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Bradykinin Receptor Localization and Cell Signaling Pathways Used by Bradykinin in the Regulation of Gonadotropin-Releasing Hormone Secretion¹

Department of Physiology and Endocrinology, Medical College of Georgia, Augusta, Georgia 30912

Address all correspondence and requests for reprints to: Dr. Darrell W. Brann, Department of Physiology and Endocrinology, Medical College of Georgia, Augusta, Georgia 30912. E-mail: dbrann{at}mail.mcg.edu

	Abstract

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In a previous publication we provided evidence of a novel neuronal pathway for the control of GnRH secretion by bradykinin. The action of bradykinin appeared to be exerted through the bradykinin B₂ receptor. In this study we demonstrated that the bradykinin B₂ receptor is densely localized in the arcuate nucleus, median eminence, organum vasculosum of the lamina terminalis, and preoptic area, regions known to be critical for the control of GnRH secretion. To determine the mechanism of action of bradykinin in stimulating GnRH release, we used immortalized GnRH (GT1–7) cells in vitro. Bradykinin stimulation of GnRH secretion from GT1–7 cells appears to involve activation of the phospholipase C signaling pathway and mobilization of extracellular and intracellular calcium stores. Evidence to support this contention was derived from the observations that incubation of the phospholipase C inhibitor, U-73122 with bradykinin, blocked the ability of bradykinin to stimulate release from GT1–7 cells. This effect was specific, as a nitric oxide synthase inhibitor and a cyclooxygenase inhibitor were found to have no effect on bradykinin-induced GnRH secretion, suggesting that nitric oxide and PGs do not mediate bradykinin effects. Pertussis toxin also had no effect on bradykinin action. This suggests that the bradykinin B₂ receptor may be coupled to a pertussis toxin-insensitive G protein in GT1–7 cells. With respect to calcium involvement in bradykinin action, fura-2 calcium indicator studies revealed that bradykinin can rapidly increase intracellular Ca²⁺ levels in GT1–7 cells. A role for intracellular Ca²⁺ in bradykinin action was further suggested by the finding that an intracellular calcium chelator, 1,2-bis(O-aminophenoxy)]ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester, significantly attenuated the effects of bradykinin on GnRH release. The elevation of intracellular calcium by bradykinin appears to be due to mobilization of calcium from the endoplasmic reticulum, as incubation of the Ca²⁺-adenosine triphosphatase inhibitor thapsigarin, which depletes endoplasmic reticulum Ca²⁺ stores, significantly attenuated bradykinin action on GnRH release. Extracellular calcium may also be involved in bradykinin action, as the L-type Ca²⁺ channel blockers verapamil and nifedipine had no effect on bradykinin-induced GnRH release, whereas the nonselective Ca²⁺ channel blocker, nickel chloride, attenuated bradykinin-induced GnRH release. Taken as a whole, these studies demonstrate that the bradykinin B₂ receptor is densely localized in key hypothalamic nuclei responsible for regulation of GnRH release, and that the mechanism of bradykinin stimulation of GnRH secretion involves activation of the phospholipase C signaling pathway, with a critical role implicated for calcium in bradykinin action in GT1–7 cells.

	Introduction

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WORK OVER THE past few decades has demonstrated that the hypothalamic releasing factor, GnRH, has a central and preeminent role in the control of reproduction due to its regulatory actions over gonadotropin secretion from the anterior pituitary (see Ref. 1 for review). GnRH is secreted from a specific group of neurosecretory neurons that are diffusely localized in the preoptic area (POA) and anterior hypothalamus (2). Abundant evidence has suggested that the activity of GnRH neurons is regulated by a complex of afferent inputs (2, 3, 4). Signals are transmitted by neurons synapsing either directly on GnRH neurons or indirectly via a multisynaptic pathway (2, 3, 4, 5, 6, 7).

