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 protein kinase C may participate in modulating increases in intracellular Ca2+that are induced by stimulation of vascular smooth muscle cells with vasopressin. Immunoblot analysis revealed that A7r5 vascular smooth muscle cells expressed conventional (α), novel (δ and ϵ), and atypical (ι/λ and μ) isoforms of protein kinase C. Stimulation of fura-2-loaded cells with 20 nM vasopressin induced a rapid transient increase in the intracellular concentration of calcium that was followed by a slowly declining component which was above baseline throughout the period of observation. Cell fractionation studies showed that the calcium response was associated with (a) transient translocation of the α and δ isoforms of protein kinase C from the cytosolic fraction to the particulate-membrane fraction, (b) sustained translocation of the ϵ isoform, and (c) no translocation of ι/λ or μ isoforms. Ratiometric and isobestic fluorescence analysis showed that vasopressin-induced Ca2+influx and release were markedly inhibited in cells that were preincubated with either 1 μM phorbol 12-myristate 13-acetate, or 10 μM 1,2 dioctanoyl-sn-glycerol, two structurally different activators of protein kinase C. In contrast, vasopressin-induced increases in intracellular Ca2+were not significantly altered following preincubation with either 1 μM 4α-phorbol or 4α-phorbol 12,13-didecanoate, analogs of phorbol 12-myristate 13-acetate that do not activate protein kinase C. Moreover, the inhibitory effects of phorbol 12-myristate 13-acetate were prevented by treatment with 1 μM GF109203X, a potent inhibitor of protein kinase C. Taken together, these results show that direct activation of protein kinase C can negatively modulate vasopressin-induced Ca2+influx and release in cultured vascular smooth muscle cells. They also show that stimulation 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 increases in intracellular Ca2+.