Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA310

Poster Communications

Epoxyeicosatrienoic acid pathway contributes to sustained hypoxic pulmonary vasoconstriction in mouse intrapulmonary arteries

I. Strielkov1, N. C. Krause1, F. Knoepp1, R. T. Schermuly1, H. A. Ghofrani1, N. Weissmann1

1. Excellence Cluster Cardiopulmonary System (ECCPS), German Center for Lung Research (DZL), Justus Liebig University Giessen, Giessen, Germany.

Under conditions of generalized alveolar hypoxia caused by a range of lung diseases or at high altitude, exaggerated sustained hypoxic pulmonary vasoconstriction (HPV) contributes to the development of pulmonary hypertension and cor pulmonale. The exact mechanisms underlying sustained HPV are not completely understood. The aim of the present study was to investigate the role of cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) in sustained HPV. Contractile responses of isolated intrapulmonary arteries (IPAs; 80-200 µm in diameter) of C57BL/6J mice were measured using wire myography. Changes in isometric tension are expressed as percentages of the maximum contraction induced by the exposure to 80 mM KCl (% TK). Values are presented as means ± S.E.M. (n=8 for each group except for siRNA experiments, where n=5 for each group), comparisons were performed using two-way ANOVA. Hypoxic exposure (80 min) induced slowly developing sustained contraction in IPAs (to 13.8±1.3 vs. 1.7±1.6 % TK in normoxia at 80 min; p<0.001). HPV was suppressed by a CYP epoxygenase inhibitor, MS-PPOH (30 µM; 5.1±2.9 % TK; p<0.001), abolished by an EET receptor blocker, 14,15-EEZE (1 µM; -0.3±2.8 % TK; p<0.001), and potentiated by TPPU (3 µM), an inhibitor of the EET-degrading enzyme, soluble epoxide hydrolase (32.2±4.6 % TK; p<0.001). All these agents did not affect IPA tone in normoxia (1.9±1.9, -0.3±2.8, and 5.4±5.1 % TK, respectively). Knockdown of the soluble epoxide hydrolase gene, Ephx2, in isolated arteries using siRNA also augmented HPV compared to non-targeting control (82.4±5.6 vs. 37.7±4.5 % TK; p<0.001). No changes in tone of the transfected IPAs were observed in normoxia (2.8±6.4 and 5.5±2.6 % TK in the siRNA and control group, respectively). Applications of exogenous EETs (5,6-, 8,9-, 11,12- and 14,15-EET; 1.5 µM) were without effect in non-preconstricted IPAs (0.7±1.1, 0.9±0.3, -0.7±0.3 and -0.2±1.1 % TK, respectively, vs. 0.3±0.9 % TK in control). By contrast, in IPAs preconstricted with KCl, 5,6-EET, unlike other regioisomers, induced pronounced contraction (20.1±3.2, -3,2±0.4, -1.9±1.3 and -1.0±1.5 vs. -0.2±0.7 % TK in control; p<0.001 for 5,6-EET vs. control). In patch-clamp experiments, hypoxia caused depolarization in isolated pulmonary artery smooth muscle cells to -25.4±2.1 mV (from -36.8±1.7 mV in normoxia; p<0.01), and 5,6-EET evoked significant inward whole-cell currents at a holding potential close to this value (-55.86±15.0 pA at -20mV vs. 16.0±16.3 pA at -40 mV; p<0.01). We speculate that hypoxia activates EET synthesis in IPAs. The resulting release of 5,6-EET, presumably in conjunction with hypoxia-induced depolarization in pulmonary artery smooth muscle cells, elicits vasoconstriction. In summary, the obtained results indicate that EET pathway contributes to sustained HPV in mouse IPA.

Where applicable, experiments conform with Society ethical requirements