Temperature and Protein Kinase C Modulation of Gonadotropin‐Releasing Hormone Receptors in Pituitary Cells from Intact or Castrated Male Rats

Abstract

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...

New Concepts of the Neuroendocrine Regulation of Gonadotropin Surges in Rats

“…One result of NPY actions on gonadotrophs is an increase in the number [101] or affinity [102] of LHRH receptors. Since unmasking of cryptic LHRH receptors has been suggested to be mediated by PKC [103], it is conceivable that the NPY receptor stimulation leads to phosphoinositiol hydrolysis and PKC activation [104], and hence, augmentation of LHRH binding site density.…”

Gonadotropin Regulation, Oestrogens and the Immune System

“…They have been more exten sively studied at the pituitary level. Oestrogens appear to modulate GnRH receptor number [20,21] as well as the coupling efficiency of its receptor on gonadotrophs [22][23][24][25][26][27]. This results particularly from alterations of mem brane transduction processes.…”

“…As represented in figure 3, they can be dependent or parallel to each other. For instance, oestradiol can influence voltage dependent Ca2+ channels of the L type [24], as well as G proteins and membrane enzymes involved in secretion-coupling mech anisms [22,23,26], Most of such steroid effects involve changes in protein kinase C (PKC) activity and in phosphorylation/dephosphorylation processes [25][26][27], PKC is not directly activated by oestradiol, but the steroid increases the amount of enzyme available, so that stimu lation of PKC following interaction of GnRH with its receptor results in a greater release of LH in cells treated with the steroid [26]. In addition, ovarian steroid induced methyltransfcrasc activity [22,23] represents another bio chemical process leading to increased sensitivity of go nadotrophs.…”