Vol. 294, Issue 3, 991-996, September 2000

Phorbol-Stimulated Ca²⁺ Mobilization and Contraction in Dispersed Intestinal Smooth Muscle Cells¹

Karnam S. Murthy , Yuen San Yee, John R. Grider and Gabriel M. Makhlouf

Departments of Medicine (K.S.M., Y.S.Y., J.R.G., G.M.M.) and Physiology (K.S.M., J.R.G.), Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia

This study examined the source of Ca²⁺ 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 [Ca²⁺]_i in muscle cells from both layers. Maximal contraction was similar to that elicited by receptor-linked agonists, whereas maximal [Ca²⁺]_i was 50% less. The increase in [Ca²⁺]_i was mediated by Ca²⁺ release in circular, and Ca²⁺ influx in longitudinal muscle cells; only the latter was abolished by methoxyverapamil and in Ca²⁺-free medium. [Ca²⁺]_i was essential for contraction in both cell types: contraction in longitudinal muscle cells was abolished by methoxyverapamil and in Ca²⁺-free medium; contraction in circular muscle cells was abolished only after depletion of Ca²⁺ stores. Contraction was abolished by the protein kinase C (PKC) inhibitor calphostin C (1 µM), but was not affected by the myosin light chain kinase inhibitor KT5926 (1 µM), 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 Ca²⁺-dependent PKC-,,, but was not affected by pretreatment with a specific PKC- antibody. This study demonstrates the ability of phorbol esters to mobilize Ca²⁺ from different sources in different smooth muscle cell types and establishes the requirement of Ca²⁺ for phorbol-induced contraction; the latter is exclusively mediated by Ca²⁺-dependent PKC isozymes.