MODULATION BY 1,25(OH)(2)-VITAMIN-D-3 OF THE ADENYLYL-CYCLASE CYCLIC-AMP PATHWAY IN RAT AND CHICK MYOBLASTS

We have previously reported that the calciotropic hormone 1,25(OH)(2)- vitamin D-3 stimulates influx of Ca2+ into cultured rat and embryonic chick myoblasts via voltage sensitive Ca2+-channels. In the present st udy, we show that this effect of 1,25(OH)(2)D-3 requires the mediation of the adenylylcyclase signalling system since the hormone-dependent Ca2+ influx is abolished by specific inhibitors of adenylylcyclase and protein kinase A and mimicked by forskolin and dibutyryl cAMP. 1,25(O H)(2)D-3-stimulated elevations in cellular cAMP paralleled increases i n Ca2+ uptake, further suggesting a coupling of adenylylcyclase activa tion and calcium influx. Fluoride and GTP gamma S mimicked 1,25(OH)(2) D-3-stimulation of calcium influx while GDP beta S suppressed the effe ct of the hormone. Cholera toxin and Bordetella pertussis toxin both i ncreased Ca-45(2+) uptake in rat and chick myoblasts. The hormone furt her increased cholera toxin actions, but was unable to modify pertussi s toxin-induced Ca2+ uptake, suggesting a similar target of action for pertussis toxin and 1.25(OH)(2)D-3. Incubation of microsomal membrane s with the sterol (10 nM, 2 min) markedly displaces (-32%) [S-35]GTP g amma S binding to the membranes. ADP-ribosylation of the pertussis tox in-sensitive 41 kDa substrate was significantly increased (+40%) in 1, 25(OH)(2)D-3-pretreated cells. These results suggest that 1,25(OH)(2)D -3-stimulated influx of Ca2+ into rat and embryonic chick cultured myo blasts sequentially requires inhibition of a pertussis toxin-sensitive G protein, accumulation of cAMP and activation of dihydropyridine-sen sitive Ca2+-channels through PKA-mediated phosphorylation events.