Agonist-induced regulation of pituitary receptors for gonadotropin-releasing hormone. Dissociation of receptor recruitment from hormone release in cultured gonadotrophs

The regulation of receptors for gonadotropin-releasing hormone (GnRH) by the homologous decapeptide ligand was analyzed in cultured rat anterior pituitary cells. Assay of GnRH receptors in both intact and disrupted cells showed that GnRH binding to gonadotrophs was rapidly followed by dose-dependent loss of sites that was maximal within 1 h. This early loss of GnRH receptors was not dependent on protein synthesis, and was attributable to ligand-induced processing of the peptide binding sites. No loss of GnRH sites was observed after receptor occupancy by a GnRH antagonist, or after target cell activation by exposure to a depolarizing concentration of KCl to stimulate luteinizing hormone release. After their initial down-regulation, GnRH receptors returned to normal and subsequently increased in concentration after 6 h of incubation. The delayed phase of receptor up-regulation was prevented by treatment with cycloheximide or actinomycin D and was calcium-dependent, being induced by 50 mM KCl and by low concentrations of the calcium ionophore, A23187. Conversely, calcium antagonists such as verapamil and MgCl2 impaired the agonist-induced increase of GnRH receptor sites. These findings have demonstrated that pituitary GnRH receptors undergo two distinct phases of regulation after interaction with the homologous ligand. The initial phase of agonist-dependent receptor loss is followed by a postsecretory phase of receptor recruitment that is dependent on protein synthesis. The expression of GnRH receptors can be completely dissociated from gonadotropin secretion, indicating that fusion of luteinizing hormone secretory granules with the plasma membrane is not a major pathway for transport of GnRH receptors to the cell surface in cultured gonadotrophs. Such changes in cell surface GnRH receptors during activation by the peptide agonist are relevant to the alterations in gonadotroph sensitivity that occur in vivo during physiological regulation of the pituitary gland by GnRH.     

Phosphatidic acid and the calcium-dependent actions of gonadotropin-releasing hormone in pituitary gonadotrophs

The stimulation of luteinizing hormone (LH) release and cyclic GMP (cGMP) production in rat anterior pituitary cells by gonadotropin-releasing hormone (GnRH) are receptor mediated and calcium dependent, and have been shown to be accompanied by increased phospholipid turnover and arachidonic acid release. The incorporation of 32Pi into the total phospholipid fraction of pituitary gonadotrophs was significantly elevated by 10(-8) M GnRH, with specific increases in the labeling of phosphatidylinositol and phosphatidic acid (PA). Since PA acts as a calcium ionophore in several cell types, its effects upon calcium-mediated gonadotroph responses were compared with those elicited by GnRH. In rat pituitary gonadotrophs prepared by centrifugal elutriation, PA stimulated LH release and cGMP production by 9-fold and 5-fold, respectively. The stimulation of LH release by 30 microM PA was biphasic in its dependence on extracellular calcium concentration, rising from zero in the absence of calcium to a maximum of 10-fold at 0.5 mM Ca2+ and declining at higher calcium concentrations. In dose-response experiments, PA was 3-fold more potent at 0.5 mM Ca2+ than at 1.2 mM Ca2+. The cGMP response to PA in cultured gonadotrophs was also calcium dependent, and was progressively enhanced by increasing Ca2+ concentrations up to 1.5 mM. The ability of PA to stimulate both LH release and cGMP formation in a calcium-dependent manner suggests that endogenous PA formed in response to GnRH receptor activation could function as a Ca2+ ionophore in pituitary gonadotrophs, and may participate in the stimulation of gonadotroph responses by GnRH and its agonist analogs.     

Actions of gonadotropin-releasing hormone analogues in pituitary gonadotrophs and their modulation by ovarian steroids

