Evidence for oxytocin receptors in cultured bovine luteal cells.

Specific receptors for oxytocin (OT) on intact luteal cells are demonstrated. Cultured cells from bovine corpora lutea (CL) at different stages (Days 3-5, 8-12, and 15-18 of the estrous cycle) were examined for OT receptors by a radioreceptor assay using the 125I-labeled OT antagonist [d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH2(9)] -vasotocin. Binding specificity was demonstrated in displacement studies with various related peptides. Scatchard analysis revealed the presence of a binding site with an association constant of Ka = 2.6 x 10(9) M-1 and a capacity of 5.9 fmol/micrograms DNA. Additionally, in 50% of the experiments (n = 6) two different binding sites were observed. The Ka of the high-affinity site was 2.6 x 10(10) M-1; its capacity was 0.73 fmol/micrograms DNA. The low-affinity site had an apparent Ka of 4.9 x 10(8) M-1 and a capacity of 8.8 fmol/micrograms DNA. Observation of one versus two binding sites related neither to the assay conditions nor to the state of the individual CL used for the cell culture and therefore appeared to reflect individual variation within the OT receptor population. Significant binding of OT was observed at all luteal stages. OT binding was maximal at the mid-luteal stage (Days 8-12). We conclude that a direct action of OT on the bovine CL is mediated by the OT receptor, supporting the hypothesis that luteal OT plays an important physiological role in the regulation of progesterone release and/or other luteal functions in a paracrine or autocrine fashion.     

Evidence for different gonadotropin-releasing hormone response sites in rat ovarian and pituitary cells

The participation of type I GnRH receptor (GnRH-R) on GnRH-II-induced gonadotropin secretion in rat pituitary cells was investigated. Furthermore, we extended the study of GnRH-II action to ovarian cells. The GnRH-II was able to mobilize inositol triphosphate (IP(3)) and to induce LH and FSH release in a dose-dependent manner in pituitary cells and in a GnRH-I-like manner. The GnRH-analog 135-18 (agonist for type II GnRH-R and antagonist for type I GnRH-R) was unable to elicit any cellular response tested in these pituitary cells. The GnRH-II responses were blocked by the type I GnRH-R-antagonists CRX or 135-18, suggesting that these effects were mediated by the type I GnRH-R. In contrast to pituitary cells, GnRH-I, but not GnRH-II, elicited an IP(3) response in superovulated ovarian cells; 135-18 also had no effect. However, GnRH-II as well as GnRH-I presented antiproliferative effects on these cells. Surprisingly, 135-18 had stronger antiproliferative effects than either GnRH peptide. The 135-18 analog, but not GnRH-I or GnRH-II, increased progesterone secretion in superovulated ovarian cells. These results strongly suggest that GnRH-II is able to stimulate rat pituitary cells through the type I GnRH-R, with no evidence for the presence of type II GnRH-R. On the other hand, our results indicate a putative GnRH-R in superovulated ovarian cells with response characteristics that differ from those of the GnRH-R in the pituitary.     

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.     

Evidence for tight coupling of thyrotropin-releasing hormone receptors to stimulated inositol trisphosphate formation in rat pituitary cells

