Characterization of Leydig cell gonadotropin-releasing hormone binding sites utilizing radiolabeled agonist and antagonist

Gonadotropin-releasing hormone (GnRH) binding sites in intact Leydig cells and in membrane preparations were investigated using 125I-labeled GnRH agonist and antagonist. Binding was saturable and involved a single class of high affinity sites. Intact Leydig cells and a membrane preparation had a higher affinity for GnRH agonist (Kd 3.0 +/- 1.7 X 10(-10) M) than for GnRH antagonist (Kd 10.0 +/- 1.8 X 10(-10) M). With anterior pituitary membranes the Kd was 2.8 +/- 0.7 X 10(-10) M for the agonist and 2.4 +/- 1.4 X 10(-10) M for the antagonist. The Kd for GnRH was similar for Leydig cells and the anterior pituitary. Chymotrypsin and trypsin digestion decreased receptor binding, but neuraminidase increased Leydig cell binding in contrast to the decrease in binding observed with pituitary receptors. The results suggest that the Leydig cell GnRH binding sites may differ from the pituitary receptor which may be related to structural differences in GnRH-like peptides recently described in extracts of rat testis.     

Localization of putative gonadotrophin releasing hormone receptor protein in the anterior pituitary

Specific binding of a fully biologically active 125I-gonadotrophin releasing hormone (GnRH) to isolated anterior pituitary cells is time dependent, saturable and the concentration dependent binding curves exhibit positive cooperativity. Binding to intact or solubilized plasma membranes and an affinity purified GnRH receptor protein reveals in all instances multiple high affinity binding sites. Thus, GnRH receptor protein appears to be an intrinsic constituent of the cell membrane, and perhaps, other membranous organelles. To investigate the latter, the binding of 125I-GnRH to various subcellular fractions was studied and its affinity and time requirements determined. GnRH binding to plasma membranes and secretory granules was to multiple high affinity sites, while that to nuclei and microsomes was to a single high affinity site. Binding was 1.83 +/- 0.07, 0.78 +/- 0.04, 0.31 +/- 0.03 and 0.27 +/- 0.03 fmol micrograms-1 protein for isolated plasma membranes, secretory granules, microsomes and nuclei, respectively, after 30 min incubation with 10(-9) M GnRH. The magnitude of binding to microsomes did not change during the incubation period. It did not show any decrease (p greater than 0.05) in isolated nuclei and plasma membranes, except for the 24 h time period, when a significant drop (p less than 0.001) was seen. Binding to the secretory granule fraction culminated at 15 min and then decreased (p less than 0.001) steadily to a non-detectable level at 24 h. Thus GnRH receptor protein or its portion may be an integral part of some membranous particles in the anterior pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional relationship between receptor binding and biological activity for analogs of mammalian and salmon gonadotropin-releasing hormones in the pituitary of goldfish (Carassius auratus)

The relationship between gonadotropin-releasing hormone (GnRH) receptor binding and biological activity in the goldfish pituitary for mammalian and salmon GnRH (sGnRH) analogs with structural modification at the C terminus involving replacement of glycine amide with an alkyl amine and replacement of the Gly6 residue with D amino acids was examined. The GnRH receptor binding data were analyzed with a computerized curve-fitting program (LIGAND) for a single as well as two classes of binding sites; analysis based on one site fit estimated binding affinity and capacity for one class of binding site, and analysis based on two-site fit estimated binding affinity and capacity for two classes of binding sites (high-affinity/low-capacity and low-affinity/high-capacity binding sites). The estimated receptor affinity values were then used to determine the correlation between binding affinity and gonadotropin (GTH)-release potency in vitro. The highest correlation between biological activity and receptor binding affinity was obtained for the high-affinity/low-capacity binding sites and GnRH analogs containing Trp7 and Leu8 residues (i.e., the salmon GnRH structural format) (R = 0.940 +/- 0.150). For the same group of GnRH analogs, there was no significant correlation between the relative GTH-release potency and binding affinity of the low-affinity/high-capacity sites (R = 0.159 +/- 0.434), or that obtained from a one-site fit (R = 0.198 +/- 0.431). Similarly, for mammalian GnRH analogs, significant correlation between binding affinity and biological activity (R = 0.406 +/- 0.049) was only obtained for the high-affinity sites, although the degree of correlation was significantly lower than that obtained for salmon GnRH analogs. The present findings provide strong support for the hypothesis that high-affinity GnRH receptors are involved in the control of GTH release in the goldfish pituitary. In addition, the results demonstrate clearly that the presence of Trp7, Leu8 residues in salmon GnRH molecule, a native peptide in goldfish, is important for recognition of the ligand by the GnRH receptors in the goldfish pituitary, and that structural modifications at positions 6 and 10 in this peptide can increase receptor binding affinity and biological activity at the pituitary level. The most active sGnRH analog identified to date is [D-Arg6, Pro9-NEt]-sGnRH.     

