The adenosine triphosphate (ATP)-sensitive potassium (KATP) channels protect myocytes during ischemia and reperfusion. This study investigated the effects of thiamylal on the activities of KATP channels in isolated rat ventricular myocytes during simulated ischemia.
Male Wistar rats were anesthetized with ether. Single, quiescent ventricular myocytes were dispersed enzymatically. Membrane currents were recorded using patch-clamp techniques. In the cell-attached configuration, KATP channel currents were assessed before and during activation of these channels by 2,4-dinitrophenol and after administration of 25, 50, and 100 mg/l thiamylal. The open probability was determined from current-amplitude histograms. In the inside-out configuration, the current-voltage relation was obtained before and after the application of thiamylal (50 mg/1).
In the cell-attached configuration, 2,4-dinitrophenol caused frequent channel opening. 2,4-Dinitrophenol-induced channel activities were reduced significantly by glibenclamide, suggesting that the channels studied were KATP channels. Open probability of KATP channels was reduced by thiamylal in a concentration-dependent manner. KATP channels could be activated in the inside-out configuration because of the absence of ATP. Thiamylal inhibited KATP channel activity without changing the single-channel conductance.
The results obtained in this study indicate that thiamylal inhibits KATP channel activities in cell-attached and inside-out patches, suggesting a direct action of this drug on these channels.
ADENOSINE triphosphate (ATP)–sensitive potassium (KATP) channels are a family of potassium channels inhibited by intracellular ATP. 1–3Because KATPchannels are gated by intracellular ATP, they are thought to link cellular metabolism with membrane excitability. 1In heart cells, KATPchannels are activated by depletion of intracellular ATP, hypoxia, or exposure to metabolic inhibitors 1,4–6and cause an increase in potassium ion outward flow. This activation of KATPchannels may be an endogenous protective mechanism against cardiac damage during myocardial ischemia. In addition, it has been reported that KATPchannels play an important role in ischemic preconditioning 7–9: A brief period of ischemia can make the heart more resistant to subsequent, more severe episodes of ischemia. As a possible mechanism of cardioprotective action of KATPchannels, the activation of sarcolemmal KATPchannels has been thought to protect the ischemic myocardium by shortening the action potential duration. That is, the shortening of action potential duration decreases calcium influx through voltage-dependent calcium channels, 10and causes a decrease in contractility, 11which is a major source of ATP consumption, and decreases in calcium-induced cell toxicity, 12thereby resulting in the protection of myocytes from further depletion of ATP and irreversible impairment of energy metabolism. 13It was suggested recently that mitochondrial rather than sarcolemmal KATPchannels might be playing an important role in the cardioprotective action of KATPchannels. 14–16
Studies of the effects of anesthetics on the KATPchannel activities in the heart have been performed. In vivo experiments revealed that volatile anesthetics, including halothane, isoflurane, and enflurane, activate KATPchan- nels and have cardioprotective effects. 17–23In contrast, using a patch-clamp technique, Ko et al. 12found that ketamine inhibits the KATPchannel activities in a concentration-dependent manner in rat ventricular myocytes. Kozlowski and Ashford 24also revealed that in CRI-G1 insulin-secreting cells thiopental inhibits KATPchannel activities if applied to cell-attached patches or excised inside-out patches. These findings suggest that ketamine and thiobarbiturates may attenuate the cardioprotective effects of KATPchannels during ischemia and reperfusion in the myocardium. Because thiobarbiturates are used a great deal in all types of anesthesia in patients who have coronary artery disease and are undergoing a variety of surgical procedures, it is important to evaluate the direct effects of these drugs on the KATPchannel activities in the ventricular myocardium during ischemia. Therefore, we evaluated the effects of thiamylal on the KATPchannel activities in isolated rat ventricular myocytes during simulated ischemia.
Materials and Methods
This study was approved by the Animal Investigation Committee of Tokushima University and followed the guidelines of the American Physiological Society (Bethesda, Maryland) for the humane use of animals in research. Fifty-one male Wistar rats (weight, 250–300 g) were anesthetized with ether, and 1.0 IU/g heparin was injected intraperitoneally 30 min before surgery. The chest was opened, and the beating heart was dissected as soon as the response to tail clamp was lost. The heart was perfused in a retrograde manner via the aorta using a Langendorff apparatus with standard Tyrode’s solution prewarmed to 37°C and saturated with a 95% oxygen and 5% carbon dioxide gas mixture. The composition of standard Tyrode’s solution (pH 7.4) was as follows: NaCl: 125 mM; NaHCO3: 24 mM; KCl: 5.4 mM; NaH2PO4: 0.47 mM; MgCl2: 1.05 mM; dextrose: 5.5 mM; and CaCl2: 1.8 mM. The heart was perfused with Ca2+-free Tyrode’s solution for approximately 5 min until the heart stopped beating and then was digested with Ca2+-free Tyrode’s solution containing collagenase (0.2 mg/ml) and pronase (0.05 mg/ml) for 15 min. After enzymatic digestion, the heart was perfused with low-Ca2+Tyrode’s solution (0.1 mM CaCl2) for several minutes to wash away the enzymes. The ventricle was cut into pieces in low-Ca2+Tyrode’s solution, and the myocytes were filtered. The cells were centrifuged, and the supernatant was removed. All cells used in these experiments were rod shaped and striated.