The effect of decreasing the concentration of receptors for thyrotropin-releasing hormone (TRH) on the surface of cloned rat pituitary (GH3) cells on TRH-stimulated inositol trisphosphate (Ins-P3) formation was investigated. Incubation of cells with dibutyryl cAMP (Bt2cAMP) for 16 h caused a decrease in [3H] TRH binding to intact cells to a minimum level 37 +/- 9.1% of control. Scatchard analysis of the concentration dependency of [3H]TRH binding showed that the effect of Bt2cAMP was to lower the receptor concentration without affecting its affinity for TRH. Similar decreases in [3H]TRH binding were found in cells incubated with 8-bromo-cAMP, cholera toxin, and sodium butyrate and, as shown previously, with TRH. In cells incubated with 1 mM Bt2cAMP for 16 h, but not for 1 h, the maximum TRH-induced increase in Ins-P3 was inhibited to 25 +/- 3.2% of that in control cells. Inhibition of TRH-induced Ins-P3 formation was also observed in cells treated with 8-bromo-cAMP, cholera toxin, and sodium butyrate for 16 h, and with TRH for 48 h. Inhibition of TRH-induced Ins-P3 formation and lowering of TRH receptor concentration caused by Bt2cAMP occurred in parallel with increasing doses of Bt2cAMP; at 16 h of exposure, half-maximal effects occurred with 0.3 mM Bt2cAMP. The concentration dependency of TRH-induced Ins-P3 formation was the same in control and Bt2cAMP-treated cells; half-maximal effects occurred with 10 nM TRH. These data demonstrate that decreases in TRH receptor concentration caused by several agents that act via different mechanisms are associated with reduced stimulation of Ins-P3 formation and suggest that the TRH receptor is tightly coupled to stimulation of hydrolysis of phosphatidylinositol 4,5-bisphosphate by a phospholipase C.     

Follicle-stimulating hormone enhances somatomedin C binding to cultured rat granulosa cells. Evidence for cAMP dependence

The ovarian granulosa cell has recently been shown to be the site of Somatomedin C (Sm-C) production, reception, and action. To further elucidate the relevance of Sm-C to granulosa cell physiology, we have undertaken to study the regulation of its receptor under in vitro conditions using a primary culture of rat granulosa cells. Granulosa cells cultured without treatment for 72 h displayed limited, albeit measurable, specific Sm-C binding. However, continuous treatment with increasing concentrations of follicle-stimulating hormone (FSH) for the duration of the 72-h incubation period resulted in dose-dependent increments in Sm-C binding (1.7-, 2.9-, 3.9-, and 3.6-fold increases over untreated controls for 50, 100, 180, and 330 ng/ml of FSH, respectively). This apparent up regulatory action of FSH proved time-dependent, with a minimal time requirement of 24-48 h. Granulosa cell Sm-C binding was similarly enhanced following elevation of the intracellular cAMP content by a series of cAMP-generating agonists, inhibition of cAMP-phosphodiesterase activity, or the provision of nondegradable cAMP analogs. Our findings further indicate that high dose forskolin, like FSH, is capable of augmenting Sm-C binding by itself, that a relatively inactive low dose of forskolin synergizes with FSH in this regard, but that combined treatment with maximal stimulatory doses of both agonists does not prove additive. Taken together, these observations indicate that FSH is capable of exerting a stimulatory effect on granulosa cell Sm-C binding and that cAMP, its purported intracellular second messenger, may play an intermediary role in this regard.     

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.     

Evidence for gonadotropin-releasing hormone receptor mRNA expression by estrogen in rat granulosa cells

The hormonal regulation of ovarian gonadotropin-releasing hormone (GnRH) receptor mRNA expression has been examined by in situ hybridization in hypophysectomized immature rats. In hypophysectomized rats, GnRH receptor mRNA expression is localized in the interstitial cells. After diethylstilbestrol treatment, most follicles grow to form early antral follicles and express GnRH receptor mRNA in the peripheral part of the granulosa layer, indicating that the expression in the growing follicles is estrogen-dependent. Only weak or no expression of the receptor mRNA is detectable in the atretic follicles of hypophysectomized rats, whereas very strong expression has been observed in the granulosa cells of atretic follicles of intact immature rats. Administration of testosterone or a GnRH agonist, both of which are atretic agents for ovarian follicles, to hypophysectomized rats markedly increases the apoptotic cell death of the granulosa cells but fails to induce GnRH receptor mRNA expression. The co-administration of these agents with diethylstilbestrol causes the granulosa cells of atretic follicles to express the receptor mRNA very strongly, suggesting that this mRNA expression in the atretic follicles is also estrogen-dependent. On the other hand, expression of the receptor mRNA in the ovarian interstitial cells is not affected by hypophysectomy or hormone treatments. All of these results clearly indicate that estrogen is essential for the expression of ovarian GnRH receptor mRNA in the granulosa cells of atretic follicles and growing follicles, whereas the expression in the interstitial cells is estrogen-independent.     

