Phorbol-Stimulated Ca Mobilization and Contraction in Dispersed Intestinal Smooth Muscle Cells

This study examined the source of Ca 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]i in muscle cells from both layers. Maximal contraction was similar to that elicited by receptor-linked agonists, whereas maximal [Ca]i was 50% less. The increase in [Ca]i was mediated by Ca 21 release in circular, and Ca influx in longitudinal muscle cells; only the latter was abolished by methoxyverapamil and in Ca-free medium. [Ca]i was essential for contraction in both cell types: contraction in longitudinal muscle cells was abolished by methoxyverapamil and in Ca-free medium; contraction in circular muscle cells was abolished only after depletion of Ca stores. Contraction was abolished by the protein kinase C (PKC) inhibitor calphostin C (1 mM), but was not affected by the myosin light chain kinase inhibitor KT5926 (1 mM), 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-dependent PKC-a,b,g, but was not affected by pretreatment with a specific PKC-e antibody. This study demonstrates the ability of phorbol esters to mobilize Ca from different sources in different smooth muscle cell types and establishes the requirement of Ca for phorbolinduced contraction; the latter is exclusively mediated by Cadependent PKC isozymes. The lipid second messenger diacylglycerol (DAG) and phorbol esters regulate the activity of Ca-dependent (a, bI, bII, g) and Ca-independent (d, e, h, u, m) isoforms of protein kinase C (PKC) (Nishizuka, 1995; Ron and Kazanietz, 1999). DAG and phorbol esters bind to the regulatory C1 domain of PKC and increase the affinity of PKC for membrane and other target proteins. A Ca-binding region in the regulatory C2 domain is present only in Ca-dependent PKC isozymes. Binding of PKC to the membrane and enzyme activation are differentially regulated by Ca: low concentrations of Ca increase the affinity for binding, whereas higher concentrations are required for PKC activation. Several studies have examined the requirement of Ca for phorbol-stimulated, PKC-dependent contraction of smooth muscle. Both phorbols and DAG analogs are known to cause contraction of vascular and visceral smooth muscle that is characteristically slow in developing and is preceded by a prolonged lag period (Chatterjee and Tejada, 1986; Nakajima et al., 1991). Only a few smooth muscle tissues, for example, bovine trachea, guinea pig tenia coli, and rat anococcygeus, do not respond directly to phorbols, but even in these, phorbols can potentiate the response to agents, such as extracellular K and Ca ionophores, that increase intracellular Ca levels (Mitsui and Karaki, 1993; Kaneda et al., 1995; Tajimi et al., 1997). In other muscle tissues, phorbol-induced contraction is sensitive to the absence of Ca from the extracellular medium and can be either partly or completely inhibited by L-type Ca channel blockers, implying a phorbol-stimulated increase in Ca influx (Gleason and Flaim, 1986; Chiu et al., 1987, 1988; Nakajima et al., 1991; Hattori et al., 1995; Masui and Wakabayashi, 1997). An increase in Ca influx has been directly measured in rabbit and rat aorta and dog splenic artery (Gleason and Flaim, 1986; Chiu et al., 1987; Khoyi et al., 1999). Measurements of cytosolic free [Ca]i have yielded conflicting results: an increase in Received for publication January 28, 2000. 1 This work was supported by Grant DK15564 from the National Institute of Diabetes and Digestive and Kidney Diseases. ABBREVIATIONS: DAG, diacylglycerol; PKC, protein kinase C; PLA2, phospholipase A2; PMA, phorbol-12-myristate-13-acetate; PDBu, phorbol 12,13-dibutyrate; D600, methoxyverapamil; KT5926, (8R*,9S*,11S*)-(2)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3,9,10-tetrahydro-8,11-epoxy,1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinden-1-one; AACOF3, arachidonyltrifluoromethyl ketone; PI, phosphoinositide; U73122, 1-[6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrole-2,5-dione; MLCK, myosin light chain kinase; CCK-8, cholecystokinin octapeptide. 0022-3565/00/2943-0991$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 294, No. 3 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics 2549/844345 JPET 294:991–996, 2000 Printed in U.S.A. 991 at A PE T Jornals on Sptem er 3, 2017 jpet.asjournals.org D ow nladed from [Ca]i has been reported in some studies of rat aorta and porcine carotid arteries (Rembold and Murphy, 1988; Nakajima et al., 1993; Kaneda et al., 1995), but not in other studies of rat and ferret aorta, and canine trachea (Jiang and Morgan, 1987; Ozaki et al., 1990). In this study we have examined the ability of phorbol esters to mobilize Ca and cause contraction of smooth muscle cells isolated separately from the circular and longitudinal muscle layers of guinea pig intestine. Smooth muscle cells from the two layers differ in the mechanisms they use to mobilize Ca in response to activation of G protein-coupled receptors. Ca mobilization in circular muscle is initiated by activation of phospholipase C-b1 or -b3 and is mediated by inositol 1,4,5-triphosphate-dependent Ca release (Murthy et al., 1991; Makhlouf and Murthy, 1997), whereas Ca mobilization in longitudinal muscle is initiated by activation of phospholipase A2 (PLA2) and arachidonic acid-mediated Ca influx that triggers both Caand cyclic ADP riboseinduced Ca release (Kuemmerle et al., 1994; Murthy et al., 1995). The results indicate that phorbol esters induce rapid, sustained contraction and increase [Ca]i in both circular and longitudinal muscle cells. The increase in [Ca]i is essential for contraction and is mediated by Ca influx in longitudinal muscle and Ca release in circular muscle. Phorbol-induced contraction, however, is entirely mediated by one or more Ca-dependent PKC isozymes. Experimental Procedures Preparation of Dispersed Intestinal Smooth Muscle Cells. Muscle cells were isolated separately from the circular and longitudinal muscle layers of guinea pig intestine by sequential enzymatic digestion, filtration, and centrifugation as described previously (Murthy et al., 1991). Briefly, muscle strips were incubated at 31°C for 30 min in HEPES medium with type II collagenase (0.1%) and soybean trypsin inhibitor (0.1%). The partly digested strips were washed and muscle cells allowed to disperse spontaneously for 30 min. The cells were harvested by filtration through 500-mm Nitex (Tetko Inc., Briarcliff Manor, NY) and centrifuged twice at 350g for