Membrane currents were recorded in the cell-attached and inside-out configurations using a patch-clamp amplifier as described by Hamill et al. 25The composition of bath solution for cell-attached mode was as follows: KCl: 140 mM; HEPES: 10 mM; dextrose: 5.5 mM; and ethyleneglycoltetracetic acid (EGTA): 0.5 mM. The pipette solution for cell-attached mode contained 140 mM KCl, 10 mM HEPES, and 5.5 mM dextrose. The composition of bath solution for inside-out mode was as follows: KCl: 140 mM; HEPES: 10 mM; dextrose: 5.5 mM; MgCl2: 1 mM; and ethyleneglycoltetracetic acid: 0.5 mM. The composition of pipette solution for the inside-out mode was the same as that for the cell-attached mode. Soft-glass pipettes prepared in an electrode puller (PP-830; Narishige, Tokyo, Japan) were used after being coated with Sylgard (Dow Corning Co., Midland, MI). The electrical resistance of the patch pipette was 5 to 7 MΩ for single-channel recording. Experiments were conducted with solution temperatures of 35–37°C. pClamp version 6.0 software (Axon Instruments, Foster, CA) was used to analyze the data for single-channel currents. The open probability (NPo) was determined from current amplitude histograms and was calculated using the following equation:
where N is the number of channels in the patch and Pn is the integrated channel opening. Open probability of KATPchannels was determined from recordings longer than 60 s in duration.
The thiamylal sodium (Yoshitomi Chemical, Osaka, Japan) ampule was opened before use. 2,4-Dinitrophenol (DNP; Sigma Chemical, St. Louis, MO) and glibenclamide (Sigma) were prepared as stock solutions. All other solutions were made daily. Collagenase (Yakult, Tokyo, Japan) and pronase (Sigma) were used for enzymatic dissociation.
Data are expressed as the mean ± SD. Differences among data sets were evaluated by the Student t test. A P value < 0.05 was considered statistically significant.
Effects of Thiamylal on KATPChannels in the Cell-attached Configuration
To investigate whether thiamylal affects the KATPchannel activities in intact ventricular myocytes, we studied single-channel KATPcurrents using the cell-attached configuration. As shown in figure 1A, we did not observe any channel openings in the standard bath solution. If 25-μM DNP, an inhibitor of mitochondrial ATP synthesis, was administered, frequent channel openings were observed (fig. 1A). DNP at a concentration of 25 μM was sufficient to identify KATPchannels; at higher concentrations, there were so many opened channels during the burst that we could not determine the number of channels. DNP-induced KATPchannel activities were inhibited by 10-μM glibenclamide, a specific inhibitor of KATPchannels (fig. 1B). This blockade by glibenclamide was reversible (fig. 1B). Figure 1Cshows the blockade of KATPchannels by thiamylal. Figure 2shows the relation between relative KATPchannel activities and the concentration of thiamylal. In 22 patches, the open probabilities were suppressed by increase the concentration of thiamylal. The relative channel activities after administration of thiamylal were 0.70 ± 0.05 (25 mg/l thiamylal; significantly different from DNP solution;P < 0.05), 0.41 ± 0.04 (50 mg/l thiamylal;P < 0.05), and 0.01 ± 0.01 (100 mg/l thiamylal;P < 0.05). These results indicate that inhibition of KATPchannels by thiamylal occurs in a dose-dependent manner. A decreased open probability of KATPchannel activities induced by thiamylal returned toward baseline values almost completely after thiamylal washout.
Effects of Thiamylal on KATPChannels in the Inside-out Configuration
We also studied whether thiamylal could block KATPchannels directly from the cytosolic side of ventricular myocytes. Because KATPchannels are inhibited by intracellular ATP, we studied the activities of these channels in the absence of ATP. In this series of experiments, bath application of thiamylal also inhibited the KATPchannel activities in the inside-out configuration, and washout of thiamylal reactivated the channel (fig. 3). The average percentage recovery of the KATPchannel activity after thiamylal washout was 89.3 ± 6.2% of open probability obtained before application of thiamylal (n = 26).
