Cellular redistribution of PKCalpha, rhoA, and ROKalpha 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 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.
Taggart
M J
MJ
Signal Transduction Group, Boston Biomedical Research Institute, Boston, Massachusets, USA. michael.j.taggart@man.ac.uk
Lee
Y H
YH
Morgan
K G
KG
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002478:Cells, Cultured; D003593:Cytoplasm / Q000187:drug effects / Q000201:enzymology; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D018613:Microscopy, Confocal; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009215:Myometrium; D010703:Phorbol Esters / Q000494:pharmacology; D011247:Pregnancy; D011493:Protein Kinase C / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011976:Receptors, Muscarinic / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein
D047908:Intracellular Signaling Peptides and Proteins; D010703:Phorbol Esters; D011976:Receptors, Muscarinic; C047907:12-deoxyphorbol-13-isobutyrate-20-acetate; D002217:Carbachol; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D002118:Calcium