Critical implication of transmembrane Phe310, possibly in conjunction with Trp279, in the rat gonadotropin-releasing hormone receptor activation.

The GnRH-R belongs to the superfamily of heptahelical GPCRs. A three-dimensional model of GnRH binding to its receptor predicted that Trp3 was the most deeply buried residue, potentially allowing it to interact with both Trp279, a highly conserved residue in the TMH 6 of GPCRs, and Phe310, present essentially in TMH 7 of GnRH-Rs. Replacement of Phe310 with Leu, the most common positional residue in GPCRs, induced a slightly decreased Bmax (1.6-fold) and affinity (3.8-fold); in addition, IP production was completely abolished. Similarly, replacement of Trp279 with Ser depressed the Bmax by 5.2-fold, the affinity by 2.3-fold, and totally abrogated IP production. The effect of the double mutation was not additive on binding, since the Bmax was reduced to the level of the Phe310Leu mutant, although the Kd was restored to a value not significantly different from that of the wild-type. The double mutant was also unable to induce IP production. Unexpectedly, no influence of any single or double substitution was noted on receptor internalization. These data provide evidence for the crucial role of Phe310, possibly in conjunction with Trp279, on GnRH transduction and suggest that the conformation for phospholipase C activation may not be required for GnRH-R internalization.

Functional importance of transmembrane helix 6 Trp(279) and exoloop 3 Val(299) of rat gonadotropin-releasing hormone receptor.

Previous studies have established that the interaction of gonadotropin-releasing hormone (GnRH) with its receptor (GnRHR) would require partial entry of the N- and C-terminal regions of ligand into the transmembrane core. The functional significance of the conserved aromatic residue Trp(279) present in the transmembrane helix 6, and Val(299) located in exoloop 3 of the rat GnRHR was investigated by mutagenesis followed by expression in Chinese hamster ovary-K1 cells. Compared with wild-type, substitution of Trp(279) with Ser or Arg resulted in a marked reduction or total abolition, respectively, of ligand binding and, in both cases, abrogation of GnRH-induced inositol phosphate production. A total absence of functionality was observed when Val(299) was simply replaced with Ala. Mention should be made that an expression of all mutated and wild-type receptor proteins was observed. Interestingly, the double mutant [Trp(279)Arg/Val(299)Ala]GnRHR restored B(max) to wild type (504 +/- 43 versus 541 +/- 41 fmol/mg protein), but with a diminished affinity (4.95 +/- 1.05 versus 0.94 +/- 0.35 nM), and GnRH failed to induce inositol phosphate. No influence of the mutations was seen on internalization of the receptor. The three-dimensional model of GnRH binding to the rat GnRHR was built predicting that Trp(279) is buried at 20 A in the transmembrane core of the receptor, directly in contact with Trp(3) of GnRH. In contrast, Val(299) is located in a region that cannot be precisely defined at the extracellular end of transmembrane helix 7. Although models cannot provide any clue concerning the observed interactivity between the two distal residues, altogether these data reveal the functional importance of both GnRHR Trp(279) and Val(299) and suggest that Trp(279), interacting with GnRH Trp(3), represents the bottom of the binding pocket.

LH down-regulates gonadotropin-releasing hormone (GnRH) receptor, but not GnRH, mRNA levels in the rat testis.

