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.

GnRH and PACAP action in gonadotropes: cross-talk between phosphoinositidase C and adenylyl cyclase mediated signaling pathways.

In order to respond appropriately to their environment, gonadotropes, like other cells, must integrate informational input from multiple ligands acting through multiple intracellular signaling pathways. In recent years, an increasing number of examples of functional interactions between the phosphoinositidase C (PIC) and adenylyl cyclase signaling pathways in gonadotropes have been described, and the discovery that these cells are targets for pituitary adenylyl cyclase activating peptide (PACAP) has provided a physiological context for earlier work on gonadotrope regulation by cAMP. It has become clear that gonadotropes possess multiple PIC-coupled receptor types, in addition to receptors activating adenylyl and guanylyl cyclases, so that the potential for both coincidence signaling and cross-talk in these cells is immense; examples of both are seen in the effects of PACAP and GnRH on Ca(2+) mobilization and adenylyl cyclase activation in alphaT3-1 cells. In these cells, GnRH, acting via PIC-coupled receptors, can dramatically inhibit adenylyl cyclase activated by PACAP, but can also alter cellular levels of protein kinase A subunits, providing a mechanism for coordinated regulation of both messenger and effector.

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.

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.

Relative roles of calcium derived from intra- and extracellular sources in dynamic luteinizing hormone release from perifused pituitary cells.

GnRH releases LH from pituitary gonadotropes by a calcium-dependent mechanism. Previous studies in static cell cultures have not revealed a role for intracellular-derived calcium during GnRH-stimulated LH release. In the present study we have reexamined this possibility using a perifusion system, which permits a more dynamic assessment of early cellular events. Chelation of extracellular calcium by EGTA and calcium channel blockade by methoxyverapamil prevented sustained LH release. A component of early LH release occurred independently of extracellular calcium mobilization. This previously unrecognized aspect of LH release was shown to be dependent upon intracellular calcium. The molecular mechanism by which this calcium-dependent signal is translated into a cellular response does not appear to be mediated by calmodulin or protein kinase C, whereas sustained LH release appears mediated by calmodulin. While calcium derived from extracellular sources is still viewed as the major messenger for sustained LH release, these experiments provide evidence for the involvement of intracellular-derived calcium during early GnRH-stimulated LH release.

Homologous down-regulation of gonadotropin-releasing hormone receptors and desensitization of gonadotropes: lack of dependence on protein kinase C.

The demonstration that GnRH provokes the accumulation of diacylglycerol and the redistribution of protein kinase C to the membrane fraction in gonadotropes suggests a role for this enzyme as a mediator of GnRH action. In the present work we have investigated the possibility that protein kinase C might mediate GnRH-stimulated receptor down-regulation and desensitization. Pretreatment of pituitary cells for 6 h with GnRH (10(-11) - 10(-6) M) caused a biphasic change in GnRH receptor number [the maximum binding (Bmax) for 125I-buserelin binding was increased by 10(-10) M GnRH and reduced by 10(-7) and 10(-6) M GnRH] and caused desensitization (pretreatment with 10(-9) - 10(-6) M GnRH reduced the proportion of cellular LH released in a subsequent challenge with GnRH). Pretreatment for 6 h with 0.2-200 nM phorbol myristate acetate (a protein kinase C-activating phorbol ester) did not cause desensitization, but at 200 nM, did reduce GnRH receptor number. As a further test of the requirement for protein kinase C for GnRH action, cells were depleted of all measurable protein kinase C (and rendered unresponsive to protein kinase C activators) by prior treatment with a high dose of phorbol myristate acetate (500 nM for 6 h followed by 12 h in plating medium). Depletion of protein kinase C did not alter the ability of GnRH to desensitize gonadotropes or down-regulate its own receptors. The demonstration that the effects of GnRH on receptor number and gonadotrope responsiveness are neither blocked by depletion of protein kinase C nor entirely mimicked by activation of protein kinase C suggests that these effects of the releasing hormone are not solely mediated by this enzyme.

Dependence of gonadotropin-releasing hormone-stimulated luteinizing hormone release upon intracellular Ca2+ pools is revealed by desensitization and thapsigargin blockade.

