Isoflurane Sensitizes the Cardiac Sarcolemmal Adenosine Triphosphate–Sensitive Potassium Channel to Pinacidil

The experimental procedures of this study were approved by the Animal Use and Care Committee of the Medical College of Wisconsin.

Single ventricular myocytes were enzymatically isolated from adult Hartley guinea pigs (either sex) weighing 150–300 g using a modified isolation method by Mitra and Morad. 20The guinea pigs were anesthetized by pentobarbital sodium (325 mg/kg, administered intraperitoneally) and injected with heparin (1,000 U/ml, administered intraperitoneally). After thoracotomy, the hearts were quickly excised, mounted on a Langendorff apparatus, and perfused retrogradely via  the aorta at a flow of 7–8 ml/min with oxygenated Joklik medium (Gibco BRL, Invitrogen, Grand Island, NY) containing 2.5 U/ml heparin. After blood has been washed out from the heart, this medium was replaced by an enzyme solution containing Joklik medium, 0.4 mg/ml collagenase type II (Gibco BRL, Invitrogen), 0.1 mg/ml protease XIV (Sigma-Aldrich, St. Louis, MO), and 1 mg/ml bovine serum albumin (Serologicals, Kankakee, IL), at pH 7.23. The temperature was maintained at 37°C, and oxygen and carbon dioxide concentrations were kept at 95% and 5%, respectively, by continuously bubbling the solution at a constant gas flow. After 14 min of enzyme treatment, the ventricular tissue was minced and incubated in the enzyme solution for additional 3–10 min in a shaker bath at 37°C. The cell suspension was filtered through a 200-μm mesh and centrifuged. The pellet was washed twice in modified Tyrode solution. Myocytes were stored in Tyrode solution at room temperature (22°C) up to 12 h.

The modified Tyrode solution had the following composition: 132 mm NaCl, 4.8 mm KCl, 1.2 mm MgCl², 1 mm CaCl², 10 mm HEPES, and 5 mm glucose, at pH 7.4 adjusted with NaOH. The intracellular–pipette solution contained the following: 60 mm l-glutamic acid, 50 mm KCl, 10 mm HEPES, 1 mm MgCl², 11 mm EGTA, 1 mm CaCl², and either 0.5 or 5°K²ATP, at pH 7.4 adjusted with KOH. The external–bath solution contained: 132 mm N -methyl-d-glucamine, 2 mm MgCl², 1 mm CaCl², and 10 mm HEPES, at pH 7.4 adjusted with HCl. Nisoldipine (Miles-Pentex, West Haven, CT) was added to the external solution at a concentration of 200 nm to block the L-type calcium channels. The 1-mm stock solution of nisoldipine was made in polyethylene glycol. The 10-mm stock solution of pinacidil, an opener of K^ATPchannels, was prepared in 0.1 N HCl. The 1-mm stock solution of glibenclamide, a blocker of K^ATPchannels, was prepared in DMSO. Wortmannin (Calbiochem-Novabiochem, San Diego, CA), an inhibitor of phosphatidylinositol kinases, was dissolved in DMSO and added to the pipette solution. After final dilution in the pipette solution, DMSO at a concentration of 0.005% had no effect on the whole cell I^KATP. Theophylline was applied in the external solution at 100 μm. At this concentration, theophylline shows high affinity for adenosine receptors and has only minimal if any effects on phosphodiesterase activity, cyclic adenosine monophosphate production, or intracellular Ca²⁺translocation. These cellular events may be affected by theophylline concentrations higher than 200 μm. Unless stated otherwise, all chemicals were purchased from Sigma (Sigma-Aldrich). Isoflurane (Abbott Laboratories, North Chicago, IL) was delivered after sonicating into the external solution. The concentrations of isoflurane in the recording chamber were measured using the flame ionization detection method and Shimadzu GC8A gas chromatograph (Shimadzu, Kyoto, Japan). The concentration of isoflurane used in this study was 0.55 mm, which is equivalent to 1.0 vol% at 22°C. The external solutions were delivered via  a set of syringe infusion pumps at a rate of 2 ml/min and were removed by vacuum suction.

