help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Krsmanovic, L. Z.
Right arrow Articles by Catt, K. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Krsmanovic, L. Z.
Right arrow Articles by Catt, K. J.
Endocrinology Vol. 139, No. 10 4037-4043
Copyright © 1998 by The Endocrine Society

ARTICLES

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

Address all correspondence and requests for reprints to: Kevin J. Catt, M.D., Ph.D., Endocrinology and Reproduction Research Branch, Building 49, Room 6A-36, NICHD, NIH, Bethesda, Maryland 20892. E-mail: catt{at}helix.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Agonist activation of cholinergic receptors expressed in perifused hypothalamic and immortalized GnRH-producing (GT1–7) cells induced 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 stimulatory and inhibitory effects on GnRH release. Whereas activation of nicotinic receptors caused a transient increase in GnRH release, activation of muscarinic receptors inhibited basal GnRH release. Nanomolar concentrations of ACh caused dose-dependent inhibition of cAMP production that was prevented by pertussis toxin (PTX), consistent with the activation of a plasma-membrane Gi protein. Micromolar concentrations of ACh also caused an increase in phosphoinositide hydrolysis that was inhibited by the M1 receptor antagonist, pirenzepine. In ACh-treated cells, immunoblot analysis revealed that membrane-associated G{alpha}q/11 immunoreactivity was decreased after 5 min but was restored at later times. In contrast, immunoreactive G{alpha}i3 was decreased for up to 120 min after ACh treatment. The agonist-induced changes in G protein {alpha}-subunits liberated during activation of muscarinic receptors were correlated with regulation of their respective 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 neurosecretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE DIVERSE actions of acetylcholine (ACh) are mediated by activation of nicotinic (ionotropic) and muscarinic (metabotropic) classes of receptors. Nicotinic receptors (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/Ca2+ signaling. The M2 and M4 subtypes are coupled to Gi/o, and mediate inhibition of cAMP production (2). Both classes of cholinergic receptors are expressed throughout the brain and are abundant in the hypothalamus, where monoaminergic neurons 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 catecholamines have been implicated in the regulation of GnRH release. In contrast, there is less evidence for a role of cholinergic 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 administration 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 secretion (6). Direct evidence for actions of ACh at the hypothalamic level was provided by studies on the release of LH and FSH from hypothalamic and pituitary tissue (7, 8, 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, 11, 12, 13, 14, 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 µm) into a 50-ml tube, sedimented by centrifugation for 10 min at 200 x 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 µg/ml gentamicin, 2.5 g/liter sodium bicarbonate, and 10% heat-inactivated FCS. Each dispersed hypothalamus yielded about 1.5 x 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 x 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 µM 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 -20 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. Individual point SDs were calculated using a power function variance model from the experimental duplicates. A 2 x 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 statistical 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 previously described (18), using a specific cAMP antiserum at a titer of 1:5000. This assay showed no cross-reaction with cGMP, 2',3'-cAMP, ADP, GDP, CTP, or IBMX.

Inositol phosphate production
Cells were labeled for 24 h in inositol-free DMEM medium containing 20 µCi/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 phosphates were extracted and analyzed by anion exchange chromatography as previously described. The combined InsP2 + 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 measured by liquid ß-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 x g, 15 min at 4 C, the pellet was resuspended in TE buffer and stored at -70 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 µm pore size. The blots were incubated with first antibody, followed by peroxidase-coupled goat-antirabbit IgG (H + 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. x area) adjusted for the background, which gives arbitrary units of adjusted volume (Adj. Volume).

Materials
125I-GnRH, [3H]inositol, ECL, and Western blotting reagents were obtained from Amersham Pharmacia Biotech (Arlington Heights, IL); collagenase (149 U/mg) was from Worthington Biochemical Corp. (Freehold, NJ); DNase I, trypsin, bacitracin, 3-isobutyl-1-methylxanthine (IBMX), CTP, GDP, cGMP, 2':3'-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 Peninsula Laboratories, Inc. (Belmont, CA); ACh, carbachol, nicotine, muscarine, methoctramine, atropine, pirenzepin were from Research Biochemicals International (Natick, MA); 125I-cAMP was from Covance Laboratories, Inc. (Vienna, VA); protein assay was from Pierce. Membrane Immobilon-P was from Millipore Corp. (Bedford, MA). Peroxidase-coupled goat-antirabbit IgG (L + H), FBS, and DMEM/F12 1:1 powder were from Gibco BRL-Life Technologies (Gaithersburg, MD). Antibodies to G{alpha}q/11, G{alpha}s G{alpha}i1, G{alpha}i1–2, and G{alpha}i3 were purchased from Calbiochem-Novabiochem Corp. (San Diego, CA), as well as the corresponding standard peptides; anti-G{alpha}o was a gift from MBL International Corporation (Watertown, MA). Other reagents, if not specified, were obtained from Sigma Chemical Co.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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 release. 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. 1Go, A and B). Such biphasic responses to ACh suggested that GnRH release was differentially regulated by the activation of individual cholinergic receptor subtypes. Treatment of perifused hypothalamic cells and GT1–7 cells with carbachol also produced an initial stimulatory response, followed by sustained inhibition of GnRH release (not shown).



