Objective: Simulated ischemia and reperfusion (IR) of coronary endothelial cells leads to cytosolic Ca2+ overload which contributes to the activation of the cellular contractile mechanism, resulting in formation of intercellular gaps. This could lead to creation of edema in vivo. It has been previously shown that ischemia induces activation of plasmalemmal KATP channels causing hyperpolarisation of the plasmalemma. We hypothesised that ischemia-induced hyperpolarisation of the plasmalemma of microvascular endothelial cells is followed by influx of Ca2+ in the cytosol and this contributes to Ca2+ overload of endothelial cells and ischemic endothelial barrier failure.
Results: During simulated ischemia a hyperpolarisation of the plasmalemma occurred which was reduced by the KATP channel inhibitors glybenclamide and HMR1098. (DiBAC4(3) fluorescence (%): Normoxia: 100 ± 1,7; end-ischemia: 57.5 ± 1.3*; end-ischemia+glybenclamide 80.64 ± 1.07; n=6, *p<0.05 vs. normoxia; end-ischemia+HMR1098 69 ± 0.9; n=6 *p<0.05 vs. normoxia. Perfusion with glybenclamide and HMR1098 during simulated ischemia and reperfusion lead to significant reduction of cytosolic Ca2+ overload (fura-2 ratio a.u.): normoxia: 0.49 ± 0.021; end-ischemia: 0.74 ± 0.012*; *p<0.05 vs. normoxia; end-ischemia+glybenclamide: 0.52 ± 0.02*; end-ischemia+HMR1098: 0.60 ± 0.02* n=6; *p<0.05 vs. end-ischemia; end-reperfusion: 0.77 ± 0.01; end- reperfusion+glybenclamide: 0.64 ± 0.09*; end-reperfusion+HMR1098 0.6 ± 0.07*; n=6; *p<0.05 vs. end-reperfusion. Application of glybenclamide and HMR1098 reduced significantly the formation of intercellular gaps: normoxia 100 %; end-ischemia: 220 % ± 25 %*; n=6; *p<0.05 vs. normoxia; end-ischemia+glybenclamide: 125 % ± 10 %*; end-ischemia+HMR1098 133.45 ± 8.96 %; n=6; *p<0.05 vs. end-ischemia; end-reperfusion: 289 % ± 20%; end-reperfusion+Glybenclamide: 182 % ± 19%*; end-reperfusion+HMR1098: 189 ± 4*; n=6; *p<0.05 vs. end-reperfusion.
Conclusion: Inhibition of the plasmalemmal KATP channels during simulated IR attenuates the ischemic hyperpolarisation of the plasmalemma and leads to reduction of the cytosolic Ca2+overload and formation of intercellular gaps in coronary endothelial cells.
