Blockade of Adenosine Triphosphate–sensitive Potassium Channels by Thiamylal in Rat Ventricular Myocytes

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 K^ATPchannels 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.

To investigate whether thiamylal affects the K^ATPchannel activities in intact ventricular myocytes, we studied single-channel K^ATPcurrents 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 K^ATPchannels; at higher concentrations, there were so many opened channels during the burst that we could not determine the number of channels. DNP-induced K^ATPchannel activities were inhibited by 10-μM glibenclamide, a specific inhibitor of K^ATPchannels (fig. 1B). This blockade by glibenclamide was reversible (fig. 1B). Figure 1Cshows the blockade of K^ATPchannels by thiamylal. Figure 2shows the relation between relative K^ATPchannel 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 K^ATPchannels by thiamylal occurs in a dose-dependent manner. A decreased open probability of K^ATPchannel activities induced by thiamylal returned toward baseline values almost completely after thiamylal washout.

We also studied whether thiamylal could block K^ATPchannels directly from the cytosolic side of ventricular myocytes. Because K^ATPchannels 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 K^ATPchannel activities in the inside-out configuration, and washout of thiamylal reactivated the channel (fig. 3). The average percentage recovery of the K^ATPchannel 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 K^ATPchannel conductances in the inside-out configuration.

The principal findings of this study are that thiamylal inhibits the K^ATPchannel 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 K^ATPchannel protein or indirectly via  alteration of the physical characteristics of the membrane lipids, thiamylal inhibits the K^ATPchannel 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, K^ATPchannels open to induce several protective responses in the heart. K^ATPchannels are responsible for an increase in potassium permeability and reduction of the action potential duration during hypoxia and ischemia. 12Thus, opening of K^ATPchannels 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 K^ATPchannels. 26 

Although Han et al.  27reported that, in rabbit ventricular myocytes, isoflurane inhibits the K^ATPchannel 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 K^ATPchannels 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 K^ATPchannels would be activated by volatile anesthetics. In contrast, we found that thiamylal directly inhibits K^ATPchannels in cardiac myocytes. These effects of thiamylal on the K^ATPchannel 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 K^ATPchannel 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 K^ATPchannels. The lag period between DNP exposure and induction of K^ATPchannel 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 K^ATPchannels 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 K^ATPchannel activities. This concentration may be sufficient to inhibit the K^ATPchannel 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 K^ATPchannel 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 K^ATPchannel activities in a membrane-delimited manner rather than through cytosolic second messengers.