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 microg/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 microg/kg bolus and 10 microg x kg-1 x 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%). CONCLUSION: Isoflurane reduces myocardial infarct size by a Gi protein-mediated mechanism in vivo.     

Sarcolemmal and Mitochondrial Adenosine Triphosphate- dependent Potassium Channels: Mechanism of Desflurane-induced Cardioprotection

Background: Volatile anesthetic-induced preconditioning is mediated by adenosine triphosphate-dependent potassium (KATP) channels; however, the subcellular location of these channels is unknown. The authors tested the hypothesis that desflurane reduces experimental myocardial infarct size by activation of specific sarcolemmal and mitochondrial KATP channels.

Methods: Barbiturate-anesthetized dogs (n = 88) were acutely instrumented for measurement of aortic and left ventricular pressures. All dogs were subjected to a 60-min left anterior descending coronary artery occlusion followed by 3-h reperfusion. In four separate groups, dogs received vehicle (0.9% saline) or the nonselective KATP channel antagonist glyburide (0.1 mg/kg intravenously) in the presence or absence of 1 minimum alveolar concentration desflurane. In four additional groups, dogs received 45-min intracoronary infusions of the selective sarcolemmal (HMR 1098; 1 [mu]g [middle dot] kg-1 [middle dot] min-1) or mitochondrial (5-hydroxydecanoate [5-HD]; 150 [mu]g [middle dot] kg-1 [middle dot] min-1) KATP channel antagonists in the presence or absence of desflurane. Myocardial perfusion and infarct size were measured with radioactive microspheres and triphenyltetrazolium staining, respectively.

Results: Desflurane significantly (P < 0.05) decreased infarct size to 10 +/- 2% (mean +/- SEM) of the area at risk as compared with control experiments (25 +/- 3% of area at risk). This beneficial effect of desflurane was abolished by glyburide (25 +/- 2% of area at risk). Glyburide (24 +/- 2%), HMR 1098 (21 +/- 4%), and 5-HD (24 +/- 2% of area at risk) alone had no effects on myocardial infarct size. HMR 1098 and 5-HD abolished the protective effects of desflurane (19 +/- 3% and 22 +/- 2% of area at risk, respectively).     

Isoflurane Preconditions Myocardium against Infarction via Release of Free Radicals

Background: Isoflurane exerts cardioprotective effects that mimic the ischemic preconditioning phenomenon. Generation of free radicals is implicated in ischemic preconditioning. The authors investigated whether isoflurane-induced preconditioning may involve release of free radicals.

Methods: Sixty-one [alpha]-chloralose-anesthetized rabbits were instrumented for measurement of left ventricular (LV) pressure (tip-manometer), cardiac output (ultrasonic flowprobe), and myocardial infarct size (triphenyltetrazolium staining). All rabbits were subjected to 30 min of occlusion of a major coronary artery and 2 h of subsequent reperfusion. Rabbits of all six groups underwent a treatment period consisting of either no intervention for 35 min (control group, n = 11) or 15 min of isoflurane inhalation (1 minimum alveolar concentration end-tidal concentration) followed by a 10-min washout period (isoflurane group, n = 12). Four additional groups received the radical scavenger N-(2-mercaptoproprionyl)glycine (MPG; 1 mg [middle dot] kg-1 [middle dot] min-1) or Mn(III)tetrakis(4-benzoic acid)porphyrine chloride (MnTBAP; 100 [mu]g [middle dot] kg-1 [middle dot] min-1) during the treatment period with (isoflurane + MPG; n = 11; isoflurane + MnTBAP, n = 9) or without isoflurane inhalation (MPG, n = 11; MnTBAP, n = 7).

Results: Hemodynamic baseline values were not significantly different between groups (LV pressure, 97 +/- 17 mmHg [mean +/- SD]; cardiac output, 228 +/- 61 ml/min). During coronary artery occlusion, LV pressure was reduced to 91 +/- 17% of baseline and cardiac output to 94 +/- 21%. After 2 h of reperfusion, recovery of LV pressure and cardiac output was not significantly different between groups (LV pressure, 83 +/- 20%; cardiac output, 86 +/- 23% of baseline). Infarct size was reduced from 49 +/- 17% of the area at risk in controls to 29 +/- 19% in the isoflurane group (P = 0.04). MPG and MnTBAP themselves had no effect on infarct size (MPG, 50 +/- 14%; MnTBAP, 56 +/- 15%), but both abolished the preconditioning effect of isoflurane (isoflurane + MPG, 50 +/- 24%, P = 0.02; isoflurane + MnTBAP, 55 +/- 10%, P = 0.001).     

