5,6-EET-induced contraction of intralobar pulmonary arteries depends on the activation of Rho-kinase

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Abstract
The mechanism mediating epoxyeicosatrienoic acid (EET)-induced contraction of intralobar pulmonary arteries (PA) is currently unknown. EET-induced contraction of PA has been reported to require intact endothelium and activation of the thromboxane/endoperoxide (TP) receptor. Because TP receptor occupation with the thromboxane mimetic U-46619 contracts pulmonary artery via Rho-kinase activation, we examined the hypothesis that 5,6-EET-induced contraction of intralobar rabbit pulmonary arteries is mediated by a Rho-kinase-dependent signaling pathway. In isolated rings of second-order intralobar PA (1–2 mm OD) at basal tension, 5,6-EET (0.3–10 μM) induced increases in active tension that were inhibited by Y-27632 (1 μM) and HA-1077 (10 μM), selective inhibitors of Rho-kinase activity. In PA in which smooth muscle intracellular Ca2+ concentration ([Ca2+]i) was increased with KCl (25 mM) to produce a submaximal contraction, 5,6-EET (1 μM) induced a contraction that was 7.0 ± 1.6 times greater than without KCl. 5,6-EET (10 μM) also contracted β-escin permeabilized PA in which [Ca2+]i was clamped at a concentration resulting in a submaximal contraction. Y-27632 inhibited the 5,6-EET-induced contraction in permeabilized PA. 5,6-EET (10 μM) increased phosphorylation of myosin light chain (MLC), increasing the ratio of phosphorylated MLC/total MLC from 0.10 ± 0.03 to 0.30 ± 0.02. Y-27632 prevented this increase in MLC phosphorylation. These data suggest that 5,6-EET induces contraction in intralobar PA by increasing Rho-kinase activity, phosphorylating MLC, and increasing the Ca2+ sensitivity of the contractile apparatus.
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