Properties of receptors for neurotoxic phospholipases A2 in different tissues

A radioiodinated derivative of OS₂ (¹²⁵I–OS₂), a neurotoxic monochain phospholipase A₂ isolated from taipan venom, was previously found to bind to a specific brain membrane receptor with very high affinity.¹²⁵I–OS₂ is now used to identify the properties of neurotoxic phospholipase receptors in other tissues. Heart, skeletal muscle, kidney, lung, liver, pancreas, and smooth muscle membranes also contain high-affinity binding sites for toxic phospholipases A₂. In most tissues, two different types of receptor sites have been characterized for¹²⁵I–OS₂ with K_d1 and K_d2 values in the 1–5 pM and the 10–50 pM range respectively. Whereas all receptors are similar in the different tissues in terms of their affinity for¹²⁵I–OS₂, maximal binding site capacities were very different, varying from 1.4 pmol/mg of protein in brain to 0.01 pmol/mg of protein in pancreaas. In brain, heart, and skeletal muscle, receptor densities vary with in vivo development. Affinity labeling experiments have identified the subunit composition of OS₂ receptors and indicated that these receptors do not have identical structures in the different tissues. Binding competition studies with OS₂ and other toxic phospholipases showed tissue-dependent pharmacological profiles. All these results taken together suggest the existence of a family of receptors for neurotoxic phospholipases.The abbreviations used are PLA₂ phospholipase A₂ - DSS suberic acid bis-N-hydroxysuccinimide ester - EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)1-piperazine ethanesulfonic acid - SDS sodium dodecyl sulfate - OS₁ Oxyuranus scutellatus scutellatus toxin 1 - OS₂ Oxyuranus scutellatus scutellatus toxin 2 Special issue dedicated to Dr. Lawrence Austin.     

Effect of luteal metoclopramide-induced hyperprolactinemia on pituitary and luteal responsiveness to gonadotropin-releasing hormone

The pituitary and corpus luteum responses to acute gonadotropin-releasing hormone (GnRH) administration at the mid-luteal phase (LP) were studied in 24 infertile women. Patients were randomly divided into two groups. In one group (n = 12) metoclopramide (MCP, 10 mg orally 3 times daily) was administered from day 0 or 1 of the LP for 7 days. On day 7 or 8 of LP blood samples were taken every 15 min for 180 min; then 25 micrograms GnRH were acutely administered intravenously and blood samples taken at 185, 195, 210, 225, 240, 255, 270, 285 and 300 min. In the other 12 patients the same experimental design was performed on day 7 or 8 of an untreated LP. Plasma prolactin (PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone and estradiol (E2) were assayed. The responsiveness of the different hormones to GnRH was evaluated as the integrated secretory area for 120 min after injection (sISA = stimulated integrated secretory area) and as the percentage increase (delta A) with respect to the area under basal conditions before GnRH administration (bISA = basal integrated secretory area). MCP-treated women showed higher basal PRL levels (p less than 0.01) and lower basal plasma concentrations and bISA (p less than 0.01) values of LH than controls. After GnRH a more marked response of LH secretion was observed in the treated group (p less than 0.01), so that the absolute values of sISA were superimposable in both groups. Basal and stimulated FSH secretion did not differ significantly in the study groups. Basal plasma and bISA values of progesterone were also decreased in MCP-treated subjects. After GnRH injection the absolute values of progesterone sISA were greater in controls (p less than 0.01), but delta A values were similar in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thyrotropin-releasing hormone and gonadotropin-releasing hormone receptors activate phospholipase C by coupling to the guanosine triphosphate-binding proteins Gq and G11.

