Regulation of G(q/11)alpha by the gonadotropin-releasing hormone receptor.

Evidence from use of pertussis and cholera toxins and from NaF suggested the involvement of G proteins in GnRH regulation of gonadotrope function. We have used three different methods to assess GnRH receptor regulation of G(q/11)alpha subunits (G(q/11)alpha). First, we used GnRH-stimulated palmitoylation of G(q/11)alpha to identify their involvement in GnRH receptor-mediated signal transduction. Dispersed rat pituitary cell cultures were labeled with [9,10-(3)H(N)]-palmitic acid and immunoprecipitated with rabbit polyclonal antiserum made against the C-terminal sequence of G(q/11)alpha. The immunoprecipitates were resolved by 10% SDS-PAGE and quantified. Treatment with GnRH resulted in time-dependent (0-120 min) labeling of G(q/11)alpha. GnRH (10(-12), 10(-10), 10(-8), or 10(-6) g/ml) for 40 min resulted in dose-dependent labeling of G(q/11)alpha compared with controls. Cholera toxin (5 microg/ml; activator of G(i)alpha), pertussis toxin (100 ng/ml; inhibitor of G(i)alpha actions) and Antide (50 nM; GnRH antagonist) did not stimulate palmitoylation of G(q/11)alpha above basal levels. However, phorbol myristic acid (100 ng/ml; protein kinase C activator) stimulated the palmitoylation of G(q/11)alpha above basal levels, but not to the same extent as 10(-6) g/ml GnRH. Second, we used the ability of the third intracellular loop (3i) of other seven-transmembrane segment receptors that couple to specific G proteins to antagonize GnRH receptor-stimulated signal transduction and therefore act as an intracellular inhibitor. Because the third intracellular loop of alpha1B-adrenergic receptor (alpha1B 3i) couples to G(q/11)alpha, it can inhibit G(q/11)alpha-mediated stimulation of inositol phosphate (IP) turnover by interfering with receptor coupling to G(q/11)alpha. Transfection (efficiency 5-7%) with alpha1B 3i cDNA, but not the third intracellular loop of M1-acetylcholine receptor (which also couples to G(q/11)alpha), resulted in 10-12% inhibition of maximal GnRH-evoked IP turnover, as compared with vector-transfected GnRH-stimulated IP turnover. The third intracellular loop of alpha2A adrenergic receptor, M2-acetylcholine receptor (both couple to G(i)alpha), and D1A-receptor (couples to G(s)alpha) did not inhibit IP turnover significantly compared with control values. GnRH-stimulated LH release was not affected by the expression of these peptides. Third, we assessed GnRH receptor regulation of G(q/11)alpha in a PRL-secreting adenoma cell line (GGH(3)1') expressing the GnRH receptor. Stimulation of GGH(3)1' cells with 0.1 microg/ml Buserelin (a metabolically stable GnRH agonist) resulted in a 15-20% decrease in total G(q/11)alpha at 24 h following agonist treatment compared with control levels; this action of the agonist was blocked by GnRH antagonist, Antide (10(-6) g/ml). Neither Antide (10(-6) g/ml, 24 h) alone nor phorbol myristic acid (0.33-100 ng/ml, 24 h) mimicked the action of GnRH agonist on the loss of G(q/11)alpha immunoreactivity. The loss of G(q/11)alpha immunoreactivity was not due to an effect of Buserelin on cell-doubling times. These studies provide the first direct evidence for regulation of G(q/11)alpha by the GnRH receptor in primary pituitary cultures and in GGH3 cells.

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 desensitization with gonadotropin-releasing hormone (GnRH) also diminishes gonadotrope responsiveness to maitotoxin: a role for the GnRH receptor-regulated calcium ion channel in mediation of cellular desensitization.

