A role for Rho-kinase in Ca-independent contractions induced by phorbol-12,13-dibutyrate.
1. Phorbol-12,13-dibutyrate (PDBu) is an activator of protein kinase C (PKC) that causes contractions in both physiological salt solutions and Ca(2+)-depleted solutions. In the present study, we tested the hypothesis that Rho-kinase plays a role in Ca(2+)-independent contractions induced by PDBu in vascular smooth muscles. 2. In Ca(2+)-free solution, 0.1 and 1 micromol/L PDBu induced contraction and myosin light chain (MLC(20)) phosphorylation, both of which were approximately 40% of responses obtained in normal Krebs' solution. Hydroxyfasudil (H1152; 1 micromol/L), an inhibitor of Rho-kinase, but not ML7 (10 micromol/L), an inhibitor of myosin light chain kinase, inhibited Ca(2+)-independent contractions induced by PDBu. 3. In Ca(2+)-free solution, PDBu increased phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and CPI-17 (PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa). This action was inhibited by H1152, with the phosphorylation of CPI-17 almost completely abolished by 1 micromol/L Ro31-8220, an inhibitor of PKC. 4. In Ca(2+)-free solution, PDBu increased the amount of GTP-RhoA (an activated form of RhoA). This increase was blocked by the PKC inhibitor Ro31-8220, but not by the Rho kinase inhibitor H1152. 5. In conclusion, RhoA/Rho-kinase plays an important role in Ca(2+)-independent contractions induced by PDBu in vascular smooth muscles. The results of the present study suggest that PDBu induces Ca(2+)-independent contractions by inhibiting myosin light chain phospatase (MLCP) through activation of GTP-RhoA and subsequent phosphorylation of MYPT1 and CPI-17.
Baek
Inji
I
Department of Pharmacology, Kyungpook National University School of Medicine, Daegu, Republic of Korea.
Jeon
Su Bun
SB
Kim
Juyoung
J
Seok
Young Mi
YM
Song
Min-Ji
MJ
Chae
Shung Chull
SC
Jun
Jae Eun
JE
Park
Wee Hyun
WH
Kim
In Kyeom
IK
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000201:enzymology; D001381:Azepines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D008297:Male; D009124:Muscle Proteins / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000201:enzymology; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D054645:Protein Phosphatase 1 / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014661:Vasoconstriction / Q000187:drug effects*; D014662:Vasoconstrictor Agents / Q000494:pharmacology*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
C459092:2-methyl-1-((4-methyl-5-isoquinolinyl)sulfonyl)homopiperazine; D001381:Azepines; D009124:Muscle Proteins; D018994:Myosin Light Chains; D009281:Naphthalenes; D010750:Phosphoproteins; C491519:Ppp1r14a protein, rat; D047428:Protein Kinase Inhibitors; D014662:Vasoconstrictor Agents; C431085:hydroxyfasudil; C070571:ML 7; D015240:Phorbol 12,13-Dibutyrate; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D054460:rho-Associated Kinases; D011493:Protein Kinase C; C499850:Ppp1r12a protein, rat; D054645:Protein Phosphatase 1; D043263:Myosin-Light-Chain Phosphatase; D020742:rhoA GTP-Binding Protein; D002118:Calcium