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.     

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).     

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).     

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).     

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.     

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 (KATP) channels. However, direct evidence for anesthetic-channel interaction is still limited, and little is known about the role KATP channel modulators play in this effect. Because pH is one of the regulators of KATP channels, the authors tested the hypothesis that intracellular pH (pHi) modulates the direct interaction of isoflurane with the cardiac KATP channel.

Methods: The effects of isoflurane on sarcolemmal KATP 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 [mu]m) inhibited KATP channels and decreased channel open probability (Po) in a concentration-dependent manner with an IC50 of 8 +/- 1.5 [mu]m, 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 IC50 shifting from 8 +/- 1.2 to 45 +/- 5.6 [mu]m. When applied to the channels activated at pHi 6.8, isoflurane (0.5 mm) increased Po and further reduced ATP sensitivity, shifting IC50 to 110 +/- 10.0 [mu]m.     

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.     

Isoflurane-induced Facilitation of the Cardiac Sarcolemmal KATP Channel

Background: Volatile anesthetics have cardioprotective effects that mimic ischemic preconditioning, including the involvement of adenosine triphosphate-sensitive potassium (KATP) channels. However, evidence for a direct effect of volatile anesthetic on the KATP channel is limited. In this study, the effects of isoflurane on the cardiac sarcolemmal KATP 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 KATP channel current.

Results: In the cell-attached patch configuration, 2,4-dinitrophenol (150 [mu]m) opened the sarcolemmal KATP 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 KATP channel activated by low ATP (0.2-0.5 mm). In addition, isoflurane had no effect on the KATP channel when activated by adenosine diphosphate, adenosine + guanosine triphosphate, bimakalim, and 2,4-dinitrophenol under inside-out patch configurations. When KATP 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 KATP current that was sensitive to glibenclamide. In contrast, isoflurane had no effect on the KATP channel activated by 12,13-dibutyrate in a cell-free environment.     

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.     

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.     

Mechanism of Cardiac Sarcolemmal Adenosine Triphosphate-sensitive Potassium Channel Activation by Isoflurane in a Heterologous Expression System

Background: Activation of the cardiac sarcolemmal adenosine triphosphate-sensitive potassium (KATP) channel during metabolic stress initiates cellular events that preserve cardiac performance. Previous studies showed that halogenated anesthetics prime KATP channels under whole cell voltage clamp and act in intracellular pH (pHi)-dependent manner on KATP channels in excised membrane patches. However, it is not known how halogenated anesthetics interact with these channels.

Methods: The authors evaluated the effect of pHi and isoflurane on the KATP channel subunits, the pore-forming inward rectifier Kir6.2, and the regulatory sulfonylurea receptor SUR2A, using HEK293 cells as a heterologous expression system. Single channel activity was recorded in the inside-out patch configuration.

Results: At pHi 7.4, isoflurane had negligible effect on activity of wild-type Kir6.2/SUR2A, but at pHi 6.8, the channel open probability was increased by isoflurane (0.177 +/- 0.077 to 0.364 +/- 0.164). By contrast, the open probability of truncated Kir6.2[DELTA]C26, which forms a functional channel without SUR2A, was attenuated by isoflurane at both pHi 7.4 and pHi 6.8. Coexpression of Kir6.2[DELTA]C26 with SUR2A restored pHi sensitivity of channel activation by isoflurane. Site-directed mutagenesis within the Walker motifs of SUR2A abolished isoflurane activation of KATP channel at pHi 6.8. In addition, the pancreatic-type channels expressing sulfonylurea receptor SUR1 could not be activated by isoflurane.     

In Vitro Electrophysiologic Effects of Morphine in Rabbit Ventricular Myocytes

Background: Morphine is widely used in patients undergoing surgical operations and is also reported to mediate cardioprotection of preconditioning. The current study determined effects of morphine at therapeutic to pharmacologic concentrations on cardiac action potential, L-type Ca2+ current (ICa.L), delayed rectifier K+ current (IK), and inward rectifier K+ current (IK1) in isolated rabbit ventricular myocytes.