The demonstration of an inhibitory effect of gonadotropin-releasing hormone (GnRH) agonists upon steroidogenesis in hypophysectomized rats and the presence of mRNA coding for GnRH and GnRH receptors (GnRH-R) in rat gonads suggests that GnRH can act locally in the gonads. To assess this hypothesis, we investigated the effects of GnRH analogs, gonadotropins and testosterone on the levels of both GnRH and GnRH-R mRNA in the rat testis. Using dot blot hybridization, we measured the mRNA levels 2 to 120 h after the administration of the GnRH agonist, triptorelin. We observed an acute reduction of both GnRH and GnRH-R mRNAs 24 h after the injection (about 38% of control). However, the kinetics for testis GnRH-R mRNA were different from those previously found for pituitary GnRH-R mRNA under the same conditions. Initially, the concentrations of serum LH and FSH peaked, then declined, probably due to the desensitization of the gonadotrope cells. In contrast, the GnRH antagonist, antarelix, after 8 h induced a 2.5-fold increase in GnRH-R mRNA, but not in GnRH mRNA, while gonadotropins levels were reduced. Human recombinant FSH had no significant effect on either GnRH or GnRH-R mRNA levels. Inversely, GnRH-R mRNA levels markedly decreased by 21% of that of control 24 h after hCG injection. Finally, 24 h after testosterone injection, a significant increase in GnRH-R mRNA levels (2.3 fold vs control) was found, but a reduction in the concentration of serum LH, probably by negative feedback on the pituitary, was observed. In contrast, GnRH mRNA levels were not significantly altered following testosterone treatment. Since LH receptors, GnRH-R and testosterone synthesis are colocalized in Leydig cells, our data suggest that LH could inhibit the GnRH-R gene expression or decrease the GnRH-R mRNA stability in the testis. However, this does not exclude the possibility that GnRH analogs could also affect the GnRH-R mRNA levels via direct binding to testicular GnRH-R. In contrast, the regulation of GnRH mRNA levels appeared to be independent of gonadotropins. Taken together, our results suggest a regulation of GnRH and GnRH-R mRNA specific for the testis.

GnRH-dependent up-regulation of nitric oxide synthase I level in pituitary gonadotrophs mediates cGMP elevation during rat proestrus.

We have recently provided evidence that the concentration and activity of the enzyme nitric oxide synthase type I (NOS I) was stimulated in gonadotrophs by GnRH, suggesting a role for the NOS I/NO pathway in the GnRH control of cell functions. To further establish the GnRH regulation of pituitary NOS I under physiological circumstances, we have examined the expression of NOS I during the 4-day estrus cycle in rats. Western blot analysis demonstrated that NOS I was present in the anterior pituitary at low levels during diestrus I (DI) and diestrus II, then was subject to a significant increase on the afternoon of proestrus to reach a maximal 3-fold increase between 19:00 and 20:00 h, after which NOS I decreased to return, during estrus, to levels within the range detected in diestrus. No such variation was apparent in the posterior pituitary lobe. NADPH-diaphorase histochemistry combined with immuno-identification of the cells revealed that active NOS I was expressed in both the gonadotrophs and the folliculo-stellate cells throughout the whole cycle but only the gonadotrophs showed an up-regulation during proestrus. A high temporal correlation was observed in the profiles of NOS I, pituitary cGMP and serum luteinizing hormone (LH) suggesting an implication of GnRH. Consistently, the administration of a potent GnRH antagonist to rats totally abolished the rise in pituitary NOS I and cGMP, in addition to suppressing, as expected, the surge in the secretion of LH. A role of NOS I as a mediator in the GnRH-induced augmentation in cGMP was further established in vitro by incubating anterior pituitaries in the presence of the NOS inhibitor, L-NMMA (1 mM). Altogether, these data demonstrate that the level and activity of NOS I is up-regulated in gonadotrophs during proestrus, in a manner consistent with a major implication of GnRH over a period during which its release from the hypothalamus, as well as gonadotroph responsiveness, are at maximum. This effect is accompanied by a NOS/NO-mediated rise in cGMP. In the absence of obvious effect on gonadotropin release, the roles of NO and cGMP in the regulation of gonadotroph functions, especially during proestrus, remain to be established.

Evidence that gonadotropin-releasing hormone stimulates gene expression and levels of active nitric oxide synthase type I in pituitary gonadotrophs, a process altered by desensitization and, indirectly, by gonadal steroids.

