Regulation of smooth muscle contraction in rabbit internal and sphincter by protein kinase C and Ins(1,4,5)P₃

We have examined the role of protein kinase C (PKC)-βII and its functional relationship to inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] and intracellular Ca²⁺ in the contraction of smooth muscle cells from the rabbit internal and sphincter (IAS). PKC-β (0.1-100 U/ml) and Ins(1,4,5)P₃ (10^-9 to 10^-6 M) caused concentration-dependent contraction of IAS smooth muscle cells permeabilized by saponin. The combination of threshold concentrations of Ins(1,4,5)P₃ (10^-9 M) and PKC (0.1 U/ml) was more than additive, causing near maximal shortening (28.2 ± 2.1% decrease in cell length from control). The response to high concentrations of Ins(1,4,5)P₃ and PKC used in combination was not greater than the response to either agent alone. The calmodulin antagonist W-7 (10^-9 M) inhibited the maximal contraction induced by Ins(1,4,5)P₃ but not contraction caused by PKC, whereas the PKC antagonist H-7 (10^-6 M) inhibited the maximal contraction induced by PKC but not contraction caused by Ins(1,4,5)P₃. Threshold doses of the ionophores A23187 (10^-9 M) and ionomycin (0.2 ng/ml) caused little contraction by themselves, but they potentiated the response elicited by a threshold concentration of PKC (0.1 U/ml), inducing maximal contraction. Preincubation of IAS cells with 4 mM Sr²⁺, which inhibits the release of intracellular Ca²⁺, abolished the potentiating effect of Ins(1,4,5)P₃ and calcium ionophores on PKC, but the calmodulin antagonist W-7 did not. These data suggest that the contractile effect of maximally effective doses of PKC is independent of the effects of Ins(1,4,5)P₃. At submaximal concentrations, however, PKC-dependent contraction is potentiated by Ins(1,4,5)P₃ or by ionophore-mediated release of intracellular Ca²⁺ without requiring calmodulin activation.