Acta Pharmacologica Sinica (2009) 30: 30: 1399–1414; doi: 10.1038/aps.2009.134

Aim: The present study investigated the possible regulatory mechanisms of redox agents and hypoxia on the K_ATP current (I_KATP) in acutely isolated rat ventricular myocytes.

Methods: Single-channel and whole-cell patch-clamp techniques were used to record the K_ATP current (I_KATP) in acutely isolated rat ventricular myocytes.

Results: Oxidized glutathione (GSSG, 1 mmol/L) increased the I_KATP, while reduced glutathione (GSH, 1 mmol/L) could reverse the increased I_KATP during normoxia.  To further corroborate the effect of the redox agent on the K_ATP channel, we employed the redox couple DTT (1 mmol/L)/H₂O₂ (0.3, 0.6, and 1 mmol/L) and repeated the previous processes, which produced results similar to the previous redox couple GSH/GSSG during normoxia.  H₂O₂ increased the I_KATP in a concentration dependent manner, which was reversed by DTT (1 mmol/L).  In addition, our results have shown that 15 min of hypoxia increased the I_KATP, while GSH (1 mmol/L) could reverse the increased I_KATP.  Furthermore, in order to study the signaling pathways of the I_KATP augmented by hypoxia and the redox agent, we applied a protein kinase C(PKC) inhibitor bisindolylmaleimide VI (BIM), a protein kinase G(PKG) inhibitor KT5823, a protein kinase A (PKA) inhibitor H-89, and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitors KN-62 and KN-93.  The results indicated that BIM, KT5823, KN-62, and KN-93, but not H-89, inhibited the I_KATP augmented by hypoxia and GSSG; in addition, these results suggest that the effects of both GSSG and hypoxia on K_ATP channels involve the activation of the PKC, PKG, and CaMK II pathways, but not the PKA pathway.

Conclusion: The present study provides electrophysiological evidence that hypoxia and the oxidizing reaction are closely related to the modulation of I_KATP.

Keywords: patch-clamp techniques; ATP-sensitive potassium channels; protein kinase C; protein kinase G; protein kinase A; Ca(2+)/calmodulin-dependent protein kinase II