Programme Contents

Reactive oxygen species generated from the mitochondria and not NAD(P)H-oxidase regulate L-type Ca²⁺ channel function during acute hypoxia in ventricular myocytes

Hypoxia and the thiol-reducing agent dithiothreitol increase the sensitivity of the L-type Ca²⁺ channel (I_Ca-L) to β-adrenergic receptor stimulation. We examined whether NAD(P)H-oxidase regulates cellular production of reactive oxygen species (ROS) and the function of I_Ca-L during hypoxia. Ventricular myocytes were isolated from hearts excised from anaesthetised guinea-pigs as approved by the Animal Ethics Committee of The University of Western Australia and in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (NHMRC). The cells were patch-clamped and current was recorded during exposure to the classic NAD(P)H-oxidase inhibitors apocynin or diphenyleneiodonium (DPI) and increasing concentrations of isoproterenol (Iso). DPI caused an increase in the sensitivity of the channel to Iso similar to that of hypoxia, but apocynin did not. In contrast, the K0.5 for activation of the channel by Iso in the presence of AngII, a potent agonist of NAD(P)H-oxidase during hypoxia was 1.7±0.4 nM which was similar to the K0.5 determined during hypoxia alone (1.6±1.1 nM; NS). We measured cellular production of superoxide anion (O₂⁻) using the fluorescent indicator dihydroethidium. Hypoxia was associated with a 41.2±5.2 % decrease in O₂⁻ (n=21; P<0.05). In addition, DPI caused a 21.3±4.7 % decrease in O₂⁻ (n=16; P<0.05). However, O₂⁻ did not increase when cells were exposed to AngII during hypoxia (n=24) or in room oxygen (n=6). When mitochondria were partially uncoupled with FCCP, there was a 31.3±4.5% decrease in O₂⁻ (n=23; P<0.05) and a significant increase in the sensitivity of I_Ca-L to Iso similar to that of hypoxia (n=7). Accounting for the effect of DPI on I_Ca-L, 10μM DPI caused a 67.2±7.3% decrease in oxygen consumption (n=6; P<0.01) and 28.8±2.1% decrease in O₂⁻ in isolated mitochondria (n=4; P<0.05) indicating that DPI is not a specific inhibitor of NAD(P)H-oxidase function. Hypoxia decreased O₂⁻ by 69.3±0.8% in isolated mitochondria (n=4; P<0.01). We conclude that a decrease in ROS generated from the mitochondria and not NAD(P)H-oxidase regulates channel function during hypoxia.