In a recent study, we provided evidence that the neuropeptide, bradykinin, plays an important role in the control of GnRH secretion (8). Bradykinin-immunoreactive neurons were demonstrated in the organum vasculosum of the lamina terminalis (OVLT) and arcuate nucleus (ARC) of the rat, two key hypothalamic nuclei for the control of GnRH secretion. Exogenous bradykinin stimulated GnRH release from rat hypothalamic fragments and from immortalized GnRH (GT1–7) neurons in vitro, an effect found to be mediated through the bradykinin B₂ receptor (8). A physiological role for bradykinin in the LH surge was implicated based on the finding that central administration of a bradykinin B₂ receptor antagonist attenuated the steroid-induced LH surge in the ovariectomized rat (8). Western blot analysis revealed that the bradykinin B₂ receptor protein is present in the hypothalamus, but detailed information concerning its distribution in the rat hypothalamus is lacking. Likewise, although a role for protein kinase C was implicated in bradykinin action on GnRH secretion in our previous study (8), the precise signaling pathway used by bradykinin remained to be elucidated. To address the above deficits in our knowledge, the current studies were designed to determine the localization of the bradykinin B₂ receptor in the rat hypothalamus by immunostaining and to investigate the possible signaling mechanisms by which bradykinin stimulates GnRH release from GT1–7 cells.

	Materials and Methods

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Reagents and animals
All drugs were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. Adult female Sprague Dawley rats were obtained from Holtzman (Madison, WI) and were housed in air-conditioned rooms under a photoperiod of 14 h of light (lights on, 0500–1900 h). Food and water were provided ad libitum. All animal studies were approved by our institutional committee for the care and use of animals in research and education in accordance with the guidelines of the NIH and USDA.

Immunohistochemical studies
Adult, random cycling, female rats (n = 5) were given an overdose of sodium pentobarbital and perfused transcardially with 2% sodium nitrite in saline to flush out the blood, followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.2) to fix the brain. The brains were then removed and kept in the same fixative for 6 h, after which they were placed in 30% sucrose solution in 0.1 M PBS until they sank. Coronal sections (24 µm) ranging from the OVLT to the median eminence (ME), corresponding to plates 18–31 of the Paxinos and Watson atlas (9) were cut and processed immediately for immunohistochemistry.

The free floating sections were washed in PBS and then incubated for 30 min at room temperature with a mixture of 30% hydrogen peroxide and absolute methanol in PBS (1:1 in 8 vol PBS) to quench the endogenous peroxidase. The sections were then washed thoroughly in PBS followed by incubation in 10% normal goat serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 6 h at 4 C in a humidity chamber to reduce nonspecific binding. The serum was then drained off, followed by incubation of the sections with a commercially available monoclonal antibody to bradykinin B₂ receptor (mouse antibradykinin B₂ receptor IgG; 1:250 dilution; Transduction Laboratories, Inc., Lexington, KY) for 48 h at 4 C. The sections were extensively washed in PBS and then incubated with biotinylated goat antimouse IgG (1:200 dilution; Jackson ImmunoResearch Laboratories, Inc.) for 2 h at room temperature followed by a 1-h incubation in avidin-biotin complex (Vectastain, ABC kit, Vector Laboratories, Inc., Burlingame, CA). The chromogen reaction was developed with the diaminobenzidene peroxidase substrate kit (Vector Laboratories, Inc.). The sections were then washed in PBS, mounted onto glass slides, dehydrated by passing through a series of gradually increased concentration of alcohol solutions, and coverslipped with Permount (Fisher Scientific, Fairlawn, NJ). Controls were included in which the antibody was preabsorbed overnight with the bradykinin B₂ receptor C-terminal. Preabsorption abolished the staining, indicating specificity of the immunostaining.

Immortalized GnRH neuron cultures
The GnRH neuronal cell line (GT1–7) was provided by Dr. Pam Mellon (University of California, San Diego, CA) (10). GT1–7 cells from passages 15–20 were cultured in DMEM (Life Technologies, Inc.) supplemented with 5% FCS, 5% horse serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.). The cultures were maintained at 37 C in a water-saturated atmosphere with 5% CO₂, and the medium was replaced every 4 days. Approximately 200,000 cells were cultured in each well of a 24-well plate until they reached approximately 70–80% confluence; at which time, the medium was replaced with Krebs-Ringer bicarbonate buffer (KRB; pH 7.4) containing 20 µM bacitracin and 0.5% BSA. The cells were preincubated for 30 min in KRB medium, the medium was then replaced with medium containing vehicle or various test compounds, and the incubation was continued for another 30 min under the same conditions. Where indicated in the figure legends, a 15-min preincubation with inhibitors was also used unless otherwise stated. At the end of the 30-min incubation period, the medium was collected and stored at -20 C until assayed for GnRH. Stimulation with 56 mM KCl was also performed at the end of the experiments to determine cell viability.