The I^KATPwas measured in the whole cell configuration of the patch clamp technique, 21using the EPC-7 patch clamp amplifier (List, Darmstadt-Eberstadt, Germany) and Digidata 1322A interface (Axon Instruments, Foster City, CA). The pClamp8 software (Axon Instruments) was used for data acquisition and analysis. Pipettes were pulled from borosilicate glass (Garner Glass, Claremont, CA) with a multistage PC-84 puller (Sutter, Novato, CA) and heat polished using a microforge MF-83 (Narishige, Japan). The pipette resistances ranged from 2 to 3 MΩ. The cells suspended in Tyrode solution were placed in the recording chamber on the stage of an inverted IMT2 microscope (Olympus, Tokyo, Japan). Only quiescent, rod-shaped cells with distinct striations were selected for experiments.

After a gigaohm seal was formed and the whole cell configuration was established by membrane rupture, the series resistance was adjusted to give the fastest possible capacitance transient without causing ringing. Whole cell currents were elicited by a 100-ms depolarizing voltage step to 0 mV from a holding potential of −40 mV applied every 15 s. Current amplitude was measured at the end of each voltage step. To allow for comparisons among cells, currents were normalized to cell capacitance and reported as current density (pA/pF).

Data were analyzed using the pClamp8 software (Axon Instruments) and Origin 6 software (OriginLab, Northampton, MA). Results are reported as mean ± SEM. Statistical analysis was performed using analysis of variance and Student t  test. Differences were considered significant at P < 0.05.

In all experiments of the current study, the sarcK^ATPchannel current, I^KATP, was elicited using a specific potassium channel opener, pinacidil. Pinacidil, a vasodilator and antihypertensive drug, activates sarcK^ATPchannels during normal physiologic conditions. 22–25During control conditions of our study, spontaneous activation of I^KATPwas not observed in the absence of pinacidil.

Because the degree of activation of the K^ATPchannel by pinacidil is dependent on the concentration of intracellular ATP, we first evaluated the effects of pinacidil on I^KATPin guinea pig ventricular myocytes at two different concentrations of intracellular ATP, 0.5 mm and 5.0 mm. Figure 1Ashows representative traces of the whole cell I^KATPactivated by pinacidil (5 μm) at 0.5 mm intracellular ATP. Pinacidil was applied in the bath solution after 30-min dialysis of each cell with the internal solution to allow for equilibration of intracellular ATP. Pinacidil elicited an outward, time-independent current that was inhibited by glibenclamide (0.5 μm). The time course of I^KATPactivation by pinacidil is shown in figure 1B. Current amplitude was measured at the end of a 100-ms test pulse to 0 mV from a holding potential of −40 mV, applied every 15 s. At 0.5 mm intracellular ATP, the density of pinacidil activated I^KATPwas 20.7 ± 3.2 pA/pF (n = 6), and the rate of current activation as determined from a linear regression fit was 5.4 ± 1.2 pA · pF⁻¹· min⁻¹(n = 5). At 5.0 mm intracellular ATP, the density of pinacidil-activated I^KATPwas 2.0 ± 0.3 pA/pF (n = 6). These results are in agreement with reports by other investigators showing that the magnitude of pinacidil activated I^KATPdepends on intracellular ATP. 23–25 