Cyclooxygenase-2 Mediates Ischemic, Anesthetic, and Pharmacologic Preconditioning In Vivo

Background: Cyclooxygenase-2 (COX-2) mediates the late phase of ischemic preconditioning (IPC), but whether this enzyme modulates early IPC, anesthetic-induced preconditioning (APC), or other forms of pharmacologic preconditioning (PPC) is unknown. The authors tested the hypothesis that COX-2 is an essential mediator of IPC, APC, and PPC in vivo.

Methods: Barbiturate-anesthetized dogs (n = 91) were instrumented for measurement of hemodynamics and randomly assigned to receive IPC (four 5-min coronary occlusions interspersed with 5-min reperfusions), APC (1.0 minimum alveolar concentration of isoflurane for 30 min), or PPC (selective mitochondrial KATP channel opener diazoxide, 2.5 mg/kg intravenous) in the presence or absence of pretreatment with oral aspirin (650 mg), the selective COX-2 inhibitor celecoxib (200 mg), or acetaminophen (500 mg) administered 24, 12, and 2 h before experimentation in 12 separate experimental groups. All dogs were subjected to a 60-min coronary artery occlusion followed by 3 h of reperfusion. Myocardial infarct size and coronary collateral blood flow were quantified with triphenyltetrazolium staining and radioactive microspheres, respectively. Myocardial 6-keto-prostaglandin F1[alpha], a stable metabolite of prostacyclin, was measured (enzyme immunoassay) in separate experiments (n = 8) before and after isoflurane administration, in the presence or absence of celecoxib.

Results: No significant differences in baseline hemodynamics or the left ventricular area at risk for infarction were observed between groups. IPC, isoflurane, and diazoxide all decreased myocardial infarct size (9 +/- 1, 12 +/- 2, and 11 +/- 1%, respectively) as compared with control (30 +/- 1%). Celecoxib alone had no effect on infarct size (26 +/- 3%) but abolished IPC (30 +/- 3%), APC (30 +/- 3%), and PPC (26 +/- 1%). Aspirin (24 +/- 3%) and acetaminophen alone (29 +/- 2%) did not alter infarct size or abolish APC-induced protection (18 +/- 1 and 19 +/- 1%, respectively). Isoflurane increased myocardial 6-keto-prostaglandin F1[alpha] to 463 +/- 267% of baseline in the absence but not in the presence (94 +/- 13%) of celecoxib.     

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.     

Sarcolemmal and mitochondrial adenosine triphosphate- dependent potassium channels: mechanism of desflurane-induced cardioprotection

BACKGROUND: Volatile anesthetic-induced preconditioning is mediated by adenosine triphosphate-dependent potassium (KATP) channels; however, the subcellular location of these channels is unknown. The authors tested the hypothesis that desflurane reduces experimental myocardial infarct size by activation of specific sarcolemmal and mitochondrial KATP channels. METHODS: Barbiturate-anesthetized dogs (n = 88) were acutely instrumented for measurement of aortic and left ventricular pressures. All dogs were subjected to a 60-min left anterior descending coronary artery occlusion followed by 3-h reperfusion. In four separate groups, dogs received vehicle (0.9% saline) or the nonselective KATP channel antagonist glyburide (0.1 mg/kg intravenously) in the presence or absence of 1 minimum alveolar concentration desflurane. In four additional groups, dogs received 45-min intracoronary infusions of the selective sarcolemmal (HMR 1098; 1 microg. kg-1. min-1) or mitochondrial (5-hydroxydecanoate [5-HD]; 150 microg. kg-1. min-1) KATP channel antagonists in the presence or absence of desflurane. Myocardial perfusion and infarct size were measured with radioactive microspheres and triphenyltetrazolium staining, respectively. RESULTS: Desflurane significantly (P < 0.05) decreased infarct size to 10 +/- 2% (mean +/- SEM) of the area at risk as compared with control experiments (25 +/- 3% of area at risk). This beneficial effect of desflurane was abolished by glyburide (25 +/- 2% of area at risk). Glyburide (24 +/- 2%), HMR 1098 (21 +/- 4%), and 5-HD (24 +/- 2% of area at risk) alone had no effects on myocardial infarct size. HMR 1098 and 5-HD abolished the protective effects of desflurane (19 +/- 3% and 22 +/- 2% of area at risk, respectively). CONCLUSION: Desflurane reduces myocardial infarct size in vivo, and the results further suggest that both sarcolemmal and mitochondrial KATP channels could be involved.     

