Gonadotropin-releasing hormone receptor signaling: biased and unbiased.

Gonadotropin-releasing hormone is a neuropeptide that acts via Gq coupled G-protein coupled receptors in the pituitary that mediate central control of reproduction. GnRH receptors (GnRHR) and GnRH ligands are also found in extra-pituitary sites including the CNS as well as reproductive tissues and cancer cells derived from such tissues. Much of the interest in the extra-pituitary receptors stems from the fact that they mediate anti-proliferative and/or pro-apoptotic effects and may therefore be directly targeted for cancer therapy. Type I mammalian GnRHR are atypical in that they do not bind to (or signal via) arrestins. In spite of this restriction on their signaling repertoire, there is good evidence for existence of multiple active GnRHR conformations and for activation of multiple upstream effectors (heterotrimeric and monomeric G-proteins). In this review GnRHR signaling is described, with emphasis on the relevance of functional selectivity for pharmacological characterization of GnRHR ligands, as well as its possible contribution to contextdependent GnRHR signaling and relevance for GnRHR-mediated effects on cell fate as well as GnRHR trafficking.

Using automated imaging to interrogate gonadotrophin-releasing hormone receptor trafficking and function.

Gonadotrophin-releasing hormone (GnRH) acts via seven transmembrane receptors on gonadotrophs to stimulate gonadotrophin synthesis and secretion, and thereby mediates central control of reproduction. Type I mammalian GnRHR are unique, in that they lack C-terminal tails. This is thought to underlie their resistance to rapid homologous desensitisation as well as their slow rate of internalisation and inability to provoke G-protein-independent (arrestin-mediated) signalling. More recently it has been discovered that the vast majority of human GnRHR are actually intracellular, in spite of the fact that they are activated at the cell surface by a membrane impermeant peptide hormone. This apparently reflects inefficient exit from the endoplasmic reticulum and again, the absence of the C-tail likely contributes to their intracellular localisation. This review is intended to cover some of these novel aspects of GnRHR biology, focusing on ways that we have used automated fluorescence microscopy (high content imaging) to explore GnRHR localisation and trafficking as well as spatial and temporal aspects of GnRH signalling via the Ca(2+)/calmodulin/calcineurin/NFAT and Raf/MEK/ERK pathways.

Encoding and decoding mechanisms of pulsatile hormone secretion.

Ultradian pulsatile hormone secretion underlies the activity of most neuroendocrine systems, including the hypothalamic-pituitary adrenal (HPA) and gonadal (HPG) axes, and this pulsatile mode of signalling permits the encoding of information through both amplitude and frequency modulation. In the HPA axis, glucocorticoid pulse amplitude increases in anticipation of waking, and, in the HPG axis, changing gonadotrophin-releasing hormone pulse frequency is the primary means by which the body alters its reproductive status during development (i.e. puberty). The prevalence of hormone pulsatility raises two crucial questions: how are ultradian pulses encoded (or generated) by these systems, and how are these pulses decoded (or interpreted) at their target sites? We have looked at mechanisms within the HPA axis responsible for encoding the pulsatile mode of glucocorticoid signalling that we observe in vivo. We review evidence regarding the 'hypothalamic pulse generator' hypothesis, and describe an alternative model for pulse generation, which involves steroid feedback-dependent endogenous rhythmic activity throughout the HPA axis. We consider the decoding of hormone pulsatility by taking the HPG axis as a model system and focussing on molecular mechanisms of frequency decoding by pituitary gonadotrophs.

Trafficking and signalling of gonadotrophin-releasing hormone receptors: an automated imaging approach.

Gonadotrophin-releasing hormone (GnRH) is a neuropeptide that mediates central control of reproduction by stimulating gonadotrophin secretion from the pituitary. It acts via 7 transmembrane region (7TM) receptors that lack C-terminal tails, regions that for many 7TM receptors, are necessary for agonist-induced phosphorylation and arrestin binding as well as arrestin-dependent desensitization, internalization and signalling. Recent work has revealed that human GnRH receptors (GnRHR) are poorly expressed at the cell surface. This apparently reflects inefficient exit from the endoplasmic reticulum, which is thought to be increased by pharmacological chaperones (non-peptide GnRHR antagonists that increase cell surface GnRHR expression) or reduced by point mutations that further impair GnRHR trafficking and thereby cause infertility. Here, we review recent work in this field, with emphasis on the use of semi-automated imaging to interrogate compartmentalization and trafficking of these unique 7TM receptors.