Methods: Ventricular myocytes were enzymatically isolated from rabbit hearts. Action potential and membrane currents were recorded in current and voltage clamp modes.

Results: Morphine at concentrations from 0.01 to 1 [mu]m significantly prolonged cardiac action potential, and at 0.1 and 1 [mu]m slightly but significantly hyperpolarized the resting membrane potential. In addition, morphine at 0.1 [mu]m significantly augmented ICa.L (at +10 mV) from 5.9 +/- 1.9 to 7.3 +/- 1.7 pA/pF (by 23%; P < 0.05 vs. control) and increased IK1 (at -60 mV) from 2.8 +/- 1.0 to 3.5 +/- 0.9 pA/pF (by 27%; P < 0.05 vs. control). Five [mu]m naltrindole (a selective [delta]-opioid receptor antagonist) or 5 [mu]m norbinaltorphimine (a selective [kappa]-opioid receptor antagonist) prevented the increase in ICa.L induced by morphine, but 5 [mu]m CTOP (a selective [mu]-opioid receptor antagonist) did not. The three types of opioid antagonists did not affect the augmentation of IK1 by morphine. Morphine had no effect on IK.     

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 (KATP) channels are considered essential in preconditioning mechanism. However, it is unclear whether KATP 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 [mu]m H2O2 and 100 [mu]m 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 KATP 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 KATP 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 KATP 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.     

Lidocaine and Mexiletine Inhibit Mitochondrial Oxidation in Rat Ventricular Myocytes

Background: Accumulating evidence suggests that mitochondrial rather than sarcolemmal adenosine triphosphate-sensitive K+ (KATP) channels may have an important role in the protection of myocardium during ischemia. Because both lidocaine and mexiletine are frequently used antiarrhythmic drugs during myocardial ischemia, it is important to investigate whether they affect mitochondrial KATP channel activities.

Methods: Male Wistar rats were anesthetized with ether. Single, quiescent ventricular myocytes were dispersed enzymatically. The authors measured flavoprotein fluorescence to evaluate mitochondrial redox state. Lidocaine or mexiletine was applied after administration of diazoxide (25 [mu]m), a selective mitochondrial KATP channel opener. The redox signal was normalized to the baseline flavoprotein fluorescence obtained during exposure to 2,4-dinitrophenol, a protonophore that uncouples respiration from ATP synthesis and collapses the mitochondrial potential.

Results: Diazoxide-induced oxidation of flavoproteins and the redox changes were inhibited by 5-hydroxydecanoic acid, a selective mitochondrial KATP channel blocker, suggesting that flavoprotein fluorescence can be used as an index of mitochondrial oxidation mediated by mitochondrial KATP channels. Lidocaine (10-3 to 10 mm) and mexiletine (10-3 to 10 mm) reduced oxidation of the mitochondrial matrix in a dose-dependent manner with an EC50 of 98 +/- 63 [mu]m for lidocaine and 107 +/- 89 [mu]m for mexiletine.     

Clinically Relevant Concentrations of Propofol Have No Effect on Adenosine Triphosphate-sensitive Potassium Channels in Rat Ventricular Myocytes

Background: Activation of adenosine triphosphate-sensitive potassium (KATP) channels produces cardioprotective effects during ischemia. Because propofol is often used in patients who have coronary artery disease undergoing a wide variety of surgical procedures, it is important to evaluate the direct effects of propofol on KATP channel activities in ventricular myocardium during ischemia.

Methods: The effects of propofol (0.4-60.1 [mu]g/ml) on both sarcolemmal and mitochondrial KATP channel activities were investigated in single, quiescent rat ventricular myocytes. Membrane currents were recorded using cell-attached and inside-out patch clamp configurations. Flavoprotein fluorescence was measured to evaluate mitochondrial oxidation mediated by mitochondrial KATP channels.

