Regulation of hormone-responsive Sertoli cell adenylyl cyclase in a cell-free system

Abstract

Homologous desensitization of either FSH- or isoproterenol-responsive adenylyl cyclases in Sertoli cell membranes can be achieved in a cell-free system. Incubation of membrane particles from cultured immature Sertoli cells with either FSH or isoproterenol resulted in a time- and concentration-dependent loss of subsequent adenylyl cyclase response to the homologous hormone. Half-maximal refractoriness was achieved within 20–30 min of incubation. The concentration of FSH required to obtain half-maximal loss of adenylyl cyclase response (400 ng/ml) was in the same order of magnitude as the apparent K m for FSH-stimulated adenylyl cyclase activity (300 ng/ml). Hormone-induced homologous desensitization was dependent on the presence of both ATP and Mg 2+. Increasing concentrations of ATP in the presence of FSH caused a concentration-dependent loss of subsequent response to the homologous hormone. Halfmaximal desensitization was achieved at an ATP concentration of 0.2 mM. Increasing concentrations of Mg 2+ in the presence of either FSH or isoproterenol caused desensitization for the homologous hormone. The concentration of Mg 2+ giving half-maximal effect was approximately 5 mM in excess of ATP and EDTA. However, higher concentrations of free Mg 2+, in the absence of hormone, caused desensitization of both FSH- and isoproterenol-sensitive adenylyl cyclase with half-maximal effect at approximately 30 mM. Hormone-specific desensitization was obtained in the presence of GTP. However, when GTP was substituted with the non-hydrolysable analogue GMPP(NH)P the hormonal activation remained constant throughout 90 min of incubation. The hormone-induced refractoriness was probably not due to cAMP, since all incubations were performed in the presence of an excess of cAMP (1 mM) to block phosphodiesterase (PDE) activity. However, the finding that both ATP and Mg 2+ were required for hormone-induced desensitization suggests that the process may be mediated via a cAMP-independent phosphorylation reaction. Furthermore, the hormone appears to accelerate desensitization of the adenylyl cyclase system by increasing its sensitivity to the actions of Mg 2+.