Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M603768200 on July 5, 2006

J. Biol. Chem., Vol. 281, Issue 35, 25231-25240, September 1, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/35/25231    most recent
M603768200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hu, L.
Right arrow Articles by Catt, K. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hu, L.
Right arrow Articles by Catt, K. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Essential Role of G Protein-gated Inwardly Rectifying Potassium Channels in Gonadotropin-induced Regulation of GnRH Neuronal Firing and Pulsatile Neurosecretion*

Lian Hu, Keiko Wada, Nadia Mores1, Lazar Z. Krsmanovic2, and Kevin J. Catt

From the Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510

Received for publication, April 4, 2006 , and in revised form, June 20, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor (LHR) in cultured hypothalamic cells and immortalized GnRH (gonadotropin-releasing hormone) neurons (GT1–7 cells) transiently stimulates and subsequently inhibits cAMP production and pulsatile GnRH release. The marked and delayed impairment of cAMP signaling and episodic GnRH release in GT1–7 cells is prevented by pertussis toxin (PTX). This, and the LH-induced release of membrane-bound G{alpha}s and G{alpha}i3 subunits, are indicative of differential G protein coupling to the LHR. Action potential (AP) firing in identified GnRH neurons also initially increased and then progressively decreased during LH treatment. The inhibitory action of LH on AP firing was also prevented by PTX. Reverse transcriptase-PCR analysis of GT1–7 neurons revealed the expression of G protein-gated inwardly rectifying potassium (GIRK) channels in these cells. The LH-induced currents were inhibited by PTX and were identified as GIRK currents. These responses indicate that agonist stimulation of endogenous LHR expressed in GnRH neurons activates GIRK channels, leading to suppression of membrane excitability and inhibition of AP firing. These findings demonstrate that regulation of GIRK channel function is a dominant factor in gonadotropin-induced abolition of pulsatile GnRH release. Furthermore, this mechanism could contribute to the suppression of pituitary function during pregnancy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The pulsatile mode of hypothalamic GnRH secretion and receptor activation in pituitary gonadotrophs is essential for the maintenance of episodic gonadotropin synthesis and secretion, and ultimately for normal reproductive function. Pioneering studies on Rhesus monkeys by Knobil et al. (1) defined the importance of episodic pituitary stimulation for optimal gonadotropin secretion in Rhesus as well as the relationship of GnRH release to electrical activity in the hypothalamus. Marked inhibition of multiunit electrical activity was observed during initiation of the preovulatory lutenizing hormone (LH)3 surge, coincident with the late follicular rise in serum estradiol concentration (2). In addition, the operation of a short feedback loop between the pituitary gland and the hypothalamus has been indicated by the ability of LH to modulate its own secretion in vivo (3, 4), and to decrease single cell firing rates in the medial preoptic area and basal hypothalamus (5). Studies in cultured hypothalamic neurons, as well as immortalized pituitary gonadotrophs and GnRH-producing neurons, have provided insights into the complex actions of neuropeptides, neurotransmitters, and pituitary hormones in the control of episodic GnRH secretion from the hypothalamus (6–8).

The functional properties of the GT1–7 cell line, which expresses receptors for GnRH, LH/hCG, and prolactin, as well as a variety of other hormones and transmitters, are closely similar to those of the native GnRH neuron (8–10). GT1–7 cells treated with LH or hCG exhibit a dose-dependent and rapid increase in cAMP production during the first 15 min, followed by a marked decrease that is prevented by pre-treatment with pertussis toxin. These data suggest that the LH/hCG receptors expressed in GT1–7 cells are sequentially coupled to adenylyl cyclase stimulatory Gs and inhibitory Gi proteins. The similarity of hCG action on pulsatile GnRH release to that of extracellular Ca2+ depletion and calcium channel antagonists, and its partial resistance to potassium-induced depolarization, have suggested that it results from inhibition of plasma-membrane ion channel activity (11). The present studies were performed to further clarify the signaling pathways, electrical activity, and secretory responses that are initiated by agonist activation of the endogenous LH/hCG receptors expressed in GT1–7 cells and native GnRH neurons.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue and Cell Culture—Hypothalamic tissue was removed from fetuses of 18-day pregnant Sprague-Dawley rats as previously described (12). The borders of the excised hypothalami were delineated by the anterior margin of the optic chiasm, the posterior margin of the mammillary bodies, and laterally by the hypothalamic sulci. The neuronal tissue was dispersed in 0.2% collagenase containing 0.4% bovine serum albumin, 0.2% glucose, and 0.05% DNase I. After incubation for 60 min, the tissue was gently triturated by repeated aspiration into a smooth-tipped Pasteur pipette, incubated for another 30 min, and again dispersed. The cell suspension was passed through sterile mesh and sedimented by centrifugation for 10 min at 200 x g, then washed once in phosphate-buffered saline and once in culture medium consisting of 500 ml of Dulbecco's modified Eagle's medium containing 0.584 g/liter L-glutamate and 4.5 g/liter glucose, mixed with 500 ml of 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 fetal bovine serum. Each dispersed hypothalamus yielded about 1.5 x 106 cells. Immortalized GnRH neurons (GT1–7 cells) were provided by Dr. Richard Weiner (University of California, San Francisco) (13) and were cultured under the same conditions as primary hypothalamic cells.

Cell Perifusion Procedure and Hormone Measurement—Bead-attached GT1–7 cells were perifused at a flow rate of 0.15 ml/min at 37 °C. Fractions were collected at 5-min intervals and stored at –20 °C prior to radioimmunoassay. GnRH was measured using 125I-GnRH (Amersham Biosciences), GnRH standards (Peninsula Laboratories, Belmont, CA) and primary antibody were donated by Dr. V. D. Ramirez (University of Illinois, Urbana, IL). The intra- and inter-assay coefficients of variation at 50% binding in standard samples (15 pg/ml) were 5 and 7%, respectively. The sensitivity of the assay, defined as twice the standard deviation at zero doses, was 0.2 pg/tube.

Cyclic AMP Production—For studies on cAMP release, GT1–7 cells and hypothalamic neurons were stimulated in serum-free medium (1:1 Dulbecco's modified Eagle's medium/F-12) containing 0.1% bovine serum albumin, 30 mg/liter bacitracin, and 1 mM isobutylmethylxanthine. Radioimmunoassay of cAMP was performed as previously described, using a specific cAMP antiserum at a titer of 1:5000 (14). The intra-assay coefficient of variation of the assay was 4% at 50% displacement.

