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

Abstract

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.

Figure 1. The effect of MLCK_(11–19)amide on noradrenaline-activated I_cat

A, an example of a control I_cat activated by bath-applied 100 μm noradrenaline, applied during the period denoted by the horizontal bar. B, I_cat activated by bath-applied noradrenaline 5 min after achieving whole-cell configuration with a pipette solution containing 5 μm MLCK_(11–19)amide. C and D show quantitatively the effects of MLCK_(11–19)amide on the peak amplitude (pA; C) and the activation rate (pA s⁻¹; D) of I_cat. In this and subsequent figures error bars represent the s.e.m. and the numbers (n) above the columns indicate the number of cells examined. Records are from different cells. **P < 0·01.

Figure 2. The effect of AV25 on noradrenaline-activated I_cat

A, control I_cat evoked by bath-applied noradrenaline (100 μm, horizontal bar). B illustrates I_cat recorded 5 min after achieving whole-cell configuration with a pipette solution containing 10 μm AV25. C and D show the effect of 10 μm AV25 on peak amplitude (C) and the activation rate (D) of I_cat. Records are from different cells. **P < 0·01.

Figure 3. Effects of wortmannin on I_cat

A, example of I_cat activated by bath application of 100 μm noradrenaline in the absence of wortmannin. B, I_cat activated by bath-applied noradrenaline 5 min after preincubation with 50 nm wortmannin. The break in the record represents about 4 min. C, in another cell bath application of 20 μm wortmannin evoked a ‘noisy’ inward current similar to the noradrenaline-activated I_cat. However, subsequent application of noradrenaline in the continued presence of wortmannin failed to activate a current. D illustrates a cell where 20 μm wortmannin did not evoke a current and subsequent application of noradrenaline (in the continued presence of wortmannin) failed to activate a current. The duration of application of noradrenaline and wortmannin is indicated by the horizontal open and filled bars, respectively.

Figure 4. Effects of ML-9 and ML-7 on I_cat

A, I_cat recorded by bath application of 100 μm noradrenaline in the absence of ML-9 and ML-7. B and C show the inhibition of I_cat by 5 μm ML-9 (B) and 5 μm ML-7 (C) applied in the continued presence of noradrenaline. D shows the concentration-dependent inhibitory effects of ML-9 and ML-7, where the current amplitude (pA) in the presence of ML-9 or ML-7 was normalized to the current amplitude immediately prior to application (Relative current). The relative current is plotted against the concentrations of ML-9 and ML-7 on a logarithmic scale. The data could be fitted with a logistic equation of the following form: y =y_max[x^n_H/(IC₅₀^n_H+x^n_H)], where x denotes the concentration of ML-9 or ML-7 and the slope (n_H) was 2.5 and 0.9 for ML-9 and ML-7, respectively. This gave estimated IC₅₀ values (the concentrations of ML-9 and ML-7 required to inhibit the current amplitude by 50 %) of 2 and 0.8 μm for ML-9 and ML-7, respectively. Each data point represents the mean of 5–6 cells.

Figure 5. Effect of ML-9 on GTPγS- and OAG-activated I_cat

A, representative trace of I_cat evoked by inclusion of 500 μm GTPγS in the pipette solution 2–4 min after achieving whole-cell configuration (denoted by *). B illustrates the effect of ML-9 applied during the sustained phase of the GTPγS-evoked I_cat. C, bath application of OAG (10 μm, horizontal bar) activated a slowly developing ‘noisy’ inward current. D illustrates the effect of ML-9 applied during the sustained phase of the OAG-activated I_cat.

Figure 6. Effect of high intracellular calcium concentration and calmodulin on noradrenaline-evoked I_cat

A, example of I_cat activated by bath application of noradrenaline 5 min after achieving whole-cell configuration (*) using a pipette solution containing a free calcium concentration of 14 nm. B, I_cat evoked with 400 nm Ca²⁺ plus 9 μm calmodulin in the pipette solution. C illustrates the activation of I_cat with a pipette solution containing 2 μm Ca²⁺ and 9 μm calmodulin. In A and B, noradrenaline (100 μm) was applied for the period denoted by the horizontal bars. The breaks in the records in A and B represent about 4 min. Note the differences in time scale.

Figure 7. Effect of AMP-PNP on I_cat

A, I_cat evoked by bath-applied noradrenaline (100 μm, horizontal bar) in the absence of AMP-PNP. B, I_cat evoked 5 min after achieving whole-cell configuration with a pipette solution containing 5 mm AMP-PNP. C and D show the quantitative effect of AMP-PNP on the peak amplitude (C) and the rate of activation (D) of I_cat. **P < 0·01.