Recent studies from our laboratory have suggested that isoflurane alone does not activate sarcK^ATPchannel in guinea pig ventricular myocytes at 0.5 mm intracellular ATP. 19However, we had also reported that volatile anesthetics may have sensitization effects on other cardiac sarcolemmal ion channels, for example, the sodium channel. 26Therefore, we tested whether isoflurane can sensitize the sarcK^ATPchannel to pinacidil. The experimental protocol included a 20-min dialysis of each cell with the pipette solution containing 0.5 or 5 mm ATP and 10-min exposure to isoflurane (0.57 ± 0.04 mm, n = 27), followed by a 10-min exposure to pinacidil (5 μm) in the continued presence of isoflurane. Glibenclamide (0.5 μm) was applied at the end of each protocol to confirm the identity of I^KATP. Figure 2Ashows sample traces of I^KATPin control, in the presence of isoflurane alone, and in the presence of isoflurane and pinacidil. A corresponding time course of current activation is depicted in figure 2B. After pretreatment and in the continued presence of isoflurane at 0.5 mm intracellular ATP, the pinacidil- activated current was markedly increased compared with anesthetic-free controls (compare with fig. 1). With isoflurane, the density of pinacidil-activated current was increased to 42.4 ± 6.2 pA/pF (n = 8;P < 0.05, isoflurane + pinacidil vs.  pinacidil alone). Furthermore, in the presence of isoflurane, the rate of current activation by pinacidil increased to 39.0 ± 7.9 pA · pF⁻¹· min⁻¹(n = 5), indicating that activation of I^KATPby pinacidil was accelerated in the presence of isoflurane. At 5 mm intracellular ATP, pretreatment with isoflurane caused an increase in pinacidil-activated I^KATPto 5.9 ± 2.3 pA/pF (n = 6), but compared with the corresponding control this change was not significant (P = 0.292). Figure 3summarizes the effects of 0.5 and 5 mm ATP on I^KATPactivation by pinacidil alone and by isoflurane + pinacidil following 10-min pretreatment of cells with anesthetic. These results suggest that at lower intracellular ATP, isoflurane may sensitize the sarcK^ATPchannel to pinacidil, resulting in a greater current density and accelerated current activation.

Experimental evidence supports an important role of adenosine signaling in myocardial protection. 27The cardioprotective effects of isoflurane may also be mediated by adenosine-triggered signaling pathway. 14,15To test whether this pathway is involved in the sensitization effect by isoflurane, we used a broad-spectrum antagonist of adenosine receptors, theophylline. The experiments were conducted at 0.5 mm intracellular ATP. Figure 4shows that extracellularly applied theophylline (100 μm) reduced I^KATPelicited by pinacidil (5 μm), and the current density was 9.4 ± 3.9 pA/pF (n = 6;P < 0.05, pinacidil + theophylline vs.  pinacidil alone). Furthermore, when present throughout the course of the experiment, theophylline prevented sensitization by isoflurane. In the presence of theophylline, isoflurane did not increase the pinacidil-activated I^KATP, and current density was 10.0 ± 2.5 pA/pF (n = 6;P < 0.05, isoflurane + pinacidil + theophylline vs.  isoflurane + pinacidil). Thus, blockade of adenosine receptors caused a decrease in magnitude of pinacidil-activated current through sarcK^ATPchannel and prevented sensitization by isoflurane.

Membrane phospholipids, and particularly phosphatidylinositol 4,5-biphosphate (PIP²), modulate the activity of sarcK^ATPchannel by increasing channel open probability and reducing sensitivity to ATP. 28,29However, recent work has also demonstrated that phospholipids may reduce sensitivity of K^ATPchannel to potassium channel openers and glibenclamide. 30The intracellular concentrations of phospholipids are up-regulated by the action of phosphatidylinositol kinases and down-regulated by phospholipid lipases and phosphatases. To test the hypothesis that sensitization by isoflurane may involve the phosphatidylinositol kinase-dependent pathway, we investigated the effects of wortmannin, an inhibitor of phosphatidylinositol kinases, on pinacidil-activated I^KATPat 0.5 mm internal ATP. Wortmannin was applied at 100 μm intracellularly in the pipette solution. Figure 5shows that wortmannin alone did not significantly alter activation of I^KATPby pinacidil (5 μm), and current density was 13.2 ± 1.7 pA/pF (n = 6;P = 0.139, pinacidil + wortmannin vs.  pinacidil alone). However, in the continued presence of wortmannin, isoflurane failed to sensitize I^KATPto pinacidil. In the presence of wortmannin and isoflurane, the density of pinacidil-activated I^KATPwas 15.8 ± 4.5 pA/pF (n = 7). Thus, current density was not different from that determined in the absence of isoflurane (P = 0.085, isoflurane + pinacidil + wortmannin vs.  pinacidil + wortmannin) but was significantly less than in the presence of isoflurane and pinacidil (P < 0.05, isoflurane + pinacidil + wortmannin vs.  isoflurane + pinacidil). These results suggest that wortmannin, an inhibitor of phosphatidylinositol kinases, may prevent isoflurane-mediated sensitization of sarcK^ATPchannel to pinacidil.