Whole Cell Recording of GnRH Neurons—Whole cell recording was performed on identified hypothalamic GnRH neurons cultured on collagen-coated coverslips and continuously perifused with artificial extracellular solution at a rate of 0.6 ml/min. The extracellular solution contained (in mM): 140 NaCl, 5 KCl, 10 HEPES, 10 D-glucose, 2 CaCl2, 1 MgCl2. pH was adjusted to 7.4 with NaOH. 30 µM Bicuculline and 5 µM 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt were used to block excitatory and inhibitory synaptic transmission. For GIRK current recording, extracellular KCl was increased to 30 mM and Na+ was substituted with 115 mM N-methyl-D-glucamine. Also, calcium current was blocked by 1.8 mM CoCl2, chloride current was blocked by 20 µM 5-nitro-2-(3-phenylpropylamino)benzoic acid, and sodium current was blocked by 0.5 µM tetrodotoxin. GnRH neurons were viewed under an inverted microscope Olympus IX70 with a x40 long working distance objective. All recordings were done at room temperature (23–25 °C) using patch pipettes (3–5 M{Omega}) pulled from thick wall borosilicate capillary glass (1.5-mm outer diameter and 0.86-mm inner diameter, WPI Inc., Sarasota, FL) on a Flaming/Brown puller model P-87 (Sutter Instruments Co., Novato, CA). The pipette solution contains (in mM): 20 KCl, 110 potassium gluconate, 10 Na2 creatine phosphate, 0.1 CaCl2, 2 MgCl2, 10 HEPES, 2 K2ATP (ATP potassium salt), 0.2 Na2 GTP, 1 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, pH was adjusted to 7.2 with KOH. 50 µM ZD7288 (4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyridinium chloride) was added in the pipette solution to block Ih current. An Ag/AgCl pellet was used as the reference electrode. Spontaneous activities were recorded under the I-clamp mode with a Multi-Clamp 700A amplifier (Axon Instruments, Foster City, CA), filtered at 2 KHz, and digitized at 10 KHz with Digidata 1320A (Axon Instruments). In voltage-clamp mode, the serial resistance was compensated by 70%, and leak current was automatically subtracted during recording by Multiclamp 700A. Data acquisition and subsequent analysis were performed using Clampex 9.0 software (Axon Instruments). Voltage and current traces and current-voltage curves were exported into Origin 7.5 software (MicroCal Software, Northampton, MA) for further plotting. Individual hypothalamic neurons were selected by differential interference contrast microscopy, which permits their morphological identification in cultured hypothalamic cells with an accuracy of more than 95%. Under differential interference contrast illumination, two types of bipolar GnRH neurons were identified based on nuclear size and localization. One of these has a large and centrally placed nucleus that contains an apparent nucleolus with subpolar localization. The nucleus is surrounded by a thin rim of cytoplasm, which extends throughout the processes. The other type of GnRH neuron was characterized by the presence of a relatively large nucleus that occupied 50% of the cross-sectional area of the cell body. In these cells, the cytoplasm contained numerous granules. Both categories of cells showed well developed thick and quite long processes. After recording, the cytoplasmic contents of each neuron were harvested under visual control and subjected to single-cell RT-PCR to confirm the presence of GnRH transcripts as reported previously (15, 16). As controls, the contents of hypothalamic cells that did not show typical GnRH neuronal morphology were also analyzed by RT-PCR.

RT-PCR Analysis of G Protein-gated Inwardly Rectifying Potassium (GIRK) Channels—Total RNA extracted from GT1–7 cells using Absolutely RNA RT-PCR Miniprep Kits (Stratagene, La Jolla, CA) was digested with DNase in a low-salt buffer to remove any remaining DNA. RT was performed using SuperScript III Reverse Transcriptase (Invitrogen). Using 5 µg of total RNA as template, first-strand cDNA was made using 500 ng of oligo(dT)12–18 and 1 µl of 10 mM dNTP Mix (Invitrogen) in a 130-µl reaction volume. After heat denaturing at 65 °C for 5 min and addition of 4 µl of 5 times First Strand Buffer, 1 µl of 0.1 M dithiothreitol, 1 µl of RNase OUT Recombinant RNase inhibitor (Invitrogen), and 200 units of SuperScript III reverse transcriptase, RT was performed at 55 °C for 50 min. RNA complementary to the cDNA was removed by addition of 1 µl of Escherichia coli RNase H and incubation at 37 °C for 20 min. An 0.5-µl aliquot of cDNA was used as template. Primers used were 5'-GCTATGGCTACCGCTACATCACAG-3' (nt 479–502) (sense) and 5'-CCAGTTCAAGTTGGTCAAGGGG-3' (nt 778–799) (antisense) for GIRK1 (accession number D45022 [GenBank] ); 5'-TCACCAGCCAAAGTTGCCTAAG-3' (nt 574–595) (sense) and 5'-AAGCAGAGACAAACCCGTTGAG-3' (nt 899–920) (antisense) for GIRK2 (accession number U11859 [GenBank] ); 5'-TTTCTCGTCTCACCTCTCGTCATC-3' (nt 939–962) (sense) and 5'-CAGCCATTGTGCTCCTTGTCAG-3' (nt 1348–1369) (antisense) for GIRK3 (accession number AF130860 [GenBank] ); 5'-GATTACATCCCCATTGCCACAG-3' (nt 91–112) (sense) and 5'-GCCGCTAAGGTTTTCAACACAAG-3' (nt 383–405) (antisense) for GIRK4 (accession number U33631 [GenBank] ); and 5'-AACGACCCCTTCATTGAC-3' (nt 152–169) (sense) and 5'-TCCACGACATACTCAGCAC-3' (nt 324–342) (antisense) for glyceraldehyde-3-phosphate dehydrogenase. The expected sizes of GIRK1, GIRK2, GIRK3, GIRK4, and glyceraldehyde-3-phosphate dehydrogenase were, respectively, 321, 347, 431, 315, and 191 bp. PCR conditions were: denaturing at 94 °C for 2 min, followed by 30 cycles of denaturing at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 60 s. PCR products were analyzed by electrophoresis on 2% agarose gels.

