Role of protein kinase Czeta and calcium entry in KCl-induced vascular smooth muscle calcium sensitization and feedback control of cellular calcium levels.
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 Ca(2+) 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 PKCzeta (PI(PKCzeta)) inhibited KCl-induced tonic F. A myristoylated pseudosubstrate inhibitor of PKCalpha/beta 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 muM GF-109203X inhibited only KCl-stimulated phosphorylation, not basal MYPT1-pT853. In fura-2-loaded tissues, GF-109203X and PI(PKCzeta) elevated basal [Ca(2+)](i) (calcium) and potentiated KCl-induced tonic increases in calcium while reducing KCl-induced tonic increases in F. Blockade by nifedipine of Ca(2+) entry through voltage-operated Ca(2+) channels reduced KCl-induced Ca(2+) sensitization and KCl-stimulated but not basal MYPT1-pT853. These data together support a model in which ROCK and PKCzeta 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 PKCzeta to feed forward and cause additional MYPT1-pT853 above that induced by constitutive ROCK, permitting Ca(2+) sensitization and strong F maintenance. Active PKCzeta also feeds back to attenuate the degree of KCl-induced increases in calcium.
Ratz
Paul H
PH
Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, School of Medicine, PO Box 980614, 1101 East Marshall Street, Richmond, VA 23298-0614, USA. phratz@vcu.edu
Miner
Amy S
AS
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D005246:Feedback / Q000187:drug effects / Q000502:physiology*; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D009543:Nifedipine / Q000494:pharmacology; D010766:Phosphorylation; D011189:Potassium Chloride / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000494:pharmacology / Q000502:physiology*; D011817:Rabbits; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors; D000099221:Protein Kinase C zeta
D002118:Calcium; D007211:Indoles; D008301:Maleimides; D043263:Myosin-Light-Chain Phosphatase; D009543:Nifedipine; D011189:Potassium Chloride; D011493:Protein Kinase C; D054460:rho-Associated Kinases; D000099221:Protein Kinase C zeta; C490372:PPP1R12A protein, human; C070515:bisindolylmaleimide I