from the experimental duplicates. A 2 3 2 cluster configuration and a
t-statistic of 2 for the upstroke and downstroke were used to maintain
false-positive and false-negative error rates below 10% (17). The statis-
tical significance of the pulse parameters was tested by one-way
ANOVA.
cAMP production and phosphoinositide hydrolysis
For studies on cAMP release, GnRH-producing cells were stimulated
in serum-free medium (1:1 DMEM/F-12) containing 0.1% BSA, 30 mg/
liter bacitracin, and 1 mm IBMX. RIA of cAMP was performed as pre-
viously described (18), using a specific cAMP antiserum at a titer of
1:5000. This assay showed no cross-reaction with cGMP, 29,39-cAMP,
ADP, GDP, CTP, or IBMX.
Inositol phosphate production
Cells were labeled for 24 h in inositol-free DMEM medium containing
20 mCi/ml [3H]inositol as described previously (19) and then washed
with inositol-free M199 medium and stimulated at 37 C in the presence
of 10 mm LiCl. Incubations were terminated by the addition of ice-cold
perchloric acid (5% (vol/vol) final concentration). The inositol phos-
phates were extracted and analyzed by anion exchange chromatography
as previously described. The combined InsP2 1 InsP3 fractions were
eluted from the columns by washing twice with 1 m ammonium formate
in 0.1 m formic acid (3 ml/wash), and their radioactivities were mea-
sured by liquid b-spectrometry.
Immunoblot analysis of membrane-associated G proteins
After stimulation, the cells were washed twice with TE buffer (Tris-
HCl 10 mm, EDTA 1 mm, pH 7.4), scraped from the plates, and lysed by
freeze-thawing. After centrifugation at 12,000 3 g, 15 min at 4 C, the
pellet was resuspended in TE buffer and stored at 270 C until assayed.
Protein contents were measured by the Pierce BCA protein assay kit
(Pierce, Rockford, IL). SDS-gel electrophoresis was performed on 12.5%
acrylamide gels (20), followed by blotting with PVDF membrane of 0.45
mm pore size. The blots were incubated with first antibody, followed by
peroxidase-coupled goat-antirabbit IgG (H 1 L), and visualized by
chemiluminescence. The immunoreactive bands were analyzed as three-
dimensional digitized images using a GS-700 Imaging Densitometer
(Bio-Rad Laboratories, Hercules, CA). The optical density (O.D.) of
images is expressed as volume (O.D. 3 area) adjusted for the back-
ground, which gives arbitrary units of adjusted volume (Adj. Volume).
Materials
125
I-GnRH, [3H]inositol, ECL, and Western blotting reagents were
obtained from Amersham Pharmacia Biotech (Arlington Heights, IL);
Endo • 1998
Vol 139 • No 10
collagenase (149 U/mg) was from Worthington Biochemical Corp. (Free-
hold, NJ); DNase I, trypsin, bacitracin, 3-isobutyl-1-methylxanthine
(IBMX), CTP, GDP, cGMP, 29:39-cAMP, and BSA were from Sigma
Chemical Co. (St. Louis, MO); cytodex-beads were from Pharmacia
Biotech (Piscataway, NJ); the perifusion system was from Acusyst-S
Cellex Biosciences (Minneapolis, MN); standard GnRH was from Pen-
insula Laboratories, Inc. (Belmont, CA); ACh, carbachol, nicotine, mus-
carine, methoctramine, atropine, pirenzepin were from Research Bio-
chemicals International (Natick, MA); 125I-cAMP was from Covance
Laboratories, Inc. (Vienna, VA); protein assay was from Pierce. Mem-
brane Immobilon-P was from Millipore Corp. (Bedford, MA). Peroxi-
dase-coupled goat-antirabbit IgG (L 1 H), FBS, and DMEM/F12 1:1
powder were from Gibco BRL-Life Technologies (Gaithersburg, MD).