Sustained GnRH-stimulated LH release requires extracellular Ca2+, but GnRH transiently increases LH release in Ca(2+)-free medium. Here we have tested the dependence of the transient effect on intracellular Ca2+ pools. In superfused pituitary cells three Ca(2+)-mobilizing stimuli (GnRH, A23187, and endothelin-1) all caused sustained increases in LH release in normal medium (plateau responses), but only transient increases in Ca(2+)-free medium (spike responses). In Ca(2+)-free medium, GnRH (10(-10) or 10(-9) M) increased LH release transiently and desensitized the cells to the LH-releasing effect of subsequent stimulation with 10(-7) M GnRH. This desensitization was reversed by brief exposure to Ca(2+)-containing medium between the two GnRH stimulation periods. Heterologous desensitization between GnRH and A23187 and between GnRH and endothelin-1 also occurred in Ca(2+)-free medium. Thapsigargin, which inhibits the endoplasmic reticulum Ca(2+)-ATPase and thereby elevates cytosolic Ca2+, stimulated LH release (EC50, approximately 20 microM) in static culture, an effect which, unlike those of GnRH and A23187, was not markedly reduced in Ca(2+)-free medium. Low doses of thapsigargin, which had no effect on LH release alone, inhibited both sustained GnRH-stimulated LH release from static cultures in normal medium and transient GnRH-stimulated LH release from cells superfused in Ca(2+)-free medium. These data suggest that the spike phase of GnRH-stimulated LH release is not only associated with but is also dependent upon the mobilization of a GnRH- and thapsigargin-sensitive intracellular Ca2+ pool and that the Ca2+ pool mediating this GnRH effect is identical to or substantially interchangeable with A23187- and endothelin-1-mobilizable intracellular Ca2+ pools. Inhibition of sustained GnRH-stimulated LH release by thapsigargin also suggests the involvement of an intracellular Ca2+ pool in this phase of GnRH action.

Oxytocin and progesterone release from bovine corpus luteal cells in culture: effects of insulin-like growth factor I, insulin, and prostaglandins.

The ruminant corpus luteum synthesizes and secretes oxytocin, but little is known of the regulation of these processes in the ovary. In the present work we describe a method for the preparation of cells from the early bovine corpus luteum (1-5 days postovulation) and their maintenance in serum-free culture. The release of oxytocin and progesterone from these cells was increased by the addition of insulin or insulin-like growth factor I (IGF-I), but not by IGF-II. Hormone release (measured between 60 and 84 h of culture) was increased approximately 5-fold (oxytocin) and 2.5-fold (progesterone) by maximally effective concentrations of IGF-I (EC50, 0.27 nM) and insulin (EC50, 1.94 nM). Sustained exposure (0-84 h) to prostaglandins (PGs) caused a dose-dependent reduction in oxytocin release in the presence of IGF-I (PGF2 alpha EC50, 31 nM; rank order of potency, PGF2 alpha greater than PGE2 greater than PGE1), but did not markedly reduce progesterone release. The inhibitory effect of PG on oxytocin production was mimicked by sustained exposure to a protein kinase-C activator (phorbol 12,13-dibutyrate), supporting the proposed role for this enzyme as a mediator of PG action. These data provide the first demonstration that oxytocin release from early bovine corpus luteal cell cultures can be regulated by insulin, IGF-I, and PGs. Since granulosa and/or luteal cells produce and respond to IGF-I and PGF2 alpha, our data indicate functional interaction of these compounds in the regulation of luteal cell activity.

Cyclic guanosine monophosphate production in the pituitary: stimulation by C-type natriuretic peptide and inhibition by gonadotropin-releasing hormone in alpha T3-1 cells.