To determine the site and mechanism of action of gonadal steroids on pituitary nitric oxide synthase type I (NOS I), present in both gonadotrophs and folliculo-stellate cells, the effects of castration and steroids were examined in male rats, in the presence of a GnRH antagonist (Antarelix). Western analysis showed a rapid and substantial increase with time, after orchidectomy, of NOS I protein, the concentration doubling in 24 h and reaching a maximal 4- to 5-fold increase after 3-7 days, followed by a progressive decline after 2 weeks. Testosterone or estradiol replacement, or administration of GnRH antagonist, totally abolished the effects of castration, demonstrating a mediation of the steroid effects via GnRH. In noncastrated rats, steroids and the GnRH antagonist also caused a reduction in the levels of NOS I (by 50-60%), consistent with inhibition of endogenous GnRH stimulation. In marked contrast, administration of a potent GnRH agonist (Triptorelin) to intact rats increased the levels of NOS I. A time-course study with a long-lasting formulation showed that rise in NOS I developed rapidly after a lag of approximately 5 h, with a 2-fold increase detectable after 8 h and a maximal 4.5-fold after 48 h. The level declined afterwards in a manner consistent with homologous desensitization that may occur in the continuous presence of GnRH; however, the profile was different and delayed compared with those of gonadotropin release. As observed for NOS I protein, NOS I messenger RNA concentration was increased by castration or GnRH agonist and reduced by steroids or GnRH antagonist. Taken together, these data demonstrate that steroids indirectly regulate NOS I messenger RNA and protein levels, through the hypothalamic modulation of GnRH, which represents the primary regulator of NOS I. No effect of steroids on NOS I was seen in the posterior lobe. NADPH-diaphorase histochemistry coupled to immuno-identification of the cells revealed that the treatments affecting the concentration of NOS I concomitantly altered the activity but exclusively in gonadotrophs and not in folliculo-stellate cells (which do not respond to GnRH), reinforcing the idea that GnRH played a major regulatory role. Expression in gonadotrophs of a GnRH-dependent NOS I and the ensuing production of nitric oxide represents a potentially novel signaling pathway for the neuropeptide in the anterior pituitary, consistent with the previously reported GnRH-induced cGMP production, the role of which remains to be evaluated.

Rat gonadotropin-releasing hormone receptor expressed in insect cells induces activation of adenylyl cyclase.

Increasing evidence exist that multiple G proteins mediate the effects of gonadotropin-releasing hormone (GnRH) on the synthesis and release of pituitary gonadotropins. In the present study, we have expressed the rat GnRH receptor (GnRH-R) in insect cells, by infection with a recombinant baculovirus. Under the conditions used, insect cells expressed, 48 h post-infection, a maximum of 7800 +/- 650 receptors/cell which bound GnRH agonist [D-Trp6]GnRH with a Kd = 0.52 +/- 0.06 nM indicating characteristics similar to those of the natural receptor. No binding was observed in non-infected cells or cells infected with wild-type baculovirus. In presence of GnRH, GnRH-R expressing cells elicited a time- and dose-dependent production of inositol trisphosphate, with a maximum level reached within 30 min and an EC50 = 5 nM. These recombinant insect cells also produced cAMP in response to GnRH. However, in contrast to other heterologous systems, or rat pituitary gonadotropes wherein GnRH induced a weak and delayed elevation of cAMP, in insect cells the rise of cAMP was comparatively rapid, attaining a maximum level after 2 h, and the EC50 was 5 nM. Finally, a clear activation of adenylyl cyclase (AC) in response to GnRH was shown for the first time by measuring the conversion of [alpha-32P]ATP into labeled cAMP, using membrane preparations from GnRH-R expressing insect cells. These data demonstrate that rat GnRH-R has the potential for dual coupling to both phosphoinositidase C and AC and suggest a major influence of the host cell for this coupling and/or its expression, probably in relation with the G protein repertoire and preference. This notion could be extended to several target cells other than pituitary gonadotropes that normally express the GnRH-R in mammals, including hippocampal, Leydig, granulosa, placental and GnRH-secreting hypothalamic cells.

Testosterone inhibits the basal and gonadotropin-releasing hormone-stimulated synthesis and release of newly synthesized alpha- and lutropin (LH) beta-subunit but not release of stored LH in cultured rat pituitary cells.

