Involvement of gap junctions between smooth muscle cells in sustained hypoxic pulmonary vasoconstriction development: a potential role for 15-HETE and 20-HETE

Abstract

In response to hypoxia, the pulmonary artery normally constricts to maintain optimal ventilation-perfusion matching in the lung, but chronic hypoxia leads to the development of pulmonary hypertension. The mechanisms of sustained hypoxic pulmonary vasoconstriction (HPV) remain unclear. The aim of this study was to determine the role of gap junctions (GJs) between smooth muscle cells (SMCs) in the sustained HPV development and involvement of arachidonic acid (AA) metabolites in GJ-mediated signaling. Vascular tone was measured in bovine intrapulmonary arteries (BIPAs) using isometric force measurement technique. Expression of contractile proteins was determined by Western blot. AA metabolites in the bath fluid were analyzed by mass spectrometry. Prolonged hypoxia elicited endothelium-independent sustained HPV in BIPAs. Inhibition of GJs by 18β-glycyrrhetinic acid (18β-GA) and heptanol, nonspecific blockers, and Gap-27, a specific blocker, decreased HPV in deendothelized BIPAs. The sustained HPV was not dependent on Ca(2+) entry but decreased by removal of Ca(2+) and by Rho-kinase inhibition with Y-27632. Furthermore, inhibition of GJs decreased smooth muscle myosin heavy chain (SM-MHC) expression and myosin light chain phosphorylation in BIPAs. Interestingly, inhibition of 15- and 20-hydroxyeicosatetraenoic acid (HETE) synthesis decreased HPV in deendothelized BIPAs. 15-HETE- and 20-HETE-stimulated constriction of BIPAs was inhibited by 18β-GA and Gap-27. Application of 15-HETE and 20-HETE to BIPAs increased SM-MHC expression, which was also suppressed by 18β-GA and by inhibitors of lipoxygenase and cytochrome P450 monooxygenases. More interestingly, 15,20-dihydroxyeicosatetraenoic acid and 20-OH-prostaglandin E2, novel derivatives of 20-HETE, were detected in tissue bath fluid and synthesis of these derivatives was almost completely abolished by 18β-GA. Taken together, our novel findings show that GJs between SMCs are involved in the sustained HPV in BIPAs, and 15-HETE and 20-HETE, through GJs, appear to mediate SM-MHC expression and contribute to the sustained HPV development.

Glycyrrhetinic Acid Antagonizes Pressure-Induced Venous Remodeling in Mice

“…In order to assess possible cytotoxic effects of this compound, the viability of human venous ECs and SMCs exposed to increasing concentrations of GA was analyzed. The selected GA concentrations were based on reports from the literature (e.g., Matchkov et al, 2004 ; Behringer et al, 2012 ; Kizub et al, 2016 ) and did not affect the cellular viability of HUVECs and HUVSMCs in culture (Figures 2A,B respectively). In addition, we tested whether GA may interfere with the cellular motility required for angiogenesis (e.g., in the context of wound healing processes) and structural maintenance of blood vessels.…”

Hypoxic pulmonary vasoconstriction in isolated mouse pulmonary arterial vessels

“…Collectively, the features of HPV described in isolated mouse SIPAs appear to be close to those found in bovine and rabbit PAs (endothelium‐independent monophasic sustained contraction without full relaxation upon reoxygenation; Kizub et al., ; Talbot et al., ) and significantly different from those in rat PAs (rapid biphasic contraction with full relaxation upon reoxygenation; Kizub et al., ; Strielkov et al., ). This is probably a result of essential differences between the properties of PASMCs of mice and rats (Manoury et al., ; Weissmann et al., ; Yoo et al., ).…”

“…We thus conclude that, at least in our model, sustained HPV depends primarily on PASMCs, with little or no influence from endothelial cells on the basic mechanism of HPV. Interestingly, no effect of endothelial denudation on sustained HPV in bovine intrapulmonary arteries has also been reported recently (Kizub et al., ). In contrast, the sustained phase of HPV is dependent on the endothelium in rat and canine PAs, which indicates a significant species difference in this regard (Hoshino, Morrison, & Vanhoutte, ; Robertson, Aaronson, & Ward, et al., 2003).…”

“…We thus conclude that, at least in our model, sustained HPV depends primarily on PASMCs, with little or no influence from endothelial cells on the basic mechanism of HPV. Interestingly, no effect of endothelial denudation on sustained HPV in bovine intrapulmonary arteries has also been reported recently (Kizub et al, 2016). In contrast, the sustained phase of HPV is dependent on Changes in isometric tension in small intrapulmonary arteries of TRPC6-deficient (TRPC6 −/− ) and wild-type (WT) mice during 80 min of incubation in hypoxia and normoxia in the absence of pretone (a) and in the presence of KCl-induced pretone (b).…”

“…In our experiments, PA segments did not have contact with capillaries, which can explain the absence of the acute, transient component of HPV. It is noteworthy that isolated bovine and rabbit IPAs also exhibit only sustained constriction in response to hypoxia (Kizub et al., ; Talbot, Robbins, & Dorrington, ).…”