Growth Hormone-Releasing Factor-Sensitive Adenylate Cyclase System of Purified Somatotrophs: Effects of Guanine Nucleotides, Somatostatin, Calcium, and Magnesium*

Abstract

The purpose of this study was to characterize the adenylate cyclase system in a purified population of normal somatotrophs derived from rat pituitary and to determine the responses of this system to GRF, somatostatin, guanine nucleotides, and cations. Additionally, experiments were performed to evaluate the interrelationships among changes in adenyalte cyclase activity, cellular cAMP levels, and GH release induced by GRF and somatostatin. The results obtained using homogenates and membrane preparations from somatotrophs included the following. 1) GRF caused guanine nucleotide-dependnet concentration-related (Ka, .apprx. 10-8 M) stimulation of adenylate cyclase activity. 2) Guanine nucleotides were effective in stimulating cyclase in the absence of GRF; the concentration of guanine nucleotide required for half-maximal stimulation was decreased more than 10-fold in the presence of GRF. 3) Adenylate cyclase activity increased with increasing concentrations of free Mg2+ (0.25-20 mM); activation by GRF and guanine nucleotide resulted in an approximately 7-fold increase in the enzyme''s affinity for free Mg2+. 4) Somatostatin, up to 10-6 M, did not alter basal or GRF-stimulated adenylate cyclase activity. 5) Ca2+ (0.5-11.9 .mu.M) produced concentration-dependent inhibition of basal (up to 28%) and GRF-stimulated (up to 47%) cyclase activities; the inhibitory effect of Ca2+ was accompanied by a decrement (2- to 3-fold) in the apparent affinities of the enzyme for both GRF and guanine nucleotide. In intact somatotrophs, GRF produced concentration-dependent stimulation of GH release (Ka, .apprx. 6 .times. 10-11 M), preceded by a marked elevation of cAMP levels. While somatostatin blocked GRF-induced GH release, the augmented cAMP levels were only slightly reduced. The above results indicate that in somatotrophs 1) adenylate cyclase is coupled to the GRF receptor via a stimulatory guanine nucleotide regulatory protein, Ns; 2) the mechanistic events underlying GRF stimulation of the cyclase are similar to those recognized for other hormone receptor-coupled adenylate cyclase systems; 3) the response characteristics of adenylate cyclase to GRF are consistent with an essential role for cAMP in GRF-mediated GH release; 4) the increase in intracellular Ca2+ resulting from activation of the adenylate cyclase-cAMP system by GRF will not only promote GH release, but may trigger inhibition of cAMP formation to attenuate the secretory response; and 5) a fractional population of the GRF-sensitive adenylate cyclase units may be coupled to somatostatin receptors in an inhibitory manner, and the primary action of somatostatin in blocking GRF-induced GH release occurs at a step(s) distal to cAMP formation, or independent of changes in cAMP levels.