TY - JOUR T1 - Phorbol-Stimulated Ca<sup>2+</sup> Mobilization and Contraction in Dispersed Intestinal Smooth Muscle Cells JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 991 LP - 996 VL - 294 IS - 3 AU - Karnam S. Murthy AU - Yuen San Yee AU - John R. Grider AU - Gabriel M. Makhlouf Y1 - 2000/09/01 UR - http://jpet.aspetjournals.org/content/294/3/991.abstract N2 - 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-α,β,γ, but was not affected by pretreatment with a specific PKC-ε 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. The American Society for Pharmacology and Experimental Therapeutics ER -