To further explore the mechanism of steroid feedback in male, the effects of testosterone (T) and gonadotropin-releasing hormone (GnRH) on the rates of alpha- and lutropin (LH)beta-chain synthesis, neosynthesized subunits and radioimmunoassayable LH release into the medium were studied in the cultures of anterior pituitary cells from orchiectomized and intact rats. Polypeptides were [35S]methionine-labeled, immunoprecipitated separately in the medium and cells, then after SDS-PAGE precisely quantified. The total (medium + cells) radioactivity incorporated in the absence of GnRH into alpha- and LH beta-subunit was increased in orchiectomized rat cells vs. intact rat cells. GnRH stimulated the synthesis of both subunits, whether cells were from normal or castrated rat. T suppressed basal and GnRH-enhanced synthesis of both subunits in castrated rat cells. The values became closed to those observed in the normal rat cells. Also release of neosynthesized subunits from castrated rat cells into the culture medium was inhibited by T. In contrast, T did not change the basal and GnRH-induced radioimmunoassayed LH release. These results show that T can inhibit directly, at the pituitary level, alpha- and LH beta-subunit synthesis and neosynthesized but not stored LH release. They could explain, at least in part, no correlation between modifications of GnRH and LH secretion observed in vivo in response to T replacement.

[Recent data on the gonadoliberin receptor and the neuropeptide control mechanisms for gene expression of gonadotropin hormones]. / Données récentes sur le récepteur de la gonadolibérine et les mécanismes de contrôle par le neuropeptide de l'expression des génes des hormones gonadotropes.

GNRH plays a pivotal role in the neurohormonal control of reproduction by promoting hte secretion of pituitary gonadotrophins, LH and FSH. GnRH also stimulates the synthesis of constitutive gonadotrophin subunits alpha and beta and its own receptor number. Gonadotrophin synthesis appears to be regulated by GnRH through various molecular mechanisms that include, in a complementary and in some cases differential manner, enhanced transcriptional activity of subunit genes and polyadenylation of transcripts. The latter is known to result in increased stability and/or translational activity of mRNAs. These effects of GnRH are mimicked by the direct activation of protein kinases A and C, two different but possibly interconnected signalling pathways that may account for the pleiotropic and concerted alterations of both synthesis and release of gonadotrophins. GnRH operates on the gonadotropic cell level via a transmembrane, G-protein coupled receptor, the structure of which has recently been determined by molecular cloning. This receptor differs from the other members of hte super-family essentially by a rather short length (only 327-328 amino acids) and a truncated carboxyterminus. Recent experiments suggest a genomic control of the GnRH receptor synthesis, especially by GnRH itself, the importance, and role of which remains to be established for the pituitary gonadotropic function.

[Glycoprotein hormones, glycosylation and biological activity]. / Hormones glycoprotéiques, glycosylation et activité biologique.

Glycoprotein hormones LH, FSH, TSH and hCG are heterodimeric molecules: each contains two subunits, a common alpha and a unique beta subunit. Each subunit bears one or two Asparagine linked carbohydrate moieties which have a biantennary complex-type or hybrid-type structure. Different technical methods as deglycosylation or molecular biology techniques have been used to study the role of carbohydrate residues in hormonal bioactivity. The carbohydrate chains are not directly involved in receptor binding events but their mechanisms of action is not fully understood. Two hypotheses are frequently emphasised: a conformational role or an involvement in the coupling of the receptor-adenylate cyclase system. At the post receptor level carbohydrate chains modulate the bioactivity in two ways: a global regulation following an all-or-none mode and slight one. The removal of the carbohydrate moieties leads to a loss of the in vitro hormonal activity. The results observed are dependent of the deglycosylation techniques and the bioactivity tests used. Hormone's deglycosylation reduces their capacity of production of cAMP and, to a lesser extent, their steroidogenic power. Deglycosylated hormones are antagonists to negative hormones although deglycosylated hCG has some agonist properties in vivo. Microheterogeneity of the glycoprotein hormones is due to slight variations in sialic acid and/or sulfate content. Glycoprotein hormones exist as several isoforms which differ in biological potency. Alkaline isoforms (less sialylated ones) are the most biologically active in vitro but have a short half live in vivo; acid isoforms are less active in vitro but have a longer circulatory half live. The polymorphism of glycoprotein hormones is a highly regulated process.(ABSTRACT TRUNCATED AT 250 WORDS)

Bihormonal cells producing gonadotropins and prolactin in a rat pituitary tumor cell line (RC-4B/C).

