Effects of H2O2 on Kv currents in rat pulmonary arterial myocytes (PAMs): the role of intracellular redox state
It has been proposed that changes in the intracellular redox state in PAMs during hypoxia could alter voltage-dependent K+ (Kv) channel function. Hydrogen peroxide (H2O2), one of the main intracellular oxidants, was implicated in this process (Archer, 2002; Schumacker, 2001). However, the effect of H2O2 on Kv currents (IKv) in PAMs has not yet been characterised. Therefore, our main aim was to investigate the effect of 300 µM H2O2 on IKv using the conventional patch clamp technique at room temperature. Such a high concentration of H2O2 was chosen to maximise possible effects. Male Wistar rats (225-300 g) were humanely killed. PAMs were isolated from small pulmonary arteries (<400 µm external diameter) using 1 mg/ml collagenase and 0.5 mg/ml papain. The external solution contained (mM): 130 NaCl, 5 KCl, 1.2 MgCl2, 1.5 CaCl2, 10 HEPES, 10 glucose. 1 µM paxilline and 10 µM glibenclamide were also added to eliminate Ca2+-activated and ATP-sensitive K+ currents, respectively and PAMs were dialysed with (mM): 140 KCl, 0.5 MgCl2, 0.5 CaCl2, 10 HEPES, 10 EGTA, pH=7.2. For perforated patch recordings (PP) pipette solution contained: 140 KCl, 10 HEPES, 1 EGTA, 100 µg/ml Amphotericin B. Relative shifts in the half-activation and half-inactivation potentials ( ΔVa and ΔVh, obtained from the Boltzmann fit of the IKv steady-state activation and availability respectively) were compared in the absence and presence of inhibitors in the same cell (Smirnov et al, 2002). All recordings were started 5 min after achieving the whole cell mode to allow adequate cell dialysis; PAMs were incubated for 5 min in H2O2 prior to any measurements. Data are expressed as mean±s.e.m. and compared using the students paired t-test (p<0.05 considered significant).
In PP 300 µM H2O2 had no effects upon activation, availability nor membrane potential. However, in the whole cell mode, it consistently reduced the whole cell IKv maximal conductance (Gmax) by 15±4% (n=28, p<0.0001). At the same time H2O2 significantly shifted the IKv steady-state activation to more negative potentials ( ΔV a=-9±2.4 mV, n=28, p<0.001) without significant effect on IKv availability ( ΔVh=-3.4±2.1 mV, n=7, p>0.05). This effect was associated with a small (3.1±2.5mV, n=4) membrane hyperpolarisation induced by H2O2 in the current clamp mode, indicating overall stimulatory effect of H2O2 on the IKv in dialysed PAMs. Cell dialysis with 1 mM reduced glutathione (GSH, one of the main intracellular antioxidants) totally blocked the H2O2-induced shift in IKv activation ( ΔVa =2.8±3.1 mV, n=6, p>0.05). No significant effect on Gmax was also observed under this condition.
Our findings, although being consistent with a stimulatory effect of H2O2 on IKv in PAMs described previously (Archer, 2002), nevertheless suggest that endogenous concentrations of intracellular antioxidants are sufficient to successfully neutralise the effect of even saturating amounts of H2O2 in intact rat small PAMs.
Archer SL et al. (2002). Circ. Res. 88:1259-1266.