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 study, 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(OH)(2)D-3-stimulated elevations in cellular cAMP paralleled increases in Ca2+ uptake, further suggesting a coupling of adenylylcyclase activation 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 effect of the hormone. Cholera toxin and Bordetella pertussis toxin both increased Ca-45(2+) uptake in rat and chick myoblasts. The hormone further increased cholera toxin actions, but was unable to modify pertussis toxin-induced Ca2+ uptake, suggesting a similar target of action for pertussis toxin and 1.25(OH)(2)D-3. Incubation of microsomal membranes with the sterol (10 nM, 2 min) markedly displaces (-32%) [S-35]GTP gamma S binding to the membranes. ADP-ribosylation of the pertussis toxin-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 myoblasts sequentially requires inhibition of a pertussis toxin-sensitive G protein, accumulation of cAMP and activation of dihydropyridine-sensitive Ca2+-channels through PKA-mediated phosphorylation events.