Oxygen-Derived Free Radical Stress Activates Nonselective Cation Current in Guinea Pig Ventricular Myocytes

Abstract

Oxygen-derived free radicals (O-Rs) cause alterations in cardiac electrical activity, including sustained depolarization, which may contribute to arrhythmic activity in reperfusion after ischemia. The ionic current(s) and cellular mechanism(s) underlying the sustained depolarization are not well defined. We used the whole-cell variant of the patch-clamp technique to study sustained depolarization in guinea pig ventricular myocytes during the extracellular application of O-Rs (generating system: dihydroxyfumaric acid, 3 to 6 mmol/L; FeCl₃/ADP, 0.05:0.5 mmol/L). Myocytes superfused with O-Rs (pipette EGTA, 0.1 mmol/L) showed (1) sustained depolarization to between −40 and −10 mV, (2) oscillations in membrane potential, and (3) triggered activity. The depolarization resulted from an increase in quasi–steady state difference current reversing at ≈−18 mV, and the oscillations were due to transient inward current. The latter were inhibited with ryanodine (10 μmol/L) or high pipette EGTA (5 mmol/L), but the steady state current was unaffected. Nonselective cation current (I_NSC) (recorded with Cs⁺, Li⁺, and Mg²⁺ replacing K⁺, Na⁺, and Ca²⁺, respectively; 20 mmol/L tetraethylammonium chloride [TEA] and 5 mmol/L BAPTA in the pipette solution and 10 mmol/L TEA, 10 μmol/L tetrodotoxin, and 10 μmol/L nicardipine in the bath solution) was activated by O-Rs; the increase in current was unaffected by preventing changes in [Ca²⁺]_i but was inhibited with dithiothreitol. Oxidizing agents (diamide and thimerosal) or caffeine (pipette EGTA, 0.1 mmol/L) produced a similar increase in membrane conductance. I_NSC activated with O-Rs, oxidizing agents, or caffeine was sensitive to SK&F 96365. O-R treatment was without effect when I_NSC was already activated with caffeine. The data suggest that (1) extracellular O-Rs activate a Ca²⁺-sensitive I_NSC in the absence of changes in [Ca²⁺]_i, (2) oxidative modification of extracellular sulfhydryl groups may be involved, and (3) this mechanism is different from the Ca²⁺-dependent activation of I_NSC by intracellular O-Rs, indicating that O-Rs may alter ion channel activity by differential mechanisms, depending on the compartment, extracellular or intracellular, in which they are present.