Using single hormone-staining immunocytochemical methods, we have recently characterized a novel cell line, RC-4B/C, established from an aged male rat pituitary adenoma. This cell line contained all known anterior pituitary cell types including gonadotropes. Gonadotropin-releasing hormone receptors were also present. A recent re-examination of the cell types using single hormone stains showed that the cell line underwent an alteration in the percentage of different cell types as compared to the data obtained 2 years ago. The proportion of follicle-stimulating hormone-beta (FSH beta), luteinizing hormone-beta (LH beta), prolactin (PRL), and adrenocorticotropic hormone cells increased significantly (p less than or equal to 0.001), while the proportion of growth hormone (GH), and thyrotropin-beta cells did not change. Dual staining of the monolayers showed that the cell line contained many bihormonal cells producing FSH beta + LH beta, FSH beta + PRL and LH beta + PRL. The proportion of bihormonal FSH beta + LH beta and FSH beta + PRL cells was preponderant over monohormonal cells, while the proportion of bihormonal LH beta + PRL cells was in the same range as that of monohormonal cells. Preliminary data with dual labeling also revealed the presence of GH and PRL in the same cell, but complete absence of a combination such as FSH beta + GH. No search for the presence of other bihormonal or multihormonal cells was performed. In short, our data show that the majority of the cells in the cell line RC-4B/C contain FSH beta, LH beta and PRL and that among these cells many bihormonal cells are present.

Gonadotropes in a novel rat pituitary tumor cell line, RC-4B/C. Establishment and partial characterization of the cell line.

An epithelial cell line (RC-4B/C) was established from a pituitary adenoma obtained from a 3-yr-old (ACI/fMai X F344/fMai)F1 male rat. Before Year 5 in vitro, RC-4B/C cells could not be viably recovered from cryogenic storage. Recovery of viable cells from cryogenic storage in Year 5 was associated with a more transformed phenotype, including the appearance of endogenous C-type rat retroviral particles. The ultrastructural appearance of the cells was similar to that of differentiated anterior pituitary cells; the cultured cells contained numerous, electron dense, secretory granules, Golgi complexes, and extended arrays of rough endoplasmic reticulum. Immunocytochemical study showed that all cell types present in the rat anterior pituitary gland were present in the cell line. The percentage of luteinizing hormone beta (LH beta) cells in the cell line was higher (19.9%) and that of growth hormone cells was lower (12.2%) than in normal male rat pituitary, whereas the cell line contained a comparable percentage of follicle stimulating hormone beta (FSH beta), prolactin (PRL), ACTH, and thyrotropin beta cells. Radioimmunoassay data demonstrated the PRL content of the cells was comparable to that of normal male rat pituitary gland, whereas the content of LH and FSH was 70- and 800-fold lower, respectively. Assay of specific receptor sites for gonadotropin releasing hormone (GnRH) using Scatchard plots of the data established the RC-4B/C cells contained GnRH receptor sites of the same affinity as in the pituitary gland, but of twofold lower capacity. These data suggest the RC-4B/C cell line warrants further study as a model for the induction and maintenance of the gonadotropic function of the pituitary gland.

[Characterization of gonadotropic cells in a new pituitary tumor cell line]. / Caractérisation de cellules gonadotropes dans une nouvelle lignée tumorale hypophysaire.

