Isoflurane sensitizes the cardiac sarcolemmal adenosine triphosphate-sensitive potassium channel to pinacidil

BACKGROUND: 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. METHODS: 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). RESULTS: 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). CONCLUSIONS: 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.     

Contribution of Reactive Oxygen Species to Isoflurane-induced Sensitization of Cardiac Sarcolemmal Adenosine Triphosphate-sensitive Potassium Channel to Pinacidil

Background: Myocardial protection by volatile anesthetics involves activation of cardiac adenosine triphosphate-sensitive potassium (KATP) channels. The authors have previously shown that isoflurane enhances sensitivity of the sarcolemmal KATP channel to the opener, pinacidil. Because reactive oxygen species seem to be mediators in anesthetic preconditioning, the authors investigated whether they contribute to the mechanism of the sensitization effect by isoflurane.

Methods: Ventricular myocytes were isolated from guinea pig hearts for the whole cell patch clamp recordings of the sarcolemmal KATP channel current (IKATP). Free radical scavengers N-acetyl-l-cysteine, carnosine, superoxide dismutase, and catalase were used to investigate whether reactive oxygen species mediate isoflurane facilitation of the channel opening by pinacidil. A possible role of the mitochondrial KATP channels was tested using a blocker of these channels, 5-hydroxydecanoate.

Results: The mean density (+/- SEM) of IKATP elicited by pinacidil (20 [mu]m) was 18.9 +/- 1.8 pA/pF (n = 11). In the presence of isoflurane (0.55 mm), the density of pinacidil-activated IKATP increased to 38.5 +/- 2.4 pA/pF (n = 9). Concurrent application of isoflurane and N-acetyl-l-cysteine decreased the sensitization effect by isoflurane in a concentration-dependent manner, whereby the densities of IKATP were 32.6 +/- 1.4 (n = 6), 26.2 +/- 2.3 (n = 6), and 19.4 +/- 2.1 pA/pF (n = 8) at 100, 250, and 500 [mu]m N-acetyl-l-cysteine, respectively. Concurrent application of isoflurane and carnosine (100 [mu]m), superoxide dismutase (100 U/ml), or catalase (100 U/ml) attenuated the densities of IKATP to 27.9 +/- 2.6, 27.2 +/- 2.9, and 25.9 +/- 2.2 pA/pF, respectively. None of the scavengers affected activation of IKATP by pinacidil alone. 5-Hydroxydecanoate (100 [mu]m) did not alter the sensitization effect by isoflurane, and the density of IKATP in this group was 37.1 +/- 3.8 pA/pF (n = 6).     

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

Background: Cardioprotective effects of isoflurane are partially mediated by the sarcolemmal adenosine triphosphate-sensitive potassium (sarcKATP) channel. The authors tested the hypothesis that isoflurane sensitizes sarcKATP channels to a potassium channel opener, pinacidil, via adenosine- and phospholipid-mediated pathways.

Methods: Activation by pinacidil of the KATP current (IKATP) 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 [mu]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 [mu]m) and the phosphatidylinositol kinase inhibitor wortmannin (100 [mu]m).

Results: The density of pinacidil-activated IKATP 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 IKATP (42.4 +/- 6.2 pA/pF) and accelerated the rate of current activation (from 5.4 +/- 1.2 to 39.0 +/- 7.9 pA [middle dot] pF-1 [middle dot] min-1). Theophylline attenuated current activation by pinacidil (9.4 +/- 3.9 pA/pF) and abolished the sensitization effect of isoflurane on IKATP (10.0 +/- 2.5 pA/pF). Wortmannin did not alter pinacidil activation of IKATP (13.2 +/- 1.7 pA/pF) but prevented sensitization by isoflurane (15.8 +/- 4.5 pA/pF).     

Preconditioning by Isoflurane Induces Lasting Sensitization of the Cardiac Sarcolemmal Adenosine Triphosphate-sensitive Potassium Channel by a Protein Kinase C-δ-mediated Mechanism

Background: Cardioprotective effects of volatile anesthetics in anesthetic-induced preconditioning involve activation of the cardiac sarcolemmal adenosine triphosphate-sensitive potassium (sarcKATP) channels. This study addressed the memory phase of anesthetic preconditioning by investigating whether brief exposure to isoflurane produces lasting sensitization of the sarcKATP channel and whether protein kinase C mediates this effect.

Methods: Whole cell sarcKATP channel current (IKATP) was monitored from single isolated rat ventricular cardiomyocytes. Pinacidil was used to open the channel, and the magnitude of activated IKATP was an indicator of channel's ability to open. Involvement of protein kinase C was investigated using chelerythrine and isoform-specific peptide inhibitors and activators of protein kinase C-[delta] and protein kinase C-[varepsilon].

