Phorbol-stimulated Ca(2+) mobilization and contraction in dispersed intestinal smooth muscle cells

This study examined the source of Ca(2+) mobilized by phorbol esters and its requirement for phorbol-induced contraction of smooth muscle cells isolated from the circular and longitudinal layers of guinea pig intestine. Phorbol-12-myristate-13-acetate caused rapid, sustained, concentration-dependent muscle contraction and increase in cystolic free [Ca(2+)](i) in muscle cells from both layers. Maximal contraction was similar to that elicited by receptor-linked agonists, whereas maximal [Ca(2+)](i) was 50% less. The increase in [Ca(2+)](i) was mediated by Ca(2+) release in circular, and Ca(2+) influx in longitudinal muscle cells; only the latter was abolished by methoxyverapamil and in Ca(2+)-free medium. [Ca(2+)](i) was essential for contraction in both cell types: contraction in longitudinal muscle cells was abolished by methoxyverapamil and in Ca(2+)-free medium; contraction in circular muscle cells was abolished only after depletion of Ca(2+) stores. Contraction was abolished by the protein kinase C (PKC) inhibitor calphostin C (1 microM), but was not affected by the myosin light chain kinase inhibitor KT5926 (1 microM), suggesting that activation of myosin light chain kinase was suppressed by phorbol-12-myristate-13-acetate or via PKC. Phorbol-induced contraction of permeabilized circular and longitudinal muscle cells was abolished by pretreatment with a common antibody to Ca(2+)-dependent PKC-alpha,beta,gamma, but was not affected by pretreatment with a specific PKC-epsilon antibody. This study demonstrates the ability of phorbol esters to mobilize Ca(2+) from different sources in different smooth muscle cell types and establishes the requirement of Ca(2+) for phorbol-induced contraction; the latter is exclusively mediated by Ca(2+)-dependent PKC isozymes.