Atrial, brain-type, and C-type natriuretic peptides (ANP, BNP, and CNP) act via receptors with intrinsic guanylate cyclase activity. The A-type and B-type ANP receptors are selectively activated by ANP and CNP, respectively. The anterior pituitary is a site of ANP production and action, suggesting a local regulatory function, and this may also hold true for CNP which is found at its highest tissue concentrations in the anterior pituitary. Here we show that these peptides all cause dose-dependent increases in cGMP accumulation in alpha T3-1 cells (a gonadotrope-derived cell line), GH3 cells, and in primary cultures of rat pituitary cells. The response to CNP is particularly robust in alpha T3-1 cells (59 +/- 9-fold increase, EC50 14 +/- 3 nM), and the rank order of potency in alpha T3-1 cells and primary cultures (CNP >> ANP > BNP) is suggestive of action exerted via B-type receptors. Although CNP did not alter GnRH-stimulated LH release or [3H]inositol phosphate accumulation, GnRH reduced CNP-stimulated cGMP accumulation dose dependently (EC50 approximately 0.1 nM). This inhibition reflects the ability of GnRH to shift the CNP dose-response curve rightward (increasing the EC50 for CNP action approximately 10-fold both with and without 3-isobutyl-1-methylxanthine). The inhibitory effect was not blocked by omission of extracellular Ca++ nor mimicked by the Ca++ ionophore A23187 but was mimicked by a protein kinase C (PKC)-activating phorbol ester (which had a comparable effect to GnRH on the EC50 for CNP action). The data imply that CNP rather than (or in addition to) ANP may have a local regulatory function within the pituitary, that although its role is currently unknown it may involve functional interaction with GnRH in gonadotropes, and that the effect of GnRH on CNP action may be PKC-mediated. Moreover, we suggest that alpha T3-1 cells may be a useful model for investigation of the cross-talk between the B-type natriuretic peptide receptor-regulated signal transduction pathway and the Ca++/PKC pathway activated by ligand-stimulated hydrolysis of inositol phospholipids.

Na+ dependence of gonadotropin-releasing hormone action: characterization of the Na+/H+ antiport in pituitary gonadotropes.

GnRH stimulates LH release from gonadotropes in a Ca2(+)-dependent manner. Because of the apparent relationship between cellular Ca2+ metabolism and Na(+)-driven antiports, we investigated their influence on GnRH action. We also assessed the influence of bicarbonate, because its transport may alter effects of Na+/H+ exchange on intracellular pH. In pituitary cell cultures without bicarbonate, GnRH-stimulated LH release was reduced by Na+ omission, by amiloride, and by amiloride analogs that selectively block Na+/H+ exchange. The Na+ dependence of amiloride action (EC50, 14 and 100 microM in medium with 20 and 135 mM NaCl, respectively, and no effect in Na(+)-free medium) and the order of potency of these analogs, indicated specific inhibition of Na+/H+ exchange. 5-(N,N-Di-methyl)amiloride (DMA; a potent Na+/H+ exchange inhibitor) reduced GnRH-stimulated LH release but not GnRH receptor binding or Ca2+ ionophore (A23187)-stimulated LH release, suggesting inhibition at a locus beyond receptor occupancy but before exocytosis. Amiloride analogs that selectively inhibit Na+/Ca2+ exchange also modestly reduced GnRH-stimulated LH release. Bicarbonate (10 mM) reduced the inhibitory effects of DMA and Na+ omission (but not the effects of the Na+/Ca2+ exchange inhibitors or of a Ca2+ channel antagonist), and the effect of bicarbonate was inhibited by a blocker of bicarbonate-dependent antiports. These observations reveal the Na+ dependence of GnRH action and that gonadotropes possess a Na+/H+ exchanger. The Na+ dependence of GnRH-stimulated LH release appears to reflect at least in part dependence upon this antiport. Prevention of the Na+/H+ exchange inhibitor effects by bicarbonate supports the specificity of their action, but suggests regulation of this antiport as an unlikely means of controlling LH release in vivo.

Oxytocin production and oxytocin messenger ribonucleic acid levels in bovine granulosa cells are regulated by insulin and insulin-like growth factor-I: dependence on developmental status of the ovarian follicle.