View larger version (31K):
[in this window]
[in a new window]
  		Figure 1. Biphasic actions of acetylcholine on GnRH release from perifused 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 prominent 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).

 
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. 1Go, C and D). The secretory action of nicotine was similar to that of K+-induced depolarization, consistent with the operation of nicotinic receptor channels that promote Na+ and Ca2+ entry, with consequent effects on GnRH release. In contrast, selective activation of muscarinic receptors inhibited GnRH release in both hypothalamic and GT1–7 cells, followed by a return to the basal pulsatile pattern (Fig. 1Go, 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. 2Go).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 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).

 
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 phosphoinositide 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. 3Go). ACh caused dose-dependent increases of inositol phosphate production in both hypothalamic cells and GT1–7 neurons (Fig. 4Go). Such increases were monophasic, with EC50 values in the micromolar range (Fig. 4BGo). Treatment of hypothalamic cells with muscarine also stimulated inositol phosphate production, but with lower potency than ACh (Fig. 4AGo). The stimulatory effect of carbachol on phosphoinositide hydrolysis was inhibited by the M1 receptor antagonist, pirenzepine. (Fig. 4CGo). Activation of nicotinic receptors had no effect on phosphoinositide hydrolysis (Fig. 4Go, A and B).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 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.

 


View larger version (15K):
[in this window]
[in a new window]
  		Figure 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) inhibited carbachol-induced stimulation of inositol phosphate production (open circles; panel C).

 
Effects of cholinergic agonists on cAMP production in GT1–7 neurons
Micromolar concentrations of ACh also induced prominent decreases in cAMP production in GT1–7 cells. The inhibitory effect of ACh (-44%) was evident after 5 min and persisted for up to 30 min but was no longer present after 2 h (Fig. 5AGo). Inhibition of cAMP production was also observed after treatment of GT1–7 cells with micromolar concentrations of muscarine and was maintained for about 2 h. The inhibitory action of ACh on cAMP production was dose dependent, with an IC50 in the nanomolar range (Fig. 5BGo). The inhibitory effect of carbachol on cAMP production was prevented by prior treatment of cells by PTX, suggesting that it results from the coupling of muscarinic receptors to adenylyl cyclase inhibitory G proteins (Fig. 5CGo). After pretreatment of GT1–7 cells with the M2 muscarinic antagonist, methoctramine (100 nM), carbachol no longer inhibited cAMP production and at higher concentrations caused a slight increase above the basal level (Fig. 5DGo). In contrast, treatment of GT1–7 cells with nicotine had no significant effect on cAMP production.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 5. Time- and dose-dependent effects of cholinegic agonists on cAMP production 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 sustained 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 circles) was prevented by prior treatment of GT1–7 neurons with pertussis toxin (closed triangles). Pertussis toxin had no effect on basal cAMP production (open triangle). D, Treatment of GT1–7 neurons with methoctramine (open squares) prevented the inhibitory actions of carbachol (open circles) on cAMP production (closed squares).

 
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 G{alpha}q/11, G{alpha}i3, G{alpha}o, and G{alpha}s revealed significant changes in G protein content. Activation of muscarinic receptors by ACh caused a dose- dependent decrease in G{alpha}q/11 immunoreactivity that was evident after 5 min (Fig. 6AGo). In contrast, there was only a minor change in G{alpha}i3 immunoreactivity during the first 5 min of ACh stimulation (Fig. 6BGo). After 30 min of exposure to 10 mM ACh, G{alpha}q/11 immunoreactivity returned to the control level (Fig. 6CGo). In contrast, immunoreactive G{alpha}i3 fell to a minimal level at this time (Fig. 6DGo), consistent with the decrease in basal cAMP production and inhibition of GnRH release. After prolonged (2 h) exposure to ACh, G{alpha}q/11 immunoreactivity remained at the control level (Fig. 6EGo), whereas G{alpha}i3 immunoreactivity was still significantly reduced (Fig. 6EGo).



