In the present study, we examined the effect of vasopressin (AVP) on phosphatidylcholine-hydrolyzing phospholipase D activity in primary cultured rat aortic smooth muscle cells. AVP stimulation of choline formation was dose dependent. The time-course was quite different from those of inositol phosphates. The effect of AVP on the formation of inositol phosphates (EC50 was 3 nM) was more potent than that on the formation of choline (EC50 was 30 nM). 12-O-Tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC), stimulated the formation of choline. However, 4α-phorbol 12,13-didecanoate, which is inactive for PKC, had little effect. Staurosporine, an inhibitor of protein kinases, which inhibited the TPA-induced formation of choline, had little effect on the AVP-induced formation of choline. Neither calphostin C, a highly specific PKC inhibitor, nor PKC down-regulation with 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 choline in a dose-dependent manner. NaF, an activator for GTP-binding protein (G-protein), stimulated the formation of choline. However, the formation of choline by a combination of AVP and NaF was not additive. Pertussis toxin had little effect on the AVP-induced formation of choline. 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-induced phospholipase D activation, and furthermore, that PKC is not essential for the activation.Key words: vasopressin, phospholipase D, protein kinase C, calmodulin, GTP-binding protein, aortic smooth muscle cells.
Arachidonic acid release from aortic smooth muscle cells induced by [Arg8]vasopressin is largely mediated by calcium-independent phospholipase A2.