GnRH RIA
The RIA of GnRH was performed as described previously by our laboratory (11). The first antibody was donated by Dr. M. Kelly (Oregon Primate Research Center, Beaverton, OR). The intra- and interassay variabilities were 10.2% and 17.6%, respectively. GnRH levels were expressed as picograms per well.

Intracellular Ca²⁺ measurement
Intracellular Ca²⁺ concentrations were determined by a method described previously by Brotto et al. (12, 13). GT1–7 cells were cultured in plastic dishes containing a glass coverslip (25-mm diameter and 0.16-mm thickness; Fisher Scientific) under the conditions described above, except a modified KRB buffer was used which was supplemented with HEPES (5 mM) and saturated with O₂, and the pH was brought to 7.4. After cell attachment to the coverslip, the coverslip was removed from the dish and put in a chamber, and the cells were gently washed three times with modified KRB buffer at room temperature. The cells were then incubated with 5 µM fura-2/AM (Molecular Probes, Inc., Eugene, OR) in 1 ml modified KRB buffer in the dark at 37 C in a rotating water bath. The cells were subsequently washed five times with modified KRB buffer without the dye and allowed to stand in the dark for 30 min to facilitate the deesterification of the dye. After the deesterification, the chamber was placed on the stage of an Olympus Corp. (IX-50, New Hyde Park, NY) inverted microscope. Fluorescence was measured with a microspectrofluorometer, projected onto a single cell via a x40 oil immersion objective. The fluorescent light was transmitted to a photomultiplier. The photomultiplier output (60 Hz) was digitized and stored in a microcomputer for data analysis. The fura-2 calcium transients were the ratio of fluorescence transients measured at 340 and 380 nm. The GT1–7 cells were illuminated with the excitation light for less than 10 min, and each coverslip was used for less than 1 h to reduce the possible effects of photobleaching and fura-2 leakage. At the end of the experiment, the GT1–7 cell was exposed to 10 µM ionomycin for the determination of the parameters R_max and ß_min, and the cell was then exposed to 25 mM EGTA to determine R_min and ß_max. Fura-2 calcium transients were then calibrated in terms of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) with the ratiometric procedure used by Grynkiewicz et al. (14), with the equation [Ca²⁺]_i = K_d x ß x (R - R_min)/(R_max - R), where K_d is the dissociation constant for fura-2 (224 nM), ß is the ratio of the fluorescence signal at 380 nm of a solution with high Ca²⁺ (ß_min) and low Ca²⁺ (ß_max), R is the ratio at 340/380 nm, R_min is the ratio at 340/380 when the cell is in the presence of EGTA (low Ca²⁺), and R_max is the ratio at 340/380 when the cell is in the presence of ionomycin (high Ca²⁺).

Statistical analysis
The results given in the study are expressed as the mean ± SEM. N = 5–6 per group for all experiments. The differences between groups were analyzed using one-way ANOVA, and comparisons between any two groups were made using the Student-Newman-Keuls multirange test. P < 0.05 was considered significant.

	Results

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Immunohistochemical localization of bradykinin B₂ receptor in the brain
To determine the localization of the bradykinin B₂ receptor in the brain, especially in the hypothalamus, rat brain sections ranging from the OVLT to the ME were immunostained for the bradykinin B₂ receptor using a monoclonal antibody raised against the C-terminal of the bradykinin B₂ receptor. As shown in Fig. 1, the bradykinin B₂ receptor was demonstrated to be densely localized in the ARC/ME (Fig. 1A), OVLT (Fig. 1C), and POA (Fig. 1D), regions known to be critical for the control of GnRH secretion. Dense immunostaining was also observed in the supraoptic nucleus (Fig. 1E), hippocampus (Fig. 1F), and cortex (data not shown). The bradykinin B₂ receptor immunostaining was predominately cellular, except in the ME, where there was moderately dense staining of neurofibers. The bradykinin B₂ receptor immunostaining was specific, because preadsorption with the bradykinin B₂ receptor C-terminal peptide (ERQIHKLQDWAGSRQ), which was used to generate the bradykinin B₂ receptor antibody, blocked the staining (Fig. 1B). Substitution of PBS for the primary antibody also completely abolished the staining (data not shown).

