VASOPRESSIN ACTIVATES PHOSPHOLIPASE-D THROUGH PERTUSSIS TOXIN-INSENSITIVE GTP-BINDING PROTEIN IN AORTIC SMOOTH-MUSCLE CELLS - FUNCTION OF CA2+ CALMODULIN/

In the present study, we examined the effect of vasopressin (AVP) on p hosphatidylcholine-hydrolyzing phospholipase D activity in primary cul tured rat aortic smooth muscle cells. AVP stimulation of choline forma tion was dose dependent. The time-course was quite different from thos e of inositol phosphates. The effect of AVP on the formation of inosit ol phosphates (EC(50) was 3 nM) was more potent than that on the forma tion of choline (EC(50) was 30 nM). 12-O-Tetradecanoylphorbol-13-aceta te (TPA), an activator of protein kinase C (PKC), stimulated the forma tion of choline. However, 4 alpha-phorbol 12,13-didecanoate, which is inactive for PKC, had little effect. Staurosporine, an inhibitor of pr otein kinases, which inhibited the TPA-induced formation of choline, h ad little effect on the AVP-induced formation of choline. Neither calp hostin C, a highly specific PKC inhibitor, nor PKC down-regulation wit h TPA affected AVP-induced formation of choline. A combination of AVP and TPA additively stimulated the formation of choline. The depletion of extracellular Ca2+ by (ethylenebis(oxyethylenenitrilo))tetraacetic acid significantly reduced the AVP-induced formation of choline. W-7, an antagonist of calmodulin, inhibited the AVP-induced formation of ch oline in a dose-dependent manner. NaF, an activator for GTP-binding pr otein (G-protein), stimulated the formation of choline. However, the f ormation of choline by a combination of AVP and NaF was not additive. Pertussis toxin had little effect on the AVP-induced formation of chol ine. These results strongly suggest that AVP stimulates phospholipase D in a Ca2+/calmodulin-dependent manner in aortic smooth muscle cells, that a pertussis-toxin-insensitive G-protein is involved in the AVP-i nduced phospholipase D activation, and furthermore, that PKC is not es sential for the activation.