Thiol-dependent Redox Mechanisms in the Modification of ATP-Sensitive Potassium Channels in Rabbit Ventricular Myocytes

Cellular redox state is known to be perturbed during ischemia and that Ca2+ and K+ channels have been shown to have functional thiol groups. In this study, the properties of thiol redox modulation of the ATP-sensitive K+ (KATP) channel were examined in rabbit ventricular myocytes. Rabbit ventricular myocytes were isolated using a Langendorff column for coronary perfusion and collagenase. Single-channel currents were measured in excised membrane patch configuration of patch-clamp technique. The thiol oxidizing agent 5, 5'-dithio-bis- (2-nitro-benzoic acid) (DTNB) inhibited the channel activity, and the inhibitory effect of DTNB was reversed by dithiothreitol (disulfide reducing agent; DTT). DTT itself did not have any effect on the channel activity. However, in the patches excised from the metabolically compromised cells, DTT increased the channel activity. DTT had no effect on the inhibitory action by ATP, showing that thiol oxidation was not involved in the blocking mechanism of ATP. There were no statistical difference in the single channel conductance for the oxidized and reduced states of the channel. Analysis of the open and closed time distributions showed that DTNB had no effect on open and closed time distributions shorter than 4 ms. On the other hand, DTNB decreased the life time of bursts and increased the interburst interval. N-ethylmaleimide (NEM), a substance that reacts with thiol groups of cystein residues in proteins, induced irreversible closure of the channel. The thiol oxidizing agents (DTNB, NEM) inhibited of the KATP channel only, when added to the cytoplasmic side. The results suggested that metabolism-induced changes in the thiol redox can also modulate KATP channel activity and that a modulatory site of thiol redox may be located on the cytoplasmic side of the KATP channel in rabbit ventricular myocytes.

The Effect of Trifluoroacetic Acid, a Metabolite of Isoflurane on the ATP-sensitive Potassium Channel in Rabbit Ventricular Myocytes / 대한마취과학회지

BACKGROUND: Activation of ATP-sensitive K+ channels (KATP channels) in the cardiac muscle produces cardioprotective effects during myocardial ischemia. Previous experimental evidence indicates that volatile anesthetics exert beneficial actions in ischemic myocardium and enhance functional recovery of stunned myocardium. More recently, volatile anesthetics have been demonstrated to produce cardioprotective effects in stunned myocardium in vivo, and these effects are blocked by a KATP channel antagonist. This finding suggests that KATP channel activation by isoflurane may mediate antiischemic effects. However, it was demonstrated that isoflurane inhibited KATP channel activity in rabbit ventricular myocytes. To explain the discrepancy, the present investigation tested the hypothesis that isoflurane and its metabolite, trifluoroacetic acid, contributes to the activation of KATP channels in rabbit ventricular myocytes. METHODS: Single ventricular myocytes were isolated from rabbit hearts by an enzymatic dissociation procedure. Single-channel currents were measured in inside-out patch configurations of the patch-clamp technique. The perfusing liquid was equilibrated with isoflurane by passing 100% O2 through a vaporizer. RESULTS: Isoflurane inhibited KATP channel activity without a change in the single-channel conductivity. Isoflurane decreased the burst duration and increased the interburst duration. In addition, isoflurane diminished the ATP sensitivity of KATP channels. Trifluoroacetic acid, a metabolite of isoflurane, enhanced the channel activity in a dose-dependent fashion. Trifluoroacetic acid increased the burst duration and decreased the interburst duration without a change in the single-channel conductivity. Isoflurane and trifluoroacetic acid diminished the ATP sensitivity of KATP channels. CONCLUSIONS: These results imply that isoflurane and its metabolite could mediate cardioprotective effects via KATP channel activation during myocardial ischemia.