The regulation of pituitary hormone secretion by TRH and GnRH proceeds through similar mechanisms which employ phosphoinositide hydrolysis to generate intracellular signals. Proximal events involve receptor activation of heterotrimeric (alpha beta gamma) GTP-binding (G) proteins which regulate phospholipase (PLC) activity. Since TRH and GnRH actions are not affected by cholera or pertussis toxin, a novel G protein (Gp) was suggested to mediate receptor regulation. The required Gp protein has not been identified and this was the focus of the present study. Recent molecular cloning and biochemical studies have characterized two novel, pertussis toxin-insensitive alpha-subunit proteins of the Gq subfamily (alpha q and alpha 11) which regulate the activity of the beta 1 isoenzyme of PLC. Gq and G11 represent the best candidates for the PLC-activating G proteins which mediate the actions of TRH and GnRH. To test this directly, an antibody to the common Gq/11 alpha-subunit carboxyterminal sequence was generated and shown to react with unique 42-kilodalton Gq alpha and 43-kilodalton G11 alpha proteins in membranes from TRH-responsive GH3 cells and GnRH-responsive alpha T3-1 pituitary cells. The Gq/11 alpha peptide antibody was shown to immunodeplete the Gp activity of GH3 cell membrane extracts measured by reconstitution of the guanine nucleotide regulation of PLC-beta 1. In addition, the immunoglobulin G fraction of Gq/11 alpha peptide immune serum specifically inhibited TRH- and GnRH-stimulated PLC activity measured in the membranes of GH3 and alpha T3-1 cells, respectively. The results indicate that TRH and GnRH activation of PLC requires receptor coupling to a Gp protein(s) which corresponds to Gq, G11 or both.     

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.     

Metabolism of high-density lipoproteins in cultured rat luteal cells

The uptake of cholesterol from high-density lipoproteins (HDL) labeled with 125I and [3H]cholesterol was examined in cultured rat luteal cells. Luteal cells were incubated with labeled HDL, following which the metabolic fate of the apolipoproteins and cholesterol moieties of the receptor-bound HDL were examined. About 50% of the originally bound HDL apolipoproteins were released into the medium in 24 h by a temperature-dependent process while only 5% of the HDL cholesterol was released unmetabolized. Inclusion of unlabeled HDL in the chase incubation resulted in increased release of apolipoprotein-derived radioactive products without significant change in the release of unmetabolized cholesterol. 60% of the apolipoprotein-derived radioactivity could be precipitated with trichloroacetic acid; the remaining trichloroacetic acid-soluble radioactive fraction was identified as [125I]iodotyrosine. Gel filtration chromatography of the chase-released material showed that the trichloroacetic acid-precipitable products, which contained no detectable amounts of cholesterol, eluted over a range of molecular sizes (9-80 kDa). No intact HDL was retroendocytosed. About 80% of trichloroacetic acid-precipitable products could be immunoadsorbed on anti-apolipoprotein A-I antibody immobilized on CNBr-activated Sepharose, suggesting the presence of fragments containing apolipoprotein A-I. This material was also capable of reassociating with native HDL. Lysosomal inhibitors were partially effective in inhibiting the amount of trichloroacetic acid-soluble products formed. The lysosomal degradation appeared to have no role in the uptake of HDL-derived cholesterol. These studies demonstrate preferential and total uptake of HDL cholesterol by luteal cells, with concomitant degradation of the lipoprotein.     

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.     

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.     