Recently, GnRH antagonists (GnRHant) like cetrorelix and ganirelix have been introduced in protocols of controlled ovarian hyperstimulation for assisted reproductive techniques to prevent premature luteinizing hormone (LH) surges. Here we tested, whether the actions of cetrorelix and the GnRH agonist (GnRHag) triptorelin in gonadotrophs are dependent on the steroid milieu. Furthermore, we characterized the actions of cetrorelix and triptorelin on LH secretion and the total LH pool. Female rat pituitary cells were treated either with 0.1 nM triptorelin for 1, 2, 4 and 6 days or for 1, 3, 5 and 6 h or with 1, 10 or 100 nM cetrorelix for 1, 2, 3 and 5 h or for 10 min. Cells were stimulated for 3h with different concentrations of GnRH (10 pM-1 microM). For analysis of the total LH pool, which is composed of stored and released LH, cells were lysed with 0.1% Triton X-100 at -80 degrees C overnight. To test, whether the steroid milieu affects the actions of cetrorelix and triptorelin, cells were incubated for 52 h with 1 nM estradiol (E) alone or with combinations of 100 nM progesterone (P) for 4 or 52 h, respectively. Cells were then treated with 0.1 nM triptorelin for 9 h or 1 nM cetrorelix for 3 h and stimulated for 3 h with different concentrations of GnRH (10 pM-1 microM). The suppressive effect of triptorelin on LH secretion was fully accomplished after 3 h of treatment, for cetrorelix only 10 min were sufficient. The concentration of cetrorelix must be at least equimolar to GnRH to block LH secretion. Cetrorelix shifted the EC50s of the GnRH dose-response curve to the right. Triptorelin suppressed total LH significantly (from 137 to 36 ng/ml) after 1 h in a time-dependent manner. In contrast, only high concentrations of cetrorelix increased total LH. In steroid treated cells the suppressive effects of triptorelin were more distinct. One nanomolar cetrorelix suppressed GnRH-stimulated LH secretion of cells not treated with steroids from 10.1 to 3.5 ng/ml. In cells, additionally treated with estradiol alone or estradiol and short-term progesterone, LH levels were higher (from 3.5 to 5.4 or 4.5 ng/ml, respectively). In cells co-treated with estradiol and progesterone for 52 h LH secretion was only suppressed from 10.1 to 9.5 ng/ml. Steroid treatments diminished the suppressive effect of cetrorelix on LH secretion. In conclusion, the depletion of the total LH pool contributes to the desensitizing effect of triptorelin. The actions of cetrorelix and triptorelin are dependent on the steroid milieu.     

Interaction of fluorescent gonadotropin-releasing hormone with receptors in cultured pituitary cells

A fluorescent derivative of the gonadotropin-releasing hormone (GnRH) agonist analog, [D-Lys6]GnRH, was synthesized for receptor studies and shown to be biologically active. The rhodamine-derivatized peptide (Rh-GnRH) retained 40% of the receptor binding activity of [D-Lys6]GnRH, and 50% of the luteinizing hormone-releasing activity assayed in cultured pituitary cells. The fluorescent analog was employed to visualize the distribution of GnRH receptors in cultured pituitary cells, using the technique of video-intensified fluorescence microscopy. The binding of Rh-GnRH was confined to the large gonadotrophs which comprised 15% of the cell population. The specificity of the binding was shown by the absence of significant fluorescence in the presence of a 100-fold excess of [D-Lys6]GnRH, or when Rh-GnRH was incubated with choriocarcinoma, neuroblastoma, or 3T3 cell lines devoid of GnRH receptors. The interaction of Rh-GnRH with living pituitary cells was characterized by an initial diffuse distribution, followed by the formation of polar aggregates that later appeared to be internalized. These observations emphasize the value of fluorescent derivatives of GnRH for elucidating the course of the interaction with specific receptors on pituitary gonadotrophs. The initial results indicate that GnRH-receptor complexes undergo aggregation during stimulation of luteinizing hormone release, and are later internalized for subsequent degradation and/ or intracellular actions.     

Effects of progesterone on gonadotropin-releasing hormone receptor concentration in cultured estrogen-primed female rat pituitary cells

Acute (0.5–4 h) treatment of estradiol (E)-primed female rat pituitary cells with progesterone (P) augments gonadotropin-releasing hormone (GnRH)-induced LH release, whereas chronic (48 h) P-treatment reduces pituitary responsiveness to the hypothalamic decapeptide. Dispersed E-primed (48 h, 1 nM) rat pituitary cells were cultured for 4 or 48 h in the presence of 100 nM P to assess the effects of the progestagen on GnRH receptors and on gonadotrope responsiveness to the decapeptide. P-treatment (4 h) significantly augmented GnRH-receptor concentrations (4.44 ± 0.6 fmol/10⁶ cells) as compared to cells treated only with E (2.6 ± 0.5fmol/10⁶ cells). Parallel significant changes in GnRH-induced LH secretion were observed. The acute increase in GnRH-receptor number was nearly maximal (180% of receptor number in cells treated with E alone) within 30 min of P addition. Chronic P-treatment (48 h) significantly reduced pituitary responsiveness to GnRH as compared to E-treatment. The GnRH-receptor concentrations (3.9 ± 0.6 fmol/10⁶ cells), however, remained elevated above those in E-primed cells. GnRH-receptor affinity was not influenced by any of the different treatments. These results indicate that the acute facilitatory P-effect on GnRH-induced LH release is at least chronologically closely related to an increase in GnRH-receptor concentration. The chronic negative P-effect on pituitary responsiveness to GnRH, however, shows no relation to changes in available GnRH receptors.     