Divalent cations regulate glucagon binding. Evidence for actions on receptor-Ns complexes and on receptors uncoupled from Ns

The effects of Mg2+ or ethylenediaminetetraacetic acid (EDTA) on 125I-glucagon binding to rat liver plasma membranes have been characterized. In the absence of guanosine 5'-triphosphate (GTP), maximal binding of 125I-glucagon occurs in the absence of added Mg2+. Addition of EDTA or Mg2+ diminishes binding in a dose-dependent manner. In the presence of GTP, maximal binding occurs in the presence of 2.5 mM Mg2+ (EC50 = 0.3 mM) while EDTA or higher concentrations of Mg2+ diminish binding. Response to exogenous Mg2+ or EDTA depends on the concentration of Mg2+ in the membranes and may vary with the method used for membrane isolation. Solubilized 125I-glucagon-receptor complexes fractionate on gel filtration columns as high molecular weight, GTP-sensitive complexes in which receptors are coupled to regulatory proteins and lower molecular weight, GTP-insensitive complexes in which receptors are not coupled to other components of the adenylyl cyclase system. In the absence of GTP, 40 mM Mg2+ or 5 mM EDTA diminishes receptor affinity for hormone (from KD = 1.2 +/- 0.1 nM to KD = 2.6 +/- 0.3 nM) and the fraction of 125I-glucagon in high molecular weight receptor-Ns complexes without affecting site number (Bmax = 1.8 +/- 0.1 pmol/mg of protein). Thus, while GTP promotes disaggregation of receptor-Ns complexes, Mg2+ or EDTA diminishes the affinity with which these species bind hormone. In the presence of GTP, hormone binds to lower affinity (KD = 9.0 +/- 3.0 nM), low molecular weight receptors uncoupled from Ns.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotropin-releasing hormone receptor binding in bovine anterior pituitary

Synthetic gonadotropin-releasing hormone (GnRH) was monoiodinated at a high specific radioactivity with 125I. The iodinated hormone retained full biological activity as assessed by the release of luteinizing hormone in vitro from bovine anterior pituitary tissue slices. Specific binding of 125I-labeled gonadotropin-releasing hormone of high affinity and low capacity was obtained using dispersed bovine anterior pituitary cells. The binding had sigmoid characteristics, compatible with the presence of more than one binding site. The subcellular fraction responsible for binding was identified with the plasma membranes. However, significant binding also occurred in the secretory granules fraction. The plasma membranes were solubilized with sodium dodecyl sulfate. Using gonadotropin-releasing hormone covalently coupled to a solid phase, a protein was purified by an affinity technique from the solubilized plasma membrane preparation which possessed similar binding propperties as plasma membranes, both intact and solubilized. The protein migrated as a single component on polyacrylamide gel in sodium dodecyl sulfate and the estimated molecular weight was 60 000. The character of the gonadotropin-releasing hormone concentration dependence binding as well as association kinetics were multiphasic and suggested the presence of more than one binding site. When analyzed by the Hill plot, the Hill coefficient of all binding curves was always greater than one which is compatible with positive cooperativity. This was further supported by the dissociation studies where the dissociation rate was inversely proportionate to both the gonadotropin-releasing hormone concentration and the time interval during which the gonadotropin-releasing hormone-gonadotropin-releasing hormone receptor protein complex was formed. Using difference chromatography, aggregation of the purified gonadotropin-releasing hormone receptor protein was demonstrated to occur upon its exposure to gonadotropin-releasing hormone. The formed macromolecular complexes bound preferentially 125I-labeled gonadotropin-releasing hormone. It is concluded that a single receptor protein is responsible for gonadotropin-releasing hormone binding in the bovine anterior pituitary. It is a part of the plasma membranes. Its interaction with gonadotropin-releasing hormone provokes transitions of the protein into different allosteric forms and this may be related to the biological effect of gonadotropin-releasing hormone on gonadotropin secretion.     