Antibodies to Gaq/11, Gas Gai1, Gai1–2, and Gai3 were purchased from
Calbiochem-Novabiochem Corp. (San Diego, CA), as well as the cor-
responding standard peptides; anti-Gao was a gift from MBL Interna-
tional Corporation (Watertown, MA). Other reagents, if not specified,
were obtained from Sigma Chemical Co.
Results
GnRH release from perifused hypothalamic cells and
GT1–7 neurons
Treatment of both hypothalamic neurons and GT1–7
cells with ACh elicited prominent changes in GnRH re-
lease. An initial rapid increase occurred during the first 15
min, followed by a decline to a transient plateau phase and
a subsequent fall to below the initial basal level. Recovery
from the late inhibitory phase was slow, and GnRH release
was suppressed for up to 60 min after the cessation of ACh
treatment (Fig. 1, A and B). Such biphasic responses to
ACh suggested that GnRH release was differentially reg-
ulated by the activation of individual cholinergic receptor
subtypes. Treatment of perifused hypothalamic cells and
GT1–7 cells with carbachol also produced an initial stim-
ulatory response, followed by sustained inhibition of
GnRH release (not shown).
Selective activation of nicotinic receptors stimulated
GnRH release in both hypothalamic and GT1–7 cells and
was followed by a return to the basal pattern of pulsatile
release (Fig. 1, C and D). The secretory action of nicotine
was similar to that of K1-induced depolarization, consis-
tent with the operation of nicotinic receptor channels that
promote Na1 and Ca21 entry, with consequent effects on
GnRH release. In contrast, selective activation of musca-
rinic receptors inhibited GnRH release in both hypotha-
lamic and GT1–7 cells, followed by a return to the basal
pulsatile pattern (Fig. 1, E and F). These results suggest
that muscarinic receptor subtypes are responsible for the
ACh-induced inhibition of GnRH secretion. Basal GnRH
release was potentiated by the nonselective cholinergic
receptor antagonist, atropine, as well as by the selective M2
antagonist, methoctramine (Fig. 2).
Stimulation of phosphoinositide hydrolysis by cholinergic
agonists in hypothalamic cells and GT1–7 neurons
Treatment of both hypothalamic cells and GT1–7 cells with
ACh caused time- and dose-dependent stimulation of phos-
phoinositide hydrolysis. The maximal stimulatory effect of 1
mm ACh was reached after 5 min, and the production of
inositol phosphate declined to the basal level after 2 h (Fig.
3). ACh caused dose-dependent increases of inositol phos-
phate production in both hypothalamic cells and GT1–7 neu-
 MUSCARINIC REGULATION OF GnRH SECRETION
FIG. 1. Biphasic actions of acetylcholine on GnRH release from peri-
fused hypothalamic cells and GT1–7 neurons. In both hypothalamic
cells (A) and GT1–7 neurons (B), treatment with acetylcholine (ACh,
closed circles) caused a transient increase in GnRH release followed
by a decline to below the basal level. Treatment of hypothalamic cells
(C) and GT1–7 neurons (D) with nicotine (closed circles) caused prom-
inent increases in GnRH release, followed by a return to the basal
level (open circles). Treatment of hypothalamic cells (E) and GT1–7
neurons (F) with muscarine (closed circles) caused inhibition of GnRH
release, followed by a return to basal pulsatile release (open circles).
rons (Fig. 4). Such increases were monophasic, with EC50
values in the micromolar range (Fig. 4B). Treatment of hy-
pothalamic cells with muscarine also stimulated inositol
phosphate production, but with lower potency than ACh
(Fig. 4A). The stimulatory effect of carbachol on phospho-
inositide hydrolysis was inhibited by the M1 receptor antag-
onist, pirenzepine. (Fig. 4C). Activation of nicotinic receptors
had no effect on phosphoinositide hydrolysis (Fig. 4, A and B).