Results: The mean density of IKATP elicited by pinacidil (5 [mu]m) in anesthetic-free conditions was 3.8 +/- 3.7 pA/pF (n = 11). After 10 min of exposure to isoflurane (0.56 mm) and 10 or 30 min of anesthetic washout, pinacidil-elicited IKATP was increased to 15.6 +/- 11.3 pA/pF (n = 12; P < 0.05) and 11.8 +/- 3.9 pA/pF (n = 6; P < 0.05), respectively. In the presence of chelerythrine (5 [mu]m), isoflurane did not potentiate channel opening, and IKATP was 6.6 +/- 4.6 pA/pF (n = 11). Application of protein kinase C-[delta] peptide inhibitor also abolished isoflurane-induced sensitization of sarcKATP channel, and IKATP was 7.7 +/- 5.4 pA/pF (n = 12). In contrast, protein kinase C-[varepsilon] peptide inhibitor did not affect channel sensitization, and pinacidil-elicited current was 14.8 +/- 9.6 pA/pF (n = 12). Interestingly, when both protein kinase C-[delta] and protein kinase C-[varepsilon] activators were applied instead of isoflurane, they sensitized the channel to the same extent as isoflurane (18.9 +/- 7.2 pA/pF, n = 11, and 18.6 +/- 11.1 pA/pF, n = 10, respectively).     

Preconditioning by isoflurane induces lasting sensitization of the cardiac sarcolemmal adenosine triphosphate-sensitive potassium channel by a protein kinase C-delta-mediated mechanism

BACKGROUND: Cardioprotective effects of volatile anesthetics in anesthetic-induced preconditioning involve activation of the cardiac sarcolemmal adenosine triphosphate-sensitive potassium (sarcKATP) channels. This study addressed the memory phase of anesthetic preconditioning by investigating whether brief exposure to isoflurane produces lasting sensitization of the sarcKATP channel and whether protein kinase C mediates this effect. METHODS: Whole cell sarcKATP channel current (IKATP) was monitored from single isolated rat ventricular cardiomyocytes. Pinacidil was used to open the channel, and the magnitude of activated IKATP was an indicator of channel's ability to open. Involvement of protein kinase C was investigated using chelerythrine and isoform-specific peptide inhibitors and activators of protein kinase C-delta and protein kinase C-epsilon. RESULTS: The mean density of IKATP elicited by pinacidil (5 microm) in anesthetic-free conditions was 3.8 +/- 3.7 pA/pF (n = 11). After 10 min of exposure to isoflurane (0.56 mm) and 10 or 30 min of anesthetic washout, pinacidil-elicited IKATP was increased to 15.6 +/- 11.3 pA/pF (n = 12; P < 0.05) and 11.8 +/- 3.9 pA/pF (n = 6; P < 0.05), respectively. In the presence of chelerythrine (5 microm), isoflurane did not potentiate channel opening, and IKATP was 6.6 +/- 4.6 pA/pF (n = 11). Application of protein kinase C-delta peptide inhibitor also abolished isoflurane-induced sensitization of sarcKATP channel, and IKATP was 7.7 +/- 5.4 pA/pF (n = 12). In contrast, protein kinase C-epsilon peptide inhibitor did not affect channel sensitization, and pinacidil-elicited current was 14.8 +/- 9.6 pA/pF (n = 12). Interestingly, when both protein kinase C-delta and protein kinase C-epsilon activators were applied instead of isoflurane, they sensitized the channel to the same extent as isoflurane (18.9 +/- 7.2 pA/pF, n = 11, and 18.6 +/- 11.1 pA/pF, n = 10, respectively). CONCLUSION: Isoflurane induces prolonged sensitization of the sarcKATP channel to opening that persists even after anesthetic withdrawal. Our results indicate that protein kinase C-delta, rather than protein kinase C-epsilon, is a likely mediator of isoflurane effects, although both protein kinase C-delta and protein kinase C-epsilon can modulate the channel function.     

The interaction of isoflurane and protein kinase C-activators on sarcolemmal KATP channels

Protein kinase C (PKC)-dependent signaling pathways may be involved in the "memory" effect of anesthetic and ischemic preconditioning, which facilitates activation of cardioprotective adenosine triphosphate (ATP)-sensitive potassium channels during later ischemic challenge and ATP depletion. Using patch-clamp techniques, we found that exposure of isolated guinea pig cardiomyocytes to 1 mM of isoflurane after phorbol ester stimulation of PKC facilitates the induction of larger (P < or = 0.05) sarcolemmal K(ATP) channel currents (IKATP) during cell dialysis with 0.5, compared to 1.0, mM of ATP in the pipette (10 +/- 5 versus 2 +/- 1 pA/pF in five and six cells, respectively). A PKC inhibitor, bisindolylmaleimide, abolished the induction of IKATP by a second brief isoflurane exposure under these conditions. A diacylglycerol PKC activator applied via the pipette elicited concentration-related activation of IKATP. The diacylglycerol alone (0.5 microM) elicited I(KATP), averaging 5 +/- 3 pA/pF in nine cells. Briefly treating myocytes on the microscope stage with isoflurane, followed by washout and patching with the same diacylglycerol solution, elicited larger (P < or = 0.01) IKATP, averaging 40 +/- 9 pA/pF (10 cells), with an onset 48 +/- 2 min after anesthetic pretreatment. Facilitation of IKATP by isoflurane during the reduction of intracellular ATP is dependent on PKC, whereas "preconditioning" myocytes with isoflurane causes persistent changes in sarcolemmal KATP channel function, which enhance the induction of IKATP by a diacylglycerol.     