Maitotoxin (MTX) stimulates gonadotropin release from pituitary cell cultures. The time course and efficacy of LH release in response to GnRH and to MTX are similar; both secretagogues require extracellular Ca2+ and are inhibited by the selective Ca2+ ion channel antagonist methoxyverapamil (D600). LH release in response to either GnRH or MTX is not measurably inhibited by two other chemical classes of Ca2+ ion channel inhibitors represented by nifedipine and by diltiazem. The two secretagogues are nonadditive in their action on LH release when presented at high doses and prior studies indicate that MTX has no endogenous ionophoretic activity. These observations indicate that MTX likely stimulates LH release due to activation of the GnRH receptor associated Ca2+-ion channel in the gonadotrope. We have therefore assessed the functional state of this channel during the development of homologous desensitization of the gonadotrope to GnRH by measuring the ability of MTX to stimulate LH release. Cells were desensitized with GnRH in the presence of 3 mM EGTA. Under these conditions, the cells become refractory to GnRH in the absence of gonadotropin release since the latter process, but not the former, requires extracellular Ca2+. Accordingly, this approach allows assessment of the degree of desensitization in the absence of the influence of gonadotropin depletion. Such desensitized cells are less responsive to GnRH. Desensitized pituitary cells also respond with diminished efficacy and potency to MTX three or more hours after GnRH treatment but not at an earlier time (1 h) when GnRH receptors are diminished. These data are consistent with a model in which homologous desensitization is viewed as developing in two phases.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Evidence that signalling pathways by which thyrotropin-releasing hormone and gonadotropin-releasing hormone act are both common and distinct.

TRH and GnRH receptors are each coupled to G proteins of the Gq/11 family. Activation of each of these receptors by their respective ligands results in the stimulation of phospholipase C activity, leading to calcium mobilization and protein kinase C activation. Thus, the effects of TRH and GnRH may be mediated through the same intracellular signal transduction pathway. To compare responses to TRH and GnRH directly within one cell type, we have stably transfected the rat pituitary GH3 lactotrope cell line, which expresses the endogenous TRH receptor, with an expression vector containing rat GnRH receptor cDNA. Transfected cells specifically bound GnRH with high affinity and responded to GnRH stimulation with an increase in PRL mRNA levels, analogous to their response to TRH stimulation. Stably transfected GH3 cells, which were then transiently transfected with luciferase reporter constructs containing either the PRL or the glycoprotein hormone alpha-subunit promoter, responded to either GnRH or TRH stimulation with an increase in luciferase activity in a time- and dose-dependent fashion. The stimulatory effects of maximally effective concentrations of TRH and GnRH were additive on PRL, but not alpha-subunit, gene expression. These data, coupled with evidence of cross-desensitization of alpha-subunit, but not PRL, promoter activity stimulation by TRH and GnRH, suggest that there may be differences in the signal transduction pathways activated by TRH and GnRH receptors in the regulation of PRL and alpha-subunit gene expression.

Addition of catfish gonadotropin-releasing hormone (GnRH) receptor intracellular carboxyl-terminal tail to rat GnRH receptor alters receptor expression and regulation.

Mammalian GnRH receptor (GnRHR) is unique among G protein-coupled seven-transmembrane segment receptors due to the absence of an intracellular C-terminal tail frequently important for internalization and/or desensitization of other G protein-coupled receptors. The recent cloning of nonmammalian (i.e. catfish, goldfish, frog, and chicken) GnRHRs shows that these contain an intracellular C terminus. Addition of the 51-amino acid intracellular C terminus from catfish GnRHR (cfGnRHR) to rat GnRHR (rGnRHR) did not affect rGnRHR binding affinity but elevated receptor expression by about 5-fold. Truncation of the added C terminus impaired the elevated receptor-binding sites by 3- to 8-fold, depending on the truncation site. In addition, introducing the C terminus to rGnRHR altered the pattern of receptor regulation from biphasic down-regulation and recovery to monophasic down-regulation. The extent of down-regulation was also enhanced. The alteration in receptor regulation due to the addition of a C terminus was reversed by truncation of the added C terminus. Furthermore, addition of the cfGnRHR C terminus to rGnRHR significantly augmented the inositol phospholipid (IP) response of transfected cells to Buserelin, but this did not result from the elevation of receptor-binding sites. Addition of the C terminus did not affect Buserelin-stimulated cAMP and PRL release. GH3 cells transfected with wild-type cfGnRHR did not show measurable Buserelin binding or significant stimulation of IP, cAMP, or PRL in response to Buserelin (10[-13]-10[-9] M). GH3 cells transfected with C terminus-truncated cfGnRHR showed no IP response to Buserelin (10[-13]-10[-7] M). These results suggest that addition of the cfGnRHR intracellular C terminus to rGnRHR has a significant impact on rGnRHR expression and regulation and efficiency of differential receptor coupling to G proteins.