View larger version (30K):
[in this window]
[in a new window]
  		Figure 6. Western blot analysis of G protein {alpha}-subunits in membranes of GT1–7 neurons. A, Activation of muscarinic receptors by acetylcholine caused a dose-dependent decrease in G{alpha}q/11 immunoreactivity that was evident after 5 min. B, In contrast, there was no change in G{alpha}i3 immunoreactivity during the first 5 min of acetylcholine stimulation. After 30 min of exposure to millimolar concentrations of acetylcholine, G{alpha}q/11 immunoreactivity returned to the control level (C), whereas G{alpha}i3 immunoreactivity fell to a minimal level (D). After prolonged exposure (2 h) to acetylcholine, G{alpha}q/11 immunoreactivity remained at the control level (E), whereas G{alpha}i3 immunoreactivity was still significantly reduced (F).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, concomitant analyses were performed on cultured hypothalamic neurons and immortalized GnRH neurons (GT1 cells) to use the complementary features of both systems (heterogeneous normal cells vs. homogeneous 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, 22, 23, 24). This property, with their spontaneous electrical activity (25), expression of ion channels (26, 27, 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 hypothalamic 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, 33, 34, 35, 36), peripheral hormones (37, 38), and feedback actions of gonadal steroids (39, 40, 41, 42).

It is clear that GnRH release is influenced by nicotinic and muscarinic receptors, which respectively mediate transient stimulatory and sustained inhibitory actions on neurosecretion. 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 stimulatory actions of these agonists are in part attributable to the activation of nicotinic receptor channels, and in part to activation of muscarinic receptors coupled to Gq with consequent stimulation of phosphoinositide hydrolysis. The stimulatory action of cholinergic agents on inositol phosphate production was elicited by micromolar agonist concentrations, with EC50 of 12 µM for ACh and 26 µM for carbachol. The ability of pirenzepine to prevent this response identified the M1 receptor as the mediator of this cholinergic stimulatory action on phosphoinositide hydrolysis. There was no significant change in inositol phosphate production in nicotine-stimulated cells, in which the transient secretory response is presumably related to calcium influx through nicotinic 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 mediator of this cholinergic inhibitory action, which is evident 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 spontaneously 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 ß{gamma} subunits from Gi (or Go), with consequent actions on plasma membrane ion channels (44) that lead to suppression of neurosecretion. 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 G{alpha}q/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, G{alpha}i3 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 G{alpha}q/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 receptors (49). Activation of G{alpha}s in mouse lymphoma cells (S49 cyc- cells) by cholera toxin, or reduction of {alpha}s GTPase activity by mutations, accelerated the rate of degradation of {alpha}s by 3- to 4-fold and induced a shift of the {alpha}s from a membrane-bound to a soluble compartment. In the same cells, the ß- adrenoceptor agonist, isoproterenol, caused a rapid (<2 min) 20% shift of {alpha}s 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 transient increase in GnRH release elicited by application of nicotine is consistent with the operation of nicotinic receptors as ion channels that mediate Na+ and Ca2+ entry. The similarity of the secretory responses during nicotinic receptor activation to that elicited by depolarization with potassium suggests that Ca2+ entry is responsible for the transient increase in GnRH release. Activation of M1 muscarinic receptors caused a transient decrease in G{alpha}q/11 immunoreactivity and stimulation of phosphoinositide hydrolysis, followed by increased GnRH secretion. In contrast, activation of M2 muscarinic receptors caused a sustained reduction in G{alpha}i3 immunoreactivity and a PTX-sensitive reduction in cAMP production, with concomitant inhibition of GnRH release. The agonist-induced down-regulation of {alpha} subunits liberated after dissociation of the heterotrimeric G proteins during receptor 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 transmembrane domain receptors that respectively influence the activities of phospholipase C and adenylyl cyclase. Thus, stimulation of nicotinic and muscarinic receptors in normal and immortalized hypothalamic neurons activates ligand-gated receptor channels and/or G protein-dependent second messenger systems and signal transduction pathways, with consequent stimulatory (nicotinic, M1) or inhibitory (M2) effects on neurosecretion.

