Cellular redistribution of PKCalpha, rhoA, and ROKalpha following smooth muscle agonist stimulation

Abstract

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 Calpha (PKCalpha), rhoA, and rho kinase (ROK). We also directly confirmed, by single cell force measurements, carbachol-induced [Ca(2+)](i) sensitization of contractility. Laser scanning confocal immunofluorescent microscopy of central smooth muscle cell sections determined that, at rest, PKCalpha, rhoA, and ROKalpha 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 PKCalpha, rhoA, and ROKalpha 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 PKCalpha, rhoA, and ROKalpha contributes to the efficacy of agonist-induced contractile activation of smooth muscle.