View larger version (165K): [in this window] [in a new window]   Figure 1. Immunohistochemical localization of the bradykinin B2 receptor in the brain of the random cycling female rat. Dense immunostaining for bradykinin B2 receptor was observed in ARC/ME (A), OVLT (C), POA (D), supraoptic nucleus (SOP; E), and hippocampus (F). Preadsorption of the antibody with the bradykinin B2 receptor C-terminal peptide resulted in a loss of staining (B). The results are from a representative animal. A total of five animals was used. Magnification: A, C, D, and E, x200; B and F, x100. V, Third ventricle; OX, optic chiasm; HP, hippocampus.

View larger version (37K): [in this window] [in a new window]   Figure 2. Effect of a NO synthase inhibitor (L-NMMA) on bradykinin-induced GnRH release from immortalized GnRH neurons. The cells were preincubated with either vehicle or 100 µM L-NMMA for 15 min, the medium was removed, and an incubation was then performed with vehicle, 50 µM bradykinin (BK), or L-NMMA alone or with bradykinin for 30 min. Groups with different subscripts are significantly different (P < 0.05). NOS, NO synthase.

View larger version (34K): [in this window] [in a new window]   Figure 3. Effect of a PG synthesis inhibitor (indomethacin) on bradykinin-induced GnRH release from immortalized GnRH neurons. The cells were preincubated with either vehicle or 100 µM indomethacin for 15 min, the medium was removed, and an incubation was then performed with vehicle, 50 µM bradykinin (BK), or indomethacin alone or with bradykinin for 30 min. Groups with different subscripts are significantly different (P < 0.05).

View larger version (38K): [in this window] [in a new window]   Figure 4. Effect of a PLC inhibitor (U-73122) on bradykinin-induced GnRH release from immortalized GnRH neurons. The cells were preincubated with either vehicle or 1 µM U-73122 for 15 min, the medium was removed, and an incubation was then performed with vehicle, 20 µM bradykinin (BK), or U-73122 alone or with bradykinin for 30 min. Groups with different subscripts are significantly different (P < 0.05).

View larger version (38K): [in this window] [in a new window]   Figure 5. Effect of pertussis toxin on bradykinin-induced GnRH release from immortalized GnRH neurons. The cells were preincubated with either vehicle or 200 ng/ml for 24 h. The medium was then removed, and an incubation was performed with vehicle, 50 µM bradykinin (BK), or pertussis toxin alone or with bradykinin for 30 min. Groups with different subscripts are significantly different (P < 0.05). PT, Pertussis toxin.

View larger version (33K): [in this window] [in a new window]   Figure 6. Effect of an intracellular Ca2+ chelator (BAPTA/AM) on bradykinin-induced GnRH release from immortalized GnRH neurons. The cells were preincubated with either vehicle or 100 µM BAPTA/AM for 15 min, the medium was removed, and an incubation was then performed with vehicle, 50 µM bradykinin (BK), or BAPTA/AM alone or with bradykinin for 30 min. Groups with different subscripts are significantly different (P < 0.05).

View larger version (30K): [in this window] [in a new window]   Figure 7. Effect of an ER Ca2+-ATPase inhibitor (thapsigargin) on bradykinin-induced GnRH release from immortalized GnRH neurons. The cells were preincubated with either vehicle or 100 nM thapsigargin for 15 min, the medium was removed, and an incubation was then performed with vehicle, 50 µM bradykinin (BK), or thapsigargin alone or with bradykinin for 30 min. Groups with different subscripts are significantly different (P < 0.05).

View larger version (81K): [in this window] [in a new window]   Figure 8. Effect of L-type Ca2+ channel blockers (verapamil and nifedipine) and a nonselective Ca2+ channel blocker (NiCl2) on bradykinin-induced GnRH release from immortalized GnRH neurons. The cells were preincubated with either vehicle or verapamil (ver; 10 and 100 µM), nifedipine (nif; 10 and 100 µM), and 50 µM NiCl2 for 15 min; the medium was removed; and an incubation was then performed with vehicle, 50 µM bradykinin (BK), or verapamil or with nifedipine or NiCl2 alone or with bradykinin for 30 min. Groups with different subscripts are significantly different (P < 0.05).

