Proceedings of The Physiological Society
University of Edinburgh (2011) Proc Physiol Soc 25, C04 and PC04
Mechanisms of O2 sensing and Ca2+ homeostasis during hypoxic pulmonary vasoconstriction in pulmonary arteries from rats
M. J. Connolly1, J. Prieto-Lloret1, J. P. Ward1, P. I. Aaronson1
1. Division of Asthma, Allergy and Lung Biology, King's College London, London, United Kingdom.
Hypoxic pulmonary vasoconstriction (HPV), the process by which hypoxia constricts pulmonary arteries (PA), requires that PA smooth muscle cells contain both oxygen sensing and downstream effector mechanisms causing contraction. Here, we characterised the roles of the electron transport chain and NADPH oxidase in oxygen sensing, and of effector mechanisms involving Ca2+ release. HPV in isolated PA and perfused lung is of low amplitude, and is therefore generally studied in the presence of agonist-induced prestimulation (‘pretone’). This approach magnifies the response, making it easier to study, but may also distort its properties. We therefore recorded HPV in the absence of pretone in second and third order PA branches from young (~250 g) Wistar rats, using conventional myography. Arteries were exposed to severe hypoxia (95% N2, 5% CO2) over three 40 minute periods, each separated by ~30 min, with the second and third HPVs being used as the control and intervention responses, respectively (eg. drugs were applied 20 min before and during the third response, which was compared to the second using paired T-tests). HPV typically exhibited a transient peak (phase 1), after which tension fell slightly and then rose gradually to a plateau (phase 2). Phase 1 was abolished but phase 2 was unaffected by pretreatment with the mitochondrial complex I inhibitor rotenone (1 μM, n = 7), whereas both phases of HPV were abolished by the complex IV inhibitor cyanide (10 μM, n = 5). Pretreatment of PA with superoxide dismutase (200 units/ml), catalase (200 units/ml), the combination of these enzymes, or with the putative NADPH oxidase inhibitor VAS 2870 (10 μM) had no effect on either phase of HPV (n = 7,7,10 and 8, respectively). Conversely, the superoxide scavenger TEMPOL (3 mM) reduced phase 1 from 0.48 ± 0.10 mN to 0.28 ± 0.06 mN (p < 0.05) and abolished phase 2 HPV (n=5). The putative two pore channel antagonist NED-19 had no effect on either phase of HPV at concentrations of either 1 or 10 μM (n = 12 and 5, respectively). Pretreatment with 100 μM ryanodine to block the ryanodine receptor reduced phase 1 from 0.51 ± 0.07 mN to 0.16 ± 0.07 mN, and phase 2 from 0.56 mN ± 0.15 mN to 0.20 ± 0.08 mN (n=6, p<0.05 for both phases), whereas pretreatment with a combination of ryanodine (10 μM) and caffeine (10 mM) to deplete SR Ca2+ abolished both phases. Application of Ca2+ -free solution containing 200 μM EGTA ablated phase 1 HPV but had no significant effect on phase 2, and re-addition of Ca2+ to the solution during phase 2 did not increase contraction. These data indicate that the oxygen sensing mechanisms for phase 1 and 2 may differ, but in neither case involve NADPH oxidase. Phase 2 HPV seems to be fully accounted for by the release of Ca2+ from the SR.
Where applicable, experiments conform with Society ethical requirements