Calcium mobilization in the pituitary gonadotrope: relative roles of intra- and extracellular sources.

GnRH stimulates pituitary gonadotropin release by a Ca+2-dependent mechanism. Indeed, while it is clear that Ca+2 fulfills the requirements of a second messenger, the relative roles of Ca+2 mobilized from intra- and extracellular sources have never been distinguished. In the present study we examined the requirements for intra- and extracellular Ca+2 by three different means. First, in static cultures we used a specific Ca+2 ion channel blocker, methoxyverapamil (D600), to block entry of extracellular Ca+2 into pituitary cell cultures to determine if brief elevation of intracellular Ca+2 (whether derived from external or internal sources) could support continued gonadotropin release. Studies over a wide range of GnRH concentrations indicated that blockade of Ca+2 entry into the gonadotrope (in the presence of continued occupancy of the GnRH receptor by the releasing hormone) resulted in termination of LH release. Second, compounds that stabilize intracellular Ca+2 (preventing its mobilization), such as 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate-HCl (TMB-8) and dantrolene (Dantrium), were shown not to alter the potency or efficacy of GnRH in stimulating LH release. Third, we used a system of perifused cells to measure the actions of D600, EGTA, or the removal of GnRH on stimulated LH release to correlate precisely the release process with access to Ca+2 in the extracellular compartment. The results of these studies suggest that LH release in response to GnRH is primarily dependent on Ca+2 mobilized from extracellular sources. Termination of accessibility to this Ca+2 pool also results in termination of release. The data are consistent with a model in which GnRH occupancy of its receptor regulates a plasma membrane Ca+2 ion channel; continued access to the extracellular Ca+2 pool is required for continued LH release.

Radiation inactivation (target size analysis) of the gonadotropin-releasing hormone receptor: evidence for a high molecular weight complex.

In the present study we used radiation inactivation (target size analysis) to measure the functional mol wt of the GnRH receptor while it is still a component of the plasma membrane. This technique is based on the observation that an inverse relationship exists between the dose-dependent inactivation of a macromolecule by ionizing radiation and the size of that macromolecule. This method demonstrates a mol wt of 136,346 +/- 8,120 for the GnRH receptor. This estimate is approximately twice that obtained (60,000) by photoaffinity labeling with a radioactive GnRH analog followed by electrophoresis under denaturing conditions and, accordingly, presents the possibility that the functional receptor consists of a high mol wt complex in its native state. The present studies indicate that the GnRH receptor is either a single weight class of protein or several closely related weight classes, such as might occur due to protein glycosylation.

Altered rate of synthesis of gonadotropin-releasing hormone receptors: effects of homologous hormone appear independent of extracellular calcium.