Oxytocin is a major peptide product of the ruminant corpus luteum, and the release of oxytocin from serum-free cultures of bovine granulosa cells is stimulated by insulin and insulin-like growth factor-I (IGF-I). Here we have assessed the effects of insulin and IGF-I on oxytocin gene expression in bovine granulosa cells and the dependence of these effects on the developmental status of the cells. When cells from individual follicles were cultured, the estradiol concentration of the follicular fluid was highly correlated with insulin-stimulated oxytocin release. Subsequently, cells were pooled from follicles selected on the basis of estradiol content, and two subsets of cells were distinguished. The first contained highly differentiated cells, as judged by the high estradiol (HE-cells) concentration of the follicular fluid (greater than 40 ng/ml), high levels of LH receptors, and high hCG-stimulated cAMP accumulation. The second subset contained cells from follicles with low estradiol (less than 1 ng/ml; LE-cells) which have fewer LH receptors and low hCG-stimulated cAMP accumulation. Oxytocin production was increased more than 50-fold by insulin (EC50, 230 +/- 57 ng/ml) and IGF-I (EC50, greater than 10 ng/ml), but only in the HE-cells. Oxytocin mRNA was also greatly increased by insulin and IGF-I in the HE-cells only. In contrast, insulin and IGF-I stimulated progesterone release from both HE- and LE-cells. Since oxytocin production is a characteristic of bovine luteal cells, our results support possible roles for IGF-I and insulin in regulation of luteinization or luteal activity. The data suggest that effects of insulin and IGF-I on oxytocin production reflect their effects on oxytocin gene transcription, and that granulosa cells must be appropriately primed (presumably by the in vivo hormonal environment) before they are able to produce oxytocin in response to these polypeptides.

Oxytocin receptor-mediated activation of phosphoinositidase C and elevation of cytosolic calcium in the gonadotrope-derived alphaT3-1 cell line.

Gonadotropes synthesize and secrete LH and FSH under the control of GnRH, which acts via phosphoinositidase C (PIC)-linked G protein coupled receptors. Additionally, gonadotropin released from the pituitary is influenced by oxytocin, a peptide that has been shown to play a role in generation of the preovulatory LH surge. Although oxytocin receptors are present in the pituitary, studies have identified their presence on lactotropes but not on gonadotropes, raising the question of which cells act as the direct target of oxytocin in gonadotrope regulation. In this study, we examined effects of oxytocin on alphaT3-1 cells, a gonadotrope-derived cell line. Oxytocin, vasopressin, and vasotocin each stimulated accumulation of [3H]inositol phosphates in cells prelabeled with [3H]inositol, indicating activation of PIC. The rank order of potency (oxytocin > vasotocin > vasopressin) and sensitivity to inhibition by oxytocin and vasopressin receptor antagonists, revealed the effect to be mediated by oxytocin-selective receptors. Like other PIC activators, these nonapeptides caused biphasic (spike-plateau) increases in the cytosolic Ca2+. The spike response to oxytocin and GnRH were both retained in Ca2+-free medium, reflecting mobilization of intracellular Ca2+, and were comparably reduced by thapsigargin, implying mobilization of Ca2+ from a shared thapsigargin-sensitive intracellular pool. Brief stimulation with oxytocin, vasopressin, or vasotocin prevented subsequent Ca2+ responses to oxytocin, but not to GnRH, suggesting that the oxytocin receptor undergoes rapid homologous desensitization and reinforcing the interpretation that the nonapeptides act via the same receptor type. Oxytocin did not increase Ca2+ in cells stimulated with GnRH, whereas GnRH caused a spike Ca2+ increase even in the presence of oxytocin, implying that different mechanisms of desensitization (Ca2+ pool depletion and receptor uncoupling) are operating for two distinct PIC-coupled receptors in these cells. The demonstration that oxytocin acts directly via PIC-linked, oxytocin-selective receptors to increase cytosolic Ca2+ in a gonadotrope-derived cell line is consistent with the possibility that oxytocin has a comparable effect on nonimmortalized gonadotropes.

Gonadotropin-releasing hormone and pituitary adenylate cyclase-activating polypeptide affect levels of cyclic adenosine 3',5'-monophosphate-dependent protein kinase A (PKA) subunits in the clonal gonadotrope alphaT3-1 cells: evidence for cross-talk between PKA and protein kinase C pathways.