Involvement of PKC alpha and G-protein-coupled receptor kinase 2 in agonist-selective desensitization of mu-opioid receptors in mature brain neurons.

BACKGROUND AND PURPOSE: The ability of an agonist to induce desensitization of the mu-opioid receptor (MOR) depends upon the agonist used. Furthermore, previous data suggest that the intracellular mechanisms underlying desensitization may be agonist-specific. We investigated the mechanisms underlying MOR desensitization, in adult mammalian neurons, caused by morphine (a partial agonist in this system) and DAMGO (a high-efficacy agonist). EXPERIMENTAL APPROACH: MOR function was measured in locus coeruleus neurons, by using whole-cell patch-clamp electrophysiology, in rat and mouse brain slices (both wild-type and protein kinase C (PKC)alpha knockout mice). Specific isoforms of PKC were inhibited by using inhibitors of the receptors for activated C-kinase (RACK), and in vivo viral-mediated gene-transfer was used to transfect neurons with dominant negative mutants (DNMs) of specific G-protein-coupled receptor kinases (GRKs). KEY RESULTS: Morphine-induced desensitization was attenuated by using RACK inhibitors that inhibit PKCalpha, but not by other isoform-specific inhibitors. Further, the PKC component of morphine-induced desensitization was absent in locus coeruleus neurons from PKCalpha knockout mice. The PKC-enhanced morphine-induced desensitization was not affected by over-expression of a GRK2 dominant negative mutant (GRK2 DNM). In contrast, DAMGO-induced MOR desensitization was independent of PKC activity but was reduced by over-expression of the GRK2 DNM but not by that of a GRK6 DNM. CONCLUSIONS AND IMPLICATIONS: In mature mammalian neurons, different MOR agonists can induce MOR desensitization by different mechanisms, morphine by a PKCalpha-mediated, heterologous mechanism and DAMGO by a GRK-mediated, homologous mechanism. These data represent functional selectivity at the level of receptor desensitization.

Internalization and desensitization of the oxytocin receptor is inhibited by Dynamin and clathrin mutants in human embryonic kidney 293 cells.

Oxytocin (OT) has long been used as an uterotonic during labor management in women, and yet responses to OT infusion remain variable and unpredictable among patients. The investigation of oxytocin receptor (OTR) regulation will benefit labor management, because the clinical practice of continuous iv infusion of OT is not optimal. As with other G protein-coupled receptors, it is likely that the OTR internalizes and/or desensitizes upon continuous agonist exposure. The mechanisms by which this might occur, however, are unclear. Here we explore OTR internalization and desensitization in human embryonic kidney cells by utilizing inhibitors of heterologous second messenger systems and recently available mutant cDNA constructs. We report rapid and extensive internalization and desensitization of the OTR upon agonist exposure. Internalization was unaffected by inhibitors of protein kinase C or Ca(2+) calmodulin-dependant kinase II but was significantly reduced after transfection with dominant-negative mutant cDNAs of G protein-coupled receptor kinase 2, beta-Arrestin2, Dynamin, and Eps15 (a component of clathrin-coated pits). Moreover, desensitization of the OTR, measured by a calcium mobilization assay, was also inhibited by the aforementioned cDNA constructs. Thus, our data demonstrate, for the first time, the importance of the classical clathrin-mediated pathway during agonist-induced OTR internalization and desensitization.

Signalling and anti-proliferative effects mediated by gonadotrophin-releasing hormone receptors after expression in prostate cancer cells using recombinant adenovirus.

