Cellular redistribution of PKCα, rhoA, and ROKα following smooth muscle agonist stimulation

Efficient receptor-coupled activation of smooth muscle requires discrete coordination of many signal transducing events from the plasma membrane to the myofilaments. Recruitment of key factors to the plasma membrane is thought to be crucial for transduction of extracellular signals leading to contractility. We investigated, therefore, for the first time in intact differentiated smooth muscle cells; the distributions of three molecules important for receptor-coupled excitation: protein kinase C alpha (PKC alpha), rhoA, and rho kinase (ROK). We also directly confirmed, by single cell force measurements, carbachol-induced [Ca2+](i) sensitization of contractility.. Laser scanning confocal immunofluorescent microscopy of central smooth muscle cell sections determined that, at rest, PKC alpha, rhoA, and ROK alpha were distributed predominantly throughout the cytosol. Muscarinic stimulation resulted in significant redistribution of each protein to the cell membrane. By digital image analysis, peripheral:cytosolic distributions of PKC alpha, rhoA, and ROK alpha were calculated as, respectively, 1.05 +/- 0.03 (8), 1.09 +/- 0.03 (5), and 1.26 +/- 0.04 (12) at rest, increasing significantly following stimulation to 2.09 +/- 0.22 (6), 2.02 +/- 0.12 (8), and 1.93 +/- 0.05 (10). It is proposed that this receptor-coupled recruitment to the cell periphery of the downstream signaling molecules PKC alpha, rhoA, and ROK alpha contributes to the efficacy of agonist-induced contractile activation of smooth muscle. (C) 1999 Academic Press.