Thyrotropin binding to and adenylate cyclase activity of porcine thyroid plasma membranes.

Plasma membranes have been purified from porcine thyroid gland homogenate by discontinuous sucrose gradient centrifugation. The preparations contained specific binding sites for thyrotropin but not for luteinizing hormone or the beta subunits of thyrotropin and luteinizing hormone. Optimum conditions of 125I-labeled thyrotropin binding were pH 6.0-6.5 and 37 degrees C. Thyrotropin binding was reduced by divalent (Ca2+, Mg2+) and monovalent cations (Na+, K+, Li+), 50% inhibition being obtained at 10 mM and 50 mM respectively. Displacement curves of 125I-labeled bovine or porcine thyrotropin by the unlabeled hormone from three species was in the order of increasing concentrations (bovine greater than porcine greater than human) which is the order of decreasing biological activity of these hormone preparations in the assay in vivo in the mouse. The validity of the results was established by controlling that porcine membranes bound the native and the 125I-labeled hormones with equal affinity. A single type of high-affinity (Kd = 0.28 nM) binding sites was detected for bovine and porcine thyrotropins. In contrast, porcine plasma membranes bound human thyrotropin with a lower affinity (Kd = 70 nM). A good correlation was found at equilibrium and in the conditions of the cyclase assay, between receptor occupancy and adenylate cyclase activation for the three hormones.     

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.     

The sites of high affinity binding of L-[3H]glutamic acid in human platelets. A new type of platelet receptor?

The total membrane fraction of human platelets was found to contain high affinity sites of L-[3H]glutamic acid binding (Kd = 100 nM, Bmax = 1.06 pmol/mg protein). The pH optimum for binding is at pH approximately 6.9 Na+ (1-150 mM) inhibit glutamate binding by platelet membranes (IC50 = 12 mM). Ca2+ (50-100 microM) stimulate the binding by 10-20% and inhibit it by 20-30% at concentrations of 1-5 mM. Monoclonal antibodies to the glutamate receptor strongly suppress the L-[3H]glutamate binding by platelet membranes (IC50 = 300 nm). The presence in human platelets of a glutamate-sensitive receptor complex similar to the central nervous system glutamate receptor is postulated.     

The pituitary gonadotropin-releasing hormone (GnRH) receptor of the female rabbit: characterization and developmental aspects.

The aim of the present study was to characterize the pituitary gonadotropin-releasing hormone (GnRH) binding site in the rabbit and investigate its possible role in sexual maturation of the female rabbit. A radioligand binding assay was established, and the presence of specific 125I-labelled D-Ala6-des-Gly10-GnRH ethylamide (125I-DAl6EA) binding sites in the anterior pituitary gland of the rabbit was demonstrated. 125I-DAla6EA binding was saturable, specific, displaceable, reversible, correlated with increasing tissue concentrations, and susceptible to physiological manipulation. 125I-DAla6EA binding indicated the presence of two binding sites in the female adult rabbit pituitary: a high affinity, low capacity site (KD = 0.3-0.4 nM; Bmax = 100-200 fmol/mg protein) and a lower affinity, high capacity site (KD = 30 nM; Bmax = 5-8000 fmol/mg protein). Ontogeny of 125I-DAl6EA binding in the female rabbit (40-120 days of age) did not show a correlation between binding site number and serum luteinizing hormone (LH). In addition, the net serum LH response in female rabbits to a subcutaneous injection of DAla6EA (10 ng, 100 ng, and 1 microgram per kilogram body weight) was not significantly different between animals 40, 75, and 120 days of age. This suggests that a decrease in pituitary responsiveness to GnRH is not associated with sexual maturation in the female rabbit. Results indicate that factors other than and (or) in addition to GnRH binding site number, such as postreceptor events, play a role in gonadotropin secretion in the female rabbit.     