We have shown previously that protein kinase A (PKA) subunit levels are regulated by activation of PKA or protein kinase C (PKC) in anterior pituitary cells. GnRH also influenced PKA subunit levels, suggesting that hormonal regulation occurs in gonadotrophs, and therefore, we have reexamined this question using the clonal gonadotrope-derived cell line (alphaT3-1 cells). Western blot analysis, using specific immunoaffinity purified immunoglobulins, revealed expression of catalytic (Cat) and regulatory type I (RI) and type II (RII) subunits of PKA in these cells. Activation of adenylyl cyclase (AC) with forskolin, or of PKC with tetradecanoyl phorbol acetate (TPA), caused a rapid (detectable at 0.5-1 h) and concentration-dependent loss of all PKA subunits. Forskolin (10-100 microM) reduced Cat and RI by 60% and RII by 30%, whereas TPA (0.1-1 microM) reduced Cat and RII by 50% and RI by 40%. Simultaneous activation of PKA and PKC caused the expected dose-dependent reductions in Cat, and the effects of forskolin or TPA were nearly additive. RI and RII were reduced similarly by 10 nM TPA, whereas 100 nM TPA tended to prevent the reduction of RI or RII caused by forskolin. GnRH, which activates phosphoinositidase C and not AC in these cells, caused a clear loss of Cat or RII at all concentrations tested and of RI at 0.1 nM. Pituitary adenylate cyclase-activating polypeptide 38, which acts via PVR-1 receptors to stimulate both phosphoinositidase C and AC in these cells, also caused a clear dose-dependent decrease in Cat, RI, and RII, although higher concentrations were needed for the latter effects. Together, the data demonstrate that catalytic and regulatory subunits of PKA are subject to both hormonal and receptor-independent regulation in alphaT3-1 cells, reinforcing the possibility that such effects occur in nonimmortalized gonadotropes. Whereas the effects of PKA activators very likely involve proteolytic degradation of the dissociated PKA holoenzyme, the effects of TPA and GnRH occur in the absence of cAMP elevation by unknown mechanisms. Whatever the mechanisms involved, the data reveal a mechanism for cross-talk between phosphoinositidase C and AC-mediated hormonal signals, in which PKC activation seems to play a pivotal role.

Pituitary ATP receptors: characterization and functional localization to gonadotropes.

We have used real-time dynamic video imaging of Fura-2 fluorescence to study the acute effects of extracellular ATP on [Ca2+]i in pituitary cells and found that ATP produced a rapid biphasic cytosolic Ca2+ increase in a dose-dependent manner in both rat pituitary gonadotropes and gonadotrope-derived alpha T3-1 cells. Removal of extracellular Ca2+ only attenuated the plateau phase of ATP-induced intracellular Ca2+ response and suramin (100 microM), a P2-purinoceptor antagonist, reduced the ATP effect by 40-60%. Further studies revealed the following rank-order of agonist potency in the Ca2+ response: -ATP = ADP = UTP > 2-methylthioATP >> beta, gamma-methylene ATP/alpha,beta-methylene ATP. Both homologous and heterologous desensitization occurred with ATP and UTP and these did not have additive effects. Together the data suggest that the intracellular Ca2+ responses of gonadotropes and alpha T3-1 cells to these nucleotides are mediated by a single class of receptor pharmacologically characterized as the P2U subtype. This is the first demonstration of functional ATP receptors in pituitary gonadotropes.

Pituitary adenylate cyclase-activating polypeptide effects in pituitary cells: modulation by gonadotropin-releasing hormone in alpha T3-1 cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) acts via type I receptors in the pituitary to stimulate cAMP production. Gonadotropes are likely target cells for PACAP action, and we have recently shown alpha T3-1 cells, a clonal gonadotrope-derived cell line, to be PACAP responsive. Here we have explored the influence of GnRH on PACAP action in alpha T3-1 cells and show that PACAP38-stimulated cAMP production is inhibited by GnRH in both the presence and the absence of a phosphodiesterase inhibitor. This effect appears not to be Ca++ mediated but is mimicked by protein kinase C activation with phorbol 12-myristate 13-acetate. However, GnRH and phorbol 12-myristate 13-acetate do not inhibit binding of [125I]PACAP27 to intact alpha T3-1 cells, nor do they inhibit forskolin- or cholera toxin-stimulated cAMP accumulation, implying that the inhibitory effects are exerted at early stages in the PACAP receptor signaling pathway but distal to receptor occupancy. When cells were preincubated with PACAP38, extensive washing failed to prevent the stimulatory effect of the polypeptide presumably because of the slow rate of receptor-ligand dissociation. However, when the time course of PACAP38-stimulated effects on intracellular cAMP was assessed, the stimulatory effect of PACAP38 could be rapidly reversed by GnRH addition, and the inhibitory effect of GnRH was rapidly be reversed by a GnRH receptor antagonist. The data provide the first demonstration of cross-talk between phospholipase C and adenylate cyclase-activating peptides in gonadotrope-derived cells and establish the potential for hormonal modulation of PACAP action. We suggest that this inhibitory effect of GnRH might enable the releasing hormone to control the kinetics of cAMP signaling in gonadotropes in vivo.

