Cardioprotective effects of isoflurane are partially mediated by the sarcolemmal adenosine triphosphate-sensitive potassium (sarcK ATP ) channel. The authors tested the hypothesis that isoflurane sensitizes sarcK ATP channels to a potassium channel opener, pinacidil, adenosine- and phospholipid-mediated pathways.
Activation by pinacidil of the K ATP current (I KATP ) was monitored in guinea pig ventricular myocytes at 0.5 and 5 mm intracellular ATP in the whole cell configuration of the patch clamp technique. The sensitization effect was evaluated by pretreating each myocyte with isoflurane (0.57 +/- 0.04 mm) before application of pinacidil (5 micro m) in the continued presence of the anesthetic. To investigate whether intracellular signaling pathways may be involved in isoflurane sensitization, the authors used the adenosine receptor antagonist theophylline (100 micro m) and the phosphatidylinositol kinase inhibitor wortmannin (100 micro m).
The density of pinacidil-activated I KATP was higher at 0.5 mm ATP (20.7 +/- 3.2 pA/pF) than at 5 mm ATP (2.0 +/- 0.3 pA/pF). At 0.5 mm ATP, pretreatment with isoflurane caused an increase in density of pinacidil-activated I KATP (42.4 +/- 6.2 pA/pF) and accelerated the rate of current activation (from 5.4 +/- 1.2 to 39.0 +/- 7.9 pA. pF(-1). min(-1) ). Theophylline attenuated current activation by pinacidil (9.4 +/- 3.9 pA/pF) and abolished the sensitization effect of isoflurane on I KATP (10.0 +/- 2.5 pA/pF). Wortmannin did not alter pinacidil activation of I KATP (13.2 +/- 1.7 pA/pF) but prevented sensitization by isoflurane (15.8 +/- 4.5 pA/pF).
These results suggest that isoflurane increases sensitivity of cardiac sarcK ATP channels to the potassium channel opener pinacidil. Blockade of adenosine receptors or phosphatidylinositol kinases abolishes the sensitization effect, suggesting that the adenosine and phospholipid signaling pathways may be involved in the actions by isoflurane.
VOLATILE anesthetic–induced preconditioning appears as effective as ischemic preconditioning (IPC) in protecting the heart against ischemia–reperfusion injury by decreasing myocardial infarct size and improving postischemic functional recovery. 1–4This important finding may in the future have an impact in clinical settings where an increasing number of surgical patients with coronary artery disease are at a high risk for perioperative myocardial ischemia. While the mechanisms underlying IPC has been a major field of investigation since the first report by Murry et al. , 5the cellular and molecular mechanisms of anesthetic-induced preconditioning are not yet defined, although the pathways involved are thought to mimic IPC.
Two distinct populations of myocardial adenosine triphosphate–sensitive potassium (KATP) channel, the sarcolemmal KATP(sarcKATP) and the mitochondrial KATP(mitoKATP) channels, contribute to IPC, but their exact roles are not elucidated. Recent evidence supports a predominant role of mitoKATPchannels in the initiation of IPC. 6–9The sarcKATPchannels are thought to mediate cardioprotection during the reoxygenation phase. 10Similarly, in anesthetic-induced preconditioning, both the sarcKATPand mitoKATPchannels have been indicated to contribute to cardioprotection, 11although their specific roles are not yet determined.
There is limited direct evidence on the interaction of volatile anesthetic with the cardiac sarcKATPchannel. Isoflurane was shown to have no significant effect on sarcKATPchannel in human atrial cells. 12Other investigators reported that isoflurane inhibited single KATPchannel current in the cell-free membrane patches from rabbit ventricular myocytes and attenuated channel sensitivity to ATP. 13The in vivo studies have suggested that the effects of volatile anesthetics on KATPchannels may involve intracellular signaling. 14It has been shown that blockade of adenosine receptors and Giproteins abolishes the cardioprotective effects of volatile anesthetics. 14,15The contribution of intracellular signaling to modulation of volatile anesthetic effects on the KATPchannel has also been demonstrated at the single cell level. 16–18
The in vitro effects of volatile anesthetics on the sarcKATPchannel have been investigated during conditions where channel activity was monitored during application of the anesthetic. It is uncertain, however, whether pretreatment with volatile anesthetic can facilitate the opening of KATPchannel, a condition relevant to anesthetic-induced preconditioning. We have previously reported that isoflurane alone is unable to elicit sarcKATPchannel opening under whole cell conditions. 19In the current study, we tested the hypothesis that pretreatment with isoflurane increases sensitivity of sarcKATPchannels to the potassium channel opener pinacidil, facilitating KATPcurrent (IKATP) activated by pinacidil. In addition, an involvement of the adenosine- and phospholipid-mediated signaling pathways to the actions by isoflurane were tested.