Synthesis and evaluation of novel gonadotropin-releasing hormone receptor-targeting peptides

The purpose of this study was to develop novel radiolabeled gonadotropin-releasing hormone (GnRH) receptor-targeting peptides for breast cancer imaging. Three novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated GnRH peptides were designed and synthesized. The radiometal chelator DOTA was conjugated to the epsilon or alpha amino group of D-lysine, or the epsilon amino group of L-lysine via an Ahx {aminohexanoic acid} linker to generate DOTA-Ahx-(D-Lys(6)-GnRH1), DOTA-Ahx-(D-Lys(6)-GnRH2) and DOTA-Ahx-(L-Lys(6)-GnRH3), respectively. The conjugation of the DOTA to the epsilon amino group of D-lysine (rather than alpha amino group of D-lysine nor epsilon amino group of L-lysine) maintained the nanomolar GnRH receptor binding affinity. The IC(50) values of DOTA-Ahx-(D-Lys(6)-GnRH1), DOTA-Ahx-(D-Lys(6)-GnRH2) and DOTA-Ahx-(L-Lys(6)-GnRH3) were 36.1 nM, 10.6 mM and 4.3 mM, respectively. Since only DOTA-Ahx-(D-Lys(6)-GnRH1) displayed nanomolar receptor binding affinity, the specific GnRH receptor binding of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) was determined in human GnRH receptor membrane preparations. Furthermore, the biodistribution and tumor imaging properties of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) were examined in MDA-MB-231 human breast cancer-xenografted nude mice. (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) exhibited specific GnRH receptor binding and rapid tumor uptake (1.76 ± 0.58% ID/g at 0.5 h postinjection) coupled with fast whole-body clearance through the urinary system. The MDA-MB-231 human breast cancer-xenografted tumor lesions were clearly visualized by single photon emission computed tomography (SPECT)/CT at 1 h postinjection of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1). The profound impact of DOTA position on the binding affinity of the GnRH peptide provided a new insight into the design of novel radiolabeled GnRH peptides. The successful imaging of MDA-MB-231 human breast cancer-xenografted tumor lesions using (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) suggested its potential as a novel imaging probe for human breast cancer imaging.     

Gonadotropin releasing hormone activation is mediated by dimerization of occupied receptors

A dinitrophenyl (DNP)-derivative of a gonadotropin releasing hormone (GnRH) antagonist was prepared by chemical modification of the epsilon amino group in position 6 of [D-pGlu1,D-Phe2,D-Trp3,D-Lys6]GnRH with 1-fluoro-2, 4-dinitrobenzene. The DNP-antagonist D-pGlu-D-Phe-D-Trp-Ser-Tyr-D-Lys(N epsilon-DNP)-Leu-Arg-Pro-Gly-NH2, retained high affinity binding to the GnRH receptor of pituitary membrane preparations and exhibited antagonistic activity when assayed in cultured pituitary cells. Both antibodies against DNP and their Fab fragments were able to bind the DNP-antagonist. However, only the addition of bivalent antibodies (and not the Fab fragments) converted the DNP-antagonist to an agonist. These results suggest that divalency is a critical factor in GnRH action.     