Steady state levels of plasma membrane receptors (measured in radioligand assays) result from processes that contribute to the production of receptors (synthesis, recycling, and unmasking) as well as those that contribute to the loss of receptors (degradation, internalization, and inactivation). Accordingly, we have previously adapted the density shift technique to determine the contribution due to synthesis of GnRH receptors. In the present study we have evaluated the ability of homologous hormone to affect the rate of synthesis of these receptors. Additionally, because of its role in other actions of the releasing hormone, the requirement for extracellular Ca2+ to mediate the effects of GnRH was assessed. Cultures of pituitary cells, prepared from female weanling rats, were used for all studies. After treatment with GnRH, a GnRH antagonist, or the calcium ionophore A23187, with or without EGTA (a calcium chelator), cells were further cultured for up to 24 h in medium containing either dense or normal amino acids. After this treatment, receptors for GnRH were covalently linked to a radiolabeled photoaffinity probe [( 125I]Tyr5-[azido-benzoyl-D-Lys6-GnRH]) then solubilized in 1% sodium dodecyl sulfate. Receptors that had incorporated the dense amino acids (i.e. newly synthesized receptors) were separated from those that had been synthesized before the addition of dense amino acids by velocity sedimentation in sucrose gradients (0-20% sucrose, 1% sodium dodecyl sulfate, and 10 mM Tris-HCl, pH 7.0; centrifuged at 156,000 x g for 24 h). After centrifugation, gradients were fractionated, and the radioactivity in each fraction was quantified. GnRH treatment (10 or 0.1 nM) increased the rate at which dense amino acids were incorporated into GnRH receptors (t 1/2 = 13 +/- 2, 15 +/- 1, and 25 +/- 2 h for 0.1 nM GnRH, 10 nM GnRH, and control values, respectively). GnRH antagonist alone did not change the rate of GnRH receptor synthesis (t 1/2 = 22 +/- 3 h) compared to the control value (t 1/2 = 25 +/- 2 h) and was able to block the effects of GnRH. The effects of GnRH were not antagonized by inclusion of 3 mM EGTA during treatment (t 1/2 = 15 +/- 1 h vs. 13 +/- 2 h for 0.1 nM GnRH in the presence and absence of 3 mM EGTA, respectively).(ABSTRACT TRUNCATED AT 400 WORDS)

Maintenance of gonadotropin-releasing hormone (GnRH)-stimulated luteinizing hormone release despite desensitization of GnRH-stimulated cytosolic calcium responses.

Involvement of ionized cytosolic calcium ([Ca2+]i) and protein kinase-C (PKC) in GnRH-stimulated LH release was assessed by correlating measurable changes in [Ca2+]i and LH release in PKC-depleted and nondepleted gonadotropes. Primary cultures of anterior pituitary cells were loaded with the calcium-sensitive fluorescent dye fura-2 and placed in a perifusion chamber. GnRH pulses were delivered to the cells, and changes in fura-2 fluorescence and LH release were determined. The level of [Ca2+]i (assessed by fura-2) increased rapidly to a maximum within 20-40 sec, followed by a slower decline over the next minute (spike phase) to a sustained intermediate value (plateau phase). GnRH-stimulated LH release was unaffected by loading cells with fura-2. Both LH release and changes in [Ca2+]i were directly dependent on GnRH concentration. Pretreatment with the GnRH antagonist Antide (50 nM; [NAcD2Nal1-DpClPhe2-D3Pal3-Ser4-NicLys5-++ +DNicLys6-Leu7-ILys8-Pro9-DAla10]NH2 ) had no effect on basal [Ca2+]i or basal LH release, but did block both GnRH-stimulated calcium mobilization and GnRH-stimulated LH release. GnRH pretreatment (3.5 nM; 10 min) blocked the calcium spike phase, but not the plateau phase occurring in response to a GnRH pulse (10 nM; 5 min) delivered immediately after pretreatment. Inhibition of the calcium spike phase was transient (recovery within 15 min) and was dependent on pretreatment concentrations of GnRH. Calcium spike phase inhibition by GnRH pretreatment prevented increased LH release from PKC-depleted cells in response to a subsequent pulse of GnRH, but not from gonadotropes with normal levels of PKC. This suggests that initial LH release is dependent on changes in [Ca2+]i, but enhancement of LH release after periods of elevated GnRH concentrations may be dependent on PKC.