Inhibitory effect of beta-endorphin on gonadotropin-releasing hormone and thyrotropin-releasing hormone releasing activity in cultured rat anterior pituitary cells

To study the effect of human beta-endorphin (beta h-End) on pituitary response to gonadotropin-releasing hormone (LH-RH) and thyrotropin-releasing hormone (TRH) in vitro, we used dispersed rat pituitary cells. When beta h-End (10(-7) M) was simultaneously added along with LH-RH, its stimulatory effect was blocked and naloxone (NAL, 10(-5) M) did not reverse the beta h-End inhibitory effect. NAL alone elicited an increase in LH release, but in the presence of both stimulants (LH-RH and NAL), LH secretion was lower than that observed with LH-RH alone. TRH stimulatory activity of TSH and PRL secretion was blunted by the presence of beta h-End (10(-7) M) and was not reversed by NAL (10(-5) and 10(-3) M). These data suggest that beta h-End directly blocks the LH, TSH- and PRL-secreting activity of both LH-RH and TRH at the pituitary level. This beta h-End effect is not reversed by the specific opiate receptor blocker NAL.     

Transient but not oscillating component of the calcium mobilizing response to gonadotropin-releasing hormone depends on calcium influx in pituitary gonadotrophs.

Gonadotropin-releasing hormone (GnRH)-stimulated changes in the cytosolic free Ca2+ concentration ([Ca2+]i) were studied in gonadotrophs cultured from 3-week ovariectomized rat pituitaries. One animal was used per cell preparation. [Ca2+]i was monitored in individual gonadotrophs by dual emission microspectrofluorimetry, using Indo-1 as the intracellular fluorescent Ca2+ probe. A short stimulation with GnRH evoked a complex concentration-dependent Ca2+ response in individual gonadotrophs. 0.1-1 nM GnRH triggered a series of sinusoidal-like [Ca2+]i oscillations superimposed upon a modest slow [Ca2+]i rise--the oscillating response mode--while 10-100 nM GnRH caused a biphasic increase in [Ca2+]i consisting of a monophasic transient and oscillations--the transient/oscillating response mode. Despite the consistency of Ca2+ responses, an inter-preparation heterogeneity of [Ca2+]i oscillations frequency was noticed. Moreover, we observed that, within a given cell preparation, the frequency of [Ca2+]i oscillations was independent of GnRH concentration whereas both peak [Ca2+]i and area under the [Ca2+]i versus time curve were concentration-dependent. Thus, in gonadotrophs, the presence of the GnRH signal would lead to [Ca2+]i oscillations, while the amplitude of the [Ca2+]i responses would code for the concentration of agonist. Both transient and oscillating components of GnRH responses depended on releasing activity of Ca(2+)-sequestering pools in as much as GnRH responses were unaffected by brief removal of external Ca2+, but suppressed by chelating intracellular free Ca2+ with BAPTA. However, prolonged exposure to a Ca(2+)-free medium suppressed the transient component while leaving the oscillating component unaffected. We therefore propose that gonadotrophs employ Ca(2+)-sequestering pools, whose maintenance depends on a slow Ca(2+)-entry, to give an amplitude-coded Ca2+ rise in response to a short GnRH stimulation.     

Increase in follicle stimulating hormone content occurs in cultured human fetal pituitary cells exposed to gonadotropin-releasing hormone

To investigate the mechanisms by which GnRH regulates FSH production in the human fetus, dispersed pituitary cells from second trimester human fetuses were cultured on surface-modified plates. Exposure of cells to GnRH [(10(-8) and 10(-7) mol/L), study I] or [D-Ala6]des-Gly10-GnRH ethylamide (DALA) [(10(-11) to 10(-7) mol/L), study II] for 48 h resulted in an elevation of total FSH which correlated with an increase in releasable, but not nonreleasable, FSH. When pituitary cells were incubated for 24, 48 and 72 h with and without 10(-8) mol/L GnRH (study III), total FSH was significantly increased in cells cultured for 48-72 h without GnRH compared to cells lysed at the beginning of the incubation (p less than 0.001). At all intervals, GnRH significantly enhanced total FSH compared to respective controls (p less than 0.05).     