The pituitary tumor cell line RC-4B/C was established in The Jackson Laboratory from an aged rat pituitary adenoma. Immunocytochemical studies of this cell line showed that all pituitary cell types were present. Approximately 20% reacted with antisera (AS) to ovine (o) LH beta, 8.6% with AS to oFSH beta, 15% with AS to rat PRL, 12% with AS to equine GH, 9% with AS to porcine TSH beta and 8.6% with AS to ACTH1-24. Using NIDDK rat kits, RIA showed about 0.38, 0.08 and 607.50 ng per 10(6) cells of LH, FSH and PRL, respectively, vs 33.9, 75.6 and 573 ng in freshly dispersed rat pituitary cells. The GnRH receptor content of the cell line was about a half that of normal rat pituitary cells but the receptor affinity was the same. A chronic treatment of the cells for about 5 months with a "sub-physiological" concentration (3.7 pM) of a GnRH agonist had 3 major effects: 1) as compared to the controls, a 3-fold increase in the cell number in the log phase; 2) an increase of the percentage of FSH beta cells from 8.6 to 21.9% whereas LH beta cells and the cell content of LH and FSH remained stationary; 3) a decrease of the percentage of PRL cells from 15 to 6.5% and an almost 250-fold decrease of PRL cell content. Incorporation studies with [35S] Met demonstrated that the alpha subunit in the cell line was only partly glycosylated. Pretreatment of the cells with 5 nM estradiol restored, at least partly, glycosylation of alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

[Isolation of a pituitary receptor for gonadoliberin (GnRH) in a highly purified state]. / Obtention du récepteur hypophysaire de la gonadolibérine (GnRH) à l'état hautement purifié.

Using a gonadoliberin (GnRH) receptor preparation from the bovine pituitaries, we purified the receptor approximately 14,000 fold as compared to the starting material, with a overall yield of about 40%. The binding capacity of a iodinated GnRH analog, used for radioreceptor assay, increased from 16 fmoles/mg of protein in the crude material to 225 pmoles/mg of protein in the final product. The affinity constant was not modified by the purification process and remained close to Ka = 10(10) M-1. Electrophoretic analysis of the purified preparation suggests an apparent molecular mass of about 60,000 Da for the receptor.

Gonadotrophin releasing hormone receptors and the response of pituitary gonadotrophs in culture.

We have investigated the effect of the time of culture on cell number, cell content of LH, cell responsiveness to gonadotrophin releasing hormone (GnRH) and binding parameters of GnRH in rat anterior pituitary cells in culture. Although a decrease in the cell number was observed during the culture period, the cell content of LH remained unchanged. The receptor affinity (Ka) in acutely dispersed cells was 0.86 X 10(7) l/mol for [3H]GnRH and 1.36 X 10(10) l/mol for a highly potent agonist, [D-Ser(But)6]GnRH(1-9)nonapeptide-ethylamide (GnRH-A). The affinity and binding capacity (0.3 fmol/10(6) cells) for iodinated GnRH-A did not change significantly during the 6-day culture period. On the contrary, the values of Ka and binding capacity (257 fmol/10(6) cells) for tritiated GnRH decreased by about 50% between days 1 and 6 of culture. Our results suggest that 125I-labelled GnRH-A binds mostly to high-affinity and low-capacity receptor sites, while [3H]GnRH, which must be used at a higher concentration, also binds to low-affinity, high-capacity binding sites and is therefore useless for the measurement of GnRH receptor binding affinity and binding capacity. Since the biological response of the cells to GnRH increased with the time of culture, it is concluded that although GnRH action is receptor-mediated, binding capacity and biological activity are not necessarily correlated.

Solubilization and partial purification of the high-affinity gonadoliberin receptor from the bovine pituitary gland.

The high-affinity gonadoliberin (GnRH) receptor contained in a membrane preparation from frozen bovine anterior pituitary glands has been solubilized in Triton X-100 and the binding properties of the solubilized product have been examined. The radioreceptor-binding assay, using the GnRH agonist [D-Ser(t-Bu)6] des-Gly10GnRH N-ethylamide (GnRH-A) as radioligand, demonstrated that the kinetics of association and dissociation, the binding constants, as well as the specificity of receptor were not altered in the solubilized receptor preparations. Affinity chromatography on a concanavalin A-Sepharose column, with elution of adsorbed material using a solution of alpha-methyl-D-mannoside, allowed a 33-fold purification of the receptor. The Ka of the receptor thus purified was of the same order as that of the starting material, although slightly higher values were found. Only about one-half of the total receptor activity applied to the column was retained in spite of several recyclings. The other half was found in the nonadsorbed fraction. It is postulated that the detergent-solubilized fraction contains two forms of the GnRH receptor. The nonadsorbed fraction probably contains a partially or totally deglycosylated form. It is possible that the detergent-solubilization process somewhat alters the physicochemical properties of a part of the GnRH receptor molecules. Electrophoretic analysis of the purified receptor preparations, with a subsequent GnRH-binding assay, suggests that the apparent molecular mass of the high-affinity GnRH receptor, or of its monomeric form, is approximately 60,000 Da.

