CALCITRIOL TRANSMEMBRANE SIGNALING - REGULATION OF RAT MUSCLE PHOSPHOLIPASE-D ACTIVITY

In rat skeletal muscle, calcitriol, the hormonal form of vitamin D-3, rapidly stimulates the biphasic formation of diacylglycerol (DAG), the second phase being independent of phosphoinositide hydrolysis driven by phospholipase C. In this work we showed that the effect of calcitri ol on the second phase of DAG formation was totally inhibited in the a bsence of extracellular Ca2+ and by the Ca2+-channel blockers nifedipi ne and verapamil, whereas the Ca2+ ionophore A23184, similar to calcit riol, increased DAG formation by 100%. GTP gamma S, which activates G protein-mediated signals, mimicked the effects of the hormone while GD P beta S, an inhibitor of G proteins, suppressed calcitriol-induced DA G formation. To elucidate the metabolic pathway of the late phase of D AG production, we examined the contribution of phospholipase D (PLD),w hich acts an phosphatidylcholine (PC) generating phosphatidic acid tha t is converted to DAG by a phosphatidate phosphohydrolase. In [H-3] ar achidonate-labeled muscle, calcitriol increased [H-3] phosphatidyletha nol (PEt) formation in the presence of ethanol, a reaction specific fo r PLD. The effects of the hormone were time-and dose-dependent with ma ximum PEt levels achieved at 10(-9) M. The phorbol ester TPA also stim ulated PEt formation. The combination of calcitriol and TPA was more e ffective than either compound alone. In rat muscle, calcitriol increas ed PKC activity in a time-dependent fashion. Bisindolymaleimide, a sel ective inhibitor of the enzyme, completely suppressed TPA-induced PEt and attenuated the effects of the hormone. These results provide the f irst evidence concerning calcitriol stimulation of the hydrolysis of P C in a mammalian tissue through a phospholipase D catalyzed mechanism involving Ca2+, protein kinase C, and G proteins.