1,25(OH)(2)-VITAMIN-D-3 STIMULATION OF PHOSPHOLIPASE-C AND PHOSPHOLIPASE-D IN MUSCLE-CELLS INVOLVES EXTRACELLULAR CALCIUM AND A PERTUSSIS-SENSITIVE G-PROTEIN

The steroid hormone 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3] activat es in chick myoblasts the breakdown of phosphoinositides by phospholip ase C and the hydrolysis of phosphatidylcholine by phospholipase D. Ex tracellular Ca2+ requirement and GTP-binding protein mediation of 1,25 (OH)(2)D-3-dependent activation of phospholipases C and D were investi gated in cells prelabelled with [H-3]glycerol or [H-3]arachidonic acid . Generation of diacylglycerol by phospholipase C and phosphatidyletha nol by phospholipase D were shown to be dependent on extracellular cal cium, since both responses were suppressed by EGTA and the Ca2+-channe l blockers nifedipine and verapamil, and were mimicked by the calcium ionophore A23187. The G-protein activators guanosine 5'-O-(3-thiotriph osphate) and AlF4- strongly enhanced diacylglycerol and phosphatidylet hanol release in myoblasts while guanosine 5'-O-(2-thiodiphosphate), w hich inhibits G-protein-mediated signals, abolished 1,25(OH)(2)D-3-dep endent diacylglycerol and phosphatidylethanol release. Bordetella pert ussis toxin pretreatment suppressed the hormone action. These results suggest that 1,25(OH)(2)D-3-stimulation of phosphoinositide-specific p hospholipase C and phospholipase D in chick myoblasts is mediated by a pertussis-sensitive GTP-binding protein(s) and the influx of extracel lular calcium.