Modulation of gonadotropin-releasing hormone receptor concentration in cultured female rat pituitary cells by estradiol treatment

Short-term (0.5-4 h) treatment of rat pituitary cells in culture with estradiol (E2) results in a significant decrease of Gonadotropin-Releasing Hormone (GnRH) induced LH-release. We studied whether changes in the concentrations of GnRH-receptors (GnRH-R) might account for this phenomenon: pituitary cells from adult female rats were incubated for 4 or 24 h in the presence or absence of 10(-9) M E2. Then saturation curves of D-Ala6-des-Gly10-GnRH ethylamide binding were obtained. In addition, binding studies were carried out in cultures incubated for 0.5, 1, 2 or 4 h with or without 10(-9) M E2 using a near saturating concentration of GnRH-analog. No changes of GnRH-R affinity occurred (4 h experiments: Ka in vehicle treated cells: 0.94 +/- 0.2 x 10(9) M-1, Ka in E2 treated cells: 1.06 +/- 0.3 x 10(9) M-1; 24 h experiments: Ka vehicle: 0.95 +/- 0.2 x 10(9) M-1, Ka E2: 0.82 +/- 0.3 x 10(9) M-1). The GnRH-R concentrations, however, were significantly reduced (44 +/- 3%; P less than 0.001) by 4 h E2 treatment and increased (by 68 +/- 8%; P less than 0.01) by 24 h of E2 treatment. The GnRH induced LH-release in aliquots of the same cell preparations was significantly reduced after 4 h and markedly increased after 24 h of E2 treatment. The experiments on the time-course of the reduction of D-Ala6-GnRH-binding by E2 treatment showed that the number of GnRH-R was significantly decreased (24 +/- 1%; P less than 0.05) already after 0.5 h of exposure to the estrogen. This is also the time period after which the negative E2-effect on GnRH-induced LH-release becomes significant. These data provide first evidence that the short-term negative E2-effect on GnRH induced LH-release by rat pituitary cells in culture could be mediated via a reduction of available GnRH-R.     

Pituitary adenylate cyclase-activating polypeptide stimulates nitric-oxide synthase type I expression and potentiates the cGMP response to gonadotropin-releasing hormone of rat pituitary gonadotrophs

Nitric-oxide synthase type I (NOS I) is expressed primarily in gonadotrophs and in folliculo-stellate cells of the anterior pituitary. In gonadotrophs, the expression and the activity of NOS I are stimulated by gonadotropin-releasing hormone (GnRH) under both experimental and physiological conditions. In the present study, we show that pituitary adenylate cyclase-activating polypeptide (PACAP) is twice as potent as GnRH at increasing NOS I levels in cultured rat anterior pituitary cells. The action of PACAP is detectable after 4-6 h and maximal at 24 h, this effect is mimicked by 8-bromo-cAMP and cholera toxin and suppressed by H89 suggesting a mediation through the cAMP pathway. Surprisingly, NADPH diaphorase staining revealed that these changes occurred in gonadotrophs exclusively although PACAP and cAMP, in contrast to GnRH, have the potential to target several types of pituitary cells including folliculo-stellate cells. There was no measurable alteration in NOS I mRNA levels after cAMP or PACAP induction. PACAP also stimulated cGMP synthesis, which was maximal within 15 min and independent of cAMP, however, only part resulted from NOS I/soluble guanylate cyclase activation implying that in contrast to GnRH, PACAP has a dual mechanism in cGMP production. Interestingly, induction of NOS I by PACAP markedly enhanced the capacity of gonadotrophs to produce cGMP in response to GnRH. The fact that PACAP may act on gonadotrophs to alter NOS I levels, generate cGMP, and potentiate the cGMP response to GnRH, suggests that cGMP could play important cellular functions.     

Coincidence of down-regulation and desensitization in pituitary gonadotrophs stimulated by gonadotropin releasing hormone

The dynamics of gonadotropin releasing hormone (GnRH) induced luteinizing hormone (LH) release was studied $\text{in}$$\text{vitro}$ by superfusion of cultured pituitary cells. Continuous exposure of the cells to GnRH resulted in desensitization of the gonadotroph responsiveness to further stimulation by the hormone. The refractory state was achieved within 4 hr of hormone introduction (10^?7 M) and was accompanied by down-regulation of GnRH receptors (50%) assayed by equilibration with [¹²⁵I]iodo-[D-Ala⁶]des-Gly¹⁰-GnRH N-ethylamide. The data indicate that GnRH can regulate the number of its own receptors, and that desensitization is accompanied by down-regulation.     

