P01 Mechanisms of hydrogen sulfide mediated contraction in rat small pulmonary arteries

Abstract

The gas H2S (hereafter ‘sulfide’) typically acts as an vasodilator in the systemic circulation, but in pulmonary arteries elicits a biphasic contraction via an unknown mechanism. This is potentially a physiologically important effect, since it has also been proposed that the concentration of sulfide increases in pulmonary arteries under hypoxic conditions, thereby acting as an oxygen sensor which is important for initiating (and possibly mediating) hypoxic pulmonary vasoconstriction. We therefore assessed the mechanism of the sulfide-induced contraction in rat 2nd order pulmonary arteries (PA), using NaHS as a source of sulfide. Techniques used included recording of isometric force development and mitochondrial membrane potential in PA rings mounted in a small vessel myograph and measurement of reactive oxygen species (ROS) levels in segments of PA using the luminescent ROS indicator LO12 (10 μM). NaHS (500 μM) evoked a complex PA contraction comprising a small contraction followed by a second and larger contraction which gradually relaxed. The contraction was abolished by the mitochondrial complex three blocker antimycin (10 μg/ml) and was greatly reduced by the anti-oxidant TEMPOL (3 mM), the RyR blocker dantrolene (50 μM,) or the Rho kinase blocker Y27632 (1 μM). On the othe hand, the sulfide contraction was little affected by treatment of arteries with the mitochondrial complex 1 blocker rotenone (1 μM) or by their incubation in Ca2+-free PSS containing 0.2 mM EGTA. Incubation of pulmonary arteries in NaHS also caused activation of the monomeric G protein RhoA (measured using a G-lisa assay) and also of its downstream effector rho kinase (as shown by an increase in MYPT phosphorylation). A 15 min incubation of control cultured pulmonary artery smooth muscle cells (PASMC) in 1 mM NaHS (1 mM), followed by a 10 min wash to remove NaHS from the solution, led to a significant increase in cellular ROS production recorded using LO12. This increase in ROS was not present in PASMC in which expression of the the mitochondrial membrane flavoprotein sulfide-quinone oxoreductase (SQR) had been knocked down using siRNA. Application of a range of concentrations (10–1000 μM) of NaHS to pulmonary arteries led to a transient depolarisation of the mitochondrial membrane potential (recorded using TMRE) which was variably preceded and/or followed by hyperpolarisation. In the presence of rotenone, which caused depolarisation of the mitochondrial potential, low concentrations of NaHS (10–100 μM) caused a transient repolarisation, whereas high concentrations caused a complex response consisting of a transient repolarisation followed by a depolarisation and a second long-lasting repolarisation. Based on these results, we conclude that sulfide causes contraction by causing Ca2+ sensitisation and the release of Ca2+ from the sarcoplasmic reticulum via ryanodine receptors. Furthermore, we speculate that these responses are mediated by ROS produced by complex 3 owing to an increased flow of electrons into the mitochondrial electron transport chain consequent upon the metabolism of sulfide by SQR.