Cholera toxin and dibutyryl cyclic adenosine 3',5'-monophosphate sensitize gonadotropin-releasing hormone-stimulated inositol phosphate production to inhibition in protein kinase-C (PKC)-depleted cells: evidence for cross-talk between a cholera toxin-sensitive G-protein and PKC.

The present study assesses the relationship between G-proteins and protein kinase-C (PKC) in the gonadotrope. Cells were depleted of PKC with 1 microM phorbol 12-myristate 13-acetate for 12 h, followed by medium 199-BSA for 6 h before treatment with vehicle, pertussis toxin (PTX), cholera toxin (CTX), or (Bu)2cAMP (dBcAMP) for 18 h. PTX (10 ng/ml) significantly decreased GnRH-stimulated inositol phosphate (IP) production over a range of 10(-8)-10(-6) M GnRH. The degree of this inhibition was the same in control cells and PKC-depleted cells. Pretreatment with CTX (0.5 microgram/ml) significantly decreased GnRH-stimulated IP production over a range of 10(-9)-10(-6) M GnRH in PKC-depleted cells. This effect was mimicked by pretreatment with 3 mM dBcAMP. Although CTX and dBcAMP both decreased GnRH-stimulated IP production in control cells, this effect was enhanced in PKC-depleted cells. CTX (0.1 microgram/ml) and dBcAMP (3 mM) both enhanced GnRH-stimulated LH release, whereas PTX (100 ng/ml) had no effect. This was observed in control as well as PKC-depleted cells. Both PKA and PKC are capable of regulating IP turnover by phosphorylating phospholipase-C at distinct sites. CTX activates a G-protein that increases cAMP. cAMP can then activate PKA. In PKC-depleted cells, CTX inhibits GnRH-stimulated IP production. This effect is mimicked by dBcAMP, which suggests a role for PKA in the gonadotrope. The results of this study provide evidence for cross-talk between a CTX-sensitive G-protein and PKC.

Luteinizing hormone release and gonadotropin-releasing hormone (GnRH) receptor internalization: independent actions of GnRH.

Three different approaches are described which provide independent and new evidence that gonadotropin-releasing hormone (GnRH) internalization and GnRH-stimulated LH release are distinct actions of the releasing hormone. 1) Removal of GnRH from medium bathing the pituitary cell cultures resulted in the prompt return of LH release to basal levels. This finding indicated that a continuous supply of externally applied GnRH is required for the stimulation of LH release. 2) Covalent immobilization of D-Lys6-des-Gly10-Pro9-ethylamide GnRH (a GnRH agonist) on agarose beads resulted in a derivative which stimulated LH release with full efficacy. At concentrations of immobilized releasing hormone analog sufficient to evoke gonadotropin release, the quantity of LH release was restricted by the number of beads added. This finding was interpreted as evidence that the attachment of immobilized agonist was stable during the bioassay and indicated that LH release could be stimulated with full efficacy without the requirement for GnRH internalization. 3) Comparative studies using image-intensified microscopy and the cell culture bioassay showed that 100 microM vinblastin markedly inhibited large scale patching and capping of the GnRH receptor (viewed by image-intensified microscopy), but did not alter the EC50 or efficacy of LH release stimulated by GnRH or the agonist described above. These observations indicated that internalization as well as large scale patching and capping of the GnRH receptor are not required for LH release.

GnRH-mediated desensitization of the pituitary gonadotrope is not calcium dependent.