Protein tyrosine kinase-dependent modulation of isoflurane effects on cardiac sarcolemmal K(ATP) channel

BACKGROUND: Cardiac adenosine triphosphate-sensitive potassium (K(ATP)) channels and protein tyrosine kinases (PTKs) are mediators of ischemic preconditioning, but the interaction of both and a role in myocardial protection afforded by volatile anesthetics have not been defined. METHODS: Whole cell and single channel patch clamp techniques were used to investigate the effects of isoflurane and the PTK inhibitor genistein on the cardiac sarcolemmal K(ATP) channel in acutely dissociated guinea pig ventricular myocytes. RESULTS: At 0.5 mm internal ATP, genistein (50 microm) elicited whole cell K(ATP) current (22.5 +/- 7.9 pA/pF). Genistein effects were concentration-dependent, with an EC50 of 32.3 +/- 1.4 microm. Another PTK inhibitor, tyrphostin B42, had a similar effect. The inactive analog of genistein, daidzein (50 microm), did not elicit K(ATP) current. Isoflurane (0.5 mm) increased genistein (35 microm)-activated whole cell K(ATP) current from 14.5 +/- 3.1 to 32.5 +/- 6.6 pA/pF. Stimulation of receptor PTKs with epidermal growth factor, nerve growth factor, or insulin attenuated genistein and isoflurane effects, and the protein tyrosine phosphatase inhibitor orthovanadate (1 mm) prevented their actions on K(ATP) current. In excised inside-out membrane patches, and at fixed 0.2 mm internal ATP, genistein (50 microm) increased channel open probability from 0.053 +/- 0.016 to 0.183 +/- 0.039, but isoflurane failed to further increase open probability (0.162 +/- 0.051) of genistein-activated channels. However, applied in the presence of genistein and protein tyrosine phosphatase 1B (1 microg/ml), isoflurane significantly increased open probability to 0.473 +/- 0.114. CONCLUSIONS: These results suggest that the PTK-protein tyrosine phosphatase signaling pathway may be one of the regulators of cardiac sarcolemmal K(ATP) channel and may play a role in modulating its responsiveness to isoflurane. Relative importance of this modulation for cardioprotection by volatile anesthetics remains to be established.     

Differential modulation of the cardiac adenosine triphosphate-sensitive potassium channel by isoflurane and halothane

BACKGROUND: The cardiac adenosine triphosphate-sensitive potassium (K(ATP)) channel is activated during pathophysiological episodes such as ischemia and hypoxia and may lead to beneficial effects on cardiac function. Studies of volatile anesthetic interactions with the cardiac K(ATP) channel have been limited. The goal of this study was to investigate the ability of volatile anesthetics halothane and isoflurane to modulate the cardiac sarcolemmal K(ATP) channel. METHODS: The K(ATP) channel current (I(KATP)) was monitored using the whole cell configuration of the patch clamp technique from single ventricular cardiac myocytes enzymatically isolated from guinea pig hearts. I(KATP) was elicited by extracellular application of the potassium channel openers 2,4-dinitrophenol or pinacidil. RESULTS: Volatile anesthetics modulated I(KATP) in an anesthetic-dependent manner. Isoflurane facilitated the opening of the K(ATP) channel. Following initial activation of I(KATP) by 2,4-dinitrophenol, isoflurane at 0.5 and 1.3 mm further increased current amplitude by 40.4 +/- 11.1% and 58.4 +/- 20.6%, respectively. Similar results of isoflurane were obtained when pinacidil was used to activate I(KATP). However, isoflurane alone was unable to elicit K(ATP) channel opening. In contrast, halothane inhibited I(KATP) elicited by 2,4-dinitrophenol by 50.6 +/- 5.8% and 72.1 +/- 11.6% at 0.4 and 1.0 mm, respectively. When I(KATP) was activated by pinacidil, halothane had no significant effect on the current. CONCLUSIONS: The cardiac sarcolemmal K(ATP) channel is differentially modulated by volatile anesthetics. Isoflurane can facilitate the further opening of the K(ATP) channel following initial channel activation by 2,4-dinitrophenol or pinacidil. The effect of halothane was dependent on the method of channel activation, inhibiting I(KATP) activated by 2,4-dinitrophenol but not by pinacidil.     