Prolactin receptors in cultured rat mammary tumor cells. Energy-dependent uptake and degradation of hormone receptors

After energy depletion by uncouplers of oxidative phosphorylation or inhibitors of electron transport, primary cultures of carcinogen-induced rat mammary tumors have a 2- to 20-fold increase in the number of cell surface prolactin receptors. When energy-depleted cells were treated with 0.15 M NaCl plus 50 mM glycine pH 3, for 1 min at 4 degrees C, 75% of the specific surface-bound 125I-labeled ovine prolactin was removed, but prolactin and its receptor were not destroyed. Using this technique, we found that receptor-bound prolactin can be internalized (becomes resistant to pH 3.0 treatment) and then degraded. The internalization of occupied receptors required energy, was completed 30-60 min before degradation, and was independent of protein synthesis. Hormone degradation (t1/2, 42 min) but not uptake was prevented by NH4Cl or lysosomotropic amines. In the presence of cycloheximide, receptors were lost (t1/2, 62 min) unless such loss was prevented by KCN. After unoccupied receptors were activated by energy depletion, surface receptors were lost when inhibitor was removed and glucose was added. Thus, both occupied and unoccupied prolactin receptors are constantly removed from the cell surface via an energy-dependent uptake mechanism. If the receptor levels are first increased by energy depletion (with or without bound ligand) or if protein synthesis is inhibited, there is a net loss of surface binding sites. Since the receptors reappeared with 15 h after cycloheximide removal, some of the receptors probably are recycled under normal steady state conditions.     

Differential desensitization and internalization of three different bullfrog gonadotropin-releasing hormone receptors

We previously demonstrated the presence of three distinct types of the gonadotropin-releasing hormone receptor (GnRHR) in a bullfrog (denoted bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). The bfGnRHRs exhibited differential tissue distribution and ligand selectivity. In the present study, we demonstrated the desensitization and internalization kinetics of these receptors in both transiently-transfected HEK293 cells and retrovirus-mediated stable cells. The time-course accumulation of the inositol phosphate in response to GnRH revealed that bfGnRHR-1 and -2 were rapidly desensitized, whereas bfGnRHR-3 was slowly desensitized. A comparison of the internalization kinetics revealed the most rapid rate and highest extent of internalization of bfGnRHR-2 among the three receptors. Interestingly, the mechanisms that underlie the receptor internalization appear to differ from each other. Internalization of bfGnRHR-1 was dependent on both dynamin and beta-arrestin, whereas those of bfGnRHR-2 and -3 were dependent on dynamin, but not on arrestin. These results, therefore, suggest that differential regulatory mechanisms for desensitization and internalization of the GnRHR are involved in diverse cellular and physiological responses to GnRH stimulation.     

Detection of three distinct phospholipases A2 in cultured mast cells.

Phospholipase A2 activity in lysates of mast cells such as rat mastocytoma RBL-2H3 cells and mouse bone marrow-derived IL-3-dependent mast cells (BMMC) was measured using phosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylserine (PS) as a substrate. Both types of cells exhibited phospholipase A2 activity with a similar pH profile; the optimum pH observed with PS as a substrate was 5.5-7.4, whereas that with PE or PC was 8.0-9.0. PE and PC bearing an arachidonate at the sn-2 position were cleaved more efficiently by PE, PC-hydrolyzing phospholipase A2 than phospholipids with a linoleate. A monoclonal antibody raised against rabbit platelet 85-kDa cytosolic phospholipase A2 absorbed the PE, PC-hydrolyzing activity. PS-hydrolyzing activity was purified from RBL-2H3 cells and BMMC by sequential heparin-Sepharose, butyl-Toyo-pearl, and reverse-phase HPLC. On reverse-phase HPLC, the PS-hydrolyzing activity of RBL cells was separated into two peaks, A and B. The peak B activity was inhibited by the anti-rat 14-kDa group II phospholipase A2 antibody, while the peak A activity was not. The partially purified peak A activity hydrolyzed PS about 10-fold more efficiently than PE at optimum pH of 5.5-7.4. No appreciable hydrolysis was observed with PC or phosphatidylinositol (PI). Thus, mast cells may express at least three distinct phospholipases A2; 14-kDa group II phospholipase A2, 85-kDa cytosolic arachidonate preferential phospholipase A2, and a novel phospholipase A2 that shows high substrate specificity for PS.