Immunoblot Analysis of Membrane-associated and Cytosolic G{alpha}s and G{alpha}i3 Subunits—GT1–7 cells were washed twice with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4), then scraped from the plates and lysed by freeze-thawing. After centrifugation at 12,000 x g for 15 min at 4 °C, pellets were resuspended in TE buffer and stored at –70 °C. For immunoblot analysis of cytosolic G{alpha}s and G{alpha}i3, GT1–7 cells were washed twice with cold phosphate-buffered saline, scraped from the culture dishes in buffer A (50 mM triethanolamine HCl, 25 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol, 10 µM each aprotinin and leupeptin, pH 7.5) in the presence of sterile 0.25 M sucrose, and kept at –70 °C. Cells were subsequently lysed by nitrogen cavitation at 800 p.s.i. for 3 min, followed by centrifugation at 500 x g for 5 min at 4 °C to remove tissue debris (first pellet). The first supernatant was then centrifuged at 2,000 x g for 15 min at 4 °C. The second pellet was saved, and its supernatant was further centrifuged at 100,000 x g for 1 h at 4 °C.The third supernatant was taken as cytosol. Protein contents were measured by the Pierce BCA protein assay kit. SDS-gel electrophoresis was performed on 8% acrylamide gels, followed by blotting with polyvinylidene difluoride membrane of 0.45-µm pore size.

G proteins were detected by incubation of blots with first antibody (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or first antibody preadsorbed with the corresponding peptide antigen (1:1000 + 10 µg of peptide), followed by peroxidase-coupled goat anti-rabbit IgG (H+L), and visualized by chemiluminescence (Invitrogen). The immunoreactive bands were analyzed as three-dimensional digitized images using a GS-700 Imaging Densitometer (Bio-Rad). The optical density (OD) of images is expressed as volume (OD x area) adjusted for the background, which gives arbitrary units of adjusted volume.

Chemicals—Oligonucleotides were obtained from Gene Probe Technologies, Gaithersburg, MD. Absolutely RNA RT-PCR Miniprep Kit was purchased form Stratagene, La Jolla, CA. SuperScript III RNase H Reverse Transcriptase, Platinum Taq DNA polymerase, pCR2.1 vector, and TOPO TA cloning kit were purchased from Invitrogen. The Wizard Plus Minipreps DNA purification system was purchased from Promega, Madison, WI. Isoproterenol and the selective 5-HT1 receptor agonist analog PAPP were purchased from Sigma. The cAMP-dependent protein kinase (PKA) peptide inhibitor was from Promega. Highly purified rat LH and hCG were obtained from Dr. A. F. Parlow (National Hormone and Peptide Program, Harbor-UCLA Medical Center, Torrance, CA).

Data Analysis—GnRH pulses were identified and their parameters determined by computerized cluster analysis (17). Individual point standard deviations 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 up stroke and down stroke were used to maintain false-positive and false-negative error rates below 10%. The pulse parameters were analyzed by one-way analysis of variance and results expressed as mean ± S.E. Statistical comparisons were performed using the Kruskal-Wallis test followed by the Mann-Whitney U test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
LH/hCG-induced Modulation of Pulsatile GnRH Release, cAMP Production, and Electrical Activity in Native and Immortalized GnRH Neurons—Perifused hypothalamic cells exhibited pulsatile GnRH release with clearly defined peaks and inter-peak intervals as determined by cluster pulse analysis (11, 17). Treatment with 2 nM hCG had an initial stimulatory effect followed by marked inhibition of pulsatile GnRH release, which returned after the cessation of hCG treatment (Fig. 1A). The inhibitory action of hCG was prevented by prior treatment with PTX (200 ng/ml, 4 h), indicating that the gonadotropin-induced suppression of pulsatile GnRH release is mediated by activation of Gi/Go proteins (Fig. 1B).

Signaling studies revealed that hCG-induced cAMP production in GT1–7 cells also exhibited an early stimulatory response and a delayed inhibitory phase. The decrease in cAMP production was prevented by treatment with pertussis toxin, consistent with the coupling of LH/hCG receptors to both Gs and Gi/o proteins (Fig. 1C). Treatment with the cAMP-dependent PKA peptide inhibitor (1 µM), prevented the time-dependent inhibition of cAMP production, indicating that activation of PKA promotes coupling of the LH/hCG receptor to Gi/Go (Fig. 1D).

Whole cell recordings from identified hypothalamic GnRH neurons consistently revealed spontaneous AP firing, with most of the cells (75%) showing irregular spiking activity (Fig. 1E). Firing of spontaneous action potentials (AP) was also evident in GT1–7 neurons, with properties similar to those of hypothalamic GnRH neurons (Fig. 1I). Treatment with 2 nM LH caused time-dependent biphasic changes in the frequency of AP firing in both identified hypothalamic GnRH neurons and GT1–7 cells. In hypothalamic GnRH neurons, the frequency of AP firing increased from the control of 0.3 ± 0.04 Hz to 1.3 ± 0.2 (p < 0.01; n = 20) during LH treatment (Fig. 1, E and F, respectively). In GT1–7 neurons, LH treatment also initially increased AP firing from 2.0 ± 0.3 Hz basal to 3.7 ± 0.4 Hz (p < 0.01; n = 20; Fig. 1, I and J, respectively). In contrast, prolonged LH treatment for up to 10 min significantly reduced AP firing frequency in both native GnRH neurons (0.3 ± 0.04 Hz basal versus 0.14 ± 0.02 Hz LH treatment; p < 0.01; n = 20; Fig. 1G) and GT1–7 neurons (2.0 ± 0.3 Hz basal versus 0.5 ± 0.03 Hz LH treatment; p < 0.01; n = 20; Fig. 1K). These LH-induced changes in AP firing were reversible, and basal firing resumed in both cell types during the washout period (Fig. 1, H, L, and M).


Figure 1
View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 1.
LH/hCG-induced modulation of pulsatile GnRH release, cAMP production, and electrical activity in native and immortalized GnRH neurons. A, activation of LH/hCG receptors in perifused hypothalamic cells causes initial stimulation and subsequent inhibition of pulsatile GnRH release (closed circles). B, PTX-induced reversal of the inhibitory action of hCG on GnRH release (closed circles). Traces for GnRH release are representative of five individual experiments. C, time-dependent biphasic effects of hCG on cAMP production (closed circles). PTX prevents hCG-induced inhibition of cAMP production (closed squares). D, reversal of the inhibitory action of hCG on cAMP production (closed circles) by protein kinase A inhibitor (closed squares). Data are mean ± S.E. of four independent experiments. E, spontaneous electrical activity of identified hypothalamic GnRH neuron. F, initial stimulation of AP firing during LH stimulation. G, inhibition of AP firing during prolonged LH treatment. H, resumption of AP firing during the washout period. I, spontaneous AP firing in an immortalized GnRH neuron. J, stimulation of AP firing by LH. K and L, inhibition of AP firing by LH in GT1–7 neurons. M, recovery of AP firing during the washout period. N, spontaneous electrical activity of a hypothalamic GnRH neuron during PTX treatment. O and P, prevention of LH-induced inhibition of spontaneous AP firing by PTX. R, decrease of AP firing during re-exposure to PTX treatment.