View larger version (22K): [in this window] [in a new window]   Figure 9. Effect of bradykinin on intracellular Ca2+ levels in immortalized GnRH neurons. The cells on glass coverslips were first loaded with 5 µM fura-2/AM, a Ca2+ indicator, for 15 min in the dark and then were subjected to bradykinin treatment (1 µM) at the time point indicated. Fluorescence from a single cell was monitored and recorded. The intracellular Ca2+ concentration is calculated from the equation described in Materials and Methods. A, Ca2+ transients in a GT1–7 cell induced by 1 µM bradykinin. B, Comparison of basal Ca2+ levels before 1 µM bradykinin treatment to peak magnitude of Ca2+ levels after bradykinin treatment. *, P < 0.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the literature, the bradykinin B₂ receptor has been localized to specific nervous nuclei only in the sheep and guinea pig (15, 16). To our knowledge, our study presents for the first time information about the distribution of the bradykinin B₂ receptor in the hypothalamus and brain of the rat. The overall pattern of the localization of the receptor in the rat central nervous system (CNS) resembles that observed in the sheep (15). In the sheep, the bradykinin B₂ receptor is localized throughout the CNS with different levels of density. Moderate levels of the receptor were detected in the OVLT and ME of the sheep and in the cortex and hypothalamus. The bradykinin B₂ receptor was demonstrated to be localized in the guinea pig hippocampus, cortex, and hypothalamus, but no specific hypothalamic nuclei localization was demonstrated (16).

The localization of the bradykinin B₂ receptor in the supraoptic nucleus, hippocampus, and cortex of the rat suggests that bradykinin may have actions in the CNS in addition to the regulation of GnRH secretion. Along these lines, intraventricular bradykinin injection has been reported to enhance vasopressin secretion (17) and induce diuretic effects (18), functions that the supraoptic nucleus regulates. With respect to possible functions of the bradykinin B₂ receptor in the hippocampus and cortex, central injection of bradykinin enhances memory formation in the 2-day-old chick (19), whereas administration of an inhibitor of bradykinin degradation also enhances memory and learning in the rat (20). Thus, bradykinin and its receptor may act in the hippocampus and cerebral cortex to regulate learning and memory. This possibility is under further investigation by our laboratory.

With respect to the mechanism of action of bradykinin in the control of GnRH secretion, the present study provides evidence for an important role of calcium. Direct measurement of calcium levels in GT1–7 cells demonstrated that bradykinin rapidly, potently, and transiently increases intracellular calcium levels. This calcium response to bradykinin in our cell system is quite similar to that observed in many other cell systems (21, 22, 23). The elevated calcium levels in response to bradykinin appear to be due to the release of calcium from intracellular calcium stores as well as mobilization of calcium from extracellular stores. The extracellular calcium appears to enter through nonselective calcium channels, as L-type calcium channel blockers had no effect on bradykinin-induced GnRH release, whereas a nonselective calcium channel blocker attenuated bradykinin-induced GnRH release. It is unclear what sequence initiates extracellular calcium entry, but it could be due to IP₄, a metabolite of IP₃, as IP₄ has been reported to induce calcium influx (24).

Intracellular calcium release from the ER also appears to be involved in mediating bradykinin-induced GnRH release, as an intracellular calcium chelator (BAPTA/AM) and a calcium-ATPase inhibitor (thapsigargin) both attenuated bradykinin-induced GnRH release. Thapsigargin has been reported to exert a stimulatory effect on hormone/peptide secretion in some systems, which could complicate interpretations, as it could deplete the ready releasable pool of the hormone/peptide. However, we observed no effect of thapsigargin alone on basal GnRH release, so it is unlikely that the ready releasable pool of GnRH was depleted in our studies. The stimulus for the release of calcium release from the ER by bradykinin most likely starts with bradykinin-induced activation of PLC, as evidenced by attenuation of the bradykinin-induced GnRH release by a PLC inhibitor (U-71322). The PLC messengers IP₃ and diacylgycerol then act to stimulate calcium release from the ER and to activate PKC, respectively. Some caution has to be used in interpreting the U-71322 results, as it may have other actions, such as to stimulate intracellular calcium release, potentiate IP₃-mediated calcium release, and directly activate ion channels. However, we did not observe any effect of U-71322 on basal GnRH release in our system. Nevertheless, one cannot totally exclude other possible modes of action of U-71322 other than the intended one of direct inhibition of PLC. Clarification of this issue awaits the development of better, more specific, and potent PLC inhibitors.