Results: In the cell-attached configuration, open probability of KATP channels was reduced by propofol in a concentration-dependent manner (EC50 = 14.2 [mu]g/ml). In the inside-out configurations, propofol inhibited KATP channel activities without changing the single-channel conductance (EC50 = 11.4 [mu]g/ml). Propofol reduced mitochondrial oxidation in a concentration-dependent manner with an EC50 of 14.6 [mu]g/ml.     

Isoflurane-induced Dilation of Porcine Coronary Microvessels Is Endothelium Dependent and Inhibited by Glibenclamide

Background: Isoflurane has been reported to cause dose-dependent constriction in isolated coronary microvessels. However, these results are inconsistent with data from in situ and in vivo heart preparations which show that isoflurane dilates the coronary vasculature. To clarify the direct effects of isoflurane on coronary tone, we measured the response of isolated porcine resistance arterioles (ID, 75 +/- 4.0 [mu]m; range, 41-108 [mu]m) to isoflurane in the presence and absence of adenosine triphosphate-sensitive and Ca2+-activated potassium channel blockers and also after endothelial removal.

Methods: Subepicardial arterioles were isolated, cannulated, and pressurized to 45 mmHg without flow in a 37[degrees]C vessel chamber filled with MOPS buffer (pH = 7.4). After all vessels developed spontaneous (intrinsic) tone, dose-dependent (0.17-0.84 mm; approximately 0.5-2.5 minimum alveolar concentration) isoflurane-mediated effects on vessel ID were studied in the presence and absence of extraluminal glibenclamide (1 [mu]m; an adenosine triphosphate-sensitive channel blocker) or iberiotoxin (100 nm; a Ca2+-activated potassium channel blocker) or before and after endothelial denudation using the nonionic detergent CHAPS (0.4%). Vessel ID was measured using an inverted microscope and videomicrometer, and vasomotor responses were analyzed by normalizing changes in arteriole ID to the dilation observed after exposure to 10-4 m sodium nitroprusside, which causes maximal dilation.

Results: Isoflurane caused dose-dependent dilation of all coronary arterioles. This vasodilation was 6.0 +/- 0.7 [mu]m at an isoflurane concentration of 0.16 mm (approximately 0.5 minimum alveolar concentration) and 25.3 +/- 2.1 [mu]m at 0.75 mm (approximately 2.5 minimum alveolar concentration). These values represent 18.1 +/- 1.7% and 74.1 +/- 3.3%, respectively, of that observed with 10-4 sodium nitroprusside (34 +/- 3 [mu]m). Glibenclamide, but not iberiotoxin, exposure affected arteriolar dilation in response to isoflurane. Glibenclamide caused a downward displacement of the isoflurane dose-response curve, reducing isoflurane-mediated dilation by an average of 36%. Denuded arterioles showed a marked (approximately 70%) reduction in their ability to dilate in response to isoflurane.     

Mechanisms of Desflurane-induced Preconditioning in Isolated Human Right Atria In Vitro

Background: The authors examined the role of adenosine triphosphate-sensitive potassium (KATP) channels, adenosine A1 receptor, and [alpha] and [beta] adrenoceptors in desflurane-induced preconditioning in human myocardium, in vitro.

Methods: The authors recorded isometric contraction of human right atrial trabeculae suspended in oxygenated Tyrode's solution (34[degrees]C; stimulation frequency, 1 Hz). Before a 30-min anoxic period, 3, 6, and 9% desflurane was administered during 15 min. Desflurane, 6%, was also administered in the presence of 10 [mu]m glibenclamide, a KATP channels antagonist; 10 [mu]m HMR 1098, a sarcolemmal KATP channel antagonist; 800 [mu]m 5-hydroxy-decanoate (5-HD), a mitochondrial KATP channel antagonist; 1 [mu]m phentolamine, an [alpha]-adrenoceptor antagonist; 1 [mu]m propranolol, a [beta]-adrenoceptor antagonist; and 100 nm 8-cyclopentyl-1,3-dipropylxanthine (DPX), the adenosine A1 receptor antagonist. Developed force at the end of a 60-min reoxygenation period was compared (mean +/- SD).