The current–voltage relation obtained from 19 patches before and after the application of thiamylal (50 mg/l) is shown in figure 4. The current–voltage curves before and after the application of thiamylal were linear in the negative potential range, with single-channel conductances of 73.4 ± 1.7 picosiemens (pS) and 75.1 ± 2.2 pS before and after 50 mg/l thiamylal, respectively. There were no statistical differences between control and thiamylal-treated series. These results suggest that thiamylal does not change the KATPchannel conductances in the inside-out configuration.
The principal findings of this study are that thiamylal inhibits the KATPchannel activities without changing the single-channel conductance in the inside-out patches and in the cell-attached patches. Although we cannot discriminate whether thiamylal acts directly on the KATPchannel protein or indirectly via alteration of the physical characteristics of the membrane lipids, thiamylal inhibits the KATPchannel activities in a membrane-delimited manner rather than through a cytosolic second messenger, which is absent in the excised inside-out patch.
Adenosine triphosphate–sensitive potassium channels are important for regulation of cellular energy metabolism associated with membrane excitability. 1During ischemia and reperfusion, KATPchannels open to induce several protective responses in the heart. KATPchannels are responsible for an increase in potassium permeability and reduction of the action potential duration during hypoxia and ischemia. 12Thus, opening of KATPchannels benefits the heart, possibly by reducing calcium influx through voltage-dependent calcium channels, slowing ATP depletion, and decreasing calcium-induced toxicity. 12In addition, with reperfusion or reoxygenation, oxygen free radicals are generated, which could trigger a chain of damaging chemical reactions resulting in “reperfusion injury.” Free radical–induced injury can be attenuated by potassium channel openers that act on KATPchannels. 26
Although Han et al. 27reported that, in rabbit ventricular myocytes, isoflurane inhibits the KATPchannel activities in the inside-out patch-clamp configurations, there are many in vivo or isolated whole-heart experiments showing that volatile anesthetics, including halothane, isoflurane, and enflurane, activate KATPchannels and have cardioprotective effects. 17–23Kanaya and Fujita 28and Kersten et al. 19,20reported that isoflurane improves regional myocardial contraction and preserves high-energy phosphate concentrations in a canine model of myocardial stunning. Boutros et al. 21found in isolated rat hearts that ischemic preconditioning, halothane, and isoflurane provide significant protection against ischemia. In addition, Kersten et al. 29demonstrated that pretreatment with isoflurane, similar to ischemic preconditioning, produces functional recovery of stunned myocardium in anesthetized dogs. Because the recovery of regional contractile function enhanced by isoflurane is abolished by pretreatment with glibenclamide, 19,20,29it has been thought that cardiac KATPchannels would be activated by volatile anesthetics. In contrast, we found that thiamylal directly inhibits KATPchannels in cardiac myocytes. These effects of thiamylal on the KATPchannel activities are similar to those of ketamine. Ko et al. 12studied the effects of ketamine on single rat ventricular myocytes and revealed that ketamine inhibits the KATPchannel activities in a concentration-dependent manner in both the inside-out and the cell-attached patch configurations. The results of the current study and findings reported by Ko et al. 12suggest that thiamylal and ketamine may increase the extent of myocardial damage during anesthesia.
Our study had several limitations. First, in our cell-attached configurations, we used DNP to simulate ischemia. Because DNP inhibits mitochondrial ATP synthesis, we could observe marked opening of KATPchannels. The lag period between DNP exposure and induction of KATPchannel current was a few minutes. This latency is considered to reflect the time for the depletion of endogenous energy sources before intracellular ATP levels decreased. However, the activation of KATPchannels by DNP may be different from that by ischemia or hypoxia. 12Second, for clinical uses in humans, the peak plasma concentration of thiamylal during induction of general anesthesia is approximately 100–150 mg/l, with an intravenous dose of 4–5 mg/kg. 30In the current investigation, 100 mg/l thiamylal completely inhibited the KATPchannel activities. This concentration may be sufficient to inhibit the KATPchannel activities and potentially reverse the antiischemic effects mediated by this channel. However, it should be noted that free drug concentration at the myocyte in situ may be much lower than that stated previously, because its affinity for plasma proteins is very high. Third, high extracellular potassium concentration (140 mM) and a resultant membrane depolarization might have altered the behavior of the channel and the sensitivity of thiamylal. 12Therefore, we should be careful in extending the current results to the human heart.
In summary, thiamylal inhibits the KATPchannel activities without affecting the channel conductances during simulated ischemia in the cell-attached and inside-out patch-clamp configurations of single rat myocytes. These results suggest that thiamylal inhibits the KATPchannel activities in a membrane-delimited manner rather than through cytosolic second messengers.