Contraction of single vascular smooth muscle cells by phenylephrine at constant [Ca2+]i

Abstract

The mechanism of alpha-adrenergic agonist-mediated force generation was investigated in single hyperpermeable vascular smooth muscle cells. By use of a previously described method, force was recorded from individual ferret aortic cells made hyperpermeable by exposure to saponin. When the cells were clamped at pCa 7, addition of protein kinase M (PKM), the constitutively active form of protein kinase C (PKC), caused a sustained increase in force, which was reversible upon addition of the PKC pseudosubstrate inhibitor peptide (PSSI) corresponding to residues 19-31 of PKC. Phenylephrine at pCa 7 caused a dose-dependent increase in contractile force of the hyperpermeable cells, which was reversible on addition of phentolamine. The phenylephrine contraction could also be inhibited by the same concentration of PSSI that was effective toward the PKM-induced contraction. The response of the cells to a constant [phenylephrine] in different Ca buffers showed a lack of Ca dependence between pCa 8.6 and 7.0. The addition of PSSI to unstimulated cells caused a small but significant drop in basal tone. Taken together, these results suggest that a fraction of the basal tone, as well as the phenylephrine contraction that occurs in the skinned cells at constant intracellular free Ca2+ concentration, is the result of activation of a Ca-independent isozyme of PKC.