Gonadotrophin-releasing hormone receptors (GnRH-Rs) are found in cancers of reproductive tissues, including those of the prostate, and gonadotrophin-releasing hormone (GnRH) can inhibit growth of cell lines derived from such cancers. Although pituitary and extra-pituitary GnRH-R transcripts appear identical, their functional characteristics may differ. Most extra-pituitary GnRH-Rs have low affinity for GnRH analogues and may not activate phospholipase C or discriminate between agonists and antagonists in the same way as do pituitary GnRH-Rs. Here we have assessed whether GnRH-Rs expressed exogenously in prostate cancer cells differ functionally from those of gonadotrophs. We found no evidence for endogenous GnRH-Rs in PC3 cells, but after infection with adenovirus expressing the GnRH-R (Ad GnRH-R) at 10 plaque forming units (p.f.u.)/cell or greater, at least 80% of the cells expressed GnRH-Rs. These sites had high affinity (K(d )for [(125)I]Buserelin 1.1+/-0.4 nM) and specificity (rank order of potency: Buserelin> GnRH>>chicken (c) GnRH-II), and mediated stimulation of [(3)H]inositol phosphate (IP) accumulation. Increasing viral titre from 3 to 300 p.f.u./cell increased receptor number (2000 to 275 000 sites/cell respectively) and [(3)H]IP responses. GnRH also caused a biphasic increase in the cytoplasmic Ca(2+) concentration in Ad GnRH-R-infected cells but not in control cells. Mobilization of Ca(2+) from intracellular stores contributed to the spike phase of this response whereas the plateau phase was dependent upon Ca(2+) entry across the plasma membrane. This effect on Ca(2+) and stimulation of [(3)H]IP accumulation were both blocked by the GnRH-R antagonist, Cetrorelix. In addition, GnRH reduced cell number (as measured in MTT activity assays) and DNA synthesis (as measured by [(3)H]thymidine incorporation) in Ad GnRH-R-infected cells (but not in control cells). This effect was mimicked by agonist analogues and inhibited by two antagonists. Thus, when exogenous GnRH-Rs are expressed at a density comparable to that in gonadotrophs, they are functionally indistinguishable from the endogenous GnRH-Rs in gonadotrophs. Moreover, expression of high affinity GnRH-Rs can facilitate a direct anti-proliferative effect of GnRH agonists on prostate cancer cells.

The gonadotrophin-releasing hormone receptor: signalling, cycling and desensitisation.

Sustained stimulation of G-protein coupled receptors (GPCRs) typically causes receptor desensitisation that is mediated by phosphorylation, often within the C-terminal tail of the receptor. The consequent binding of beta-arrestin not only prevents the receptor from activating its G-protein (causing desensitisation) but can also target it for internalisation via clathrin-coated vesicles and can mediate signalling to proteins regulating endocytosis and mitogen-activated protein kinase (MAPK) cascades. GnRH acts via phospholipase C coupled GPCRs on pituitary gonadotrophs. The type I GnRH-receptors (GnRH-Rs) found only in mammals, are unique in that they lack C-terminal tails and apparently do not undergo agonist-induced phosphorylation or bind beta-arrestin. They are therefore resistant to receptor desensitisation and internalise slowly. In contrast, the type II GnRH-Rs, found in numerous vertebrates, possess such tails and show rapid desensitisation and internalisation with concomitant receptor phosphorylation (within the C-terminal tails) and/or binding of beta-arrestin. The binding to beta-arrestin may also be important for association with dynamin, a GTPase that controls cleavage of endosomes from the plasma membrane. Using recombinant adenovirus to express GnRH-R, we have found that blockade of dynamin-dependent endocytosis inhibits internalisation of type II (Xenopus) GnRH-Rs but not type I (human) GnRH-Rs, revealing the existence of functionally distinct routes through which these receptors are internalised. Although type I GnRH-R do not rapidly desensitise, sustained activation of GnRH receptors does cause desensitisation of gonadotrophin secretion, an effect which must therefore involve adaptive responses distal to the receptor. One such response is the GnRH-induced down regulation of inositol 1, 4, 5 trisphosphate receptors that apparently underlies desensitisation of Ca2+ mobilisation in a gonadotroph-derived cell line. Although activation of other GPCRs can down-regulate inositol 1, 4, 5 trisphosphate receptors, the effect of GnRH is atypically rapid and pronounced, presumably because of the receptor's atypical resistance to desensitisation. GnRH-Rs are also expressed in several extra-pituitary sites and these may mediate direct inhibition of proliferation of hormone-dependent cancer cells. Infection with type I GnRH-R expressing adenovirus facilitated expression of high affinity, PLC-coupled GnRH-R in mammary and prostate cancer cells and these mediated pronounced antiproliferative effects of receptor agonists. No such effect was seen in cells transfected with a type II GnRH-R, implying that it is mediated most efficiently by a non-desensitising receptor. Thus it appears that the GnRH-Rs have undergone a period of rapidly accelerated molecular evolution that is of functional relevance to GnRH-R signalling in pituitary and extra-pituitary sites.