Effects of cholinergic agonists on cAMP production in
GT1–7 neurons
Micromolar concentrations of ACh also induced promi-
nent decreases in cAMP production in GT1–7 cells. The in-
hibitory effect of ACh (244%) was evident after 5 min and
persisted for up to 30 min but was no longer present after 2 h
(Fig. 5A). Inhibition of cAMP production was also observed
after treatment of GT1–7 cells with micromolar concentra-
tions of muscarine and was maintained for about 2 h. The
inhibitory action of ACh on cAMP production was dose
4039
FIG. 2. Effects of atropine and methoctramine on GnRH release from
perifused GT1–7 neurons. Treatment of GT1–7 neurons with atropine
(A) potentiated basal GnRH release (closed circles). The selective M2
receptor antagonist, methoctramine, (B) also potentiated basal GnRH
release (closed circles).
dependent, with an IC50 in the nanomolar range (Fig. 5B). The
inhibitory effect of carbachol on cAMP production was pre-
vented by prior treatment of cells by PTX, suggesting that it
results from the coupling of muscarinic receptors to adenylyl
cyclase inhibitory G proteins (Fig. 5C). After pretreatment of
GT1–7 cells with the M2 muscarinic antagonist, methoctra-
mine (100 nm), carbachol no longer inhibited cAMP produc-
tion and at higher concentrations caused a slight increase
above the basal level (Fig. 5D). In contrast, treatment of
GT1–7 cells with nicotine had no significant effect on cAMP
production.
Identification of G proteins coupled to cholinergic receptors
in GT1–7 neurons
Western blot analysis of membrane preparations from
GT1–7 cells with specific antibodies to Gaq/11, Gai3, Gao, and
Gas revealed significant changes in G protein content. Acti-
vation of muscarinic receptors by ACh caused a dose-
dependent decrease in Gaq/11 immunoreactivity that was
evident after 5 min (Fig. 6A). In contrast, there was only a
minor change in Gai3 immunoreactivity during the first 5 min
of ACh stimulation (Fig. 6B). After 30 min of exposure to 10
mm ACh, Gaq/11 immunoreactivity returned to the control
level (Fig. 6C). In contrast, immunoreactive Gai3 fell to a
minimal level at this time (Fig. 6D), consistent with the de-
crease in basal cAMP production and inhibition of GnRH
release. After prolonged (2 h) exposure to ACh, Gaq/11 im-
munoreactivity remained at the control level (Fig. 6E),
whereas Gai3 immunoreactivity was still significantly re-
duced (Fig. 6E).
Discussion
In the present study, concomitant analyses were per-
formed on cultured hypothalamic neurons and immortalized
4040 MUSCARINIC REGULATION OF GnRH SECRETION
FIG. 3. Time-course of phosphoinositide hydrolysis in acetylcholine-
treated hypothalamic cells and GnRH neurons. In both hypothalamic
cells (A) and GT1–7 neurons (B), acetylcholine-stimulated inositol
phosphate production peaked at 5 min and declined to the basal level
after 2 h.
GnRH neurons (GT1 cells) to use the complementary features
of both systems (heterogeneous normal cells vs. homoge-
neous transformed cells). Cultured hypothalamic cells and
immortalized GT1 neurons exhibit pulsatile GnRH release at
a frequency similar to that of GnRH secretion in vitro (21–24).
This property, with their spontaneous electrical activity (25),
expression of ion channels (26 –28), and coexpression of
GnRH and its receptors (29), suggest that GnRH neurons can
operate as an elemental endogenous GnRH oscillator. The
modulation of GnRH pulse amplitude and frequency by
GnRH agonist and antagonist analogs in cultured hypotha-
lamic neurons and GT1–7 cells indicates the potential role of
autocrine control of GnRH release. The in vivo operation of
the GnRH oscillator is influenced by neuropeptides (30, 31),
neurotransmitters (32–36), peripheral hormones (37, 38), and
feedback actions of gonadal steroids (39 – 42).
