PROTEIN-KINASE-C - MODULATION OF VASOPRESSIN-INDUCED CALCIUM INFLUX AND RELEASE IN A7R5 VASCULAR SMOOTH-MUSCLE CELLS

This study was guided by the hypothesis that specific isoforms of prot ein kinase C may participate in modulating increases in intracellular Ca2+ that are induced by stimulation of vascular smooth muscle cells w ith vasopressin. Immunoblot analysis revealed that A7r5 vascular smoot h muscle cells expressed conventional (alpha), novel (delta and epsilo n), and atypical (iota/lambda and mu) isoforms of protein kinase C, St imulation of fura-2-loaded cells with 20 nM vasopressin induced a rapi d transient increase in the intracellular concentration of calcium tha t was followed by a slowly declining component which was above baselin e throughout the period of observation. Cell fractionation studies sho wed that the calcium response was associated with (a) transient transl ocation of the alpha and delta isoforms of protein kinase C from the c ytosolic fraction to the particulate-membrane fraction, (b) sustained translocation of the epsilon isoform, and (c) no translocation of iota /lambda or mu isoforms, Ratiometric and isobestic fluorescence analysi s showed that vasopressin-induced Ca2+ influx and release were markedl y inhibited in cells that were preincubated with either 1 mu M phorbol la-myristate 13-acetate, or 10 mu M 1,2 dioctanoyl-sn-glycerol, two s tructurally different activators of protein kinase C, In contrast, vas opressin-induced increases in intracellular Ca2+ were not significantl y altered following preincubation with either 1 mu M 4 alpha-phorbol o r 4 alpha-phorbol 12,13-didecanoate, analogs of phorbol 12-myristate 1 3-acetate that do not activate protein kinase C, Moreover, the inhibit ory effects of phorbol la-myristate 13-acetate were prevented by treat ment with 1 mu M GF109203X, a potent inhibitor of protein kinase C, Ta ken together, these results show that direct activation of protein kin ase C can negatively modulate vasopressin-induced Ca2+ influx and rele ase in cultured vascular smooth muscle cells. They also show that stim ulation with vasopressin induces translocation of specific isoforms of protein kinase C, an observation suggesting that one or more of these isoforms may participate in modulation of vasopressin-induced increas es in intracellular Ca2+. (C) 1998 Academic Press.