Evolutionary aspects of gonadotropin-releasing hormone and its receptor

Summary 1. Gonadotropin-releasing hormone (GnRH) was originally isolated as a hypothalamic peptide hormone that regulates the reproductive system by stimulating the release of gonadotropins from the anterior pituitary. However, during evolution the peptide was subject to gene duplication and structural changes, and multiple molecular forms have evolved.2. Eight variants of GnRH are known, and at least two different forms are expressed in species from all vertebrate classes: chicken GnRH II and a second, unique, GnRH isoform.3. The peptide has been recruited during evolution for diverse regulatory functions: as a neurotransmitter in the central and sympathetic nervous systems, as a paracrine regulator in the gonads and placenta, and as an autocrine regulator in tumor cells.4. Evidence suggests that in most species the early-evolved and highly conserved chicken GnRH II has a neurotransmitter function, while the second form, which varies across classes, has a physiologic role in regulating gonadotropin release.5. We review here evolutionary aspects of the family of GnRH peptides and their receptors.     

Activation of translation in pituitary gonadotrope cells by gonadotropin-releasing hormone

The neuropeptide GnRH is a central regulator of mammalian reproductive function produced by a dispersed population of hypothalamic neurosecretory neurons. The principal action of GnRH is to regulate release of the gonadotropins, LH and FSH, by the gonadotrope cells of the anterior pituitary. Using a cultured cell model of mouse pituitary gonadotrope cells, alphaT3-1 cells, we present evidence that GnRH stimulation of alphaT3-1 cells results in an increase in cap-dependent mRNA translation. GnRH receptor activation results in increased protein synthesis through a regulator of mRNA translation initiation, eukaryotic translation initiation factor 4E-binding protein, known as 4EBP or PHAS (protein, heat, and acid stable). Although the GnRH receptor is a member of the rhodopsin-like family of G protein-linked receptors, we show that activation of translation proceeds through a signaling pathway previously described for receptor tyrosine kinases. Stimulation of translation by GnRH is protein kinase C and Ras dependent and sensitive to rapamycin. Furthermore, GnRH may also regulate the cell cycle in alphaT3-1 cells. The activation of a signaling pathway that regulates both protein synthesis and cell cycle suggests that GnRH may have a significant role in the maintenance of the pituitary gonadotrope population in addition to directing the release of gonadotropins.     

Effects of gonadotropin-releasing hormone on growth hormone secretion and gene expression in common carp pituitary

Using radioimmuno- and ribonuclease protection assays, we examined the effects of gonadotropin-releasing hormone and its analogs on the growth hormone mRNA level and growth hormone secretion in common carp (Cyprinus carpio) pituitary fragments with static incubation. After a 24 h treatment, sGnRH ([Trp(7),Leu(8)]-LHRH) and sGnRH-A ([D-Arg(6),Pro(9)]-LHRH) (0.1 nM-1 microM) elevated the GH mRNA level and stimulated the GH secretion in a dose-dependent manner, with a higher potency for sGnRH-A. In a time-course experiment, the function of sGnRH and sGnRH-A (10 nM) on GH secretion was observed after 6 h incubation, while no action on the GH mRNA level were noted until 12 h after treatment. Comparing mammalian GnRH, avian GnRH and piscine GnRH, sGnRH and sGnRH-A showed the highest potency in increasing GH mRNA level and GH-release, followed by cGnRH-II ([His(5),Tyr(8)]-LHRH), and finally LHRH and LHRH-A([D-Trp(6), Pro(9)]-LHRH). These findings, taken together, suggest that GnRH not only can influence GH release, but also play a role in the regulation of GH synthesis.     

Solubilization and purification of rat pituitary gonadotropin-releasing hormone receptor

Gonadotropin-releasing hormone (GnRH) receptors were solubilized from rat pituitary membrane preparations in an active form by using the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid). The solubilized receptor exhibits high affinity, saturability, and specificity. The soluble supernatant retained 100% of the original binding activity when stored at 4 or -20 degrees C in the presence of 10% glycerol. The receptors were resolved into two components on the basis of chromatography on wheat germ agglutinin-agarose. Homogeneous receptor preparation was obtained by two cycles of affinity chromatography on immobilized avidin column coupled to [biotinyl-D-Lys6]GnRH. The overall recovery of the purified receptor was 4-10% of the initial activity in the CHAPS extract, and the calculated purification -fold was approximately 10,000 to 15,000. Analysis of iodinated purified GnRH receptors by autoradiography indicated the presence of two bands, Mr = 59,000 and 57,000. This was confirmed by photoaffinity labeling of the partially purified receptors and suggests that both components can specifically bind the hormone.     

