Evidence for myosin light chain kinase mediating noradrenaline-evoked cation current in rabbit portal vein myocytes.

The role of myosin light chain kinase (MLCK) in the activation of the noradrenaline-evoked non-selective cation current (Icat) was examined with the whole-cell recording technique in single rabbit portal vein smooth muscle cells. Intracellular dialysis with 5 microM MLCK(11-19)amide, a substrate-specific peptide inhibitor of MLCK, markedly reduced the amplitude and rate of activation of noradrenaline-evoked Icat. A similar result was obtained when the cells were dialysed with 10 microM AV25, which also inhibits MLCK by an action at the auto-inhibitory domain of MLCK. Inhibitors of binding of ATP to MLCK, wortmannin and synthetic naphthalenesulphonyl derivatives (ML-7 and ML-9), at micromolar concentrations, also reduced the amplitude of noradrenaline-evoked Icat. ML-7 and ML-9 (both at 5 microM) reduced the amplitude of Icat induced by both guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) and 1-oleoyl-2-acetyl-sn-glycerol (OAG). MLCK(11-19)amide, AV25 and ML-9 did not inhibit the noradrenaline-evoked Ca2+-activated potassium current at a holding potential of 0 mV. In addition, MLCK(11-19)amide and AV25 did not reduce the non-selective cation current induced by ATP in rabbit ear artery cells. Intracellular dialysis with 2 microM Ca2+ and 9 microM calmodulin activated Icat, which developed over a period of about 5 min. Intracellular dialysis with the non-hydrolysable analogue of ATP, 5'-adenylylimidodiphosphate (AMP-PNP), reduced the amplitude and rate of activation of noradrenaline-evoked Icat. The results indicate that MLCK mediates noradrenaline-activated Icat in rabbit portal vein smooth muscle cells.