It is clear that GnRH release is influenced by nicotinic and
muscarinic receptors, which respectively mediate transient
stimulatory and sustained inhibitory actions on neurosecre-
tion. Thus, nonselective cholinergic agonists such as ACh
and carbachol exerted sequential stimulatory and inhibitory
actions on GnRH release from both normal and immortalized
GnRH neurons. These actions were separable into individual
stimulatory and inhibitory responses in cells treated with
nicotinic and muscarinic agonists, respectively. The stimu-
latory actions of these agonists are in part attributable to the
activation of nicotinic receptor channels, and in part to ac-
tivation of muscarinic receptors coupled to Gq with conse-
quent stimulation of phosphoinositide hydrolysis. The stim-
ulatory action of cholinergic agents on inositol phosphate
production was elicited by micromolar agonist concentra-
Endo • 1998
Vol 139 • No 10
FIG. 4. Dose-dependent effects of acetylcholine on phosphoinositide
hydrolysis in hypothalamic cells and GnRH neurons. Acetylcholine
(open circles) caused dose-dependent increases of inositol phosphate
production in both hypothalamic cells (A) and GT1–7 neurons (B).
Treatment of hypothalamic cells (A) with muscarine (closed circles)
also stimulated inositol phosphate production, but with lower potency
than acetylcholine. Activation of nicotinic receptors by nicotine
(closed squares) had no effect on phosphoinositide hydrolysis (A and
B). The M1 receptor antagonist, pirenzepine, (closed triangles) inhib-
ited carbachol-induced stimulation of inositol phosphate production
(open circles; panel C).
tions, with EC50 of 12 mm for ACh and 26 mm for carbachol.
The ability of pirenzepine to prevent this response identified
the M1 receptor as the mediator of this cholinergic stimula-
tory action on phosphoinositide hydrolysis. There was no
significant change in inositol phosphate production in nic-
otine-stimulated cells, in which the transient secretory re-
sponse is presumably related to calcium influx through nic-
otinic receptor-channels.
The inhibitory action of muscarinic agonists on cAMP
release was evident at much lower concentrations than those
required to stimulate inositol phosphate production. Also, it
was prevented by treatment with pertussis toxin, consistent
with its mediation by M2 or M4 receptors coupled to Gi
regulatory proteins. The ability of methoctramine to prevent
this response identified the M2 receptor as the predominant
 MUSCARINIC REGULATION OF GnRH SECRETION 4041

FIG. 5. Time- and dose-dependent ef-

fects of cholinegic agonists on cAMP pro-
duction in GT1–7 neurons. A, Treatment
of GT1–7 neurons with acetylcholine
caused inhibition of cAMP production
(open circles) that was maximal after 30
min, and was no longer evident after 2 h.
Muscarine (closed circles) also inhibited
cAMP production, and its effect was sus-
tained for about 2 h. B, The inhibitory
action of acetylcholine (open circles) on
cAMP production was dose dependent,
with IC50 in the nanomolar range. C, The
inhibitory effect of carbachol (open cir-
cles) was prevented by prior treatment of
GT1–7 neurons with pertussis toxin
(closed triangles). Pertussis toxin had no
effect on basal cAMP production (open tri-
angle). D, Treatment of GT1–7 neurons
with methoctramine (open squares) pre-
vented the inhibitory actions of carbachol
(open circles) on cAMP production (closed
squares).

mediator of this cholinergic inhibitory action, which is evi-
dent at nanomolar concentrations of ACh. The increase in
basal GnRH release during application of the nonselective
cholinergic antagonist atropine, as well as the selective M2
muscarinic receptor antagonist methoctramine, indicate that
M2 muscarinic receptors exert a tonic inhibitory action on
GnRH release. This may occur as a consequence of its spon-
taneously active conformation (43), or of the local production
of ACh as observed in both hypothalamic cells and GT1–7
neurons (unpublished data). The rapid inhibitory action of
ACh on pulsatile GnRH release from hypothalamic cells and
GT1–7 neurons could also be related to the release of bg
subunits from Gi (or Go), with consequent actions on plasma
membrane ion channels (44) that lead to suppression of neu-
rosecretion. Such effects could include both inhibition of
voltage-dependent calcium channels (45, 46) and activation
of inwardly rectifying potassium channels (47).
