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

Abstract

Abstract Binding constants of [(125) I]Des-Gly(10)-(D-Ala(6))-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 1-5-(isoquinolinyl-sulfoxyl) 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 degrees C for 25 min, the Scatchard plot was not linear and calculation of the Hill coefficient (N(H)) was indicative of positive cooperativity (N(H)= 1.26 +/- 0.02). Such non-linearity was not observed when cells were incubated at 0.5 degrees C for 3 h. In that condition the maximal number of binding sites measured at equilibrium (B(max)) increased (15.1 +/- 0.05 versus 9.3 +/- 0.5 fmoles x mg(-1) proteins at 21 degrees C). Two control experiments permitted us to rule out the possibility that lower B(max) at 21 degrees C might reflect internalization: 1) Cells were first incubated with the ligand at 21 degrees C for 25 min and subsequently for 3 additional hours at 0.5 degrees C. Preincubation did not affect the B(max) obtained at 0.5 degrees 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 0.5 degrees C and 21 degrees C. When the cells were incubated with PMA, the Scatchard plot was linearized and the B(max) recorded at 21 degrees C increased by 50% over control cells (13 +/- 0.7 fmoles x mg(-1) 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 degrees or 21 degrees C, with B(max) values of 14.4 +/- 0.3 and 15 +/- 0.34 fmoles x mg(-1) proteins, respectively. PKC activation did not affect binding in that model, but H7 decreased the number of sites (B(max)= 10.7 +/- 0.9) and induced appearance of positive cooperativity (N(H)= 1.36 +/- 0.07). 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 positive cooperativity, either on normal cells or on cells from castrates after protein kinase inhibition, suggesting that such a cooperativity is not related to a GnRH-dependent phosphorylation.