Previous studies have shown that the secretion rate of secretory granules is proportional to intracellular calcium levels (25, 26). Our finding that bradykinin-induced GnRH release is a calcium-dependent event is consistent with this observation. Likewise, this calcium-dependent mechanism of bradykinin action is quite similar to that of endothelin (27), glutamate (28, 29), and -aminobutyric acid (29) in the stimulation of GnRH release. However, the channels by which calcium enters into the cells are different. For instance, glutamate and -aminobutyric acid use L-type calcium channels, whereas bradykinin appears to use non-L-type calcium channels. The reason for this difference is as yet unclear and deserves further investigation.

Although a role for the PLC signaling pathway was implicated in bradykinin-induced GnRH release, no evidence was found to support involvement of NO or PGs in bradykinin action. Bradykinin had been reported to stimulate PG and NO production in other cell systems (30), and bradykinin itself yields arginine upon metabolism, which could serve as a substrate for NO production by the enzyme NO synthase. However, treatment with N-methyl-L-arginine, a NO synthase inhibitor, had no effect on bradykinin-induced GnRH release. In other experiments, a 100-µM dose of another NO synthase inhibitor, N^G-nitro-L-arginine, also had no effect on bradykinin stimulation of GnRH release (data not shown). Thus, NO does not appear to mediate the effects of bradykinin on GnRH release. Likewise, a cyclooxygenase inhibitor (indomethacin) had no effect on bradykinin-induced GnRH secretion, suggesting that PGs also are not involved in the mediation of bradykinin action on GnRH release. Furthermore, using a pertussis toxin protocol previously shown to block acetylcholine effects mediated by inhibitory G proteins in GT1–7 cells (31), we observed no effect of 100 or 200 nM pertussis toxin on bradykinin-induced GnRH release. This suggests that the bradykinin B₂ receptor may be coupled to a pertussis toxin-insensitive G protein in GT1–7 cells.

Finally, it should be mentioned that the K_d of the bradykinin B₂ receptor is in the low nanomolar range. Effective concentrations for stimulation of GnRH release by bradykinin range from 1–50 µM in this and previous studies using GT1–7 cells and hypothalamic fragments (8). As bradykinin has a half-life of only seconds due to active metabolism by converting enzymes, only a small fraction of the applied bradykinin may actually reach and interact with the receptor. Nevertheless, it is difficult to conclude whether the results are physiological, as bradykinin was applied exogenous without any determination of the role of endogenous bradykinin, and our studies used an artificial in vitro model (GT1–7 cells). In a previous study, we did address this issue and demonstrated a potential physiological role for endogenous bradykinin in the steroid-induced LH surge, as central (third ventricle) administration of a specific bradykinin B₂ receptor antagonist significantly attenuated the LH surge, whereas a bradykinin B₁ receptor antagonist had no effect (8). Furthermore, bradykinin also stimulated GnRH release from male and female hypothalami in vitro (8). Thus, endogenous bradykinin appears to have a physiological role in the control of GnRH and LH release in the rat.

In summary, the present study demonstrates that the bradykinin B₂ receptor is strategically localized in hypothalamic nuclei critical for the control of GnRH secretion. Bradykinin stimulation of GnRH secretion from GT1–7 cells appears to be mediated by the PLC pathway. IP₃ evokes calcium release from the ER, and simultaneously, extracellular calcium enters into cells through non-L-type calcium channels by as yet unknown mechanisms. Collectively, these two sources of calcium dramatically increase cytoplasmic calcium levels, which facilitates the exocytosis of GnRH.

	Footnotes

 
¹ This work was supported by Research Grant HD-28964 from NICHHD, NIH.

Received February 26, 1999.

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