Gonadotropin releasing-hormone receptors: photoaffinity labeling with an antagonist

A photoaffinity antagonist of gonadotropin releasing hormone (GnRH), D pGlu-D-Phe-D-Trp-Ser-D-Lys6(N epsilon-azidobenzoyl)-Leu-Arg-Pro-Gly-NH2 (photoaffinity antagonist) was prepared by reacting [D-pGlu1, D-Phe2, D-Trp3, D-Lys6]GnRH with the N-hydroxysuccinimide ester of 4-azidobenzoic acid. The analog appeared homogeneous when analyzed by thin-layer chromatography and its photoreactivity was demonstrated by spectral changes when exposed to light. The photoaffinity antagonist retained high affinity binding to the GnRH receptor of pituitary membrane preparations and exhibited antagonistic activity when assayed in vitro in whole pituitaries. Pituitary membrane preparations were incubated with the radioactive photoaffinity GnRH antagonist and irradiated with light. Sodium dodecyl sulfate gel electrophoresis after solubilization and reduction showed the specific labeling of a single specific protein with an apparent molecular weight of 60,000 daltons. These results indicate that GnRH agonists and antagonists bind to the same receptor.     

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.     

A gonadotropin-releasing hormone-binding inhibitor from bovine ovaries. Purification and identification as histone H2A

Bovine, ovine, rat, and human ovaries contain a protein defined as gonadotropin-releasing hormone (GnRH)-like because it reversibly inhibits the high affinity binding of GnRH to rat ovarian membranes, but these same tissues contain little, if any, detectable GnRH. In the present study this GnRH-binding inhibitor (GnRH-BI) was purified from bovine ovaries by a combination of reversed-phase high pressure liquid chromatography, cation-exchange, and gel filtration chromatography. Purification was monitored by analysis of GnRH-like activity in the highly specific rat ovarian membrane receptor assay. Amino acid composition and partial sequence analysis indicated that the purified ovarian GnRH-BI is histone H2A. Calf thymus histone H2A and the purified ovarian protein showed identical dose-dependent (ID50 = 2 microM), competitive, and reversible inhibitory effects on GnRH binding to rat ovarian membranes. The inhibitory effects could not be explained by charge interactions alone since spermine and spermidine at 10-fold higher concentrations did not inhibit GnRH binding. Furthermore, the effects showed specificity since EGF binding to rat ovarian membranes was not inhibited. It is possible that the inhibition of GnRH binding by the purified ovarian fraction was due to low level contamination by a highly active binding inhibitor. However, based on the variety of different purification procedures, the identical effects seen with histone H2A, and the absence of other proteins on gel electrophoresis, we conclude that the ovarian GnRH-BI is probably histone H2A.     

Mechanisms for the antigonadotropic action of the ovarian gonadotropin-releasing hormone-binding inhibitor protein/histone H2A on ovarian cells.

A GnRH-binding inhibitor (GnRH-BI) was recently purified from bovine ovaries. On the basis of amino acid composition and partial sequence analysis this antigonadotropic GnRH-BI was identified as histone H2A. In the present study the mechanism for the antigonadotropic action of histone H2A was examined and compared to that of GnRH and poly-L-lysine. The potential sites examined were the receptor-coupled pathway of second message synthesis including receptor binding of hormone, G protein activation, and adenylyl cyclase activation. Histone H2A inhibited (ID50 = 2 microM) the binding of hCG by membrane receptors from luteinized rat ovaries in a noncompetitive and dose-dependent manner. The binding of FSH by membrane receptors from immature rat ovaries was not inhibited by histone H2A. Binding of GnRH by pituitary membrane receptors was inhibited by histone H2A, and the ID50 of 8 microM was similar to that previously observed for GnRH binding sites in rat ovarian membranes. No high-affinity binding of histone H2A by rat ovarian membranes was detected. Near-maximal doses of histone H2A (7 microM), poly-L-lysine (10 microM), and GnRH (1 microM) inhibited LH-stimulated cAMP production in isolated rat luteal cells. Inhibition by H2A and poly-L-lysine was larger than by GnRH. Furthermore, histone H2A and poly-L-lysine inhibited cholera toxin (CT)-stimulated cAMP production, but GnRH did not. Like GnRH, neither histone H2A nor poly-L-lysine inhibited forskolin (FK)-stimulated cAMP production. In isolated rat granulosa cells, histone H2A and poly-L-lysine inhibited FSH-, CT-, and FK-stimulated cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotropin-releasing hormone binding inhibitors in the ovary

