Protein kinase-C activation stimulates synthesis of gonadotropin-releasing hormone (GnRH) receptors, but does not mediate GnRH-stimulated receptor synthesis.

Abstract

Gonadotropes respond to GnRH with LH synthesis and release, desensitization, changes in GnRH receptor number, and GnRH receptor synthesis. Activation of protein kinase-C (PKC) appears to be involved in LH beta gene expression, but is not required for acute LH release, desensitization, or receptor down-regulation. The present studies were conducted to determine whether PKC mediates GnRH-stimulated receptor synthesis. We have adapted the density shift technique to measure the synthesis of GnRH receptors in pituitary culture. Pituitary cells from female weanling rats were exposed to medium containing treatments, dense amino acids (greater than 95% 13C, 15N, and 2H), dialyzed horse serum (10%, vol/vol), and fetal calf serum (2.5%, vol/vol). Treatments consisted of medium alone, phorbol myristate acetate (PMA), phorbol dibutyrate (PdBu), or GnRH. To deplete cells of PKC, cultures were exposed for 8-16 h to 1 microM PMA. Short term treatment with PKC activators (PMA or PdBu, 1 microM) or GnRH (0.1 nM) was given for 30 min. After treatment, GnRH receptors were covalently linked to [125I]Tyr5-azidobenzoyl-D-Lys6-GnRH and solubilized. Newly synthesized (densely labeled) GnRH receptors were separated from normal receptors by velocity sedimentation (156,000 X g; 24 h; 0-20% sucrose) and quantified by gamma-spectroscopy. Treatment with GnRH significantly stimulated the synthesis of GnRH receptors. Treatment of pituitary cell cultures with PMA (8-16 h) also stimulated the synthesis of GnRH receptors, although to a lesser extent than that observed after GnRH treatment. The synthesis of GnRH receptors in response to 0.1 nM GnRH was not different in cells with a normal complement of PKC compared to those depleted of PKC activity. This indicates that the ability of GnRH to stimulate the synthesis of its own receptor is not mediated by PKC. Short term treatment of cell cultures with 1 microM PMA or PdBu (30 min) stimulated GnRH receptor synthesis similar to treatment with 0.1 nM GnRH. When PMA and GnRH were administered simultaneously, GnRH receptor synthesis was stimulated to a greater extent than with either agent alone, suggesting differing mechanisms of action. These results indicate that although activators of PKC can stimulate the synthesis of GnRH receptors, PKC does not mediate the effects of GnRH on homologous receptor synthesis.