Increasing concentrations of LHRH or its active analog des-gly¹⁰ (D-ala⁶) LHRH induced a bimodal pattern of FSH and LH release from incubated male rat pituitaries. A low amplitude response (40% increase of LH over baseline levels) was observed for concentrations of LHRH and the agonist in the range of 10^–12 and 10^–13 M respectively. After a plateau of gonadotropin stimulation, a further high amplitude response (180–240% increase over baseline levels) occurred between 3.10^–10 and 10^–8 for LHRH and 3.10¹¹ and 3.10⁹ M for des-gly¹⁰ (D-ala⁶) LHRH. Corresponding half maximal effective concentrations (EDso) were 2 and 0.3 nM respectively. Stimulation of FSH release closely paralleled that of LH. The low amplitude, high apparent affinity response was only obtained when the peptides were diluted in low concentrations of acidic tissue extracts. A preliminary study indicated that this method of dilution minimized peptide loss by adsorption. In addition, the low amplitude, high affinity response was never observed on pituitaries sampled from castrates. These experiments suggest the presence of two distinct populations of LHRH recognition sites on pituitary gonadotrophs. Under our experimental conditions, the appearance of the higher affinity response was dependent upon prior exposure to sex steroids. This could be due to a direct action of the steroid on the expression of a high affinity LHRH receptor.

Unite de recherches sur la dynamique des ensembles neuroendocriniens, U 159 INSERM, 2ter rue d'AIesia, 75014 Paris, France.t Pharmacologie Neuro-lmmuno-Endocrinienne (CNRS UA 11 13), lnstitut Pasteur, 28 rue du Dr Roux, 75015 Paris, France.Key words: gonadotropin-releasing hormone receptors, protein kinase C, pituitary cell culture, castration. AbstractBinding constants of ['251]Des-Gly,0-(D-Ala,)-gonadotropin-releasing hormone-ethylamide (GnRHa) to dispersed pituitary cells were evaluated in a 4-day culture. Cells were sampled either from intact or from castrated male rats and binding was measured at various temperatures before or after treatment with phorbol-12-myristate-13-acetate (PMA) or l-5-(isoquinolinyl-sulfc.xyl) 2-methylpiperazine (H7), which activate or inhibit, respectively, protein kinase C (PKC). In cells from intact rats incubated with increasing concentrations of ligand at 21 "C for 25 rnin, the Scatchard plot was not linear and calculation of the Hill coefficient (NH) was indicative of positive cooperativity (NH= 1.26_+0.02). Such non-linearity was not observed when cells were incubated at 0.5 "C for 3 h. In that condition the maximal number of binding sites measured at equilibrium (Bmax) increased (15.1 t0.05 versus 9.3k0.5 fmoles x mg-' proteins at 21 "C). Two control experiments permitted us to rule out the possibility that lower B , , , at 21 "C might reflect internalization: 1) Cells were first incubated with the ligand at 21 "C for 25 min and subsequently for 3 additional hours at 0.5 "C. Preincubation did not affect the B , , , obtained at 0.5 "C; 2) when the radioligand bound to the cell surface was washed out with an acidic buffer, only 13% of the specific radioactivity was retained irrespective of the ligand concentration applied, a much lower value than the 40% binding difference observed between 05°C and 21°C. When the cells were incubated with PMA, the Scatchard plot was linearized and the B, , , recorded at 21 "C increased by 50% over control cells (13k0.7 fmolesxrng-' proteins). Conversely, inhibition of PKC by H7, a preferential PKC inhibitor, was ineffective. In contrast, cells sampled from castrates exhibited linear and comparable Scatchard plots at either 0.5 or 21 "C, with B , , , values of 14.4k0.3 and 15k0.34 fmoles x mg-' proteins, respectively. PKC activation did not affect binding in that model, but H7 decreased the number of sites (B,,,= 10.7t0.9) and induced appearance of positive cooperativity (NH= 1.36k0.07). Taken together, these experiments reveal a pool of GnRH receptors in the pituitary of intact rats that recognizes the ligand in a phosphorylation-dependent manner. These binding sites also became evident when biological properties of the membrane were modified by temperature or cell homogenization. After castration, PKC activation was no longer a prerequisite for recruitment of the total population of receptors whereas protein kinase inhibition resulted in a reduction of maximal binding. Finally, our observations demonstrate that GnRH binding can exhibit positiv...