Abstract
We have recently demonstrated in vascular smooth muscle (VSM) that membrane depolarization by high KCl induces Ca2+-dependent Rho activation and myosin phosphatase (MLCP) inhibition (Ca2+-induced Ca2+-sensitization) through the mechanisms involving phosphorylation of myosin-targeting protein 1 (MYPT1) and 17-kDa protein kinase C (PKC)-potentiated inhibitory protein of PP1 (CPI-17). In the present study, we investigated whether and how cAMP affected Ca2+-dependent MLCP inhibition by examining the effects of forskolin, cell-permeable dibutyryl cAMP (dbcAMP), and isoproterenol. Forskolin, but not its inactive analog 1,9-dideoxyforskolin, inhibited KCl-induced contraction and the 20-kDa myosin light chain (MLC) phosphorylation without inhibiting Ca2+ mobilization in rabbit aortic VSM. dbcAMP mimicked these forskolin effects. We recently suggested that Ca2+-mediated Rho activation is dependent on class II α-isoform of phosphoinositide 3-kinase (PI3K-C2α). Forskolin inhibited KCl-induced stimulation of PI3K-C2α activity. KCl-induced membrane depolarization stimulated Rho in a manner dependent on a PI3K but not PKC and stimulated phosphorylation of MYPT1 at Thr850 and CPI-17 at Thr38 in manners dependent on both PI3K and Rhokinase, but not PKC. Forskolin, dbcAMP, and isoproterenol inhibited KCl-induced Rho activation and phosphorylation of MYPT1 and CPI-17. Consistent with these data, forskolin, isoproterenol, a PI3K inhibitor, or a Rho kinase inhibitor, but not a PKC inhibitor, abolished KCl-induced diphosphorylation of MLC. These observations indicate that cAMP inhibits Ca2+-mediated activation of the MLCP-regulating signaling pathway comprising PI3K-C2α, Rho, and Rho kinase in a manner independent of Ca2+ and point to the novel mechanism of the cAMP actions in the regulation of vascular smooth muscle contraction.
Footnotes
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This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan, the Japan Society for the Promotion of Science, and Novartis Pharma.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.111443.
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ABBREVIATIONS: MLCK, myosin light-chain kinase; MLCP, myosin phosphatase; MYPT1, myosin-targeting protein 1; CPI-17, 17-kDa PKC-potentiated inhibitory protein of PP1; VSM, vascular smooth muscle; MLC, 20-kDa myosin light chain; PI3K-C2α, phosphoinositide 3-kinase class II α isoform; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; FSK, forskolin; dd-forskolin, 1,9-dideoxy-forskolin; PDBu, phorbol 12,13-dibutyrate; dbcAMP, dibutyryl cAMP; ISO, isoproterenol; GF109203X, 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide; Y27632, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2HCl; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; ILK, integrin-linked kinase; PK, protein kinase.
- Received July 23, 2006.
- Accepted November 15, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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