Desensitization of gonadotropin-releasing hormone action in the gonadotrope-derived alpha T3-1 cell line.

Sustained exposure of gonadotropes to GnRH causes a pronounced desensitization of GnRH-stimulated gonadotropin release, but the mechanisms involved are poorly understood. Recent studies have suggested, however, that GnRH-stimulated phosphoinositidase C (PIC) activity does not undergo rapid ( < 5 min) homologous desensitization in alpha T3-1 cells, and we have, therefore, used this cell line to address the question of whether desensitization occurs distal to PIC activity and/or in an intermediate time frame. We show that GnRH stimulates a rapid increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3; maximum at 10-20 sec with a modest reduction thereafter] and that the GnRH-stimulated accumulation of [3H]IPs (in cells stimulated in the presence of LiCl) increases linearly over 5-300 sec. This clearly indicates that desensitization of PIC does not occur within this period and that the dramatic reduction in the rate of Ins(1,4,5)P3 accumulation (10-30 sec) is due to its metabolism, rather than to a reduction in Ins(1,4,5)P3 generation. Pretreatment for 60 min with 10(-7)M GnRH reduced cell surface GnRH receptor number by 48% (without measurably altering Kd). The pretreatment also reduced maximal GnRH-stimulated [3H]IP accumulation (to 66% of the control) and increased the EC50 for GnRH-stimulated [3H]IP accumulation approximately 3-fold, demonstrating that desensitization of GnRH-stimulated [3H]IP accumulation can, indeed, occur within 60 min, but that this may be attributable to receptor loss (without appreciable uncoupling of residual receptors from their immediate effector system). Pretreatment for 60 min with GnRH also caused a dose-dependent reduction in both spike and plateau phases of the GnRH effect on cytosolic Ca2+. This effect could not be overcome by stimulation with high concentrations of GnRH and appears, therefore, to reflect not only receptor loss, but, also, an additional inability of agonist-occupied GnRH receptors to elevate cytosolic Ca2+. The effect of KCl on cytosolic Ca2+ was similarly reduced by GnRH pretreatment, suggesting that desensitization of voltage-operated Ca2+ channels mediates desensitization of the plateau phase Ca2+ response to GnRH. Such a mechanism could not, however, explain desensitization of the spike phase of the Ca2+ response to GnRH seen in normal or Ca2+ -free medium. Accordingly, the data reveal a novel mechanism for homologous desensitization to GnRH in which agonist-occupied GnRH receptors are rendered unable to mobilize intracellular Ca2+ and imply that desensitization of GnRH-stimulated Ins(1,4,5)P3 production and/or action occurs.

Enhanced responsiveness of gonadotropes after protein kinase-C activation: postreceptor regulation of gonadotropin releasing hormone action.

GnRH stimulates the release of LH from pituitary gonadotropes in a Ca2+-and calmodulin-dependent manner. Although GnRH also appears to activate protein kinase-C in gonadotropes, the role of this enzyme in GnRH action remains undetermined. In the present work we have assessed the effect of pretreatment of pituitary cell cultures with a protein kinase-C-activating phorbol ester on gonadotrope responsiveness to GnRH. Pretreatment for 6 h with phorbol 12-myristate 13-acetate (PMA) reduced the EC50 for GnRH-stimulated LH release approximately 8-fold without altering the maximum proportion of total cellular LH release. This increase in the potency of GnRH occurred in the absence of any measurable change in receptor affinity. Subsequent studies revealed that PMA pretreatment did not alter the EC50 for GnRH-stimulated [3H]inositol phosphate accumulation (an indicator of phosphoinositide hydrolysis), but did cause a modest reduction (approximately 2-fold) in the EC50 for LH release in response to the Ca2+ ionophore A23187 and the Ca2+ channel-activating compound maitotoxin. These observations demonstrate that the efficiency of coupling of the GnRH receptor to LH release can be regulated at a postreceptor locus by activation of protein kinase-C and that an increased responsiveness of Ca2+-regulated effector systems to mobilized Ca2+ appears to contribute to the observed effect.