Received April 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sargent PB 1993 The diversity of neuronal nicotinic acetylcholine receptors. Ann Rev Neurosci 16:403–443[Medline]
  2. Caulfield MP 1993 Muscarinic receptors-characterization, coupling and function. Pharmacol Ther 58:319–379[CrossRef][Medline]
  3. Kizer JS, Palkovits M, Tappaz J, Kebabian J, Brownstein MJ 1976 Distribution of releasing factors, biogenic amines, and related enzymes in the bovine median eminence. Endocrinology 98:685–695[Abstract]
  4. Everett JW, Sawyer CH, Markee JE 1949 A neurogenic timing factor in control of the ovulatory discharge of luteinizing hormone in the cyclic rat. Endocrinology 44:234–250[Medline]
  5. Markee JE, Everett JW, Sawyer CH 1952 The relationship of the nervous system to the release of gonadotropin and regulation of the sex cycle. Recent Prog Horm Res 7:139–157
  6. Libertun C, McCann SM 1973 Blockade of the release of gonadotropins and prolactin by subcutaneous or intraventricular injection of atropine in male and female rats. Endocrinology 92:1714–1724[Medline]
  7. Simonovic I, Motta M, Martini L 1974 Acetylcholine and the release of the follicle-stimulating hormone-releasing factor. Endocrinology 95:1373–1379[Medline]
  8. Fiorindo RP, Martini L 1975 Evidence for a cholinergic component in the neuroendocrine control of luteinizing hormone (LH) secretion. Neuroendocrinology 18:322–332[Medline]
  9. Libertun C, McCann SM 1976 Blocade of the postorchidectomy increase in gonadotropins by implants of atropine into the hypothalamus. Proc Soc Exp Biol Med 152:143–146[Abstract]
  10. Kalash J, Romita V, Billiar RB 1989 Third ventricular injection of alpha-bungarotoxin decreases pulsatile luteinizing hormone secretion in the ovariectomized rat. Neuroendocrinology 49:462–470[Medline]
  11. Kalra SP, Kalra PS 1983 Neural regulation of luteinizing hormone secretion in the rat. Endocr Rev 4:311–351[CrossRef][Medline]
  12. Billiar RB, Kalash J, Romita V, Tsuji K, Kosuge T 1988 Neosurugatoxin: CNS acetylcholine receptors and luteinizing hormone secretion in ovariectomized rats. Brain Res Bull 20:315–322[CrossRef][Medline]
  13. Richardson SB, Prasad JA, Hollander CS 1982 Acetylcholine, melatonin, and potassium depolarization stimulate release of luteinizing hormone-releasing hormone from rat hypothalamus in vitro. Proc Natl Acad Sci USA 79:2686–2689[Abstract/Free Full Text]
  14. Koren D, Egozi Y, Sokolovsky M 1992 Muscarinic involvement in the regulation of gonadotropin-releasing hormone in the cyclic rat. Mol Cell Endocrinol 90:87–93[CrossRef][Medline]
  15. Negro-Vilar A 1982 The median eminence as a model to study presynaptic regulation of neural peptide release. Peptides 3:305–310[CrossRef][Medline]
  16. Kow L-M, Pfaff DW 1988 Transmitter and peptide actions on hypothalamic neurons in vitro: Implications for lordosis. Brain Res Bull 20:857–861[CrossRef][Medline]
  17. Urban RJ, Johnson ML, Veldhuis JD 1989 In vivo biological validation and biophysical modeling of the sensitivity and positive accuracy of endocrine peak detection. Endocrinology 124:2541–2547[Abstract]
  18. Fujita K, Aguilera G, Catt KJ 1979 The role of cyclic AMP in aldosterone production by isolated zona glomerulosa cell. J Biol Chem 254:8567–8574[Abstract/Free Full Text]
  19. Arora KK, Cheng Z, Catt KJ 1997 Mutations of the conserved DRS motif in the second intracellular loop of the gonadotropin-releasing hormone receptor affect expression, activation, and internalization. Mol Endocrinol 11:1203–1212[Abstract/Free Full Text]
  20. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
  21. Krsmanovic LZ, Stojilkovic SS, Catt KJ 1996 Pulsatile gonadotropin-releasing hormone release and its regulation. Trends Endocrinol Metab 7:56–59
  22. Wetsel WC, Valenca MM, Merchenthaler I, Liposits Z, Lopez FJ, Weiner RI, Mellon PL, Negro-Vilar A 1992 Intrinsic pulsatile secretory activity of immortalized luteinizing hormone-releasing hormone-secreting neurons. Proc Natl Acad Sci USA 89:4149–4153[Abstract/Free Full Text]
  23. Weiner RI 1996 Cellular basis of the GnRH pulse generator. Nippon Sanka Fujinka Gakkai Zasshi 48:573–577[Medline]