This study investigated the effects of isoflurane on the sensitivity of cardiac sarcK^ATPchannels to a potassium channel opener, pinacidil, as measured by changes in the whole cell I^KATP. The results show that, although isoflurane alone may not activate whole cell I^KATPin guinea pig ventricular cells, pretreatment with isoflurane increases sensitivity of these channels to pinacidil, as reflected in the increased current density and accelerated rate of I^KATPactivation. Furthermore, the sensitization effect is abolished by theophylline and wortmannin. This is a novel finding showing that a volatile anesthetic can enhance sensitivity of the cardiac sarcK^ATPchannel to its opener via  adenosine-triggered signaling cascade and membrane phospholipids.

Sensitization effect of isoflurane was greater at 0.5 mm than at 5 mm intracellular ATP. This suggests that impaired metabolic conditions that may cause a local depletion of ATP could facilitate isoflurane actions, an important finding regarding cardioprotection under compromised cellular function. A possibility of isoflurane modulation of ATP sensitivity of K^ATPchannel has been suggested by the study showing that isoflurane may decrease ATP sensitivity despite increasing channel closed time. 13However, it has also been reported that trifluoroacetic acid, a metabolite of isoflurane, may modulate ATP sensitivity of K^ATPchannels. 31Taking under account a very low metabolism of isoflurane in vivo , and our in vitro  experimental conditions where single myocytes are directly exposed to isoflurane, the results of the current study suggest that isoflurane itself rather than its metabolite sensitizes the channel. Since pinacidil effects are ATP-dependent, whereby a decrease in ATP leads to a greater activation of I^KATP, 24,25the sensitization of sarcK^ATPchannel to pinacidil by isoflurane may, in part, result from its ability to alter ATP sensitivity.

Released under metabolic stress, adenosine may exert cardioprotective effects by activating A¹and A³receptors coupled via  G^o/Gⁱproteins to multiple effectors. These include phospholipases C and D, phosphoinositides, protein kinases such as protein kinase C (PKC), and the K^ATPchannel. 27Recent studies demonstrated that in cardioprotection, the triggering action of adenosine is not dependent on K^ATPchannel activation or free radical production, but rather results from a direct activation of the kinases. 32Although adenosine was not used in our study, an antagonist of adenosine receptors, theophylline, prevented sensitization by isoflurane. This suggests that isoflurane may alter activity of adenosine receptors, since it has been demonstrated that isoflurane does not increase the production or release of adenosine. 33Isoflurane may act on other components of the adenosine signaling pathway, including not only PKC, but also the phospholipases or phosphatidylinositol kinase activity upstream of PKC. Attenuation of I^KATPactivation by pinacidil in the presence of theophylline could be explained by a decreased potency of pinacidil resulting from adenosine receptor blockade, since it is known that activation of adenosine receptors can enhance the potency of potassium channel openers nicorandil and levcromakalim in the arterial smooth muscle. 34 