 
Consistent with Gi/o coupling of the LH/hCG receptors expressed in native hypothalamic GnRH neurons, pretreatment with 200 ng/ml PTX for 4 h prevented the time-dependent inhibition of AP firing and instead caused a sustained increase in AP firing and GnRH secretion. The rate of AP firing increased from 0.3 ± 0.05 Hz, PTX alone to 0.7 ± 0.06 Hz PTX + LH (1.5 min; p < 0.01, n = 12, Fig. 1O) and fell to 1.4 ± 0.12 Hz during prolonged treatment (p < 0.01, n = 10, Fig. 1P). During the washout period, AP firing frequency returned to the basal level (Fig. 1R).

The roles of increased cAMP production in neurosecretion and electrical activity were also examined in cultured hypothalamic cells during treatment with forskolin in the absence and presence of hCG. In static cultures, direct activation of adenylyl cyclase by forskolin caused a monotonic and time-dependent increase in GnRH release. In contrast, treatment with hCG caused an initial increase, followed by a marked decrease during sustained treatment (Fig. 2A). Production of cAMP also showed a monotonic increase during treatment with forskolin (Fig. 2B). In contrast to GnRH release, cAMP production remained continuously elevated during combined treatment with hCG and forskolin, indicating that inhibition of GnRH release is mediated by mechanisms that are not primarily dependent on cAMP production (Fig. 2B).

Patch-clamp recordings from identified hypothalamic GnRH neurons showed that treatment with 10 µM forskolin caused a time-dependent and sustained increase in AP firing. The frequency of AP firing increased from the control of 0.3 ± 0.02 to 0.9 ± 0.04 Hz during initial stimulation (Fig. 2, C and D, respectively, p < 0.01; n = 20). This persisted during prolonged treatment (Fig. 2E, p < 0.01; n = 10), and the AP firing rate returned to the control level during washout (Fig. 2F). The forskolin-induced increase in AP firing (Fig. 2, G and H) was markedly inhibited during concomitant treatment with LH, with a decrease from 2.1 ± 0.3 Hz forskolin treated to 0.4 ± 0.06 Hz (p < 0.01, n = 8) (Fig. 2, H and I). The increased rate of AP firing was restored during further treatment with forskolin (Fig. 2J).

The Gs-coupled beta2-adrenergic receptor agonist, isoproterenol, also caused a sustained increase in AP firing in identified GnRH neurons (Fig. 2, K–N). In contrast, selective activation of the Gi-coupled 5HT1 receptor by PAPP inhibited the rate of AP firing in hypothalamic GnRH neurons (Fig. 2, O–S).

Identification of G Proteins Coupled to LH Receptors in GT1–7 Neurons—Western blot analysis of membrane preparations and cytosolic fractions from GT1–7 cells with specific antibodies to G{alpha}s and G{alpha}i3 revealed significant changes in the cellular distribution of activated G protein {alpha}-subunits during hCG treatment. Membrane-bound G{alpha}s immunoreactivity was significantly reduced after treatment with hCG for 30 min (5.2 ± 0.6 in control versus 2.76 ± 0.2 in hCG treated, n = 3, p < 0.05, Fig. 3A). In contrast, cytosolic G{alpha}s increased during hCG treatment, consistent with the agonist-induced redistribution of activated G protein subunits (0.7 ± 0.08 in control versus 1.76 ± 0.2 in hCG treated, n = 3, p < 0.05, Fig. 3B). After prolonged exposure (4 h) to 2 nM hCG, membrane-bound G{alpha}i3 immunoreactivity decreased (3.9 ± 0.4 in control versus 2.2 ± 0.3 in hCG treated, n = 3, p < 0.05, Fig. 3C) and cytosolic immunoreactivity increased, consistent with the time-dependent reduction in cAMP production (0.54 ± 0.06 in control versus 1.46 ± 0.2 in hCG treated, n = 3, p < 0.05, Fig. 3D). The specificity of G protein {alpha} subunit antibodies was validated by blotting recombinant G protein {alpha} subunit standards with the corresponding antibodies. As shown in Fig. 3E, the immunoreactive G{alpha}s signal increased in a dose-dependent manner. Similarly, dose-dependent increases in G{alpha}s immunoreactivity were observed during incubation of G{alpha}s antibody with increasing amounts of cell membrane proteins derived from GT1–7 cells (Fig. 3F). For further validation of the G{alpha}s and G{alpha}i3 antibodies, GT1–7 cells were treated with toxins that selectively stimulate G{alpha}s and inhibit G{alpha}i3, respectively. As shown in Fig. 3G, membrane-bound G{alpha}s immunoreactivity probed with G{alpha}s antibody was significantly reduced during treatment with cholera toxin (1 ng/ml, 4 h) to stimulate Gs, but was unchanged during treatment with PTX (Fig. 3G). In contrast, membrane-bound G{alpha}i3 immunoreactivity probed with G{alpha}i3 antibody was significantly increased during treatment PTX (200 ng/ml, 4 h). However, treatment with CTX had no effect on G{alpha}i3 immunoreactivity (Fig. 3H).

Expression of GIRK mRNAs in GT1–7 Neurons—RT-PCR analysis of total RNA derived from cultured GT1–7 neurons using gene-specific primers based on sequences of GIRK channels subunits gave the expected fragment sizes of 321 for GIRK1 (Fig. 4A), 347 for GIRK2 (Fig. 4B), 431 for GIRK3 (Fig. 4C), and 315 for GIRK4 (Fig. 4D) base pairs. No such products were obtained in the absence of reverse-transcribed mRNA, indicating that the RNA preparation was free of genomic DNA contamination.