Protein kinase C-epsilon primes the cardiac sarcolemmal adenosine triphosphate-sensitive potassium channel to modulation by isoflurane

BACKGROUND: Cardioprotection by volatile anesthetic-induced preconditioning is known to involve intracellular signaling pathways. Recent studies have shown that protein kinase C (PKC) plays an important role in anesthetic-induced preconditioning. In this study, the effects of the activation of specific isozymes of PKC, specifically PKC-epsilon and -delta, on the modulation of the sarcolemmal adenosine triphosphate-sensitive potassium (sarcKATP) channel by isoflurane were investigated. METHODS: The sarcKATP current was measured in ventricular myocytes isolated from guinea pig hearts using the whole cell configuration of the patch clamp technique. Peptides that induced the translocation of specific PKC isozymes were used to activate PKC-epsilon and PKC-delta. RESULTS: Under whole cell conditions, isoflurane alone was unable to elicit the opening of the sarcKATP channel. Pretreatment with the specific PKC-epsilon activator, PP106, primed the sarcKATP channel to open in the presence of isoflurane. The resulting sarcKATP current densities in the presence of 0.88 mm isoflurane were 6.5 +/- 6.0 pA/pF (n = 7) and 40.4 +/- 18.2 pA/pF (n = 7) after pretreatment with 100 and 200 nm PP106, respectively. The PKC-epsilon antagonist PP93 abolished this effect. A scrambled peptide of the PKC-epsilon activator PP105 did not prime the sarcKATP channel. The PKC-delta activator PP114 was significantly less effective in priming the sarcKATP channel. 5-Hydroxydecanoate significantly attenuated the effect of the PKC-epdsilon activator on the sarcKATP channel. In addition, immunohistochemical analysis showed that the PKC-epsilon isoform translocated to both the mitochondria and sarcolemma after anesthetic-induced preconditioning, whereas the PKC-delta isoform translocated to the mitochondria. CONCLUSION: The PKC-epsilon isozyme primed the sarcKATP channel to open in the presence of isoflurane. The PKC-delta isozyme was significantly less effective in modulating the isoflurane effect on this channel.     

Effects of propofol and thiamylal on nicorandil induced ATP-sensitive potassium channel activities in cultured rat aortic smooth muscle cells

BACKGROUND: Nicorandil, a hybrid ATP-sensitive potassium (K(ATP)) channel opener and nitrate compound, is used clinically for the treatment of angina pectoris. In the present study, we investigated the effects of propofol and thiamylal on sarcolemmal K(ATP) channels activities induced by nicorandil in cultured rat aortic smooth muscle cells. METHODS: We used inside-out patch clamp configurations to investigate the effects of propofol and thiamylal on nicorandil induced K(ATP) channel activities. RESULTS: K(ATP) channel was not spontaneously activated by patch excision in the absence of intracellular ATP. Application of nicorandil (100 microM) induced a marked activation of KATP channel currents, which was completely blocked by 3 microM glibenclamide, the sulfonylurea that blocks K(ATP) channels. Nicorandil induced KATP channel currents were not significantly inhibited by application of 10 and 100 microM propofol to intracellular surface. However, application of 100 and 300 microM thiamylal to intracellular surface significantly inhibited the nicorandil induced K(ATP) channel currents, with relative channel activities decreasing to 0.65 +/- 0.08 and 0.46 +/- 0.10 of control, respectively. CONCLUSIONS: Propofol had no effect on nicorandil induced sarcolemmal KATP channel activities in rat aortic smooth muscle cells, whereas thiamylal significantly inhibited these channel activities at clinically relevant concentrations.     

Distinct roles for sarcolemmal and mitochondrial adenosine triphosphate-sensitive potassium channels in isoflurane-induced protection against oxidative stress

BACKGROUND: Cardiac preconditioning, including that induced by halogenated anesthetics, is an innate protective mechanism against ischemia-reperfusion injury. The adenosine triphosphate-sensitive potassium (K(ATP)) channels are considered essential in preconditioning mechanism. However, it is unclear whether K(ATP) channels are triggers initiating the preconditioning signaling, and/or effectors responsible for the cardioprotective memory and activated during ischemia-reperfusion. METHODS: Adult rat cardiomyocytes were exposed to oxidative stress with 200 microM H(2)O(2) and 100 microM FeSO4. Myocyte survival was determined based on morphologic characteristics and trypan blue exclusion. To induce preconditioning, the myocytes were pretreated with isoflurane. The involvement of sarcolemmal and mitochondrial K(ATP) channels was investigated using specific inhibitors HMR-1098 and 5-hydroxydecanoic acid. Data are expressed as mean +/- SD. RESULTS: Oxidative stress induced cell death in 47 +/- 14% of myocytes. Pretreatment with isoflurane attenuated this effect to 26 +/- 8%. Blockade of the sarcolemmal K(ATP) channels abolished the protection by isoflurane pretreatment when HMR-1098 was applied throughout the experiment (50 +/- 21%) or only during oxidative stress (50 +/- 12%), but not when applied during isoflurane pretreatment (29 +/- 13%). Inhibition of the mitochondrial K(ATP) channels abolished cardioprotection irrespective of the timing of 5-hydroxydecanoic acid application. Cell death was 42 +/- 23, 45 +/- 23, and 46 +/- 22% when 5-hydroxydecanoic acid was applied throughout the experiment, only during isoflurane pretreatment, or only during oxidative stress, respectively. CONCLUSION: The authors conclude that both sarcolemmal and mitochondrial K(ATP) channels play essential and distinct roles in protection afforded by isoflurane. Sarcolemmal K(ATP) channel seems to act as an effector of preconditioning, whereas mitochondrial K(ATP) channel plays a dual role as a trigger and an effector.     