An analysis of the changes in membrane-associated Gaq/11
subunit levels during activation of the M1 receptor by ACh
revealed a dose-dependent decrease at 5 min, with a return
to control levels at 30 and 120 min. In contrast, Gai3 subunits
showed a more extensive and sustained reduction that was
evident for up to 120 min at high ACh concentrations. These
changes in G proteins are consistent with the initial activation
of phosphoinositide hydrolysis via M1 receptors and the
subsequent M2 receptor-mediated inhibitory action of ACh
on GnRH release from both cultured hypothalamic neurons
and GT1 cells. The down-regulation of Gaq/11 by ACh in
GT1–7 cells is qualitatively similar to results obtained for
Chinese hamster ovarian cells (CHO cells) expressing M1
muscarinic receptors (48) and human M3 muscarinic recep-
tors (49). Activation of Gas in mouse lymphoma cells (S49
cyc2 cells) by cholera toxin, or reduction of as GTPase activity
by mutations, accelerated the rate of degradation of as by 3-
to 4-fold and induced a shift of the as from a membrane-
bound to a soluble compartment. In the same cells, the b-
adrenoceptor agonist, isoproterenol, caused a rapid (,2 min)
20% shift of as from the membrane-bound to the soluble
compartment (50).
Our data indicate that ACh modulates GnRH release from
normal and immortalized hypothalamic neurons by acting
on both nicotinic and muscarinic receptor subtypes. The tran-
sient increase in GnRH release elicited by application of
nicotine is consistent with the operation of nicotinic receptors
as ion channels that mediate Na1 and Ca21 entry. The sim-
ilarity of the secretory responses during nicotinic receptor
activation to that elicited by depolarization with potassium
suggests that Ca21 entry is responsible for the transient in-
crease in GnRH release. Activation of M1 muscarinic recep-
tors caused a transient decrease in Gaq/11 immunoreactivity
and stimulation of phosphoinositide hydrolysis, followed by
increased GnRH secretion. In contrast, activation of M2 mus-
carinic receptors caused a sustained reduction in Gai3 im-
munoreactivity and a PTX-sensitive reduction in cAMP pro-
duction, with concomitant inhibition of GnRH release. The
agonist-induced down-regulation of a subunits liberated af-
ter dissociation of the heterotrimeric G proteins during re-
ceptor activation may reflect their release from the plasma
membrane and/or degradation by specific proteases. Such
down-regulation also provides an indication of the mode(s)
of G protein coupling used by individual seven transmem-
brane domain receptors that respectively influence the ac-
tivities of phospholipase C and adenylyl cyclase. Thus, stim-
ulation of nicotinic and muscarinic receptors in normal and
immortalized hypothalamic neurons activates ligand-gated
4042 MUSCARINIC REGULATION OF GnRH SECRETION Endo • 1998
Vol 139 • No 10

FIG. 6. Western blot analysis of G pro-

tein a-subunits in membranes of GT1–7
neurons. A, Activation of muscarinic re-
ceptors by acetylcholine caused a dose-
dependent decrease in Gaq/11 immuno-
reactivity that was evident after 5 min.
B, In contrast, there was no change in
Gai3 immunoreactivity during the first
5 min of acetylcholine stimulation. Af-
ter 30 min of exposure to millimolar
concentrations of acetylcholine, Gaq/11
immunoreactivity returned to the con-
trol level (C), whereas Gai3 immunore-
activity fell to a minimal level (D). After
prolonged exposure (2 h) to acetylcho-
line, Gaq/11 immunoreactivity remained
at the control level (E), whereas Gai3
immunoreactivity was still signifi-
cantly reduced (F).

receptor channels and/or G protein-dependent second mes-
senger systems and signal transduction pathways, with con-
sequent stimulatory (nicotinic, M1) or inhibitory (M2) effects
on neurosecretion.
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