Voltage-step commands (–160 to –20 mV; 200 ms) delivered to identified hypothalamic GnRH neurons were used to elicit GIRK currents. The magnitude of the basal GIRK current was hyperpolarization-dependent, and was maximal at a test potential of –160 mV (Fig. 4, E and F, n = 5). The GIRK currents decreased in a hyperpolarization-dependent manner and were close to zero at –40 mV (Fig. 4, E and F). Treatment of identified GnRH neurons with 2 nM LH during the voltage ramp caused a substantial rise in GIRK current, which increased by 102.6 ± 12.6% (p < 0.01; n = 5) for all voltage steps negative to potassium equilibrium (Fig. 4, E and F). During the washout period the GIRK current fell almost to the basal level (Fig. 4, E and F). In all cells, the hyperpolarizing component of the K+ current was significantly reduced (p < 0.03; n = 5) when Ba2+ (200 µM) was added to the perfusion medium, consistent with its identity as a GIRK current (Fig. 4, E and F). Pretreatment of identified GnRH neurons with PTX (200 ng/ml) for 4 h had no significant effect on the basal GIRK current elicited by voltage-step potentials (Fig. 4, G and H, n = 5). In contrast, the LH-induced increase in GIRK current was prevented by prior treatment with PTX, indicating that beta{gamma} subunits Gi/o proteins mediate the significant increase in GIRK current of identified GnRH neurons (Fig. 4, G and H).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The G protein-coupled LHR is highly expressed in the testis and ovary, and has been extensively studied in several mammalian species including rodents, ungulates, primates, and humans. LHRs have also been identified in several tissues that are not recognized as primary gonadotropin target sites, such as uterus, placenta, fallopian tubes, uterine vessels, umbilical cord, brain, and lymphocytes (18, 19). The LHR is primarily coupled to Gs and on activation stimulates adenylyl cyclase activity and cAMP production. In addition to Gs coupling, in some tissues the agonist-activated LHR also couples to Gi/o leading to inhibition of cAMP production (11) and/or activation of phospholipase C (20, 21).


Figure 2
View larger version (47K):
[in this window]
[in a new window]
  		FIGURE 2.
cAMP-induced modulation of GnRH release and electrical activity in hypothalamic GnRH neurons. A, time-dependent stimulatory effect of forskolin on GnRH production in cultured hypothalamic cells (open circles) and reversal of the stimulatory action of forskolin on GnRH production by hCG (closed squares). Data are mean ± S.E. of four independent experiments. B, time-dependent stimulatory effect of forskolin on cAMP production in static cultures of hypothalamic cells (open circles) and potentiation of the forskolin stimulatory action on cAMP production by hCG (closed circles). Data are mean ± S.E. of four independent experiments. C, spontaneous electrical activity of an identified hypothalamic GnRH neuron. D and E, forskolin-induced increase in spontaneous action potential firing in cultured hypothalamic GnRH neurons. F, recovery of AP firing during the washout period. G, spontaneous electrical activity of identified hypothalamic GnRH neuron. H, forskolin-induced increase in spontaneous action potential firing. I, inhibition of forskolin-stimulated spontaneous AP firing by LH. J, resumption of fast AP firing during re-exposure to forskolin.

 
The hypothalamic expression of LHR, as well as the presence of measurable quantities of bioactive LH, indicate that LH originating by local production (22), retrograde pituitary stalk blood flow (23), or via the blood-brain barrier (18) could be involved in regulation of GnRH neuronal function. To further analyze the possible role of LH in GnRH secretion, we studied fetal hypothalamic cells in static cultures and during column perifusion. As previously observed, hCG had both stimulatory and inhibitory effects on GnRH release. Such a GnRH secretory profile is attributable to LH/hCG receptor coupling to two or more G proteins (24–28). The initial stimulatory effect on GnRH release is caused by activation of the cAMP signaling pathway, and its short duration is consistent with the absence of a sustained increase in cAMP production. In contrast, the prolonged forskolin-induced cAMP increase causes a sustained and prominent elevation of GnRH release (11, 29, 30). This is also evident in static cultures, where forskolin elicits a dose-dependent monotonic increase in both cAMP production and GnRH secretion (Fig. 2, A and B). The inhibition of pulsatile GnRH release observed during perifusion with hCG was prevented by prior treatment with PTX, indicating that activation of Gi/o protein(s) mediates the inhibitory action of LH on GnRH secretion. Furthermore, PTX treatment prevented the characteristic time-dependent decrease in cAMP production, and caused a prolonged and sustained increase in cAMP production and a substantial increase in GnRH secretion. The time-dependent LH-induced decrease in cAMP production was also prevented by a PKA inhibitor. This observation suggests that, as proposed for the beta2-adrenergic receptor (31), agonist-induced increases in cAMP production and consequent activation of PKA cause a switch of LHR coupling from Gs to Gi/o.


Figure 3
View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 3.
Cellular distribution of G protein coupled to luteinizing hormone receptors in GT1–7 neurons. A, activation of LH/hCG receptors by hCG causes a decrease in membrane-bound G{alpha}s immunoreactivity that is evident after 30 min. B, cytosolic G{alpha}s increases during treatment with hCG. C, membrane-bound G{alpha}i3 immunoreactivity decreases during treatment with hCG. D, cytosolic G{alpha}i3 increases during treatment with hCG. Data are mean ± S.E. of three independent experiments.

 
In addition to its effects on intracellular signaling, previous electrophysiological studies have shown that LH also modulates hypothalamic multiunit electrical activity (32, 33). In our studies, the spontaneous electrical activity of identified hypothalamic GnRH neurons was transiently increased during treatment with LH. The initial increase in spontaneous AP firing was associated with membrane depolarization, increased bursting activity, and the appearance of lower amplitude broad APs. The increase in AP firing could be attributable to increased cAMP production and activation of cyclic nucleotide-gated channels expressed in both native and immortalized GnRH neurons (34). Furthermore, treatment with forskolin, a direct activator of adenylyl cyclase that promotes a 10-fold increase in cAMP production (Fig. 2B), and isoproterenol, a beta2-adrenergic receptor agonist, caused significant increases in AP firing that are also consistent with activation of cyclic nucleotide-gated channels (35, 36). It is evident from our data, and studies by others, that cAMP signaling from Gs-coupled receptors expressed in hypothalamic GnRH neurons increases membrane excitability and promotes prominent and sustained increases in GnRH secretion (28, 30).

In both hypothalamic cells and GT1–7 neurons, treatment with LH activates time-dependent stimulatory and inhibitory changes in cAMP synthesis and GnRH release. Such changes in cAMP and GnRH production are similar to these elicited by activation of Gi/o-coupled muscarinic M2 (37) and 5-HT1 serotonin receptors expressed in GnRH neurons (28). These findings indicate that the actions of LH/hCG are mediated by activation of multiple signaling pathways that can account for its diverse actions on GnRH release.

