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Modulation of K(ATP) currents in rat ventricular myocytes by hypoxia and a redox reaction.

Yan XS, Ma JH, Zhang PH - Acta Pharmacol. Sin. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Cardio-Electrophysiological Research Laboratory, Medical College, Wuhan University of Science and Technology, Wuhan 430065, China.

ABSTRACT

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 Ca2+/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).

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Effect of GSH on KATP channel activities induced by GSSG. The individual current trace was evoked by a voltage step to +80 mV from a holding potential of −40 mV in a cell-attached patch. (A–C) show the original current records, and (D–F) show the all-point histograms. (A, D) Control; (B, E) 15 min after perfusion with 1 mmol/L GSSG; (C, F) 1 mmol/L GSSG+1 mmol/L GSH. Note the scale.
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fig2: Effect of GSH on KATP channel activities induced by GSSG. The individual current trace was evoked by a voltage step to +80 mV from a holding potential of −40 mV in a cell-attached patch. (A–C) show the original current records, and (D–F) show the all-point histograms. (A, D) Control; (B, E) 15 min after perfusion with 1 mmol/L GSSG; (C, F) 1 mmol/L GSSG+1 mmol/L GSH. Note the scale.

Mentions: The individual current traces were evoked by a 500 ms voltage pulse to +80 mV from a holding potential of −40 mV in a cell attached patch. In six cell-attached patches, the IKATP was very poor or even invisible before the application of GSSG (Figure 2A). After perfusion with 1 mmol/L GSSG, the IKATP increased markedly and reached a maximum at about 15 min, but the current amplitudes remained unchanged. Figure 2B shows a burst of IKATP after 15 min of perfusion with 1 mmol/L GSSG. Application of 1 mmol/L GSH (applied after 15 min of perfusion with 1 mmol/L GSSG) depressed the GSSG-increased IKATP (Figure 2C). Figure 2 illustrates the typical currents recorded from one patch. The application of 1 mmol/L GSSG increased the mean open probability of single KATP channels from the control value of 0.008±0.0007 to 0.463±0.038 (n=6, P<0.01 vs control). It was reduced to 0.154±0.009 after exposure to 1 mmol/L GSH (n=6, P<0.01 vs 1 mmol/L GSSG). Figure 2D–2F presents an example of the corresponding all-point histograms from another cell-attached patch. To further corroborate the effect of the redox agent on the KATP channel, we employed the redox couple DTT/H2O2. Similarly, in another six cell-attached patches, after perfusion with 0.3 mmol/L H2O2, the IKATP increased remarkably, and the current type gradually changed from the original background currents to burst currents (Figure 3B). After perfusion with 0.6 mmol/L H2O2, the KATP channel activity increased more significantly, but the current amplitudes remained unchanged (Figure 3C). After the application of 1 mmol/L DTT, the KATP channel activities were reversed (Figure 3D). Figure 3 illustrates the typical currents recorded from one patch. H2O2 increased the mean open probability of single KATP channels from the control value of 0.007±0.0006 to 0.364±0.032 (0.03 mmol/L H2O2, n=6, P<0.01 vs control) and to 0.649±0.060 (0.06 mmol/L H2O2, n=6, P<0.01 vs 0.03 mmol/L H2O2). The mean open probability was reduced to 0.215±0.017 after exposure to 1 mmol/L DTT (0.06 mmol/L H2O2+1 mmol/L DTT, n=6, P<0.01 vs 0.06 mmol/L H2O2). Figure 3E-H shows a representative example of the corresponding all-point histograms from another cell-attached patch. Consequently, we can conclude from the results that the action of the redox couples was bidirectional: the reducing agents decreased the KATP channel activity, whereas the oxidizing agents increased the activity.


Modulation of K(ATP) currents in rat ventricular myocytes by hypoxia and a redox reaction.

Yan XS, Ma JH, Zhang PH - Acta Pharmacol. Sin. (2009)

Effect of GSH on KATP channel activities induced by GSSG. The individual current trace was evoked by a voltage step to +80 mV from a holding potential of −40 mV in a cell-attached patch. (A–C) show the original current records, and (D–F) show the all-point histograms. (A, D) Control; (B, E) 15 min after perfusion with 1 mmol/L GSSG; (C, F) 1 mmol/L GSSG+1 mmol/L GSH. Note the scale.
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fig2: Effect of GSH on KATP channel activities induced by GSSG. The individual current trace was evoked by a voltage step to +80 mV from a holding potential of −40 mV in a cell-attached patch. (A–C) show the original current records, and (D–F) show the all-point histograms. (A, D) Control; (B, E) 15 min after perfusion with 1 mmol/L GSSG; (C, F) 1 mmol/L GSSG+1 mmol/L GSH. Note the scale.
Mentions: The individual current traces were evoked by a 500 ms voltage pulse to +80 mV from a holding potential of −40 mV in a cell attached patch. In six cell-attached patches, the IKATP was very poor or even invisible before the application of GSSG (Figure 2A). After perfusion with 1 mmol/L GSSG, the IKATP increased markedly and reached a maximum at about 15 min, but the current amplitudes remained unchanged. Figure 2B shows a burst of IKATP after 15 min of perfusion with 1 mmol/L GSSG. Application of 1 mmol/L GSH (applied after 15 min of perfusion with 1 mmol/L GSSG) depressed the GSSG-increased IKATP (Figure 2C). Figure 2 illustrates the typical currents recorded from one patch. The application of 1 mmol/L GSSG increased the mean open probability of single KATP channels from the control value of 0.008±0.0007 to 0.463±0.038 (n=6, P<0.01 vs control). It was reduced to 0.154±0.009 after exposure to 1 mmol/L GSH (n=6, P<0.01 vs 1 mmol/L GSSG). Figure 2D–2F presents an example of the corresponding all-point histograms from another cell-attached patch. To further corroborate the effect of the redox agent on the KATP channel, we employed the redox couple DTT/H2O2. Similarly, in another six cell-attached patches, after perfusion with 0.3 mmol/L H2O2, the IKATP increased remarkably, and the current type gradually changed from the original background currents to burst currents (Figure 3B). After perfusion with 0.6 mmol/L H2O2, the KATP channel activity increased more significantly, but the current amplitudes remained unchanged (Figure 3C). After the application of 1 mmol/L DTT, the KATP channel activities were reversed (Figure 3D). Figure 3 illustrates the typical currents recorded from one patch. H2O2 increased the mean open probability of single KATP channels from the control value of 0.007±0.0006 to 0.364±0.032 (0.03 mmol/L H2O2, n=6, P<0.01 vs control) and to 0.649±0.060 (0.06 mmol/L H2O2, n=6, P<0.01 vs 0.03 mmol/L H2O2). The mean open probability was reduced to 0.215±0.017 after exposure to 1 mmol/L DTT (0.06 mmol/L H2O2+1 mmol/L DTT, n=6, P<0.01 vs 0.06 mmol/L H2O2). Figure 3E-H shows a representative example of the corresponding all-point histograms from another cell-attached patch. Consequently, we can conclude from the results that the action of the redox couples was bidirectional: the reducing agents decreased the KATP channel activity, whereas the oxidizing agents increased the activity.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Cardio-Electrophysiological Research Laboratory, Medical College, Wuhan University of Science and Technology, Wuhan 430065, China.

ABSTRACT

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 Ca2+/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).

Show MeSH
Related in: MedlinePlus