ALTERATIONS IN ELECTRICAL-ACTIVITY AND MEMBRANE CURRENTS INDUCED BY INTRACELLULAR OXYGEN-DERIVED FREE-RADICAL STRESS IN GUINEA-PIG VENTRICULAR MYOCYTES

Oxygen-derived free radicals (O-Rs) are thought to induce alterations in cardiac electrical activity; however, the underlying membrane ionic currents affected by O-Rs and the mechanisms by which O-Rs induce the ir effects on ion channels in the heart are not well defined. In this study, we investigated the time-dependent changes in resting membrane potential and action potential configuration and changes in steady-sta te membrane currents in guinea pig ventricular myocytes after intracel lular application of an O-R-generating system. O-Rs were generated fro m the combination of dihydroxyfumaric acid (3 mM) and FeCl3: ADP (0.05 :0.5 mM) added to the pipette solution that was used to record membran e potential and currents via the whole-cell variant of the patch-clamp technique. Intracellular exposure of myocytes to the O-R-generating s olution induced three stages of changes: 1) an early depolarization (5 -10 mV) and an increase in action potential duration accompanied by a decrease in resting inward rectifying K+ current conductance, 2) delay ed afterdepolarizations and triggered activity caused by the activatio n of transient inward current mediated by Na+-Ca2+ exchange, with fail ure to repolarize and sustained depolarization between -35 and -20 mV, reflecting the stimulation of nonselective cation current, and 3) a l ate stage of marked decline in action potential duration, hyperpolariz ation, and loss of excitability accompanied by activation of the outwa rd current through ATP-sensitive K+ channels. These alterations in ele ctrical activity and membrane currents could be prevented by pretreatm ent with N-(2-mercaptopropionyl)glycine (500 muM), a scavenger of hydr oxyl free radicals. The alterations associated with stages 1 and 2 but not stage 3 were completely abolished on intracellular Ca2+ chelation (5 mM EGTA in the pipette solution) or disruption of sarcoplasmic ret iculum Ca2+ handling with ryanodine (10 muM). This study shows that in tracellular O-R stress causes specific alterations in membrane ionic c urrents, leading to changes in resting membrane potential and action p otential configuration. Moreover, the data indicate that an elevation in intracellular Ca2+ due to abnormal Ca2+ handling by the sarcoplasmi c reticulum is a cause of some of the alterations in membrane currents during O-R stress.