  24. Lopez FJ, Merchenthaler IJ, Moretto M, Negro-Vilar A 1998 Modulating mechanisms of neuroendocrine cell activity: the LHRH pulse generator. Cell Mol Neurobiol 18:125–146
  25. Kusano K, Fueshko S, Gainer H, Wray S 1995 Electrical and synaptic properties of embryonic luteinizing hormone-releasing hormone neurons in explant cultures. Proc Natl Acad Sci USA 92:3918–3922[Abstract/Free Full Text]
  26. Spergel DJ, Catt KJ, Rojas E 1996 Immortalized GnRH neurons express large-conductance calcium-activated potassium channels. Neuroendocrinology 63:101–111[Medline]
  27. Bosma MM 1993 Ion channel properties and episodic activity in isolated immortalized gonadotropin-releasing hormone (GnRH) neurons. J Membr Biol 136:85–96[Medline]
  28. Charles AC, Hales TG 1995 Mechanisms of spontaneous calcium oscillations and action potentials in immortalized hypothalamic (GT1–7) neurons. J Neurophysiol 73:56–64[Abstract/Free Full Text]
  29. Krsmanovic LZ, Stojilkovic SS, Mertz LM, Tomic M, Catt KJ 1993 Expression of gonadotropin-releasing hormone receptors and autocrine regulation of neuropeptide release in immortalized hypothalamic neurons. Proc Natl Acad Sci USA 90:3908–3912[Abstract/Free Full Text]
  30. Maggi R, Pimpinelli F, Martini L, Piva F 1995 Inhibition of luteinizing hormone-releasing hormone secretion by delta-opioid agonists in GT1–1 neu-ronal cells. Endocrinology 136:5177–5181[Abstract]
  31. Dragatsis I, Zioudrou C, Gerozissis K 1995 Specific delta-opioid antagonists exert an agonist-independent inhibitory effect, similar to the agonist, on the release of GnRH in vitro. Cell Mol Neurobiol 15:389–400[CrossRef][Medline]
  32. Uemura T, Nishimura J, Yamaguchi H, Hiruma H, Kimura F, Minaguchi H 1997 Effects of noradrenaline on GnRH-secreting immortalized hypothalamic (GT1–7) neurons. Endocr J 44:73–78[Medline]
  33. Kalra SP, Horvath T, Naftolin F, Xu B, Pu S, Kalra PS 1997 The interactive language of the hypothalamus for the gonadotropin releasing hormone (GnRH) system. J Neuroendocrinol 9:569–576[CrossRef][Medline]
  34. Bourguignon JP, Gerard ML, Alvarez Gonzalez M-L, Purnelle G, Franchimont P 1995 Endogenous glutamate involvement in pulsatile secretion of gonadotropin-releasing hormone: evidence from effect of glutamine and developmental changes. Endocrinology 136:911–916[Abstract]
  35. Ping L, Mahesh VB, Bhat GK, Brann DW 1997 Regulation of gonadotropin-releasing hormone and luteinizing hormone secretion by AMPA receptors. Neuroendocrinology 66:246–253[Medline]
  36. Spergel DJ, Krsmanovic LZ, Stojilkovic SS, Catt KJ 1995 L-type Ca2+ channels mediate joint modulation by gamma-amino-butyric acid and glutamate of [Ca2+]i and neuropeptide secretion in immortalized gonadotropin-releasing hormone neurons. Neuroendocrinology 61:499–508[Medline]
  37. Mores N, Krsmanovic LZ, Catt KJ 1996 Activation of LH receptors expressed in GnRH neurons stimulates cyclic AMP production and inhibits pulsatile neuropeptide release. Endocrinology 137:5731–5734[Abstract]
  38. Milenkovic Lj, D’Angelo G, Kelly PA, Weiner RI 1994 Inhibition of gonadotropin-releasing hormone release by prolactin from GT1 neuronal cell lines through prolactin receptors. Proc Natl Acad Sci USA 91:1244–1247[Abstract/Free Full Text]
  39. Dluzen DE, Ramirez VD 1986 In vivo LH-RH output of ovariectomized rats following estrogen treatment. Neuroendocrinology 43:459–465[Medline]
  40. Attardi B, Klatt B, Hoffman GE, Smith MS 1997 Facilitation or inhibition of the estradiol-induced gonadotropin surge in the immature rat by progesterone: regulation of GnRH and LH messenger RNAs and activation of GnRH neurons. J Neuroendocrinol 9:589–599[CrossRef][Medline]
  41. Wiebe JP 1997 Nongenomic actions of steroids on gonadotropin release. Recent Prog Horm Res 52:71–99
  42. Levine JE 1997 New concepts of the neuroendocrine regulation of gonadotropin surges in rats. Biol Reprod 56:293–302[Abstract]
  43. Jakubik J, Bacakova L, el-Fakahany EE, Tucek S 1995 Constitutive activity of the M1–M4 subtypes of muscarinic receptors in transfected CHO cells and of muscarinic receptors in the heart cells revealed by negative antagonists. FEBS Lett 377:275–279[CrossRef][Medline]
  44. Wickman K, Clapham DE 1995 Ion channel regulation by G proteins. Physiol Rev 75:865–885[Abstract/Free Full Text]