The membrane phospholipids regulate the sarcK^ATPchannels by modifying its sensitivity to ATP, sulfonylureas, and potassium channel openers. 30Phosphatidylinositol phosphates, and particularly PIP², greatly reduce sensitivity to inhibition by ATP that is mediated via  the pore-forming Kir6.2 subunit of K^ATPchannel. 28,30In addition, sensitivities to the openers and glibenclamide that are mediated by the SUR2A subunit 35,36are also decreased by PIP². The levels of PIP²are up-regulated via  a rapid activation of phosphatidylinositol kinases and phosphorylation of phosphatidylinositol and PI-4-P, and down-regulated by phospholipid lipase–mediated hydrolysis or the action of phospholipid phosphatases. 29Stimulation of phosphatidylinositol kinases would therefore be expected to increase PIP²concentrations and thus alter channel sensitivity to its openers. Consequently, either inhibition of phosphatidylinositol kinases to prevent PIP²synthesis, or activation of phospholipases to enhance PIP²breakdown, would be expected to have an enhancing effect on sensitivity of the sarcK^ATPchannel to specific potassium channel openers. It is possible that isoflurane sensitization results from altered activity of some of these enzymes. Previous studies demonstrated that volatile anesthetics may increase activity of phospholipase C in erythrocyte membranes 37or skeletal muscle. 38Isoflurane may also interact with the PKC signaling pathway where adenosine and PIP²pathways merge, since it has been suggested that elements distal to PIP²may be involved in the PIP²-induced modification of ATP sensitivity. 39 

To test whether isoflurane sensitization involves modulation of phosphatidylinositol kinases, we used wortmannin, an inhibitor of phosphatidylinositol-3 and -4 kinases. 40,41In the presence of wortmannin, isoflurane failed to sensitize the K^ATPchannel to pinacidil. This suggests that modulation of pinacidil effects by isoflurane may occur via  phosphatidylinositol kinases. The phosphatidylinositol kinases, particularly phosphatidylinositol-3 kinase, are key signaling enzymes mediating activation of other kinases such as protein kinase B, p70 kinase, numerous PKC isoforms, and endothelial nitric oxide synthase. 42Thus, we cannot exclude a possibility that isoflurane effects involves one of these pathways. Nevertheless, the wortmannin experiments suggest that isoflurane sensitizes the cardiac sarcK^ATPchannel to pinacidil by a mechanism that is upstream of PKC and may involve phospholipid-mediated control of the channel.

Compared with other K^ATPchannel subtypes, the cardiac sarcK^ATPchannel composed of Kir6.2 and SUR2A subunits shows high sensitivity to potassium channel openers. 43Binding of pinacidil to the identified binding sites on the SUR2A subunit 35is modulated by nucleotides and requires ATP hydrolysis to induce a conformational change that stabilizes the pinacidil-activated state. 44Thus, we cannot disregard a possibility of allosteric modulation by isoflurane of channel protein, which could cause an increase in sensitivity to pinacidil possibly by enhancing the accessibility of channel opener to its binding sites on the SUR2A subunit.

The current study and the study by Kohro et al.  45suggest that isoflurane may have differential effects on the sarcK^ATPand mitoK^ATPchannels in ventricular myocytes. Isoflurane alone does not open sarcK^ATPchannel, but enhances activity of the channels activated by specific channel openers or metabolic inhibitors. 16,19In contrast, isoflurane appears to directly activate the mitoK^ATPchannel, as measured by an increase in flavoprotein oxidation. 45Isoflurane may also enhance the mitoK^ATPchannel activated by diazoxide. These findings suggest differential sensitivity of sarcolemmal and mitochondrial channels to isoflurane. However, isoflurane may enhance activity of both types of K^ATPchannels previously opened by specific channel activators.

In summary, this study is the first to show that pretreatment with a volatile anesthetic, isoflurane, enhances sensitivity of the cardiac sarcK^ATPchannel to pinacidil. Isoflurane sensitization is modulated by intracellular ATP and may involve components of adenosine and phospholipid signaling pathways. Sensitization of cardiac K^ATPchannel to specific openers may be one of the cellular mechanisms by which isoflurane and other volatile anesthetics produce myocardial protection.