In the present work we describe homologous desensitization of gonadotropin release from pituitary cells in response to short-term exposure to GnRH. Like the release process itself, desensitization requires receptor occupancy by an agonist. In contrast, however, inhibition of LH release by chelation of extracellular calcium or blockade of the calcium ion channel does not inhibit desensitization. Further, stimulation of gonadotropin release by ionophore A23187 (which mobilizes calcium without GnRH receptor occupancy) does not lead to desensitization. These findings indicate that the decrease in pituitary sensitivity to GnRH due to an initial exposure to the peptide can be uncoupled from LH release. The data provide the first evidence of a calcium-independent biological effect of the releasing hormone in cultured pituitary cells.

Activin modulates the intracellular signaling system activated by gonadotropin-releasing hormone: dual effect on calcium messenger system and protein kinase-C pathway.

This study was performed to assess the effects of activin on intracellular mechanisms involved in GnRH action. When rat pituitary cell cultures were pretreated with activin-A (5-80 ng/ml) for 3 days, subsequent FSH and LH release (percentage of total cellular FSH and LH released during 4 h) in response to GnRH (10(-10)-10(-6) M) was not significantly different from that in cells pretreated with medium alone. In contrast, activin pretreatment increased the potency of both A23187 (Ca2+ ionophore) and phorbol 12-myristate 13-acetate [a protein kinase-C (PKC) activator] as secretagogues for FSH and LH release. FSH or LH release in response to another Ca(2+)-mobilizing secretagogue, maitotoxin (an activator of the GnRH receptor-associated Ca2+ channel), was not increased by activin. Although PKC is capable of influencing the actions of Ca2+, which is believed to be the second messenger for GnRH action, neither GnRH- nor maitotoxin-stimulated gonadotropin release was increased by activin even when the influence of activin on PKC was eliminated by the addition of a PKC inhibitor (staurosporine; 100 nM) during the final 30 min of the 3-day pretreatment period. These results indicate that although activin does not influence GnRH action with regard to gonadotropin release, it increases the sensitivity of the system regulating gonadotropin release to increases in cytosolic Ca2+ concentrations and PKC activation. Furthermore, activin appears to exhibit an inhibitory effect(s) at some point(s) in GnRH action in a PKC-independent manner, which could be responsible for opposing the increased sensitivity of the gonadotrope to Ca2+. The differential effects of activin on gonadotropin release in response to Ca(2+)-mobilizing secretagogues (ionophore and maitotoxin) raise the possibility that the activity of the GnRH receptor-associated Ca2+ channel may be suppressed by activin.

Gonadotropin-releasing hormone-mediated desensitization of cultured rat anterior pituitary cells can be uncoupled from luteinizing hormone release.

GnRH stimulates LH release from pituitary gonadotropes. Prolonged exposure of these cells to GnRH results in decreased sensitivity to further stimulation by the releasing hormone both in vivo and in vitro. Chelation of extracellular Ca++ with EGTA blocks GnRH-stimulated LH release but does not prevent subsequent desensitization. Desensitization occurs when cells are preincubated in EGTA containing 10(-7) M GnRH for a variety of times (20 min to 12 h) or when cells are preincubated for 3 h in EGTA with 10(-10), 10(-9), or 10(-8) M GnRH. A GnRH antagonist does not cause desensitization to GnRH and blocks desensitization in response to GnRH in the Ca++-free medium. Preincubation in EGTA containing 10(-7) M GnRH for 3 h did not alter sensitivity of cells to sn 1,2 dioctanoylglycerol (a protein kinase C activator), Ca++ ionophore A23187, or veratridine (an activator of endogenous ion channels). These results suggest that desensitization results from occupancy of the GnRH receptor by an agonist and may be uncoupled from LH release.

The relationship between gonadotropin-releasing hormone-stimulated luteinizing hormone release and inositol phosphate production: studies with calcium antagonists and protein kinase C activators.