  45. Ikeda SR 1996 Voltage-dependent modulation of N-type calcium channels by G-protein ß{gamma} subunits. Nature 380:255–258[CrossRef][Medline]
  46. Hertlitze S, Garcia DE, Mackie K, Hille B, Scheuer T, Catterall WA 1996 Modulation of Ca2+ channels by G-protein ß{gamma} subunits. Nature 380:258–262[CrossRef][Medline]
  47. Hille B 1994 Modulation of ion-channel function by G-protein-coupled receptors. Trends Neurosci 17:531–535[CrossRef][Medline]
  48. Mullaney I, Mitchell FM, McCallum JF, Buckley NJ, Milligan G 1993 The human muscarinic M1 acetylcholine receptor, when expressed in CHO cells, activates and downregulates Gq{alpha} and G11{alpha} equally and non-selectively. FEBS Lett 324:241–245[CrossRef][Medline]
  49. van de Westerlo E, Yang J, Logsdon C, Williams JA 1995 Down-regulation of the G-proteins Gq alpha and G11 alpha by transfected human M3 muscarinic acetylcholine receptors in Chinese hamster ovary cells is independent of receptor down-regulation. Biochem J 310:559–563
  50. Levis MJ, Bourne HR 1992 Activation of the alpha subunit of Gs in intact cells alters its abundance, rate of degradation, and membrane avidity. J Cell Biol 119:1297–1307[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
L. Hu, K. Wada, N. Mores, L. Z. Krsmanovic, and K. J. Catt
Essential Role of G Protein-gated Inwardly Rectifying Potassium Channels in Gonadotropin-induced Regulation of GnRH Neuronal Firing and Pulsatile Neurosecretion
J. Biol. Chem., September 1, 2006; 281(35): 25231 - 25240.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
K. Wada, L. Hu, N. Mores, C. E. Navarro, H. Fuda, L. Z. Krsmanovic, and K. J. Catt
Serotonin (5-HT) Receptor Subtypes Mediate Specific Modes of 5-HT-Induced Signaling and Regulation of Neurosecretion in Gonadotropin-Releasing Hormone Neurons
Mol. Endocrinol., January 1, 2006; 20(1): 125 - 135.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
C.-M. Yeung, B.-S. An, C. K. Cheng, B. K.C. Chow, and P. C.K. Leung
Expression and transcriptional regulation of the GnRH receptor gene in human neuronal cells
Mol. Hum. Reprod., November 1, 2005; 11(11): 837 - 842.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. A. Andric, D. Zivadinovic, A. E. Gonzalez-Iglesias, A. Lachowicz, M. Tomic, and S. S. Stojilkovic
Endothelin-induced, Long Lasting, and Ca2+ Influx-independent Blockade of Intrinsic Secretion in Pituitary Cells by Gz Subunits
J. Biol. Chem., July 22, 2005; 280(29): 26896 - 26903.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
A. Cariboni, F. Pimpinelli, S. Colamarino, R. Zaninetti, M. Piccolella, C. Rumio, F. Piva, E. I. Rugarli, and R. Maggi
The product of X-linked Kallmann's syndrome gene (KAL1) affects the migratory activity of gonadotropin-releasing hormone (GnRH)-producing neurons
Hum. Mol. Genet., November 15, 2004; 13(22): 2781 - 2791.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
V. Trkulja, V. Crljen-Manestar, H. Banfic, and Z. Lackovic
Involvement of the Peripheral Cholinergic Muscarinic System in the Compensatory Ovarian Hypertrophy in the Rat
Experimental Biology and Medicine, September 1, 2004; 229(8): 793 - 805.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
L. Z. Krsmanovic, N. Mores, C. E. Navarro, K. K. Arora, and K. J. Catt
An agonist-induced switch in G protein coupling of the gonadotropin-releasing hormone receptor regulates pulsatile neuropeptide secretion
PNAS, March 4, 2003; 100(5): 2969 - 2974.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. H. Shah, J.-W. Soh, and K. J. Catt
Dependence of Gonadotropin-releasing Hormone-induced Neuronal MAPK Signaling on Epidermal Growth Factor Receptor Transactivation
J. Biol. Chem., January 24, 2003; 278(5): 2866 - 2875.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
M. Tomic', F. Van Goor, M.-L. He, D. Zivadinovic, and S. S. Stojilkovic
Ca2+-Mobilizing Endothelin-A Receptors Inhibit Voltage-Gated Ca2+ Influx through Gi/o Signaling Pathway in Pituitary Lactotrophs
Mol. Pharmacol., June 1, 2002; 61(6): 1329 - 1339.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. Roy and D. D. Belsham
Melatonin Receptor Activation Regulates GnRH Gene Expression and Secretion in GT1-7 GnRH Neurons. SIGNAL TRANSDUCTION MECHANISMS
J. Biol. Chem., January 4, 2002; 277(1): 251 - 258.
[Abstract] [Full Text]