C-type natriuretic peptide (CNP) in the pituitary: is CNP an autocrine regulator of gonadotropes?

Natriuretic peptides act via receptors with intrinsic guanylate cyclase activity to stimulate cGMP production and are thought to be important regulators of neuroendocrine systems. C-Type natriuretic peptide (CNP) is of particular interest in this regard because the highest tissue concentrations of CNP occur in the anterior pituitary, where it is a highly potent stimulator of cGMP production. Here we show that pituitaries of rats and mice contain abundant CNP prohormone messenger RNA (mRNA), but no atrial natriuretic peptide or B-type natriuretic peptide prohormone mRNAs. Using reverse transcriptase-polymerase chain reaction, both A- and B-type natriuretic peptide receptor (GC-A and GC-B, respectively) transcripts were detected in rat and mouse pituitaries, although only the GC-B mRNA was measurable by Northern blotting. Immunohistochemistry revealed CNP-positive cells in the anterior, but not posterior, pituitaries of rats, and the vast majority of these cells were identified as gonadotropes by colocalization of CNP and LH immunoreactivities. Targeted toxicity using GnRH conjugated to the ricin-A chain was used to test whether gonadotropes are also direct targets for GnRH action. The conjugate dose dependently inhibited the proliferation of alpha T3-1 cells (gonadotrope-derived cells with GnRH receptors), but had no such effect on GH3 cells (which do not have GnRH receptors). Culture of rat pituitary cells with the conjugate caused comparable reductions in CNP-stimulated cGMP production, GnRH-stimulated LH release, and CA2+ ionophore (A23187)-stimulated LH release, but did not measurably alter cAMP production in response to pituitary adenylate cyclase-activating polypeptide. We conclude that CNP is synthesized in the pituitary, where it is located predominantly in gonadotropes, and GC-B receptors expressed in the pituitary mediate the direct effects of CNP in gonadotropes. Together with the recent demonstration of CNP synthesis and action in alpha T3-1 cells, the data suggest CNP to be a novel autocrine regulator of gonadotropes.

Effects of pituitary adenylate cyclase-activating polypeptide in the pituitary: activation of two signal transduction pathways in the gonadotrope-derived alpha T3-1 cell line.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is thought to play a hypophysiotropic role, but little is known of the identity of PACAP-stimulated cells in the pituitary, the nature of the PACAP receptors on specific cell types, and the effector systems for these receptors. Here we describe the effects of PACAP in alpha T3-1 cells, a gonadotrope-derived cell line. In these cells, PACAP38 causes concentration-dependent increases in cAMP accumulation (EC50, 3 nM), [3H]inositol phosphate ([3H]IP) production (EC50, 20 nM), and the cytosolic Ca2+ concentration. The Ca2+ response is biphasic and is sustained only in Ca(2+)-containing medium. Intact alpha T3-1 cells possess a single class of [125I]PACAP27-binding sites (Kd, 3.3 nM; binding capacity, 35 fmol/10(6) cells). The rank orders of potencies for stimulation of cAMP and [3H]IP production and for inhibition of [125I] PACAP27 binding by three related peptides are identical (PACAP38 = PACAP27 > > vasoactive intestinal peptide). In addition to stimulation of LH release from primary cultures of rat pituitary cells and [3H] IP accumulation in alpha T3-1 cells, PACAP38 synergizes with low GnRH concentrations in the production of these effects. Moreover, long term exposure to PACAP38 stimulates [3H]thymidine incorporation and increases steady state levels of the gonadotropin alpha-subunit in alpha T3-1 cells. We conclude that alpha T3-1 cells possess type I PACAP receptors which mediate the observed effector system responses, and demonstration of the effects of PACAP on this gonadotrope-derived cell line provides further evidence that gonadotropes are direct targets for PACAP action. The data imply that stimulation of phospholipase-C by PACAP is responsible (at least in part) for the observed increase in cytosolic Ca2+, which, in turn, probably mediates the effects of PACAP on LH release. We suggest, however, that in gonadotropes, the effects of PACAP on cell replication and gonadotropin synthesis may prove more important than the peptide's modest effects on LH release.

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.