The coupling between GnRH-stimulated phosphoinositide (PI) turnover and LH release has been investigated in rat pituitary cell cultures. Accumulation of [3H]inositol phosphates ([3H]IPs) formed by hydrolysis of PIs was measured in cells that had been preloaded with [3H]myo-inositol. GnRH stimulated both LH release and incorporation of [3H]inositol into total [3H]IPs with similar dose and time dependencies. [3H] IP production in response to GnRH could be blocked by a GnRH antagonist, but was stimulated by a compound that provokes receptor microaggregation. GnRH-stimulated IP production persisted in the presence of either the Ca2+ channel blocker D600 or the calmodulin antagonist pimozide at concentrations that reduced LH release to 60% and 20% of control, respectively. Stimulated [3H]IP production was inhibited at higher concentrations of D600. In 1-h incubations, GnRH-stimulated [3H]IP production, but not LH release, was markedly inhibited by the protein kinase C activators phorbol myristate acetate and 1,2-dioctanoylglycerol. These findings indicate that in the gonadotrope, GnRH-stimulated LH release and [3H]IP production are closely coupled to receptor activation by an agonist; Ca2+ antagonists uncouple stimulated LH release from [3H]IP production; and protein kinase C activators uncouple stimulated PI turnover from LH release. Thus, GnRH-stimulated production of PI metabolites, as measured by [3H]IP accumulation, is apparently not sufficient to support LH release in the absence of Ca2+. In addition, GnRH-stimulated LH release is apparently not dependent on full expression of the PI response.

Agents that decrease gonadotropin-releasing hormone (GnRH) receptor internalization do not inhibit GnRH-mediated gonadotrope desensitization.

Exposure of pituitary cell cultures to GnRH causes gonadotropin release, receptor capping, internalization, and loss as well as altered responsiveness of the target cell. In the present study, the relationship between loss of gonadotrope secretory responsiveness to GnRH (desensitization) and internalization of the GnRH-receptor complex was examined. Pituitary cell cultures were pretreated (30 min) with vinblastine (100 microM, a concentration that prevents measurable receptor internalization) or with medium containing carrier only, incubated with 10(-7) M GnRH (a desensitizing concentration) with or without vinblastine or with medium alone for 60 min, and finally washed and rechallenged for 3 h with increasing concentrations of GnRH to assess the degree of desensitization as determined by LH release. Results indicate that vinblastine had no measurable effect on the ability of GnRH to stimulate LH release or desensitize the cells. In a second series of studies, a GnRH analog (D-Lys6-GnRH) was immobilized to a cross-linked agarose matrix. The covalent link was shown to be stable by biological, immunological, and physical criteria. This product bound to the GnRH receptor and provoked LH release, but was not internalized, as determined by GnRH receptor binding assays. Cultured cells were treated with either 10(-9) M free analog or an equivalent concentration of coupled analog (as measured by LH release) for 3 h. Cells were washed, then rechallenged with GnRH to assess desensitization. Both the free and coupled analogs provoked an equivalent degree of desensitization. While a significant degree of desensitization also occurred in the presence of 3 mM EGTA (conditions that totally inhibited GnRH-stimulated LH release), the loss of responsiveness was not as great as in the absence of EGTA, indicating that partial depletion of available LH may play a role in GnRH-stimulated gonadotrope desensitization. The present findings suggest that GnRH receptor internalization and LH release can be uncoupled and that loss of the GnRH receptor by internalization is not a sufficient explanation for GnRH-mediated desensitization of the gonadotrope.

Transcriptional activation of gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: involvement of multiple signal transduction pathways.

