Involvement of protein kinase C in the modulation of 1 alpha,25-dihydroxy-vitamin D-3-induced Ca-45(2+) uptake in rat and chick cultured myoblasts

The calciotropic hormone 1 alpha,25-dihydroxy-vitamin D-3 (1,25(OH)(2)D-3) has been shown to stimulate both rat and chick myoblast Ca-45(2+) uptake via modulation of dihydropyridine-sensitive L-type calcium channels through phosphorylation by the cAMP/protein kinase A pathway. We further investigated the involvement of protein kinases in 1,25(OH)(2)D-3-signal transduction on cultured myoblasts. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) was found to rapidly stimulate myoblast Ca-45(2+) uptake, mimicking 1,25(OH)(2)D-3. The effects of PMA were time- (1-5 min) and dose (50-100 nM)-dependent, were mimicked by 1,2-dioctanoylglycerol (DOG) and were specific, since the inactive analogue 4 alpha-phorbol was without effect. Analogously to the hormone, PMA-enhanced Ca-45(2+) uptake was suppressed by the Ca2+-channel blocker nifedipine (5 mu M). 1-(5-isoquinolynsulfonyl)-2-methylpiperazine (H-7), a PKC inhibitor, and down-regulation of PKC by prolonged exposure to PMA (1 mu M, 24 h), abolished both PMA and hormone effects on rat and chick cells. As in chick myoblasts, 1,25(OH)(2)D-3 activated PKC in rat myoblasts, with translocation of activity from the cytosol to the cell membrane. Treatment of myoblasts with PMA (100 nM) plus 1,25(OH)(2)D-3 (1 nM) greatly potentiated Ca-45(2+) uptake than either agent alone. PMA also increased myoblast cAMP content. These results suggest the involvement of PKC in the mechanism by which 1,25(OH)(2)D-3 rapidly stimulates calcium uptake in both mammalian and avian myoblasts.