In addition to its initial transient stimulation of AP firing in identified hypothalamic GnRH neurons, LH subsequently caused pronounced and time-dependent inhibition of spontaneous AP firing. This effect was reversible, and spontaneous firing of APs was recovered during the washout of LH. The ability of pertussis toxin to prevent the inhibitory action of LH receptor activation on spontaneous AP firing in hypothalamic GnRH neurons could be related to the release of beta{gamma} subunits from Gi (or Go). Furthermore, all subunits that constitute GIRK channels were found to be expressed in hypothalamic GnRH neurons. Consistent with coupling to Gi/o, the activation of LH/hCG receptors expressed in native GnRH neurons also inhibited forskolin-induced increases in AP firing despite the increase in cAMP production. This indicates that beta{gamma} subunits derived from PTX-sensitive G proteins, rather than decreases in cAMP production, are largely responsible for the inhibitory action of LH on AP firing. The mechanism of these effects could include both inhibition of voltage-dependent calcium channels (38) and activation of inwardly rectifying potassium channels (39, 40).


Figure 4
View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 4.
Expression and LH-induced activation of GIRK channels in GT1–7 neurons. A, expression of GIRK1 mRNA in GT1–7 neurons. B, expression of GIRK2 mRNA in GT1–7 neurons. C, expression of GIRK3 mRNA in GT1–7 neurons. D, expression of GIRK4 mRNA in GT1–7 neurons. +RT, with reverse transcriptase. –RT, without reverse transcriptase. E, basal GIRK current in identified GnRH neurons at voltage step command of –120 mV (circles, shown are representative traces recorded from five individual GnRH neurons). LH-induced potentiation of GIRK current (rectangles, n = 5). Recordings were taken 15 min after start of LH treatment. Decrease of LH-induced current during the washout period (triangles, n = 5) and Ba2+-induced inhibition of LH-activated GIRK current (horizontal triangles, n = 5). Recordings were taken 15 min after the start of LH treatment. F, current-voltage relationships of basal GIRK current (circles), LH-activated (squares), during washing (triangles), and blockade of GIRK current by Ba2+ (horizontal triangles). Current-voltage curves were derived from native GnRH neurons recorded in panel E. G, basal GIRK current in identified GnRH neurons at voltage step command of –120 mV (circles, n = 5). Traces were recorded 15 min after the start of LH treatment. LH-induced potentiation of GIRK current in identified GnRH neurons (rectangles, n = 5). Currents were recorded 15 min after the start of LH treatment. Effects of PTX (200 ng/ml, 4 h) on voltage step-induced GIRK current (triangles, n = 5). Recordings were taken 15 min after start of LH treatment. PTX-induced inhibition of LH-activated GIRK current in hypothalamic GnRH neurons (diamonds, n = 5). Traces were recorded 15 min after the start of LH treatment. H, current-voltage relationships of basal GIRK current (circles), LH-activated (squares), during treatment with LH plus PTX (triangles), PTX-mediated inhibition of LH-induced GIRK current (diamonds), and blockade of GIRK current by Ba2+ (horizontal triangles). Current-voltage curves were derived from native GnRH neurons recorded in panel F.

 
The pulsatile secretion of GnRH from GT1 neurons, fetal hypothalamic neurons, and early embryonic olfactory neurons is dependent on the presence of extracellular Ca2+. Incubation of GT1–7 neurons in Ca2+-depleted medium reduces the basal intracellular calcium concentration ([Ca2+]i) in single cells and abolishes pulsatile GnRH secretion in perifused cells (9, 41, 42). In GT1 neurons, high membrane depolarization induced by extracellular K+ increases [Ca2+]i and promotes GnRH secretion (41). Activation of voltage-gated calcium channels by K2-induced membrane depolarization also facilitates GnRH secretion from perfused hypothalamic explants (43, 44), cultured hypothalamic neurons (9), and embryonic neurons (45). In addition, facilitation of voltage-gated Ca2+ entry by Bay K8644, an L-type Ca2+ channel agonist, increases [Ca2+]i and GnRH secretion. Conversely, L-type Ca2+ channel antagonists reduce spontaneous and K+-simulated [Ca2+]i and GnRH secretion (41). The voltage-gated Na+ channel blocker, tetrodotoxin, and several K+ channel antagonists also modulate [Ca2+]i and GnRH secretion (46–48), indicating that action potential firing promotes GnRH secretion. Furthermore, GnRH neurons derived from embryonic olfactory placodes exhibit synchronized [Ca2+]i oscillations that occur at the same frequency as the concomitant pulsatile GnRH release (45). These results indicate that GnRH neurons are spontaneously active, and that voltage-gated Na+, Ca2+, and K+ channels participate in this activity. Furthermore, action potential-driven Ca2+ entry through L-type Ca2+ channels is critical in maintaining pulsatile GnRH secretion from native and immortalized GnRH neurons (49, 50). In addition to this process, studies on the role of 5-HT-liberated Gbeta{gamma}-subunits in synaptic transmission have revealed another inhibitory action of Gbeta{gamma} on neurosecretion that is distal to Ca2+ entry and cAMP signaling, and acts directly on the exocytotic fusion machinery (51). This mechanism involves binding of beta{gamma}-subunits to the C terminus of SNAP25 and interference with the Ca2+-induced soluble N-ethylmaleimide-sensitive factor attachment protein receptor machinery for vesicle fusion and secretory granule exocytosis (52, 53).

Depression of neuronal firing in raphe nuclei cells during activation of 5-HT1 receptors involves membrane hyperpolarization elicited by increased K+ conductance (54). At postsynaptic sites in the hippocampus, 5-HT1A receptor activation elicits hyperpolarization by enhancing K+ channel activity (55, 56). Cardiac and neuronal GIRKs are activated by G protein-coupled receptors selectively coupled to pertussis toxin (PTX)-sensitive G{alpha}i/o proteins. The time course for GIRK channel activation elicited by application of receptor agonists can be influenced by the multiple intervening steps of the G protein cycle. These begin with agonist binding to the GPCR and end with Gbeta{gamma} binding to the GIRK channel subunits that promote gating transitions to the open state. In addition to the G protein activation steps, signal termination by GTP hydrolysis by G{alpha} subunit and Gbeta{gamma} reassociation also influence the kinetics and amplitude of agonist-activated GIRK currents (57, 58). Stimulation of M2 muscarinic receptors by acetylcholine in cardiac pacemaker cells activates Gi2 and Gi3 and leads, through their beta{gamma} dimers, to the increase of a K+ current (IKAch) involving GIRK1 and GIRK4 (Kir3.1 and Kir3.4) channels. This causes slowing of the heart rate due to hyperpolarization of the pacemaker cells. In neurons, GIRK/Kir3 channels are also activated by several neurotransmitters, including somatostatin, GABA, and {alpha}2-adrenergic agonists (59).

