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

1. The role of myosin light drain kinase (MLCK) in the activation of the noradrenaline-evoked non-selective cation current (I-cat) was examined with the whole-cell recording technique in single rabbit; portal vein smooth muscle cells. 2. Intracellular dialysis with 5 mu M MLCK((11-19))amide, a substrate-specific peptide inhibitor of MLCK, markedly reduced the amplitude and rate of activation of noradrenaline-evoked I-cat. A similar result was obtained when the cells were dialysed with 10 mu M AV25, which also inhibits MLCK by an action at the auto-inhibitory domain of MLCK. 3. 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 I-cat. 4. ML-7 and ML-9 (both at 5 mu M) reduced the amplitude of I-cat induced by both guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and 1-oleoyl-2-acetyl-sn-glycerol (OAG). 5. 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. 6. Intracellular dialysis with 2 mu M Ca2+ and 9 mu M calmodulin activated I-cat, which developed over a period of about 5 min. 7. Intracellular dialysis with the non-hydrolysable analogue of ATP, 5'-adenylylimido-diphosphate (AMP-PNP), reduced the amplitude and rate of activation of noradrenaline evoked I-cat. 8. The results indicate that MLCK mediates noradrenaline-activated I-cat in rabbit portal vein smooth muscle cells.