Protein Kinase C-ε Primes the Cardiac Sarcolemmal Adenosine Triphosphate-sensitive Potassium Channel to Modulation by Isoflurane

Background: Cardioprotection by volatile anesthetic-induced preconditioning is known to involve intracellular signaling pathways. Recent studies have shown that protein kinase C (PKC) plays an important role in anesthetic-induced preconditioning. In this study, the effects of the activation of specific isozymes of PKC, specifically PKC-[epsilon] and -[delta], on the modulation of the sarcolemmal adenosine triphosphate-sensitive potassium (sarcKATP) channel by isoflurane were investigated.

Methods: The sarcKATP current was measured in ventricular myocytes isolated from guinea pig hearts using the whole cell configuration of the patch clamp technique. Peptides that induced the translocation of specific PKC isozymes were used to activate PKC-[epsilon] and PKC-[delta].

Results: Under whole cell conditions, isoflurane alone was unable to elicit the opening of the sarcKATP channel. Pretreatment with the specific PKC-[epsilon] activator, PP106, primed the sarcKATP channel to open in the presence of isoflurane. The resulting sarcKATP current densities in the presence of 0.88 mm isoflurane were 6.5 +/- 6.0 pA/pF (n = 7) and 40.4 +/- 18.2 pA/pF (n = 7) after pretreatment with 100 and 200 nm PP106, respectively. The PKC-[epsilon] antagonist PP93 abolished this effect. A scrambled peptide of the PKC-[epsilon] activator PP105 did not prime the sarcKATP channel. The PKC-[delta] activator PP114 was significantly less effective in priming the sarcKATP channel. 5-Hydroxydecanoate significantly attenuated the effect of the PKC-[epsilon] activator on the sarcKATP channel. In addition, immunohistochemical analysis showed that the PKC-[epsilon] isoform translocated to both the mitochondria and sarcolemma after anesthetic-induced preconditioning, whereas the PKC-[delta] isoform translocated to the mitochondria.     

Effects of the anesthetic gases xenon, halothane, and isoflurane on calcium and potassium currents in human atrial cardiomyocytes

BACKGROUND: Negative inotropic and proarrhythmic side effects on the heart are well known for the volatile anesthetics halothane and isoflurane but not for the noble gas xenon. We investigated the effects of halothane, isoflurane, and xenon on calcium and potassium currents in human atrial myocytes to elucidate the cellular and molecular basis of their cardiac actions. METHODS: Atrial myocytes were prepared from the right auricles obtained from patients undergoing heart surgery. Ion currents were measured with the whole cell patch clamp technique during superfusion of the cells with solutions that contained halothane, isoflurane, or xenon at concentrations corresponding to their respective minimum alveolar concentration (MAC); gas concentrations were determined with the head space-gas chromatography/mass spectrometry/selected ion monitoring method. RESULTS: L-type calcium currents were significantly depressed by 31.9 +/- 4.1%, from -1.8 +/- 0.3 to -1.2 +/- 0.4 picoampere (pA)/picofarad (pF) (n = 4; P < 0.05) at 1 MAC halothane and by 21.7 +/- 9.2%, from -1.6 +/- 0.7 to -1.2 +/- 0.6 pA/pF (n = 7; P < 0.05) at 1 MAC isoflurane, but not affected by 70% xenon (1 MAC). Inwardly rectifying potassium currents were not influenced by any anesthetic. Halothane (1 MAC) significantly inhibited the transient as well as the sustained part of voltage-gated potassium outward currents, by 19.4 +/- 6.7%, from 6.7 +/- 2.1 to 5.4 +/- 1.6 pA/pF (n = 8; P < 0.05), and by 8.6 +/- 4.8%, from 5.5 +/- 1.7 to 5.0 +/- 1.5 pA/pF (n = 8; P < 0.05), respectively. Transient K+ outward currents were even more inhibited, by 25.8 +/- 4.8%, from 9.8 +/- 3.1 to 7.3 +/- 2.1 pA/pF (n = 5; P < 0.05) at 1 MAC isoflurane, whereas xenon evoked only a slight (albeit significant) inhibition, by 6.1 +/- 3.7%, from 8.2 +/- 6.0 to 7.7 +/- 5.8 pA/pF (n = 10; P < 0.05). Isoflurane and xenon did not affect sustained potassium currents. All effects of the anesthetics were fully reversible after washout. CONCLUSIONS: Halothane and isoflurane exhibited considerable inhibitory effects on voltage-gated cardiac Ca2+ and K+ currents important for the duration of action potentials and the repolarization. Xenon, in contrast, did not affect Ca2+ currents and only slightly inhibited transient K+ outward currents, in line with the almost absent cardiac side effects of the noble gas.     

Protein Tyrosine Kinase-Dependent Modulation of Isoflurane Effects on Cardiac Sarcolemmal KATP Channel

Background: Cardiac adenosine triphosphate-sensitive potassium (KATP) channels and protein tyrosine kinases (PTKs) are mediators of ischemic preconditioning, but the interaction of both and a role in myocardial protection afforded by volatile anesthetics have not been defined.