It is evident from our data, and studies by others, that signaling from Gi/o-coupled receptors expressed in native GnRH neurons and GT1–7 cells leads to activation of GIRK channels. This reduces membrane excitability and the rate of AP firing, and profoundly inhibits pulsatile GnRH release. The episodic activation of this process is a critical factor in the genesis of pulsatile neuropeptide secretion, which is an essential component of the neuronal regulation of the mammalian reproductive system.


   FOOTNOTES
 
* This work was supported by the Intramural Research Program of the National Institutes of Health, NICHD. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 On leave from the Dept. of Pharmacology, Catholic University of the Sacred Heart, 00168 Rome, Italy. Back

2 To whom correspondence should be addressed: Endocrinology and Reproduction Research Branch, Bldg. 49, Rm. 6A-36 NICHD, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-2136; Fax: 301-480-8010; E-mail: lazar{at}mail.nih.gov.

3 The abbreviations used are: LH, luteinizing hormone; hCG, human chorionic gonadotropin; PAPP, 4-[2-[4-[3-(trifluoromethyl)phenyl]-1-piperazinyl] ethyl]benzeneamine p-aminophenethyl-m-trifluoromethylphenyl piperazine; AP, action potentials; GIRK, G protein-gated inwardly rectifying potassium; RT, reverse transcriptase; nt, nucleotide(s); PKA, protein kinase A; PTX, pertussis toxin. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Knobil, E. (1988) Hum. Reprod. 3, 29–31[Free Full Text]
  2. Ordog, T., and Knobil, E. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5813–5816[Abstract/Free Full Text]
  3. Martini, L., Fraschini, F., and Motta, M. (1968) in Endocrinology and Human Behavior (Michael, P. R., ed) pp. 175–185, Oxford University Press, London
  4. Turgeon, J. L., and Barraclough, C. A. (1976) Endocrinology 98, 639–644[Abstract/Free Full Text]
  5. Wuttke, W. (1974) Exp. Brain Res. 19, 205–216[Medline] [Order article via Infotrieve]
  6. Terasawa, E. (1998) Gen. Comp. Endocrinol. 112, 283–295[CrossRef][Medline] [Order article via Infotrieve]
  7. Kalra, S. P., Horvath, T., Naftolin, F., Xu, B., Pu, S., and Kalra, P. S. (1997) J. Neuroendocrinol. 9, 569–576[CrossRef][Medline] [Order article via Infotrieve]
  8. Milenkovic, L., D'Angelo, G., Kelly, P. A., and Weiner, R. I. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1244–1247[Abstract/Free Full Text]
  9. Krsmanovic, L. Z., Martinez-Fuentes, A. J., Arora, K. K., Mores, N., Navarro, C. E., Chen, H. C., Stojilkovic, S. S., and Catt, K. J. (1999) Endocrinology 140, 1423–1431[Abstract/Free Full Text]
  10. AL-Hader, A. A., Lei, Z. M., and Rao, C. V. (1997) Biol. Reprod. 56, 1071–1076[Abstract]
  11. Mores, N., Krsmanovic, L. Z., and Catt, K. J. (1996) Endocrinology 137, 5731–5734[Abstract]
  12. Krsmanovic, L. Z., Stojilkovic, S. S., Balla, T., al-Damluji, S., Weiner, R. I., and Catt, K. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11124–11128[Abstract/Free Full Text]
  13. Mellon, P. L., Windle, J. J., Goldsmith, P. C., Padula, C. A., Roberts, J. L., and Weiner, R. I. (1990) Neuron 5, 1–10[CrossRef][Medline] [Order article via Infotrieve]
  14. Fujita, K., Aguilera, G., and Catt, K. J. (1979) J. Biol. Chem. 254, 8567–8574[Abstract/Free Full Text]
  15. Martinez-Fuentes, A. J., Hu, L., Krsmanovic, L. Z., and Catt, K. J. (2004) Mol. Endocrinol. 18, 1808–1817[Abstract/Free Full Text]
  16. Skynner, M. J., Sim, J. A., and Herbison, A. E. (1999) Endocrinology 140, 5195–5201[Abstract/Free Full Text]
  17. Urban, R. J., Johnson, M. L., and Veldhuis, J. D. (1989) Endocrinology 124, 2541–2547[Abstract/Free Full Text]
  18. Lukacs, H., Hiatt, E. S., Lei, Z. M., and Rao, C. V. (1995) Horm. Behav. 29, 42–58[CrossRef][Medline] [Order article via Infotrieve]
  19. Dufau, M. L. (1998) Annu. Rev. Physiol. 60, 461–496[CrossRef][Medline] [Order article via Infotrieve]
  20. Gudermann, T., Birnbaumer, M., and Birnbaumer, L. (1992) J. Biol. Chem. 267, 4479–4488[Abstract/Free Full Text]
  21. Herrlich, A., Kuhn, B., Grosse, R., Schmid, A., Schultz, G., and Gudermann, T. (1996) J. Biol. Chem. 271, 16764–16772[Abstract/Free Full Text]
  22. Emanuele, N., Anderson, J., Baker, G., McDonald, D., Kirsteins, L., and Lawrence, A. M. (1984) Brain Res. 309, 271–276[CrossRef][Medline] [Order article via Infotrieve]
  23. Porter, J. C., Barnea, A., Cramer, O. M., and Parker, C. R., Jr. (1978) Clin. Obstet. Gynecol. 5, 271–282
  24. Stanislaus, D., Ponder, S., Ji, T. H., and Conn, P. M. (1998) Biol. Reprod. 59, 579–586[Abstract/Free Full Text]
  25. Grosse, R., Schmid, A., Schoneberg, T., Herrlich, A., Muhn, P., Schultz, G., and Gudermann, T. (2000) J. Biol. Chem. 275, 9193–9200[Abstract/Free Full Text]
  26. McArdle, C. A., Franklin, J., Green, L., and Hislop, J. N. (2002) Arch. Physiol. Biochem. 110, 113–122[Medline] [Order article via Infotrieve]
  27. Krsmanovic, L. Z., Mores, N., Navarro, C. E., Arora, K. K., and Catt, K. J. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2969–2974[Abstract/Free Full Text]
  28. Wada, K., Hu, L., Mores, N., Navarro, C. E., Fuda, H., Krsmanovic, L. Z., and Catt, K. J. (2006) Mol. Endocrinol. 20, 125–135[Abstract/Free Full Text]