Home page
 J. Appl. Physiol.Home page
P. Berger, J. M. Tunon-De-Lara, J.-P. Savineau, and R. Marthan
Signal Transduction in Smooth Muscle: Selected Contribution: Tryptase-induced PAR-2-mediated Ca2+ signaling in human airway smooth muscle cells
J Appl Physiol, August 1, 2001; 91(2): 995 - 1003.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
A. Charles, R. Weiner, and J. Costantin
cAMP Modulates the Excitability of Immortalized Hypothalamic (GT1) Neurons via a Cyclic Nucleotide-Gated Channel
Mol. Endocrinol., June 1, 2001; 15(6): 997 - 1009.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
L. Z. Krsmanovic, N. Mores, C. E. Navarro, M. Tomic, and K. J. Catt
Regulation of Ca2+-Sensitive Adenylyl Cyclase in Gonadotropin-Releasing Hormone Neurons
Mol. Endocrinol., March 1, 2001; 15(3): 429 - 440.
[Abstract] [Full Text]

Home page
 EndocrinologyHome page
R. Maggi, F. Pimpinelli, L. Molteni, M. Milani, L. Martini, and F. Piva
Immortalized Luteinizing Hormone-Releasing Hormone Neurons Show a Different Migratory Activity in Vitro
Endocrinology, June 1, 2000; 141(6): 2105 - 2112.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
B. Shi, G. Bhat, V. B. Mahesh, M. Brotto, T. M. Nosek, and D. W. Brann
Bradykinin Receptor Localization and Cell Signaling Pathways Used by Bradykinin in the Regulation of Gonadotropin-Releasing Hormone Secretion
Endocrinology, October 1, 1999; 140(10): 4669 - 4676.
[Abstract] [Full Text]

Home page
 EndocrinologyHome page
L. Z. Krsmanovic, A. J. Martinez-Fuentes, K. K. Arora, N. Mores, C. E. Navarro, H.-C. Chen, S. S. Stojilkovic, and K. J. Catt
Autocrine Regulation of Gonadotropin-Releasing Hormone Secretion in Cultured Hypothalamic Neurons
Endocrinology, March 1, 1999; 140(3): 1423 - 1431.
[Abstract] [Full Text]

Home page
 EndocrinologyHome page
Editorial: More Pieces of the Puzzle in Place, Even More Discovered Missing
Endocrinology, October 1, 1998; 139(10): 4035 - 4035.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Krsmanovic, L. Z.
Right arrow Articles by Catt, K. J.
Right arrow Search for Related Content
PubMed