Abstract
The degree of tonic force (F) maintenance induced in vascular smooth muscle upon K+ depolarization with 110 mM KCl can be greatly reduced by inhibition of rhoA kinase (ROCK). We explored the possibility that a protein kinase C (PKC) isotype may also play a role in causing KCl-induced Ca2+ sensitization. In isometric rings of rabbit artery, the PKC inhibitors, Go-6983 (3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione), GF-109203X (2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide), and a cell-permeable (myristoylated) pseudosubstrate inhibitor of PKCζ (PIPKCζ) inhibited KCl-induced tonic F. A myristoylated pseudosubstrate inhibitor of PKCα/β that inhibited phorbol dibutyrate-induced F slightly potentiated KCl-induced tonic F and attenuated 30 mM KCl-induced F. Although the ROCK inhibitor, H-1152 [(S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)-sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride], reduced basal phosphorylation of myosin light-chain phosphatase-targeting subunit at Thr853 (MYPT1-pT853), 3 and 10 μM GF-109203X inhibited only KCl-stimulated phosphorylation, not basal MYPT1-pT853. In fura-2-loaded tissues, GF-109203X and PIPKCζ elevated basal [Ca2+]i (calcium) and potentiated KCl-induced tonic increases in calcium while reducing KCl-induced tonic increases in F. Blockade by nifedipine of Ca2+ entry through voltage-operated Ca2+ channels reduced KCl-induced Ca2+ sensitization and KCl-stimulated but not basal MYPT1-pT853. These data together support a model in which ROCK and PKCζ are constitutively active and function in “resting” muscle to regulate the basal levels of MYPT1-pT853 and calcium, respectively. In this model, KCl-induced increases in calcium activate PKCζ to feed forward and cause additional MYPT1-pT853 above that induced by constitutive ROCK, permitting Ca2+ sensitization and strong F maintenance. Active PKCζ also feeds back to attenuate the degree of KCl-induced increases in calcium.
Footnotes
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This work was supported by the National Institutes of Health [Grant R01-HL61320] and by the American Heart Association.
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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.108.142422.
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ABBREVIATIONS: F, isometric force; MLCK, myosin light-chain kinase; MLCp, myosin light-chain phosphorylation; MLCP, myosin light-chain phosphatase; GPCR, G protein-coupled receptor; ROCK, rhoA kinase; PKC, protein kinase C; VOCC, voltage-operated calcium channel; GF-109203X, 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide; Y-27632, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecar boxamide dihydrochloride; HA-1077, 1-(5-isoquinolinesulfonyl)homopiperazine hydrochloride; H-1152, (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride; PSS, physiological saline solution; MOPS, 4-morpholinepropanesulfonic acid; Lo, optimal length for muscle contraction; Fo, maximal force at the optimal length for muscle contraction; MYPT1-pT853, myosin light-chain phosphatase-targeting subunit Thr853 phosphorylation; PBS, phosphate-buffered saline; BSA, bovine serum albumin; Go-6983, 3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione; Go-6976, 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile; DMSO, dimethyl sulfoxide; PIPKC, myristoylated pseudosubstrate inhibitor of PKC; PE, phenylephrine; PD98059, 2′-amino-3′-methoxyflavone; VSM, vascular smooth muscle; CPI-17, 17-kDa protein kinase C-dependent MLCP inhibitor.
- Received June 16, 2008.
- Accepted November 13, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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