A protein present in ovaries and other tissues of many species competitively and reversibly inhibits high affinity binding of gonadotropin-releasing hormone (GnRH) to rat ovarian membranes, but this protein is not GnRH. This protein has been partially purified and characterized from bovine ovaries. The absence of GnRH binding inhibitory (GBI) activity in plasma and follicular fluid indicates that this protein may act in a localized manner within or near its site of production or release. The bovine ovarian GBI protein evokes antigonadotropic activity in ovarian cells from both the rat and the bovine. The biological effect of GBI may occur independently of interaction with high affinity binding sites for GnRH, since these are absent from the bovine ovary. Thus, the GBI protein may abrogate gonadotropin-dependent responses in ovarian cells by mechanisms separate from interaction with GnRH receptors. A complete characterization of the GBI protein and evaluation of its mechanism of action in ovarian and pituitary cells will dictate conclusions on the physiological importance of this protein.     

Homologous desensitization of gonadotropin-releasing hormone (GnRH) receptors in the goldfish pituitary: effects of native GnRH peptides and a synthetic GnRH antagonist.

Gonadotropin-releasing hormone (GnRH) stimulates release of gonadotropin hormone (GTH) through interaction with high affinity receptors in the goldfish pituitary. In the present study, we investigated desensitization of two native GnRH peptides, [Trp7, Leu8]-GnRH (sGnRH) and [His5, Trp7, Tyr8]-GnRH (cGnRH-II), using superfused fragments of goldfish pituitary in vitro. Pulsatile treatment with either sGnRH or cGnRH-II (2-min pulses given every 60 min) resulted in dose-dependent secretion of GTH from the goldfish pituitary; cGnRH-II had a greater GTH release potency and displayed a greater receptor binding affinity than sGnRH. Both sGnRH and cGnRH-II-induced GTH release were partially inhibited by concomitant treatment with either [D-Phe2, Pro3, D-Phe6]-GnRH or [D-pGlu1, D-Phe2, D-Trp3.6]-GnRH. These antagonists had greater receptor binding affinities than the native peptides, with no stimulatory action on GTH release in the absence of the GnRH agonists. Continuous treatment with either sGnRH or cGnRH-II (10(-7) M), rapidly desensitized pituitary GTH release in a biphasic fashion; initially there was a rapid increase in GTH release of approximately 10-20-fold (phase 1), followed by a sharp decline in GTH release, reaching a stable concentration 2-3-fold above the basal level (phase 2). Further stimulation of the pituitaries with sGnRH or cGnRH-II (10(-7) M) (second treatment) after 60 min recovery resulted in a significantly lower sGnRH or cGnRH-II-induced GTH release compared to that observed during the initial treatment period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Desensitization of pituitary gonadotropes by mediators of LH release

Desensitization of pituitary gonadotropes by exposure to 10 nM gonadotropin-releasing hormone (GnRH) for 6 h severely impaired the luteinizing hormone (LH) response to a second 3-h treatment with GnRH, and reduced the secretory responses to 50 microM arachidonic acid (AA), 100 nM tetradecanoyl phorbol-13-acetate (TPA), and AA + TPA. Pretreatment with AA blocked subsequent responses to AA but not to other secretagogues. Pretreatment with TPA attenuated the LH response to TPA, but not to GnRH, AA, and AA + TPA. After exposure to AA + TPA, all subsequent responses were abolished. Each of the secretagogues reduced GnRH receptor binding, but only GnRH-induced receptor loss and desensitization were reversed by simultaneous incubation with a GnRH antagonist. Similar results were obtained when 16-h pretreatment periods were used, or when the data were normalized for the concomitant reduction of cellular LH content. These findings indicate that GnRH-receptor loss and depletion of LH content are not the sole causes of GnRH-induced desensitization. Receptor uncoupling and impairment of AA- and protein kinase C-dependent pathways may also be involved in this process.     

