Limits...
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).

Show MeSH

Related in: MedlinePlus

Conductance, kinetic properties and effect of the KATP channel currents, stimulation of KATP current by GSSG in cell-attached patch. (A) KATP channel currents recorded in cell-attached patch (symmetrical 140 mmol/L K+) from rat ventricular cells at various membrane potentials. After 15 min perfusion with 1 mmol/L GSSG, KATP channel activity increased remarkably. (B) Current-voltage relationship of the KATP channel. The slope conductance of unitary inward current was 80±3.6 pS (n=6). (C) Open-time histograms of the unitary KATP channel currents at −80 mV. The histogram at −80 mV was fitted by a single exponential curve with a time constant of 1.321±0.078 ms (n=7). (D–F) Effect of Glibenclamide 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. (D) Control. (E) 15 min after perfusion with 1 mmol/L GSSG. (F) 1 mmol/L GSSG+5 μmol/L glibenclamide. Data were filtered at 2 kHz and sampled at 10 kHz. Note the scale.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4007324&req=5

fig1: Conductance, kinetic properties and effect of the KATP channel currents, stimulation of KATP current by GSSG in cell-attached patch. (A) KATP channel currents recorded in cell-attached patch (symmetrical 140 mmol/L K+) from rat ventricular cells at various membrane potentials. After 15 min perfusion with 1 mmol/L GSSG, KATP channel activity increased remarkably. (B) Current-voltage relationship of the KATP channel. The slope conductance of unitary inward current was 80±3.6 pS (n=6). (C) Open-time histograms of the unitary KATP channel currents at −80 mV. The histogram at −80 mV was fitted by a single exponential curve with a time constant of 1.321±0.078 ms (n=7). (D–F) Effect of Glibenclamide 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. (D) Control. (E) 15 min after perfusion with 1 mmol/L GSSG. (F) 1 mmol/L GSSG+5 μmol/L glibenclamide. Data were filtered at 2 kHz and sampled at 10 kHz. Note the scale.

Mentions: After 15 min of perfusion with 1 mmol/L GSSG to obtain stable KATP channel activity, the single-channel K+currents were recorded, and glibenclamide was subsequently applied to confirm these results in the cell-attached patch mode (symmetrical 140 mmol/L K+) from the rat ventricular cells at various membrane potentials (Figure 1A). The current-voltage relation for this channel is shown in figure 1B, with the conductance of the unitary inward current being 80±3.6 pS (n=6); in addition, a slight inward rectification was observed at the positive membrane potentials. The open-time histograms (at −80 mV) were fitted by a single exponential curve with a time constant of 1.321±0.078 ms (n=7, Figure 1C). The conductance and kinetic properties of this K+ channel were similar to those previously reported for the KATP channel for cardiac cells30, 31. The effects of GSSG on these KATP channels were examined under various conditions. 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. The bath application of 1 mmol/L GSSG stimulated the KATP channels, which were previously abolished by 5 μmol/L glibenclamide (Figure 1D–1F). After perfusion with 1 mmol/L GSSG, the IKATP increased markedly and reached its maximum at about 15 min, while the current amplitudes remained unchanged. The application of 5 μmol/L glibenclamide (applied after 15 min perfusion with 1 mmol/L GSSG) abolished the GSSG-increased IKATP. On the other hand, 1 mmol/L GSSG increased the mean open probability of single KATP channels from the control value of 0.006±0.0007 to 0.496±0.044 (n=6, P<0.01 vs control), which was reduced to 0.018±0.0012 after exposure to 1 mmol/L GSH (n=6, P<0.01 vs 1mmol/L GSSG).


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)

Conductance, kinetic properties and effect of the KATP channel currents, stimulation of KATP current by GSSG in cell-attached patch. (A) KATP channel currents recorded in cell-attached patch (symmetrical 140 mmol/L K+) from rat ventricular cells at various membrane potentials. After 15 min perfusion with 1 mmol/L GSSG, KATP channel activity increased remarkably. (B) Current-voltage relationship of the KATP channel. The slope conductance of unitary inward current was 80±3.6 pS (n=6). (C) Open-time histograms of the unitary KATP channel currents at −80 mV. The histogram at −80 mV was fitted by a single exponential curve with a time constant of 1.321±0.078 ms (n=7). (D–F) Effect of Glibenclamide 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. (D) Control. (E) 15 min after perfusion with 1 mmol/L GSSG. (F) 1 mmol/L GSSG+5 μmol/L glibenclamide. Data were filtered at 2 kHz and sampled at 10 kHz. Note the scale.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4007324&req=5

fig1: Conductance, kinetic properties and effect of the KATP channel currents, stimulation of KATP current by GSSG in cell-attached patch. (A) KATP channel currents recorded in cell-attached patch (symmetrical 140 mmol/L K+) from rat ventricular cells at various membrane potentials. After 15 min perfusion with 1 mmol/L GSSG, KATP channel activity increased remarkably. (B) Current-voltage relationship of the KATP channel. The slope conductance of unitary inward current was 80±3.6 pS (n=6). (C) Open-time histograms of the unitary KATP channel currents at −80 mV. The histogram at −80 mV was fitted by a single exponential curve with a time constant of 1.321±0.078 ms (n=7). (D–F) Effect of Glibenclamide 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. (D) Control. (E) 15 min after perfusion with 1 mmol/L GSSG. (F) 1 mmol/L GSSG+5 μmol/L glibenclamide. Data were filtered at 2 kHz and sampled at 10 kHz. Note the scale.
Mentions: After 15 min of perfusion with 1 mmol/L GSSG to obtain stable KATP channel activity, the single-channel K+currents were recorded, and glibenclamide was subsequently applied to confirm these results in the cell-attached patch mode (symmetrical 140 mmol/L K+) from the rat ventricular cells at various membrane potentials (Figure 1A). The current-voltage relation for this channel is shown in figure 1B, with the conductance of the unitary inward current being 80±3.6 pS (n=6); in addition, a slight inward rectification was observed at the positive membrane potentials. The open-time histograms (at −80 mV) were fitted by a single exponential curve with a time constant of 1.321±0.078 ms (n=7, Figure 1C). The conductance and kinetic properties of this K+ channel were similar to those previously reported for the KATP channel for cardiac cells30, 31. The effects of GSSG on these KATP channels were examined under various conditions. 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. The bath application of 1 mmol/L GSSG stimulated the KATP channels, which were previously abolished by 5 μmol/L glibenclamide (Figure 1D–1F). After perfusion with 1 mmol/L GSSG, the IKATP increased markedly and reached its maximum at about 15 min, while the current amplitudes remained unchanged. The application of 5 μmol/L glibenclamide (applied after 15 min perfusion with 1 mmol/L GSSG) abolished the GSSG-increased IKATP. On the other hand, 1 mmol/L GSSG increased the mean open probability of single KATP channels from the control value of 0.006±0.0007 to 0.496±0.044 (n=6, P<0.01 vs control), which was reduced to 0.018±0.0012 after exposure to 1 mmol/L GSH (n=6, P<0.01 vs 1mmol/L GSSG).

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