1-ALPHA,25-(OH)(2)-VITAMIN D-3 STIMULATES THE ADENYLYL-CYCLASE PATHWAY IN MUSCLE-CELLS BY A GTP-DEPENDENT MECHANISM WHICH PRESUMABLY INVOLVES PHOSPHORYLATION OF G(ALPHA-I)

To further understand the mechanism underlying 1,25(OH)(2)D-3 activati on of the cAMP pathway, the effect of the hormone on adenylyl cyclase (AC), GTPase and protein kinase A (PKA) activities as well as on the p hosphorylation of G(alpha i) was studied in membranes from chick skele tal muscle cells. The sterol stimulated AC activity in a dose (0.1-10 nM) and time (1-5 min.) dependent fashion, provided GTP (10 mu M) was present in the assay. High affinity GTPase activity was unaffected by the hormone. In the absence of GTP or in the presence of Mn2+ (20 mM), 1,25(OH)(2)D-3 effects on AC were abolished. PKA activity was increas ed (+1.20%) in cells pretreated (1 nM, 5 min.) with the sterol. Moreov er, immunoprecipitation of G(alpha i) from [P-32]-labeled myoblast mem branes showed that 5 min. exposure to 1 nM 1,25(OH)(2)D-3 increased (1 .5-2 fold) the phosphorylation of its alpha subunit. The present data suggest that in muscle cells, 1,25(OH)(2)D-3 activates AC by a non dir ect, GTP-dependent action which could imply amelioration of G(i) funct ion by sterol-induced alpha(i) phosphorylation. (C) 1997 Academic Pres s.