Photoaffinity labeling of gonadotropin releasing hormone receptors in control and desensitized pituitary cells

Gonadotropin releasing hormone (GnRH), preincubated with cultured rat pituitary cells, induced down regulation of GnRH receptors in a time- and dose-dependent manner. The specific binding was inhibited by 50% after 30 min and maximal inhibition (70%) was obtained after 75 min preincubation with 1 microM GnRH. Preincubation of the cells for 2 h with 10 nM GnRH inhibited the specific binding by 20%, reaching a plateau of 70% inhibition with 0.1 microM GnRH. Concomitantly, exposure of the cells to GnRH caused a time- and dose-dependent desensitization of LH release. The responsiveness of the desensitized cells was not parallel to the binding capacity and was inhibited to a greater extent (93%). Photoactivation of GnRH receptors with iodinated [azidobenzoyl-D-Lys6]GnRH in control and desensitized cells resulted in the identification of a single specific band with the same apparent molecular weight of 60K daltons. These results indicate that structural alterations of GnRH receptors are not associated with GnRH-induced desensitization. Therefore, desensitization may involve conformational changes in the receptor or more likely a post-receptor mechanism.     

Cyclic oxidation-reduction reactions regulate thromboxane A2/prostaglandin H2 receptor number and affinity in human platelet membranes

The radiolabeled thromboxane A2/prostaglandin H2 (TXA2/PGH2) agonist 125I-BOP bound to the TXA2/PGH2 receptor on human platelet membranes. Scatchard analysis showed that pretreatment of platelet membranes with the reducing agent dithiothreitol (DTT) (10 mM) for 10 min decreased maximal 125I-BOP binding (Bmax) from 1.51 +/- 0.11 pmol/mg to 0.51 +/- 0.05 pmol/mg (p = 0.001) and increased the affinity of the remaining binding sites (Kd = 647 +/- 64 pM (untreated), 363 +/- 46 pM (treated), p = 0.006). Prolonged incubation of membranes with DTT (10 mM) for 40 min further reduced the Bmax to 0.23 +/- 0.08 pmol/mg (p = 0.001 from untreated), and the binding affinity remained elevated (Kd = 334 +/- 117 pM, p = 0.035 from untreated). Kinetic analysis of 125I-BOP binding indicated that the apparent increase in binding affinity after DTT treatment was due exclusively to an increase in the rate of ligand-receptor association with no change in dissociation rate. The effects of DTT on 125I-BOP binding were dose-dependent with an EC50 of 8.1 +/- 0.2 mM. DTT inactivation of TXA2/PGH2 receptors was time-dependent with a second order rate constant (k2) of 0.123 M-1 s-1 at 20 degrees C. The platelet membrane 125I-BOP binding site was partially protected from DTT inactivation by prior occupation with the ligand. TXA2/PGH2 receptor protection by I-BOP was dose-dependent and linearly related (r = 0.97, p = 0.002) to the proportion of receptors occupied, but was incomplete since agonist occupation of 89% of the total number of receptors resulted in only a 38% protective effect. Inhibition of 125I-BOP binding after reduction with DTT could be made permanent by addition of the sulfhydryl alkylating agent N-ethylmaleimide (25 mM), but was completely reversed by reoxidation with dithionitrobenzoic acid (DTNB) (5 mM). Oxidation of untreated receptors with DTNB resulted in a 64% increase in 125I-BOP binding sites from 1.65 +/- 0.12 pmol/mg to 2.70 +/- 0.08 pmol/mg (p = 0.013) without affecting binding affinity. DTNB-induced increases in 125I-BOP binding were concentration-dependent with an EC50 of 668 +/- 106 microM and occurred in less than 1 min at 37 degrees C. In the absence of DTT, alkylation of free sulfhydryl groups with N-ethylmaleimide reduced 125I-BOP Bmax in platelet membranes to 0.85 +/- 0.08 pmol/mg (p = 0.003), but did not change the affinity of the remaining receptors. The EC50 for N-ethylmaleimide inactivation of TXA2/PGH2 receptors was 139 +/- 8 mM, and the k2 in time course experiments was 0.067 M-1 s-1 at 20 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)