1,25(OH)(2)-VITAMIN D-3 SIGNAL-TRANSDUCTION IN CHICK MYOBLASTS INVOLVES PHOSPHATIDYLCHOLINE HYDROLYSIS

1,25-Dihydroxyvitamin D-3 (1,25(OH)(2)D-3) rapidly stimulates the biph asic formation of diacylglycerol (DAG) in chick myoblasts. Neomycin (0 .5 mM), an inhibitor of phosphoinositide hydrolysis, abolished the fir st phase (1 min) but had no effect on the second 1,25(OH)(2)D-3-induce d DAG peak (5 min). In myoblasts prelabeled with [H-3]choline, 1,25(OH )(2)D-3 increased the release of [H-3]choline (maximally at 5 min), wi th a concomitant decrease in phosphatidylcholine and the absence of si gnificant changes in phosphocholine. 1,25(OH)(2)D-3 caused a significa nt increase in phosphatidylethanol (PEt) formation in myoblasts in the presence of 1.5% ethanol. The effects of 1,25(OH)(2)D-3 were time- an d dose-dependent (10(-11) to 10(-8) M) and specific as 25OHD(3) and 24 ,25(OH)(2)D-3 failed to accumulate PEt. 12-O-Tetradecanoylphorbol-13-a cetate also stimulated PEt formation. The combination of 1,25(OH)(2)D- 3 and 12-O-tetradecanoylphorbol-13-acetate was more effective than eit her compound alone. Neither the PKC inhibitor H-7 nor PKC down-regulat ion blocked the hormone-induced increase in PEt. The effects of 1,25(O H)(2)D-3 were, however, inhibited in the absence of extracellular Ca2 (+EGTA) and by nifedipine and verapamil, whereas the Ca2+ ionophore A 23181 also increased PEt generation. The data support the notion that 1,25(OH)(2)D-3 triggers the hydrolysis of phosphatidylcholine in myobl asts through a Ca2+-dependent, PKC-independent, phospholipase D-cataly zed mechanism.