Materials and Methods
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 KATPchannels, was prepared in 0.1 N HCl. The 1-mm stock solution of glibenclamide, a blocker of KATPchannels, 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 IKATP. 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.
Electrophysiologic Recordings and Data Analysis
The IKATPwas 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.
Effects of Pinacidil on Whole Cell Adenosine Triphosphate–sensitive Potassium Current
In all experiments of the current study, the sarcKATPchannel current, IKATP, was elicited using a specific potassium channel opener, pinacidil. Pinacidil, a vasodilator and antihypertensive drug, activates sarcKATPchannels during normal physiologic conditions. 22–25During control conditions of our study, spontaneous activation of IKATPwas not observed in the absence of pinacidil.
Because the degree of activation of the KATPchannel by pinacidil is dependent on the concentration of intracellular ATP, we first evaluated the effects of pinacidil on IKATPin 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 IKATPactivated 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 IKATPactivation 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 IKATPwas 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 IKATPwas 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 IKATPdepends on intracellular ATP. 23–25
Sensitization of Sarcolemmal Adenosine Triphosphate–sensitive Potassium Channel to Pinacidil by Isoflurane
Recent studies from our laboratory have suggested that isoflurane alone does not activate sarcKATPchannel 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 sarcKATPchannel 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 IKATP. Figure 2Ashows sample traces of IKATPin 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 IKATPby pinacidil was accelerated in the presence of isoflurane. At 5 mm intracellular ATP, pretreatment with isoflurane caused an increase in pinacidil-activated IKATPto 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 IKATPactivation 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 sarcKATPchannel to pinacidil, resulting in a greater current density and accelerated current activation.
Involvement of Adenosine Signaling in the Sensitization by Isoflurane
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 IKATPelicited 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 IKATP, 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 sarcKATPchannel and prevented sensitization by isoflurane.
Involvement of the Membrane Phospholipids in Isoflurane Sensitization
Membrane phospholipids, and particularly phosphatidylinositol 4,5-biphosphate (PIP2), modulate the activity of sarcKATPchannel by increasing channel open probability and reducing sensitivity to ATP. 28,29However, recent work has also demonstrated that phospholipids may reduce sensitivity of KATPchannel 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 IKATPat 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 IKATPby 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 IKATPto pinacidil. In the presence of wortmannin and isoflurane, the density of pinacidil-activated IKATPwas 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 sarcKATPchannel to pinacidil.
This study investigated the effects of isoflurane on the sensitivity of cardiac sarcKATPchannels to a potassium channel opener, pinacidil, as measured by changes in the whole cell IKATP. The results show that, although isoflurane alone may not activate whole cell IKATPin 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 IKATPactivation. 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 sarcKATPchannel 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 KATPchannel 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 KATPchannels. 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 IKATP, 24,25the sensitization of sarcKATPchannel 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 Go/Giproteins to multiple effectors. These include phospholipases C and D, phosphoinositides, protein kinases such as protein kinase C (PKC), and the KATPchannel. 27Recent studies demonstrated that in cardioprotection, the triggering action of adenosine is not dependent on KATPchannel 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 IKATPactivation 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 sarcKATPchannels 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 KATPchannel. 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 sarcKATPchannel 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 KATPchannel 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 sarcKATPchannel to pinacidil by a mechanism that is upstream of PKC and may involve phospholipid-mediated control of the channel.
Compared with other KATPchannel subtypes, the cardiac sarcKATPchannel 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 sarcKATPand mitoKATPchannels in ventricular myocytes. Isoflurane alone does not open sarcKATPchannel, but enhances activity of the channels activated by specific channel openers or metabolic inhibitors. 16,19In contrast, isoflurane appears to directly activate the mitoKATPchannel, as measured by an increase in flavoprotein oxidation. 45Isoflurane may also enhance the mitoKATPchannel activated by diazoxide. These findings suggest differential sensitivity of sarcolemmal and mitochondrial channels to isoflurane. However, isoflurane may enhance activity of both types of KATPchannels 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 sarcKATPchannel to pinacidil. Isoflurane sensitization is modulated by intracellular ATP and may involve components of adenosine and phospholipid signaling pathways. Sensitization of cardiac KATPchannel to specific openers may be one of the cellular mechanisms by which isoflurane and other volatile anesthetics produce myocardial protection.