Methods: Whole cell and single channel patch clamp techniques were used to investigate the effects of isoflurane and the PTK inhibitor genistein on the cardiac sarcolemmal KATP channel in acutely dissociated guinea pig ventricular myocytes.

Results: At 0.5 mm internal ATP, genistein (50 [mu]m) elicited whole cell KATP current (22.5 +/- 7.9 pA/pF). Genistein effects were concentration-dependent, with an EC50 of 32.3 +/- 1.4 [mu]m. Another PTK inhibitor, tyrphostin B42, had a similar effect. The inactive analog of genistein, daidzein (50 [mu]m), did not elicit KATP current. Isoflurane (0.5 mm) increased genistein (35 [mu]m)- activated whole cell KATP current from 14.5 +/- 3.1 to 32.5 +/- 6.6 pA/pF. Stimulation of receptor PTKs with epidermal growth factor, nerve growth factor, or insulin attenuated genistein and isoflurane effects, and the protein tyrosine phosphatase inhibitor orthovanadate (1 mm) prevented their actions on KATP current. In excised inside-out membrane patches, and at fixed 0.2 mm internal ATP, genistein (50 [mu]m) increased channel open probability from 0.053 +/- 0.016 to 0.183 +/- 0.039, but isoflurane failed to further increase open probability (0.162 +/- 0.051) of genistein-activated channels. However, applied in the presence of genistein and protein tyrosine phosphatase 1B (1 [mu]g/ml), isoflurane significantly increased open probability to 0.473 +/- 0.114.     

Effects of the Anesthetic Gases Xenon, Halothane, and Isoflurane on Calcium and Potassium Currents in Human Atrial Cardiomyocytes

Background: Negative inotropic and proarrhythmic side effects on the heart are well known for the volatile anesthetics halothane and isoflurane but not for the noble gas xenon. We investigated the effects of halothane, isoflurane, and xenon on calcium and potassium currents in human atrial myocytes to elucidate the cellular and molecular basis of their cardiac actions.

Methods: Atrial myocytes were prepared from the right auricles obtained from patients undergoing heart surgery. Ion currents were measured with the whole cell patch clamp technique during superfusion of the cells with solutions that contained halothane, isoflurane, or xenon at concentrations corresponding to their respective minimum alveolar concentration (MAC); gas concentrations were determined with the head space-gas chromatography/mass spectrometry/selected ion monitoring method.

Results: L-type calcium currents were significantly depressed by 31.9 +/- 4.1%, from -1.8 +/- 0.3 to -1.2 +/- 0.4 picoampere (pA)/picofarad (pF) (n = 4;P < 0.05) at 1 MAC halothane and by 21.7 +/- 9.2%, from -1.6 +/- 0.7 to -1.2 +/- 0.6 pA/pF (n = 7;P < 0.05) at 1 MAC isoflurane, but not affected by 70% xenon (1 MAC). Inwardly rectifying potassium currents were not influenced by any anesthetic. Halothane (1 MAC) significantly inhibited the transient as well as the sustained part of voltage-gated potassium outward currents, by 19.4 +/- 6.7%, from 6.7 +/- 2.1 to 5.4 +/- 1.6 pA/pF (n = 8;P < 0.05), and by 8.6 +/- 4.8%, from 5.5 +/- 1.7 to 5.0 +/- 1.5 pA/pF (n = 8;P < 0.05), respectively. Transient K+ outward currents were even more inhibited, by 25.8 +/- 4.8%, from 9.8 +/- 3.1 to 7.3 +/- 2.1 pA/pF (n = 5;P < 0.05) at 1 MAC isoflurane, whereas xenon evoked only a slight (albeit significant) inhibition, by 6.1 +/- 3.7%, from 8.2 +/- 6.0 to 7.7 +/- 5.8 pA/pF (n = 10;P < 0.05). Isoflurane and xenon did not affect sustained potassium currents. All effects of the anesthetics were fully reversible after washout.     

Opening of mitochondrial ATP-sensitive potassium channels enhances cardioplegic protection

