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.