The circus in the pituitary.

Actions and Interactions at the Pituitary was a Satellite Meeting of the 34th Congress of the International Union of Physiological Sciences held in Christchurch, New Zealand from 24-25 August 2001.

Signaling and antiproliferative effects mediated by GnRH receptors after expression in breast cancer cells using recombinant adenovirus.

GnRH receptors (GnRH-Rs) are found in human cancers, including those of the breast, and GnRH can inhibit the growth of cell lines derived from such cancers. Although pituitary and extrapituitary GnRH-R transcripts appear identical, their functional characteristics may differ. Most extrapituitary GnRH-Rs have low affinity for GnRH analogs and may not activate PLC or discriminate between agonists and antagonists in the same way as pituitary GnRH-Rs. Here we have assessed whether GnRH-Rs expressed exogenously in breast cancer cells differ from those in gonadotropes. We found no evidence for endogenous GnRH-Rs in MCF7 cells, but after infection with adenovirus expressing the GnRH-R (Ad GnRH-R) at a multiplicity of infection of 10 or greater, at least 80% expressed GnRH-Rs. These had high affinity (K(d) for [(125)I]buserelin, 1.4 nM) and specificity (rank order of potency, buserelin>GnRH>>chicken GnRH-II) and mediated stimulation of [(3)H]IP accumulation. Increasing viral titer [from multiplicity of infection, 3-300] increased receptor number (10,000-225,000 sites/cell) and [(3)H]IP responses. GnRH stimulated ERK2 phosphorylation in Ad GnRH-R-infected cells, and this effect, like stimulation of [(3)H]IP accumulation, was blocked by GnRH-R antagonists. GnRH also inhibited [(3)H]thymidine incorporation into Ad GnRH-R-infected cells (but not control cells). This effect was mimicked by agonist analogs and inhibited by two antagonists. Thus, when exogenous GnRH-Rs are expressed at density comparable to that in gonadotropes, they are functionally indistinguishable from the endogenous GnRH-Rs in gonadotropes, and increasing expression of high affinity GnRH-Rs can dramatically enhance the direct antiproliferative effect of GnRH agonists on breast cancer cells.

Differential internalization of mammalian and non-mammalian gonadotropin-releasing hormone receptors. Uncoupling of dynamin-dependent internalization from mitogen-activated protein kinase signaling.

Desensitization and internalization of G-protein-coupled receptors can reflect receptor phosphorylation-dependent binding of beta-arrestin, which prevents G-protein activation and targets receptors for internalization via clathrin-coated vesicles. These can be pinched off by a dynamin collar, and proteins controlling receptor internalization can also mediate mitogen-activated protein kinase signaling. Gonadotropin-releasing hormone (GnRH) stimulates internalization of its receptors via clathrin-coated vesicles. Mammalian GnRH receptors (GnRH-Rs) are unique in that they lack C-terminal tails and do not rapidly desensitize, whereas non-mammalian GnRH-R have C-terminal tails and, where investigated, do rapidly desensitize and internalize. Using recombinant adenovirus expressing human and Xenopus GnRH-Rs we have explored the relationship between receptor internalization and mitogen-activated protein kinase signaling in HeLa cells with regulated tetracycline-controlled expression of wild-type or a dominant negative mutant (K44A) of dynamin. These receptors were phospholipase C-coupled and had appropriate ligand affinity and specificity. K44A dynamin expression did not alter human GnRH-R internalization but dramatically reduced internalization of Xenopus GnRH-R (and epidermal growth factor (EGF) receptor). Blockade of clathrin-mediated internalization (sucrose) abolished internalization of all three receptors. Both GnRH-Rs also mediated phosphorylation of ERK 2 and for both receptors, this was inhibited by K44A dynamin. The same was true for EGF- and protein kinase C-mediated ERK 2 phosphorylation. ERK 2 phosphorylation was also inhibited by a protein kinase C inhibitor but not affected by an EGF receptor tyrosine kinase inhibitor. We conclude that a) desensitizing and non-desensitizing GnRH-Rs are targeted for clathrin-coated vesicle-mediated internalization by functionally distinct mechanisms, b) GnRH-R signaling to ERK 2 is dynamin-dependent and c) this does not reflect a dependence on dynamin-dependent GnRH-R internalization.