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.     

Isoflurane-enhanced Recovery of Canine Stunned Myocardium: Role for Protein Kinase C?

Background: Isoflurane enhances the functional recovery of postischemic, reperfused myocardium by activating adenosine A1 receptors and adenosine triphosphate-regulated potassium channels. Whether protein kinase C is involved in this process is unknown. The authors tested the hypothesis that inhibition of protein kinase C, using the selective antagonist bisindolylmaleimide, attenuates isoflurane-enhanced recovery of stunned myocardium in dogs.

Methods: Fifty dogs were randomly assigned to receive intracoronary vehicle or bisindolylmaleimide (2 or 8 [mu]g/min) in the presence or absence of isoflurane (1 minimum alveolar concentration). Five brief (5 min) coronary artery occlusions interspersed with 5-min reperfusion periods followed by 180 min of final reperfusion were used to produce myocardial stunning. Hemodynamics, regional segment shortening, and myocardial blood flow (radioactive microspheres) were measured at selected intervals.

Results: There were no differences in baseline hemodynamics, segment shortening, or coronary collateral blood flow between groups. Isoflurane significantly (P < 0.05) decreased heart rate, mean arterial pressure, rate pressure product, and the maximum rate of increase of left ventricular pressure (+dP/dtmax) in the presence or absence of bisindolylmaleimide. Sustained contractile dysfunction was observed in dogs that received vehicle (recovery of segment shortening to 12 +/- 8% of baseline), in contrast to those that received isoflurane (75 +/- 7% recovery). Bisindolylmaleimide at a dose of 2 [mu]g/min alone enhanced recovery of segment shortening (50 +/- 7% of baseline) compared with vehicle-pretreated dogs, and isoflurane in the presence of 2 [mu]g/min bisindolylmaleimide further enhanced recovery of contractile function (79 +/- 8% of baseline). In contrast, 8 [mu]g/min bisindolylmaleimide alone (32 +/- 12%) or combined with isoflurane (37 +/- 17%) did not enhance recovery of segment shortening compared with vehicle-pretreated dogs.     

N-acetylcysteine restores isoflurane-induced preconditioning against myocardial infarction during hyperglycemia

BACKGROUND: Hyperglycemia generates reactive oxygen species and prevents isoflurane-induced preconditioning. The authors tested the hypothesis that scavenging reactive oxygen species with N-acetylcysteine will restore protection against myocardial infarction produced by isoflurane in vivo. METHODS: Barbiturate-anesthetized dogs (n = 45) were instrumented for measurement of systemic hemodynamics. Myocardial infarct size and coronary collateral blood flow were measured with triphenyltetrazolium staining and radioactive microspheres, respectively. All dogs were subjected to a 60-min left anterior descending coronary artery occlusion followed by 3 h of reperfusion. Dogs were randomly assigned to receive an infusion of 0.9% saline or 15% dextrose in water to increase blood glucose concentrations to 600 mg/dl (hyperglycemia) in the absence or presence of isoflurane (1.0 minimum alveolar concentration) with or without pretreatment with N-acetylcysteine (150 mg/kg i.v.) in six experimental groups. Isoflurane was discontinued, and blood glucose concentrations were allowed to return to baseline values before left anterior descending coronary artery occlusion. RESULTS: Myocardial infarct size was 27 +/- 2% (n = 8) of the left ventricular area at risk in control experiments. Isoflurane significantly (P < 0.05) decreased infarct size (13 +/- 2%; n = 7). Hyperglycemia alone did not alter infarct size (29 +/- 3%; n = 7) but abolished the protective effect of isoflurane (25 +/- 2%; n = 8). N-Acetylcysteine alone did not affect infarct size (28 +/- 2%; n = 8) but restored isoflurane-induced cardioprotection during hyperglycemia (10 +/- 1%; n = 7). CONCLUSIONS: Acute hyperglycemia abolishes reductions in myocardial infarct size produced by isoflurane, but N-acetylcysteine restores these beneficial effects. The results suggest that excessive quantities of reactive oxygen species generated during hyperglycemia impair isoflurane-induced preconditioning in dogs.     

Sevoflurane Reduces Myocardial Infarct Size and Decreases the Time Threshold for Ischemic Preconditioning in Dogs

Background: Recent evidence indicates that volatile anesthetics exert protective effects during myocardial ischemia and reperfusion. The authors tested the hypothesis that sevoflurane decreases myocardial infarct size by activating adenosine triphosphate-sensitive potassium (KATP) channels and reduces the time threshold of ischemic preconditioning necessary to protect against infarction.