Previous studies have shown that GnRH activates transcriptional activity of its own receptor (GnRHR) gene in part through the cAMP signal transduction pathway. In the present study we explored the possible involvement of multiple signal transduction pathways in GnRH regulation of GnRHR gene transcription; these studies relied upon a luciferase reporter gene vector (GnRHR-pXP2) containing a 1226-bp promoter fragment (-1164 to +62, relative to the major transcription start site) of the mouse GnRHR gene in GGH3 cells (GH3 cells stably expressing rat GnRHR). Activation of protein kinase C (PKC) by phorbol myristic acid significantly stimulated GnRHR-luciferase reporter gene (GnRHR-Luc) activity, but did not potentiate the stimulation of GnRHR-Luc activity by the GnRH agonist, buserelin (GnRH-A). Inhibition of PKC by PKC inhibitor (GF 109203X) or depletion of PKC blocked phorbol myristic acid- or GnRH-A-stimulated GnRHR-Luc activity, but did not affect (Bu)2cAMP-stimulated GnRHR-Luc activity. In addition, GnRH-A-stimulated GnRHR-Luc activity was inhibited by preventing external Ca2+ influx with the external Ca2+ chelator EGTA or the Ca2+ ion channel antagonist, D600. Surprisingly, overexpression of the mitogen-activated protein kinase (MAPK) kinase kinase (Raf-1) inhibited GnRHR-Luc activity and partially blocked GnRH-A-stimulated GnRHR-Luc activity. In contrast, inhibition of MAPK activity by MAPK kinase inhibitor (PD 98059) or by overexpression of kinase-deficient MAPKs activated basal and GnRH-A-stimulated GnRHR-Luc activity. These results suggested that PKC- and Ca2+-dependent signal transduction pathways participate in the GnRH activation of GnRHR promoter activity, and that the MAPK cascade is involved in the negative regulation of basal and GnRH-stimulated GnRHR transcriptional activity conferred by the 1226-bp promoter fragment.

The role of protein kinases A and C pathways in the regulation of mitogen-activated protein kinase activation in response to gonadotropin-releasing hormone receptor activation.

There is convincing evidence that mitogen-activated protein kinase (MAPK) activation is coupled to both receptor tyrosine kinase and G protein-coupled receptors. The presence of the epidermal growth factor (EGF) receptor and the GnRH receptor on the surface of GGH(3)1' cells makes this cell line a good model for the assessment of MAPK activation by receptor tyrosine kinases and G protein-coupled receptors. In this study, to assess the activated and total (i.e. activated plus inactivated) MAPK, the phosphorylation state of p44 and p42 MAPKs was examined using antisera that distinguish phospho-p44/42 MAPK (Thr202/Tyr204) from p44/42 MAPK (phosphorylation state independent). The data show that both EGF (200 ng/ml) and Buserelin (a GnRH agonist; 10 ng/ml) provoke rapid activation of MAPK (within 5 and 15 min, respectively) after binding to their receptors. The role of protein kinase A (PKA) and protein kinase C (PKC) signal transduction pathways in mediating MAPK activation was also assessed. Both phorbol ester (phorbol 12-myristate 13-acetate; 10 ng/ml) and (Bu)2cAMP (1 mM) trigger the phosphorylation of MAPK, suggesting potential roles for PKC and PKA signaling events in MAPK activation in GGH(3)1' cells. Treatment of PKC-depleted cells with Buserelin activated MAPK, suggesting involvement of PKC-independent signal transduction pathways in MAPK activation in response to GnRH. Similarly, treatment of PKC-depleted cells with forskolin (50 microM) or cholera toxin (100 ng/ml) stimulated MAPK activation, whereas pertussis toxin (100 ng/ml) had no measurable effect. To further assess the role of PKA in response to EGF and Buserelin, cells were treated with EGF (200 ng/ml) for 3 min or with Buserelin (10 ng/ml) for 10 min after pretreatment with 3-isobutyl-1-methylxanthine (0.5 mM), forskolin (50 microM), or (Bu)2cAMP (1 mM) for 15 min. The results show that MAPK can be activated in a PKA-dependent manner in GGH(3)1' cells. Consistent with previous reports, the current data support the view that MAPK activation can be achieved via both PKC- and PKA-dependent signaling pathways triggered by the GnRH receptor that couples to G(q/11) and Gs alpha-subunit proteins. In contrast, G(i/o)alpha does not appear to participate in MAPK activation in GGH(3)1' cells.