ATP-mediated killing of Mycobacterium bovis bacille Calmette-Guérin within human macrophages is calcium dependent and associated with the acidification of mycobacteria-containing phagosomes.

We previously demonstrated that extracellular ATP stimulated macrophage death and mycobacterial killing within Mycobacterium bovis Bacille Calmette-Guérin (BCG)-infected human macrophages. ATP increases the cytosolic Ca(2+) concentration in macrophages by mobilizing intracellular Ca(2+) via G protein-coupled P2Y receptors, or promoting the influx of extracellular Ca(2+) via P2X purinoceptors. The relative contribution of these receptors and Ca(2+) sources to ATP-stimulated macrophage death and mycobacterial killing was investigated. We demonstrate that 1) ATP mobilizes Ca(2+) in UTP-desensitized macrophages (in Ca(2+)-free medium) and 2) UTP but not ATP fails to deplete the intracellular Ca(2+) store, suggesting that the pharmacological properties of ATP and UTP differ, and that a Ca(2+)-mobilizing P2Y purinoceptor in addition to the P2Y(2) subtype is expressed on human macrophages. ATP and the Ca(2+) ionophore, ionomycin, promoted macrophage death and BCG killing, but ionomycin-mediated macrophage death was inhibited whereas BCG killing was largely retained in Ca(2+)-free medium. Pretreatment of cells with thapsigargin (which depletes inositol (1,4,5)-trisphosphate-mobilizable intracellular stores) or 1,2-bis-(2-aminophenoxy)ethane-N, N, N',N'-tetraacetic acid acetoxymethyl ester (an intracellular Ca(2+) chelator) failed to inhibit ATP-stimulated macrophage death but blocked mycobacterial killing. Using the acidotropic molecular probe, 3-(2,4-dinitroanilino)-3'-amino-N-methyl dipropylamine, it was revealed that ATP stimulation promoted the acidification of BCG-containing phagosomes within human macrophages, and this effect was similarly dependent upon Ca(2+) mobilization from intracellular stores. We conclude that the cytotoxic and bactericidal effects of ATP can be uncoupled and that BCG killing is not the inevitable consequence of death of the host macrophage.

Rapid down-regulation of the type I inositol 1,4,5-trisphosphate receptor and desensitization of gonadotropin-releasing hormone-mediated Ca2+ responses in alpha T3-1 gonadotropes.

Despite no evidence for desensitization of phospholipase C-coupled gonadotropin-releasing hormone (GnRH) receptors, we previously reported marked suppression of GnRH-mediated Ca(2+) responses in alphaT3-1 cells by pre-exposure to GnRH. This suppression could not be accounted for solely by reduced inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) responses, thereby implicating uncoupling of Ins(1,4,5)P(3) production and Ca(2+) mobilization (McArdle, C. A., Willars, G. B., Fowkes, R. C., Nahorski, S. R., Davidson, J. S., and Forrest-Owen, W. (1996) J. Biol. Chem. 271, 23711-23717). In the current study we demonstrate that GnRH causes a homologous and heterologous desensitization of Ca(2+) signaling in alphaT3-1 cells that is coincident with a rapid (t((12)) < 20 min), marked, and functionally relevant loss of type I Ins(1,4,5)P(3) receptor immunoreactivity and binding. Furthermore, using an alphaT3-1 cell line expressing recombinant muscarinic M(3) receptors we show that the unique resistance of the GnRH receptor to rapid desensitization contributes to a fast, profound, and sustained loss of Ins(1,4,5)P(3) receptor immunoreactivity. These data highlight a potential role for rapid Ins(1,4,5)P(3) receptor down-regulation in homologous and heterologous desensitization and in particular suggest that this mechanism may contribute to the suppression of the reproductive system that is exploited in the major clinical applications of GnRH analogues.