  29. Martinez de la Escalera, G., Choi, A. L., and Weiner, R. I. (1995) Neuroendocrinology 61, 310–317[Medline] [Order article via Infotrieve]
  30. Vitalis, E. A., Costantin, J. L., Tsai, P. S., Sakakibara, H., Paruthiyil, S., Iiri, T., Martini, J. F., Taga, M., Choi, A. L., Charles, A. C., and Weiner, R. I. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 1861–1866[Abstract/Free Full Text]
  31. Daaka, Y., Luttrell, L. M., and Lefkowitz, R. J. (1997) Nature 390, 88–91[CrossRef][Medline] [Order article via Infotrieve]
  32. Beyer, C., Ramirez, V. D., Whitmoyer, D. I., and Sawyer, C. H. (1967) Exp. Neurol. 18, 313–326[CrossRef][Medline] [Order article via Infotrieve]
  33. Terasawa, E., Whitmoyer, D. I., and Sawyer, C. H. (1969) Am. J. Physiol. 217, 1119–1126[Free Full Text]
  34. El Majdoubi, M., and Weiner, R. I. (2002) Endocrinology 143, 2441–2444[Abstract/Free Full Text]
  35. Pun, R. Y., and Kleene, S. J. (2003) Biophys. J. 84, 3425–3435[Medline] [Order article via Infotrieve]
  36. Charles, A., Weiner, R., and Costantin, J. (2001) Mol. Endocrinol. 15, 997–1009[Abstract/Free Full Text]
  37. Krsmanovic, L. Z., Mores, N., Navarro, C. E., Saeed, S. A., Arora, K. K., and Catt, K. J. (1998) Endocrinology 139, 4037–4043[Abstract/Free Full Text]
  38. Ikeda, K., Kobayashi, T., Kumanishi, T., Niki, H., and Yano, R. (2000) Neurosci. Res. 38, 113–116[CrossRef][Medline] [Order article via Infotrieve]
  39. Hille, B. (1994) Trends Neurosci. 17, 531–536[CrossRef][Medline] [Order article via Infotrieve]
  40. Hill, R. H., Svensson, E., Dewael, Y., and Grillner, S. (2003) Eur. J. Neurosci. 18, 2919–2924[CrossRef][Medline] [Order article via Infotrieve]
  41. Krsmanovic, L. Z., Stojilkovic, S. S., Merelli, F., Dufour, S. M., Virmani, M. A., and Catt, K. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 8462–8466[Abstract/Free Full Text]
  42. Martinez-Fuentes, A. J., Krsmanovic, L. Z., Saito, S., Chen, H.-C., and Catt, K. J. (2000) in Program of the 82nd Annual Meeting of the Endocrine Society, Chevy Chase, MD, June 21–24, 2000, p. 21, Toronto, Canada
  43. Ramirez, V. D., Gallardo, E., and Hartter, D. (1980) J. Endocrinol. Investig. 3, 29–37[Medline] [Order article via Infotrieve]
  44. Drouva, S. V., Epelbaum, J., Hery, M., Tapia-Arancibia, L., Laplante, E., and Kordon, C. (1981) Neuroendocrinology 32, 155–162[Medline] [Order article via Infotrieve]
  45. Terasawa, E., Schanhofer, W. K., Keen, K. L., and Luchansky, L. (1999) J. Neurosci. 19, 5898–5909[Abstract/Free Full Text]
  46. Weiner, R. I., Wetsel, W., Goldsmith, P., Martinez de la Escalera, G., Windle, J., Padula, C., Choi, A., Negro-Vilar, A., and Mellon, P. (1992) Front. Neuroendocrinol. 13, 95–119[Medline] [Order article via Infotrieve]
  47. Lewis, D. L., and Ikeda, S. R. (1997) Neuroendocrinology 66, 235–245[Medline] [Order article via Infotrieve]
  48. Hiruma, H., Uemura, T., and Kimura, F. (1997) J. Neuroendocrinol. 9, 835–840[CrossRef][Medline] [Order article via Infotrieve]
  49. Van Goor, F., Krsmanovic, L. Z., Catt, K. J., and Stojilkovic, S. S. (1999) Mol. Endocrinol. 13, 587–603[Abstract/Free Full Text]
  50. Van Goor, F., Krsmanovic, L. Z., Catt, K. J., and Stojilkovic, S. S. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4101–4106[Abstract/Free Full Text]
  51. Blackmer, T., Larsen, E. C., Bartleson, C., Kowalchyk, J. A., Yoon, E. J., Preininger, A. M., Alford, S., Hamm, H. E., and Martin, T. F. (2005) Nat. Neurosci. 8, 421–425[Medline] [Order article via Infotrieve]
  52. Blackmer, T., Larsen, E. C., Takahashi, M., Martin, T. F., Alford, S., and Hamm, H. E. (2001) Science 292, 293–297[Abstract/Free Full Text]
  53. Gerachshenko, T., Blackmer, T., Yoon, E. J., Bartleson, C., Hamm, H. E., and Alford, S. (2005) Nat. Neurosci. 8, 597–605[CrossRef][Medline] [Order article via Infotrieve]
  54. Hamon, M., Lanfumey, L., el Mestikawy, S., Boni, C., Miquel, M. C., Bolanos, F., Schechter, L., and Gozlan, H. (1990) Neuropsychopharmacology 3, 349–360[Medline] [Order article via Infotrieve]
  55. Zgombick, J. M., Beck, S. G., Mahle, C. D., Craddock-Royal, B., and Maayani, S. (1989) Mol. Pharmacol. 35, 484–494[Abstract]
  56. Araneda, R., and Andrade, R. (1991) Neuroscience 40, 399–412[CrossRef][Medline] [Order article via Infotrieve]
  57. Zhang, Q., Pacheco, M. A., and Doupnik, C. A. (2002) J. Physiol. 545, 355–373[Abstract/Free Full Text]
  58. Leaney, J. L., Benians, A., Brown, S., Nobles, M., Kelly, D., and Tinker, A. (2004) Am. J. Physiol. 287, C182–C191
  59. Sadja, R., Alagem, N., and Reuveny, E. (2003) Neuron 39, 9–12[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
E.-J. Yang, B. T. Nasipak, and D. B. Kelley
Direct action of gonadotropin in brain integrates behavioral and reproductive functions
PNAS, February 13, 2007; 104(7): 2477 - 2482.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/35/25231    most recent
M603768200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hu, L.
Right arrow Articles by Catt, K. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hu, L.
Right arrow Articles by Catt, K. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement