Involvement of myosin light-chain kinase in chloride-sensitive Ca2+ influx in porcine aortic endothelial cells.

OBJECTIVES

This study was designed to investigate the involvement of myosin light-chain kinase (MLCK) in bradykinin- and thapsigargin-induced changes in intracellular Cl- and Ca2+ concentrations ([Cl-]i; [Ca2+]i) in porcine aortic endothelial cells.

METHODS

Using the fluorescent probes N-ethoxycarbonylmethyl-6-methoxyquinolinium bromide (MQAE) and fura-2/AM, the effects of different MLCK inhibitors on bradykinin- and thapsigargin-induced changes in [Cl-]i and [Ca2+]i were assessed.

RESULTS

Bradykinin and thapsigargin significantly decreased the MQAE fluorescence intensity, which indicates increased [Cl-]i; these changes were reversed by removal of extracellular chloride (Cl-o) and were significantly inhibited by Cl(-)-channel inhibitor N-phenylanthranilic acid but not by Na(+)-K(+)-Cl- cotransport inhibitor furosemide. Pretreatment with ML-9 and wortmannin, two different selective inhibitors of MLCK, significantly reduced these changes in a dose-dependent manner. The inhibitory effects of ML-9 and wortmannin on the Cl- responses were not significantly different and were not additive. Bradykinin and thapsigargin provoked large increases in [Ca2+]i, which were significantly diminished by removal of Cl-o and by pretreatment with the Cl(-)-channel inhibitor N-phenylanthranilic acid.

CONCLUSIONS

The study shows that an increase in [Cl-]i may be involved in the Ca2+ influx in response to bradykinin and thapsigargin and that MLCK might be involved in the Cl- response. We suggest that MLCK might be involved in the Cl(-)-sensitive endothelial Ca2+ responses to bradykinin and thapsigargin.