BACKGROUND: Mitochondrial and sarcolemmal ATP-sensitive potassium channels have been implicated in cardioprotection; however, the role of these channels in magnesium-supplemented potassium (K/Mg) cardioplegia during ischemia or reperfusion is unknown. METHODS: Rabbit hearts (n = 76) were used for Langendorff perfusion. Sham hearts were perfused for 180 minutes. Global ischemia hearts received 30 minutes of global ischemia and 120 minutes of reperfusion. K/Mg hearts received cardioplegia before ischemia. The role of ATP-sensitive potassium channels in K/Mg cardioprotection during ischemia and reperfusion was investigated, separately using the selective mitochondrial ATP sensitive potassium and channel blocker, 5-hydroxydecanoate, and the selective sarcolemmal ATP-sensitive potassium channel blocker HMR1883. Separate studies were performed using the selective mitochondrial ATP-sensitive potassium channel opener, diazoxide, and the nonselective ATP-sensitive potassium channel opener pinacidil. RESULTS: Infarct size was 1.9%+/-0.4% in sham, 3.7%+/-0.5% in K/Mg, and 27.8%+/-2.4% in global ischemia hearts (p < 0.05 versus K/Mg). Left ventricular peak-developed pressure (percent of equilibrium) at the end of 120 minutes of reperfusion was 91%+/-6% in sham, 92% +/-2% in K/Mg, and 47%+/-6% in global ischemia (p < 0.05 versus K/Mg). Blockade of sarcolemmal ATP-sensitive potassium channels in K/Mg hearts had no effect on infarct size or left ventricular peak-developed pressure. However, blockade of mitochondrial ATP-sensitive potassium channels before ischemia significantly increased infarct size to 23%+/-2% in K/Mg hearts (p < 0.05 versus K/Mg; no statistical significance [NS] as compared to global ischemia) and significantly decreased left ventricular peak-developed pressure to 69%+/-4% (p < 0.05 versus K/Mg). Diazoxide when added to K/Mg cardioplegia significantly decreased infarct size to 1.5%+/-0.4% (p < 0.05 versus K/Mg). CONCLUSIONS: The cardioprotection afforded by K/Mg cardioplegia is modulated by mitochondrial ATP-sensitive potassium channels. Diazoxide when added to K/Mg cardioplegia significantly reduces infarct size, suggesting that the opening of mitochondrial ATP-sensitive potassium channels with K/Mg cardioplegic protection would allow for enhanced myocardial protection in cardiac operations.     

K(ATP) channel opener protects neonatal rabbit heart better than St. Thomas' solution

OBJECTIVES: Myocardial protection with ATP-sensitive potassium channel (K(ATP) channel) openers is as effective as St. Thomas' cardioplegia (StTCP) in adult rabbit hearts. This study compares the effectiveness of the K(ATP) channel opener pinacidil to StTCP in protecting neonatal rabbit hearts exposed to global ischemia. METHODS: Seventeen neonatal rabbit hearts (7-9 days old) perfused with Krebs-Henseleit buffer (KHB) on a Langendorff apparatus underwent 90 min of normothermic ischemia. Six (ischemia control) received no pretreatment before or during ischemia. Six others (pinacidil) received a 3-min infusion of 50 microM pinacidil in KHB without StTCP at the onset of ischemia. Five others (StTCP) received a 3-min infusion of StTCP at the onset of ischemia. After 60 min of KHB reperfusion, recovery of left ventricular (LV) performance and coronary flow (CF) were measured and compared to preischemia. A paired t test was used for comparison between drug-treated and untreated groups. RESULTS: Pinacidil-treated hearts had significantly better recovery of left ventricular developed pressure (47 +/- 3.8 mmHg vs 32 +/- 2.5 mmHg, P < 0.05), contractility (+dP/dt(max); 885.4 +/- 74 mmHg vs 643.7 +/- 65 mmHg, P < 0.05), left ventricular end diastolic pressure (10.5 +/- 0.9 mmHg vs 17.4 +/- 1.2 mmHg P < 0.05), compliance (-dP/dt(max); 994.2 +/- 86 mmHg vs 673.6 +/- 69 mmHg, P < 0.05), and CF (5.9 +/- 0.4 ml/min vs 4.2 +/- 0.2 ml/min, P < 0.05) compared to ischemic control. StTCP only improved the recovery of -dP/dt(max) (877.4 +/- 73 mmHg/s vs 673.6 +/- 69 mmHg/s, P < 0.05) and CF (5.7 +/- 0.3 ml/min vs 4.2 +/- 0.2 ml/min, P < 0.05) compared to control. CONCLUSIONS: Pinacidil pretreatment provided superior recovery of systolic performance compared to St. Thomas' cardioplegia solution in neonatal hearts. Myocardial protection by pretreatment with the K(ATP) channel opener pinacidil may be a new strategy for myocardial protection during pediatric cardiac surgery.     

Isoflurane decreases ATP sensitivity of guinea pig cardiac sarcolemmal KATP channel at reduced intracellular pH

BACKGROUND: Volatile anesthetics can protect the myocardium against ischemic injury by opening the adenosine triphosphate (ATP)-sensitive potassium (K(atp)) channels. However, direct evidence for anesthetic-channel interaction is still limited, and little is known about the role K(atp) channel modulators play in this effect. Because pH is one of the regulators of K(atp) channels, the authors tested the hypothesis that intracellular pH (pHi) modulates the direct interaction of isoflurane with the cardiac K(atp) channel. METHODS: The effects of isoflurane on sarcolemmal K(atp) channels were investigated at pHi 7.4 and pHi 6.8 in excised inside-out membrane patches from ventricular myocytes of guinea pig hearts. RESULTS: At pHi 7.4, intracellular ATP (1-1,000 microm) inhibited K(atp) channels and decreased channel open probability (Po) in a concentration-dependent manner with an IC(50) of 8 +/- 1.5 microm, and isoflurane (0.5 mm) either had no effect or decreased channel activity. Lowering pHi from 7.4 to 6.8 enhanced channel opening by increasing Po and reduced channel sensitivity to ATP, with IC shifting from 8 +/- 1.2 to 45 +/- 5.6 microm. When applied to the channels activated at pHi 6.8, isoflurane (0.5 mm) increased Po and further reduced ATP sensitivity, shifting IC(50) to 110 +/- 10.0 microm. CONCLUSIONS: Changes in pHi appear to modulate isoflurane interaction with the cardiac K(atp) channel. At pHi 6.8, which itself facilitates channel opening, isoflurane enhances channel activity by increasing Po and reduces sensitivity to inhibition by ATP without changing the unitary amplitude of single channel current or the conductance. These results support the hypothesis of direct isoflurane-K(atp) channel interaction that may play a role in cardioprotection by volatile anesthetics.     

