Ca2+ signaling in hypoxic pulmonary vasoconstriction: effects of myosin light chain and Rho kinase antagonists

Abstract

Antagonists of myosin light chain (MLC) kinase (MLCK) and Rho kinase (ROK) are thought to inhibit hypoxic pulmonary vasoconstriction (HPV) by decreasing the concentration of phosphorylated MLC at any intracellular Ca2+ concentration ([Ca2+]i) in pulmonary arterial smooth muscle cells (PASMC); however, these antagonists can also decrease [Ca2+]i. To determine whether MLCK and ROK antagonists alter Ca2+ signaling in HPV, we measured the effects of ML-9, ML-7, Y-27632, and HA-1077 on [Ca2+]i, Ca2+ entry, and Ca2+ release in rat distal PASMC exposed to hypoxia or depolarizing concentrations of KCl. We performed parallel experiments in isolated rat lungs to confirm the inhibitory effects of these agents on pulmonary vasoconstriction. Our results demonstrate that MLCK and ROK antagonists caused concentration-dependent inhibition of hypoxia-induced increases in [Ca2+]i in PASMC and HPV in isolated lungs and suggest that this inhibition was due to blockade of Ca2+ release from the sarcoplasmic reticulum and Ca2+ entry through store- and voltage-operated Ca2+ channels in PASMC. Thus MLCK and ROK antagonists might block HPV by inhibiting Ca2+ signaling, as well as the actin-myosin interaction, in PASMC. If effects on Ca2+ signaling were due to decreased phosphorylated myosin light chain concentration, their diversity suggests that MLCK and ROK antagonists may have acted by inhibiting myosin motors and/or altering the cytoskeleton in a manner that prevented achievement of required spatial relationships among the cellular components of the response.