Desensitization and internalization of human and xenopus gonadotropin-releasing hormone receptors expressed in alphaT4 pituitary cells using recombinant adenovirus.

Nonmammalian vertebrates express at least two forms of GnRH and distinct forms of GnRH receptor (GnRH-R) have coevolved with their ligands. Mammalian and nonmammalian GnRH-R have key structural differences (notably the lack of C-terminal tails in mammalian GnRH-R) and comparative studies are beginning to reveal their functional relevance. However, cellular context and receptor density influence G protein-coupled receptor function and may be important variables in such work using heterologous expression systems. Here we report a comparative study using alphaT4 cells (gonadotrope progenitors that lack endogenous GnRH-R) transfected with a mammalian (human) or nonmammalian (Xenopus laevis type I) GnRH-R. Because conventional transfection strategies proved inefficient, recombinant adenovirus expressing these receptors were constructed, enabling controlled and efficient GnRH-R expression. When expressed in alphaT4 cells at physiological density, these GnRH-Rs retain the pharmacology of their endogenous counterparts (as judged by ligand specificity in radioligand binding and inositol phosphate accumulation assays) but do not activate adenylyl cyclase and are not constitutively active. Moreover, the Xenopus GnRH-R rapidly desensitizes and internalizes in these cells, whereas the human GnRH-R does not, and the internalization rates are not dependent upon receptor number. These data extend studies in COS, HEK, and GH3 cells showing that other GnRH-R with C-terminal tails desensitize and internalize rapidly, whereas tail-less mammalian GnRH-R do not. Retention of these distinctions at physiological receptor density in gonadotrope lineage cells, supports the argument that the evolution of nondesensitizing mammalian GnRH-Rs is functionally relevant and related to the development of mammalian reproductive strategies.

C-type natriuretic peptide: an important neuroendocrine regulator?

In the decade since its discovery, C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, has been shown to be produced by most of the major endocrine glands, including the hypothalamus and anterior pituitary. The relative abundance of its guanylyl cyclase-containing GC-B receptor in these glands suggests that CNP might be a local neuroendocrine regulator. Here, we review this possibility, emphasizing signalling and integration with other regulatory systems in the neuroendocrine control of reproduction.

C-type natriuretic peptide (CNP) effects in anterior pituitary cell lines: evidence for homologous desensitisation of CNP-stimulated cGMP accumulation in alpha T3-1 gonadotroph-derived cells.

C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, has been found at its highest tissue concentrations in the anterior pituitary, where it is localised in gonadotrophs. Its specific guanylyl cyclase-containing receptor, GC-B, is also expressed on several anterior pituitary cell types, and CNP potently stimulates cGMP accumulation in rat pituitary cell cultures and pituitary cell lines. The mouse gonadotroph-derived alpha T3-1 cell line has been shown to express CNP as well as GC-B (but not GC-A) receptors, suggesting that CNP may well be an autocrine regulator of gonadotrophs. Comparing effects of three natriuretic peptides (atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and CNP) on cGMP accumulation in four pituitary cell lines (alpha T3-1, TtT-GF, AtT-20 and GH(3)) we find that CNP is most potent and effective in alpha T3-1 cells. In these cells, CNP-stimulated cGMP accumulation was found to desensitise during a 30 min exposure to CNP. Pretreatment with CNP for up to 6 h also caused a significant reduction in the ability of CNP to subsequently stimulate cGMP accumulation. This effect was receptor specific, because pretreatment with sodium nitroprusside (an activator of nitric oxide-sensitive guanylyl cyclase), or with ANP or BNP, did not cause desensitisation of CNP-stimulated cGMP accumulation. Protein kinase C activation with phorbol esters also inhibited CNP-stimulated cGMP accumulation and such inhibition was also seen in cells desensitised by pretreatment with CNP. Thus it appears that the endogenous GC-B receptors of alpha T3-1 cells are subject to both homologous and heterologous desensitisation, that the mechanisms underlying these forms of desensitisation are distinct, and that cGMP elevation alone is insufficient to desensitise GC-B receptors.