Demonstration of a nonsteroidal factor in human follicular fluid that attenuates the self-priming action of gonadotropin-releasing hormone on pituitary gonadotropes

The self-priming effect of gonadotropin-releasing hormone (GnRH) on luteinizing hormone (LH) release can be demonstrated in vitro by perfusing pituitary tissue with a continuous GnRH stimulus. A characteristic biphasic response is produced. We have used this system to investigate whether or not human follicular fluid (hFF) contains a nonsteroidal substance that can attenuate the GnRH-induced LH secretion in perfused rat pituitary glands. Steroid-extracted hFF, added to the perfusing medium, attenuated the self-priming action of GnRH in a dose-dependent manner. This was not abolished by selectively depleting the inhibin content of hFF by 97% on an immunoaffinity column. Furthermore, the biological activity of the substance was resistant to heating and was removed by dialysis. It is concluded that hFF contains a nonsteroidal factor, distinct from inhibin, that can attenuate the self-priming action of GnRH on pituitary gonadotropes.     

Neutrophil cathepsin G increases calcium flux and inositol polyphosphate production in cultured endothelial cells

Exposure of endothelial cells (ENDO) to human neutrophil cathepsin G (CG) increases albumin flux across the endothelial monolayer. Since calcium influences cell shape and barrier function of ENDO monolayers, the current study was designed to determine if CG acted through alterations in Ca2+ homeostasis in ENDO. The role of Ca2+ in the increased permeability of ENDO monolayers to albumin after exposure to CG was studied by using ENDO monolayers cultured on polycarbonate filters. Exposure of ENDO monolayers to CG in the presence of the Ca2+-antagonist lanthanum partially prevented the increase in albumin flux, but exposure in the presence of agents that block voltage-regulated calcium channels did not block the increase in albumin flux. To monitor the effect of CG on Ca2+-flux, ENDO were labeled with 45Ca2+ and changes in Ca2+ flux were monitored by the release of 45Ca2+. From 1 to 15 minutes after exposure of ENDO to CG, there was increased release of 45Ca2+ compared with control cells. Calcium channel blocking agents did not inhibit the increased release of 45Ca2+, but lanthanum partially blocked the increase. The increased release of Ca2+ appeared to be due, at least in part, to activation of phospholipase C because there was an increase both in inositol polyphosphate species and in diglycerides after incubation of ENDO with CG. These studies support the hypothesis that CG increases the flux of calcium in ENDO, that this increase in Ca2+ flux may result from activation of phospholipase C, and that this system may be involved in the decreased barrier properties of the ENDO after CG exposure.     

Mechanisms of gonadotropin-releasing hormone and prostaglandin action on luteal cells

Both gonadotropin-releasing hormone (GnRH) and prostaglandin F2 alpha (PGF2 alpha) can inhibit cAMP and progesterone production in the corpus luteum; however, their mechanism of action is not known. GnRH or PGF2 alpha causes a rapid and marked increase of labelling of phosphatidylinositol (PI) and phosphatidic acid (PA) in rat luteal cells in culture. The incorporation of radioactivity is increased as early as 2 and 5 min into PA and PI, respectively. The labelling of the other phospholipids is not affected. GnRH and PGF2 alpha exert their stimulatory effects on PA-PI turnover at a mean effective dose value of ca. 15 and 100 nM, respectively. Their effects appeared to be additive when both agents were present in the same incubations. Interestingly, addition of the calcium ionophore A23187 also causes a dramatic increase of PA-PI turnover in luteal cells. By contrast, human chorionic gonadotropin and isoproterenol, agents that stimulate cAMP and progesterone production in luteal cells, as well as PGE2 (1 microM), all fail to alter phospholipid labelling; dibutyryl or 8-bromo-cAMP (2-5 mM) actually attentuates the GnRH or PGF2 alpha effect on PI and PA. A very similar PA-PI response to GnRH and PGF2 alpha has also been observed using rat granulosa cells in culture. It seems that following their binding to membrane receptors, GnRH and PGF2 alpha may share a common mechanism in the ovarian cell, possibly involving the stimulation of PA-PI metabolism.