Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility.
Myosin light chain phosphatase (MLCP) plays a pivotal role in smooth muscle contraction by regulating Ca(2+) sensitivity of myosin light chain phosphorylation. A smooth muscle phosphoprotein called CPI-17 specifically and potently inhibits MLCP in vitro and in situ and is activated when phosphorylated at Thr-38, which increases its inhibitory potency 1000-fold. We produced a phosphospecific antibody for this site in CPI-17 and used it to study in situ phosphorylation of endogenous CPI-17 in arterial smooth muscle in response to agonist stimulation. In the intact femoral artery, CPI-17 phosphorylation was negligible at the resting state and was not increased during contraction induced by K(+) depolarization. The Ca(2+)-sensitizing agonists histamine and phenylephrine induced nearly equivalent contractions, but histamine generated significantly higher levels of CPI-17 phosphorylation. In alpha-toxin-permeabilized strips at pCa 6.7, contractile force and CPI-17 phosphorylation were proportional in response to histamine, guanosine 5'-O-(gamma-thiotriphosphate), and histamine plus guanyl-5'-yl thiophosphate, implying that histamine increased CPI-17 phosphorylation through activation of G proteins. Inhibitors of Rho-kinase (Y27632) and protein kinase C (PKC; GF109203X) reduced contraction and CPI-17 phosphorylation in parallel, suggesting that CPI-17 functions downstream of Rho kinases and PKC. The results show that agonists such as histamine signal through phosphorylation of CPI-17 to produce Ca(2+) sensitization of smooth muscle contraction.
Kitazawa
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Department of Physiology and Biophysics, Georgetown University Medical Center, Washington, DC 20007, USA. tkitaz01@gusun.georgetown.edu
Eto
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Woodsome
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Brautigan
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D000577:Amides / Q000494:pharmacology; D000818:Animals; D002463:Cell Membrane Permeability / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005263:Femoral Artery / Q000187:drug effects / Q000502:physiology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008301:Maleimides / Q000494:pharmacology; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D009124:Muscle Proteins / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D043263:Myosin-Light-Chain Phosphatase; D010656:Phenylephrine / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011725:Pyridines / Q000494:pharmacology; D011817:Rabbits; D010738:Type C Phospholipases / Q000494:pharmacology; D054460:rho-Associated Kinases
D000577:Amides; C491521:CPI-17 protein, Oryctolagus cuniculus; D004791:Enzyme Inhibitors; D007211:Indoles; D047908:Intracellular Signaling Peptides and Proteins; D008301:Maleimides; D009124:Muscle Proteins; D010750:Phosphoproteins; D011725:Pyridines; C108830:Y 27632; D010656:Phenylephrine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006632:Histamine; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; C070515:bisindolylmaleimide I