GABAA receptor mediated elevation of Ca2+ and modulation of gonadotrophin-releasing hormone action in alphaT3-1 gonadotropes.

gamma-amino butyric acid (GABA) is the major inhibitory neurotransmitter in the CNS, mediating fast inhibitory synaptic transmission, by activating GABAA receptors. However, these GABA-gated Cl- channels can also be excitatory, causing depolarization, and increasing Ca2+ entry via voltage-operated Ca2+ channels (VOCCs). Evidence exists for excitatory ionotropic GABA receptors in anterior pituitary cells, including gonadotropes, but these have not been directly characterized and their pharmacology remains controversial. Here we have measured the cytosolic Ca2+ concentration ([Ca2+]i) in alphaT3-1 gonadotropes, to test for expression of excitatory GABA receptors. The GABAA agonists, GABA and muscimol, both caused rapid, robust and dose-dependent increases in [Ca2+]i (EC50 values 2.7 and 1 microM), whereas the GABAB agonist, baclofen, did not. The GABAA antagonist, bicuculline, inhibited muscimol's effect, whereas the GABAB antagonist, phaclofen, did not. The neuroactive steroid 5alpha-pregnan-3alpha-ol-11,20-dione (an allosteric activator of GABAA receptors) increased [Ca2+]i, and this effect, like that of muscimol, was inhibited by picrotoxin. The muscimol effect on [Ca2+]i was blocked by the VOCC antagonist, nifedipine, or by Ca2+-free medium. When cells were pretreated with muscimol this increased the spike phase of the [Ca2+]i response to subsequent stimulation with gonadotropin-releasing hormone (GnRH). Similar amplification was seen in muscimol-pretreated cells stimulated with GnRH in Ca2+-free medium, but not when cells were pretreated with muscimol in Ca2+-free medium. The amplification was not, however, GnRH receptor-specific, because the spike response to ionomycin was also increased by muscimol pretreatment. These data provide the first direct evidence for expression of excitatory GABAA receptors, and the first demonstration of acute steroid effects, on GnRH-responsive pituitary cells. They also reveal a novel mechanism by which GABAA activation modulates GnRH action, raising the possibility that this may also influence gonadotrophin secretion from non-immortalized gonadotropes.

Oestradiol is a potent mitogen and modulator of GnRH signalling in alphaT3-1 cells: are these effects causally related?

GnRH acts via phospholipase C (PLC) activating G-protein coupled receptors to stimulate secretion of gonadotrophins from gonadotrophs. These cells are also regulated by gonadal steroids, which act centrally to influence GnRH secretion, and peripherally to modulate GnRH action. We have shown that oestradiol can stimulate proliferation and modulate GnRH-stimulated [(3)H]inositol phosphate ([(3)H]IP(x)) accumulation (used as a measure of PLC activity) in a gonadotroph-derived cell line (alphaT3-1). Here we show that when alphaT3-1 cells were incubated in medium with 2% foetal calf serum (FCS), [(3)H]thymidine incorporation was not stimulated by oestradiol but was reduced to <2% of control by the oestrogen antagonist, raloxifene. The inhibitory effect of 10 or 1000 nM raloxifene was reversed competitively by oestradiol. A similar pattern of effects was seen when effects of oestradiol and raloxifene on the proportion of cells in the S-phase of the cell cycle (as measured by flow cytometry of propidium iodide-labelled cells) and on oestrogen receptor activity (as measured by trans-activation of the oestrogen-response elements in the vitellogenin promoter) were quantified. In addition, RT-PCR revealed expression of alpha and beta (but not beta2) subtypes of oestrogen receptors. Thus, oestrogen is an essential mitogen for alphaT3-1 cells, its mitogenic effect is oestrogen receptor mediated and is associated with a marked alteration of cell cycle distribution, and the full extent of these effects are best revealed in the presence of raloxifene. Using this strategy, we found that cells cultured for 4 days with 10 nM raloxifene expressed GnRH receptors (K(d) for (125)I-buserelin 4.33 nM) and that their activation by GnRH caused a concentration-dependent increase in [(3)H]IP(x) (in cells labelled with [(3)H]inositol) and inositol 1,4,5 trisphophate (in unlabelled cells). Addition of 10 nM oestradiol (to overcome receptor blockade by raloxifene) reduced GnRH receptor number by 31% but increased maximal effects on [(3)H]IP(x) and Ins(1,4,5)P(3) approximately 4-fold. The effects of oestradiol on GnRH receptor number and signalling were not, however, mimicked by culture for 2 days in medium with 10% FCS and the S-phase blocker, thymidine (15 mM). This treatment increased the proportion of cells in the S-phase 2- to 3-fold but did not alter GnRH receptor number or signalling. Other treatments which altered cell cycle transition (hydroxyurea, colcemid, methotrexate) also failed to alter GnRH receptor number or signalling and no correlation was seen between GnRH receptor number or GnRH-stimulated [(3)H]IP(x) accumulation and the proportion of cells in the S-phase or G2/M-phases of the cell cycle. Thus, oestradiol has pronounced effects on GnRH signalling, proliferation and cell cycle distribution in alphaT3-1 cells, but these trophic effects do not underlie the modulation of GnRH signalling.

