Protein kinase C isoform-dependent modulation of ATP-sensitive K+ channels during reoxygenation in guinea-pig ventricular myocytes

Abstract

ATP-sensitive K+ (KATP) channels activated by glucose-free anoxia close immediately upon reoxygenation in single guinea-pig ventricular myocytes, while KATP channels open persistently during reperfusion in coronary-perfused guinea-pig ventricular myocardium. To investigate the reasons behind this discrepancy, we investigated whether protein kinase C (PKC) modulates the opening of KATP channels during anoxia-reoxygenation and ischaemia-reperfusion. Exposure of guinea-pig ventricular cells to glucose-free anoxia shortened the action potential duration at 90% repolarisation (APD90) and evoked the glibenclamide-sensitive robust outward current (IK,ATP). Subsequent reoxygenation caused an immediate prolongation of APD90 and a decrease in IK,ATP within approximately 20 s. When the novel (Ca2+-independent) PKC was activated by applying 1,2-dioctanoyl-sn-glycerol (1,2DOG, 20 M) with EGTA (20 mM) in the pipette, the APD90 restored gradually after reoxygenation and the extent of recovery was appoximately 80% of the pre-anoxic value. Moreover, IK,ATP decreased slowly and remained opened for up to approximately 4 min after reoxygenation. These results suggest persistent opening of KATP channels during reoxygenation. The persistent activation of KATP channels was augmented when both novel and conventional (Ca2+-dependent) isoforms of PKC were activated by applying 1,2DOG without EGTA in the pipette. In coronary-perfused right ventricular myocardium, APD90 remained shortened for up to approximately 30 min of reperfusion. The gradual restoration of APD90 after ischaemia-reperfusion was facilitated by the KATP channel blocker glibenclamide and by the potent PKC inhibitor chelerythrine. Our results provide the first evidence that PKC activation contributes to the persistent opening of KATP channels during reoxygenation and reperfusion. We also conclude that both novel and conventional PKC isoforms co-operatively modulate the opening of KATP channels during the early phase of reoxygenation.

Figure 3. Representative effects of c,nPKC on APD₉₀ and membrane current during anoxia-reoxygenation

1,2DOG (20 μm) was perfused from 5 min before the onset of anoxia until the end of the experiment. The pipette solution was free of EGTA. A, time course of changes in the APD₉₀ during anoxia-reoxygenation. The points designated by letters in the upper panel correspond respectively to the action potential configurations shown in the lower panel. The superimposed action potentials (d→e) are composed of 10 representative action potentials recorded from the start to 90 s of reoxygenation. B and C, serial changes in whole-cell current measured at the end of test pulse. The points designated by letters in the upper panels correspond respectively to the current traces shown in the lower panels. Glibenclamide (1 μm) was applied in early (C) and late (B) phases of reoxygenation, and during the periods indicated by horizontal bars.

Figure 1. Representative effects of anoxia-reoxygenation on APD₉₀ and membrane current of single ventricular myocytes

A, time course of changes in APD₉₀ during anoxia-reoxygenation. The points designated by letters in the upper panel correspond respectively to the action potential configurations shown in the lower panel. The superimposed action potentials (d→e) are composed of 10 representative action potentials recorded from the start to 90 s of reoxygenation. B, serial changes in whole-cell current measured at the end of test pulse. The points designated by letters in upper panel correspond respectively to the current traces shown in the lower panel.

Figure 2. Representative effects of nPKC on APD₉₀ and membrane current during anoxia-reoxygenation

1,2DOG (20 μm) was perfused from 5 min before the onset of anoxia until the end of the experiment. The pipette solution contained 20 mm EGTA. A, time course of changes in APD₉₀ during anoxia- reoxygenation. The points designated by letters in the upper panel correspond respectively to the action potential configurations shown in the lower panel. The superimposed action potentials (d→e) are composed of 10 representative action potentials recorded from the start to 90 s of reoxygenation. B, serial changes in whole-cell current measured at the end of the test pulse. The points designated by letters in the upper panel correspond respectively to the current traces shown in the lower panel.

Figure 4. Effect of PKC-inactive 1,3DOG on APD₉₀ during anoxia-reoxygenation

1,3DOG (20 μm) was perfused from 5 min before the onset of anoxia until the end of the experiment. The points designated by letters in the upper panel correspond respectively to the action potential configurations shown in the lower panel. The superimposed action potentials (d→e) are composed of 10 representative action potentials recorded from the start to 90 s of reoxygenation.

Figure 5. Summary of recovery of APD₉₀ and I_K,ATP after reoxygenation

A, comparative changes in APD₉₀ after reoxygenation. APD₉₀ is expressed as a percentage of the pre-anoxic value. Lengthening time was defined as the requisite period from the onset of reoxygenation until APD₉₀ reached 50 % and 80 % of pre-anoxic value. B, time courses of decay in anoxia-induced I_K,ATP after reoxygenation. Current is expressed as a percentage of the anoxia-induced outward currents. For all three panels data are means ±s.e.m.*P < 0.05vs. control group. †P < 0.05vs. nPKC group.

Figure 6. Effects of glibenclamide and chelerythrine on APD₉₀ during 10 min of no-flow ischaemia and 60 min of reperfusion in coronary-perfused right ventricular myocardium

Glibenclamide (10 μm) or chelerythrine (1 μm) was applied from 20 min before ischaemia until the end of the experiment. The time course of APD₉₀ is expressed as a percentage of the pre-ischaemic value. Data are means ±s.e.m.