Isoflurane-induced facilitation of the cardiac sarcolemmal K(ATP) channel

BACKGROUND: Volatile anesthetics have cardioprotective effects that mimic ischemic preconditioning, including the involvement of adenosine triphosphate-sensitive potassium (K(ATP)) channels. However, evidence for a direct effect of volatile anesthetic on the K(ATP) channel is limited. In this study, the effects of isoflurane on the cardiac sarcolemmal K(ATP) channel were investigated. METHODS: Single ventricular myocytes were enzymatically isolated from guinea pig hearts. Whole cell and single-channel configurations, specifically the cell-attached and inside-out patch mode, of the patch clamp technique were used to monitor sarcolemmal K(ATP) channel current. RESULTS: In the cell-attached patch configuration, 2,4-dinitrophenol (150 microm) opened the sarcolemmal K(ATP) channel. Isoflurane (0.5 mm) further increased channel open probability and the number of active channels in the patch. In contrast, in the inside-out patch experiments, isoflurane had no significant effect on the K(ATP) channel activated by low ATP (0.2-0.5 mm). In addition, isoflurane had no effect on the K(ATP) channel when activated by adenosine diphosphate, adenosine + guanosine triphosphate, bimakalim, and 2,4-dinitrophenol under inside-out patch configurations. When K(ATP) current was monitored in the whole cell mode, isoflurane alone was unable to elicit channel opening. However, during sustained protein kinase C activation by 12,13-dibutyrate, isoflurane activated the K(ATP) current that was sensitive to glibenclamide. In contrast, isoflurane had no effect on the K(ATP) channel activated by 12,13-dibutyrate in a cell-free environment. CONCLUSIONS: Isoflurane facilitated the opening of the sarcolemmal K(ATP) channel in the intact cell, but not in an excised, inside-out patch. The isoflurane effect was not due to a direct interaction with the K(ATP) channel protein, but required an intracellular component, likely including the translocation of specific protein kinase C isoforms. This suggests that the sarcolemmal K(ATP) channel may have a significant role in anesthetic-induced preconditioning.     

The role of mitochondrial and sarcolemmal K(ATP) channels in canine ethanol-induced preconditioning in vivo

Chronic consumption of small doses of ethanol protects myocardium from ischemic injury. We tested the hypothesis that mitochondrial and sarcolemmal adenosine triphosphate-dependent potassium (K(ATP)) channels mediate these beneficial effects. Dogs (n = 76) were fed with ethanol (1.5 g/kg) or water mixed with dry food bid for 6 or 12 wk, fasted overnight before experimentation, and instrumented for measurement of hemodynamics. Dogs received intracoronary saline (vehicle), 5-hydroxydecanoate (a mitochondrial K(ATP) channel antagonist; 6.75 mg/kg over 45 min), or HMR-1098 (a sarcolemmal K(ATP) channel antagonist; 45 microg/kg over 45 min) and were subjected to a 60 min coronary artery occlusion followed by 3 h of reperfusion. A final group of dogs was pretreated with ethanol and chow for 6 wk before occlusion and reperfusion. Myocardial infarct size and transmural coronary collateral blood flow were measured with triphenyltetrazolium chloride staining and radioactive microspheres, respectively. The area at risk of infarction was similar between groups. A 12-wk pretreatment with ethanol significantly reduced infarct size to 13% +/- 2% (mean +/- SEM; n = 8) of the area at risk compared with control experiments (25% +/- 2%; n = 8), but a 6-wk pretreatment did not (21% +/- 2%; n = 8). 5-hydroxydecanoate and HMR-1098 abolished the protective effects of 12-wk ethanol pretreatment (24% +/- 2% and 29% +/- 3%, respectively; n = 8 for each group) but had no effect in dogs that did not receive ethanol (22% +/- 2% and 23% +/- 4%, respectively; n = 8 for each group). No differences in hemodynamics or transmural coronary collateral blood flow were observed between the groups. The results indicate that mitochondrial and sarcolemmal K(ATP) channels mediate ethanol-induced preconditioning in dogs independent of alterations in systemic hemodynamics or coronary collateral blood flow. IMPLICATIONS: Mitochondrial and sarcolemmal K(ATP) channels mediate ethanol-induced preconditioning independent of alterations in systemic hemodynamics or coronary collateral perfusion in vivo.