[A micromethod allowing the preparation and translation of messenger ribonucleic acid from adenohypophysis cells in culture]. / Une microméthode permettant de préparer et de traduire l'acide ribonucléique messager à partir de cellules adénohypophysaires en culture.

We have developed a microscaled method for the extraction and translation of mRNA from 2 x 10(5)-2 x 10(6) anterior pituitary cells. This method applied to a fraction of enriched gonadotrophs enabled us to confirm our previous results suggesting that (1) Rat LH subunits were synthesized in a cell-free system as precursors, from separate mRNAs; (2). The relative molecular mass of precursors, as determined by SDS-polyacrylamide gel electrophoresis, was 17-17,5 kD for LH alpha and 18,5-19 kD for LH beta. This procedure resulted in a 5-6 fold increase in the proportion of in vitro synthesized precursors in LH subunits.

[Hypophyseal GnRH receptors. A minireview (author's transl)]. / Récepteurs hypophysaires du GnRH. Mise en évidence et paramètres physico-chimiques.

The preliminary step in the involvement of gonadoliberin (GnRH) in the cellular mechanism for the secretion of the gonadotropic hormones LH and FSH, consists of a reversible binding of GnRH with specific site on plasma membranes of the pituitary gland. The parameters of this interaction have been determined in vitro with the aid of biologically active preparations of synthetic GnRH labelled with [3H] or [125I], and homogenates or preparations of plasma membranes, from the pituitary glands, of rat, sheep or beef. According to several authors, one or two types of sites are involved in GnRH-receptor binding. At 0-4 degrees C, the equilibrium association constants of the high affinity sites vary from 0.77 X 10(8) M-1 to 2.33 X 10(10) M-1. At 37 degrees C, we have obtained a value of 1.1 X 10(8) M-1 for this constant. Low affinity binding sites have been found in pituitaries and in other tissues; according to CLAYTON (1979), they may be structurally bound to enzymes which degrade GnRH. Other authors feel that a correlation may exist between the biological activity of agonistic analogues of GnRH and their capacity for binding to high affinity sites. Modulation of the binding of GnRH to low and high affinity sites by steroids and/or peptidases has been considered. The existence of intracellular receptors and internalisation of the GnRH-receptor complex have also been reported.

Binding of gonadotropin-releasing hormone (LH-RH) to the pituitary plasma membranes and the problem of adenylate cyclase stimulation.

Specific binding studies of tritium-labeled LH-RH to sheep anterior pituitary membranes at 37 degrees C showed a maximum of binding capacity of 110 +/- 20 mol/mg protein with an association rate constant of 2 +/- 1 X 10(5) M-1 S-1 and a dissociation rate constant of 0.7 +/- 0.4 X 10(-2) S-1. The Scatchard plot data showed a single type of binding site with Kass = 1.1 +/- 0.4 X 10(8) M-1 in good agreement with the kinetic studies. Various doses of LH-RH in the presence or absence of Ca2+, were unable to stimulate adenylate cyclase either in the rat anterior pituitary homogenates, or in the purified sheep anterior pituitary plasma membranes. To explain these results, it may be argued that the proportion of gonadotrophs in the pituitary gland is too small to show a significant increase in the LH-RH-induced adenylate cyclase activity. Another possibility is the disconnection of the hormonal receptor from the site of activation of adenylate cyclase during the preparation of plasma membranes. Finally, cAMP may not be involved in LH release by LH-RH.