A new photoreactive antagonist cross-links to the N-terminal domain of the gonadotropin-releasing hormone receptor.

A new photoreactive gonadotropin-releasing hormone (GnRH) antagonist [Ac-(4-azidobenzoyl)-D-Lys1, D-4-Cl-Phe2, D-Trp3, D-Arg6, D-Ala10]GnRH (PAnt-1) was synthesized and shown to bind covalently to mouse and human GnRH receptors after ultraviolet irradiation. PAnt-1 exhibited high binding affinity (Ki = 3.1 +/- 0.8 nM), and high crosslinking efficiency as shown by loss of 78% of binding sites following crosslinking at saturating concentration. Crosslinking resulted in irreversible receptor blockade as shown by inhibition of GnRH-stimulated inositol phosphate production. PAnt-1 has a photoreactive group at residue 1 of the peptide, a region believed to be critical in determining antagonist versus agonist properties of GnRH analogues. The attachment site of PAnt- to the receptor was localized between residues 11 and 19 of the extracellular N-terminal domain of the receptor by peptide mapping studies using natural sequence differences between human, mouse and sheep GnRH receptors, as well as a panel of GnRH receptor constructs with a series of engineered protease cleavage sites. A disulphide bridge between Cys14 and Cys200 was cleaved during crosslinking, suggesting that Cys14 is the crosslinked residue. These results suggest that peptide GnRH antagonists bind to the receptor with the N-terminal end of the peptide positioned in a site comprising the constrained regions of the N-terminal domain and second extracellular loop in the vicinity of the Cys14-Cys200 disulphide bridge.

C-type natriuretic peptide (CNP) effects on intracellular calcium [Ca2+]i in mouse gonadotrope-derived alphaT3-1 cell line.

C-type natriuretic peptide (CNP), the third member of the atrial natriuretic peptide family, acts via guanylyl cyclase containing GC-B receptors to stimulate cyclic guanosine 3',5' monophosphate (cGMP) accumulation in the gonadotrope-derived alphaT3-1 cell line and rat pituitary cells. This effect is inhibited by concomitant activation of the phospholipase C (PLC)-coupled gonadotrophin hormone-releasing hormone (GnRH) receptors in these cells. Since GnRH stimulates gonadotrophin secretion from gonadotropes by increasing the cytosolic Ca2+ concentration ([Ca2+]i) and natriuretic peptides have been found to influence PLC/Ca2+ signalling in other systems, we have investigated whether CNP can alter basal or GnRH-stimulated changes in [Ca2+]i in alphaT3-1 cells. In Ca 2+-containing medium, 10(-7) M CNP modestly, but significantly increased [Ca2+]i over several min, but subsequently inhibited the elevation of [Ca2+]i in response to 10(-7) M GnRH in both Ca2+-containing and Ca2+-free medium. This inhibitory effect was mimicked by 10(-6) M 8-Br-cGMP, but not by ANP, indicating mediation by cyclic GMP and the CNP-specific GC-B receptor. However, basal and GnRH-stimulated inositol (1,4,5) trisphosphate (Ins(1,4,5)P3) generation were not measurably affected by CNP, and CNP failed to affect thapsigargin-induced capacitative Ca2+ entry. Thus, it appears that the cross-talk between CNP and GnRH in these cells is reciprocal in that GnRH modulates CNP effects on cGMP generation, whereas, CNP modulates GnRH effects on Ca2+ mobilisation.