Results: Desflurane at 3% (95 +/- 13% of baseline), 6% (86 +/- 6% of baseline), and 9% (82 +/- 6% of baseline) enhanced the recovery of force after 60 min of reoxygenation as compared with the control group (50 +/- 11% of baseline). Glibenclamide (60 +/- 12% of baseline), 5-HD (57 +/- 21% of baseline), DPX (63 +/- 19% of baseline), phentolamine (56 +/- 20% of baseline), and propranolol (63 +/- 13% of baseline) abolished desflurane-induced preconditioning. In contrast, HMR 1098 (85 +/- 12% of baseline) did not modify desflurane-induced preconditioning.     

Isoflurane Preconditions Myocardium Against Infarction via Activation of Inhibitory Guanine Nucleotide Binding Proteins

Background: Isoflurane-induced myocardial protection during ischemia is mediated by adenosine triphosphate-regulated potassium (KATP) channels; however, the intracellular signal transduction cascade responsible for this process has been incompletely evaluated. The authors tested the hypothesis that isoflurane reduces myocardial infarct size through a Gi protein-mediated process.

Methods: Forty-eight hours after pretreatment with vehicle (0.9% saline) or the Gi protein inhibitor pertussis toxin (10 [mu]g/kg intravenously), barbiturate-anesthetized dogs (n = 43) were instrumented for measurement of aortic and left ventricular pressures and maximum rate of increase of left ventricular pressure. All dogs were subjected to a 60-min left anterior descending coronary artery occlusion followed by 3-h reperfusion. In four separate groups, vehicle- or pertussis toxin-pretreated dogs were studied with or without administration of 1 minimum alveolar concentration isoflurane. In two additional groups, dogs received the direct KATP channel agonist nicorandil (100 [mu]g/kg bolus and 10 [mu]g [middle dot] kg-1 [middle dot] min-1 intravenous infusion) in the presence or absence of pertussis toxin pretreatment. Myocardial perfusion and infarct size were measured with radioactive microspheres and triphenyltetrazolium staining, respectively.

Results: Isoflurane significantly (P < 0.05) decreased infarct size to 7 +/- 2% of the area at risk compared with control experiments (26 +/- 2%). Pertussis toxin pretreatment alone had no effects on myocardial infarct size (31 +/- 4%) but blocked the beneficial effects of isoflurane (21 +/- 3%). Nicorandil decreased infarct size (11 +/- 2%), but, in contrast to isoflurane, this effect was independent of pertussis toxin pretreatment (11 +/- 1%).     

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.     

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 (K(ATP)) channels. The authors have previously shown that isoflurane enhances sensitivity of the sarcolemmal K(ATP) 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 K(ATP) channel current (I(KAPT)). 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 K(ATP) channels was tested using a blocker of these channels, 5-hydroxydecanoate. RESULTS: The mean density (+/- SEM) of I(KAPT) elicited by pinacidil (20 microM) was 18.9 +/- 1.8 pA/pF (n = 11). In the presence of isoflurane (0.55 mM), the density of pinacidil-activated I(KAPT) 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 I(KAPT) 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 microM N-acetyl-L-cysteine, respectively. Concurrent application of isoflurane and carnosine (100 microM), superoxide dismutase (100 U/ml), or catalase (100 U/ml) attenuated the densities of I(KAPT) to 27.9 +/- 2.6, 27.2 +/- 2.9, and 25.9 +/- 2.2 pA/pF, respectively. None of the scavengers affected activation of I(KAPT) by pinacidil alone. 5-Hydroxydecanoate (100 microM) did not alter the sensitization effect by isoflurane, and the density of I(KAPT) in this group was 37.1 +/- 3.8 pA/pF (n= 6). CONCLUSION: These results suggest that reactive oxygen species contribute to the mechanism by which isoflurane sensitizes the cardiac sarcolemmal K(ATP) channel to the opener, pinacidil.