Abstract
Acute hypoxia dilates systemic arteries leading to increased tissue perfusion. We showed that at high stimulus conditions, porcine coronary artery smooth muscle (PCASM) was relaxed by hypoxia without a change in [Ca2+]i. CDHR was validated in permeabilized PCASM in which hypoxia decreased force and myosin regulatory light chain phosphorylation (p-MRLC) despite fixed [Ca2+]. Rho kinase-dependent phosphorylation of the myosin binding subunit MYPT1 (p-MYPT1) is associated with decreased MRLC phosphatase (MLCP) activity, and increased Ca2+-sensitivity of both p-MRLC and force. We tested the hypothesis that hypoxia induces CDHR via inhibition of Rho kinase leading to dephosphorylation of p-MYPT1, consequently increasing MLCP activity thus decreasing p-MRLC and force. Alpha-toxin permeabilized PCASM pretreated with ATPγS did not relax to hypoxia. Moreover, when MRLC but not MYPT1 was protected from ATPγS thiophosphorylation by the MRLC kinase inhibitor ML7 (300 μM), hypoxia remained ineffective against 1 μM Ca2+ contractures. In contrast, further addition of the Rho kinase inhibitor Y27632 (1 μM) to also attenuate the thiophosphorylation of MYPT1 restored CDHR of Ca2+ contractures; changes in p-MRLC, and p-MYPT1 at T696 and T853 paralleled changes in force. Thus no kinases, other than kinases inhibited by ML7 and Y27632, (nor their associated thiophosphoproteins) can be part of the CDHR mechanism. Importantly, hypoxia during ATPγS treatment inhibited Rho kinase thiophosphorylation of MYPT1. We conclude that CDHR involves inhibition of Rho kinase leading to dephosphorylation of p-MYPT1.
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