Activation of ATP-sensitive potassium channels by the predominant metabolite of isoflurane in rabbit ventricular myocytes

Background: Recent in vivo experimental evidence suggests that isoflurane-induced cardioprotection may involve KATP channel activation. However, it was demonstrated that isofluran inhibited KATP channel activities in the inside-out patch mode. To explain this discrepancy, the present investigation tested the hypothesis that a metabolite of isoflurane, trifluoroacetic acid (TFA), contributes to isoflurnae-induced cardioprotection via KATP channel activation during myocardial ischemia and reperfusion. Methods: Single ventricular myocytes were isolated from rabbit hearts by an enzymatic dissociation procedure. Patch-clamp techniques were used to record single-channel currents. KATP channel activities were assessed before and after the application of TFA with the inside-out patch mode. Results: TFA enhanced channel activity in a concentration-dependent fashion. The concentration of TFA for half-maximal activation and the Hill coefficient were 0.03 mM and 1.2, respectively. TFA did not affect the single channel conductance of KATP channels. Analysis of open and closed time distributions showed that TFA increased burst duration and decreased the interburst interval without changes in open and closed time distributions shorter than 5 ms. TFA diminished ATP sensitivity of KATP channels in a concentration-response relationship for ATP. Conclusions: TFA, a metabolite of isoflurane, enhanced KATP channel activity in a concentration-dependent fashion. These results imply that TFA could mediate isoflurane-induced cardioprotection via KATP channel activation during myocardial ischemia and reperfusion.

The effects of intracellular monocarboxylates on the ATP-sensitive potassium channels in rabbit ventricular myocytes

A regulating mechanism of the ATP-sensitive potassium channels (KATP channels) is yet to fully explained. This study was carried out to investigate the effects of intracellular application of monocarboxylates (acetate, formate, lactate, and pyruvate) on KATP channels in isolated rabbit ventricular myocytes. Single channel currents of KATP channels were recorded using the excised inside-out or permeabilized attached (open-cell) patch-clamp technique at room temperature. Intracellular application of acetate, formate and pyruvate led to an inhibition of channel activity, whereas intracellular application of lactate increased channel activity. These effects were reversible upon washout. Analysis of single channel kinetics showed that monocarboxylates did not affect open-time constant and close-time constant. These results suggest that monocarboxylates participate in modulating KATP channels activity in cardiac cells and that modulation of KATP channels activity may resolve the discrepancy between the low Ki in excised membrane patches and high levels of intracellular ATP concentration during myocardial ischemia or hypoxia.

Increase of L-type calcium current by cGMP-dependent protein kinase regulates in rabbit ventricular myocytes

BACKGROUND: We have previously reported that not only cGMP but also 8-Br-cGMP or 8-pCPT-cGMP, specific and potent stimulators of cGMP-dependent protein kinase (cGMP-PK), increased basal L-type calcium current (ICa) in rabbit ventricular myocytes. Our findings in rabbit ventricular myocytes were entirely different from the earlier findings in different species, suggesting that the activation of cGMP-PK is involved in the facilitation of ICa by cGMP. However, there is no direct evidence that cGMP-PK can stimulate ICa in rabbit ventricular myocytes. In this report, we focused on the direct effect of cGMP-PK an ICa in rabbit ventricular myocytes. METHODS AND RESULTS: We isolated single ventricular myocytes of rabbit hearts by using enzymatic dissociation. Regulation of ICa by cGMP-PK was investigated in rabbit ventricular myocytes using whole-cell voltage clamp method. ICa was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Extracellular 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP), potent stimulator of cGMP-dependent protein kinase (cGMP-PK), increased basal ICa. cGMP-PK also increased basal ICa. The stimulation of basal ICa by cGMP-PK required both 8-Br-cGMP in low concentration and intracellular ATP to be present. The stimulation of basal ICa by cGMP-PK was blocked by heat inactivation of the cGMP-PK and by bath application of 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-pCPT-cGMP), a phosphodiesterase-resistant cGMP-PK inhibitor. When ICa was increased by internal application of cGMP-PK, IBMX resulted in an additional stimulation of ICa. In the presence of cGMP-PK, already increased ICa was potentiated by bath application of isoprenaline or forskolin or intracellular application of cAMP. CONCLUSIONS: We present evidence that cGMP-PK stimulated basal ICa by a direct phosphorylation of L-type calcium channel or associated regulatory protein in rabbit ventricular myocytes.