Methods: Barbiturate-anesthetized dogs (n = 75) were instrumented for measurement of aortic and left ventricular pressures and maximum rate of increase of left ventricular pressure and were subjected to a 60-min left anterior descending (LAD) coronary artery occlusion followed by 3-h reperfusion. In four separate groups, dogs received vehicle or the KATP channel antagonist glyburide (0.1 mg/kg intravenously), and 1 minimum alveolar concentration sevoflurane (administered until immediately before coronary artery occlusion) in the presence or absence of glyburide. In three additional experimental groups, sevoflurane was discontinued 30 min (memory) before the 60-min LAD occlusion or a 2-min LAD occlusion as an ischemic preconditioning stimulus was used with or without subsequent sevoflurane (with memory) pretreatment. Regional myocardial perfusion and infarct size were measured with radioactive microspheres and triphenyltetrazolium staining, respectively.

Results: Vehicle (23 +/- 1% of the area at risk; mean +/- SEM) and glyburide (23 +/- 2%) alone produced equivalent effects on myocardial infarct size. Sevoflurane significantly (P < 0.05) decreased infarct size (13 +/- 2%). This beneficial effect was abolished by glyburide (21 +/- 3%). Neither the 2-min LAD occlusion nor sevoflurane followed by 30 min of memory were protective alone, but together, sevoflurane enhanced the effects of the brief ischemic stimulus and profoundly reduced infarct size (9 +/- 2%).     

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).     

Anesthetic-induced Preconditioning: Previous Administration of Isoflurane Decreases Myocardial Infarct Size in Rabbits

Background: Experimental evidence suggests that ATP-sensitive potassium channels are involved in myocardial ischemic preconditioning. Because some pharmacologic effects of isoflurane are mediated by KATP channels, the authors tested the hypothesis: Isoflurane administration, before myocardial ischemia, can induce or mimic myocardial preconditioning.

Methods: Myocardial infarct size was measured in three groups of propofol-anesthetized rabbits, each subjected to 30 min of anterolateral coronary occlusion followed by 3 h of reperfusion. Groups differed in their pretreatment: Group 1 (control, N = 13) no pretreatment, Group 2 (ischemic preconditioning, N = 8), 5 min of coronary occlusion and 15 min of reperfusion; Group 3 (isoflurane pretreatment; N = 15), 15 min of isoflurane (1.1% end-tidal) and 15 min of washout. Hemodynamics were monitored serially. Myocardial infarct size and the area at risk were defined using triphenyltetrazolium chloride staining and fluorescent microspheres, respectively, and both were measured using computerized planimetry.

Results: Infarct size expressed as a percentage of area at risk was 23.4 +/- 8.5% (mean +/- SD) in the isoflurane group compared with 33.1 +/- 13.3% in controls, and 8.7 +/- 6.2% in the ischemia-preconditioned group. Analysis for coincidental regressions, followed by tests for equality of slope and elevation, showed that the linear relationship between infarct size and area at risk was significantly (P < 0.05) different in all three groups because of differences in line elevation. Minor differences in hemodynamic variables were found between groups, which were unlikely to account for the significant differences in infarct size.     

N-Acetylcysteine Restores Isoflurane-induced Preconditioning against Myocardial Infarction during Hyperglycemia

Background: Hyperglycemia generates reactive oxygen species and prevents isoflurane-induced preconditioning. The authors tested the hypothesis that scavenging reactive oxygen species with N-acetylcysteine will restore protection against myocardial infarction produced by isoflurane in vivo.

Methods: Barbiturate-anesthetized dogs (n = 45) were instrumented for measurement of systemic hemodynamics. Myocardial infarct size and coronary collateral blood flow were measured with triphenyltetrazolium staining and radioactive microspheres, respectively. All dogs were subjected to a 60-min left anterior descending coronary artery occlusion followed by 3 h of reperfusion. Dogs were randomly assigned to receive an infusion of 0.9% saline or 15% dextrose in water to increase blood glucose concentrations to 600 mg/dl (hyperglycemia) in the absence or presence of isoflurane (1.0 minimum alveolar concentration) with or without pretreatment with N-acetylcysteine (150 mg/kg IV) in six experimental groups. Isoflurane was discontinued, and blood glucose concentrations were allowed to return to baseline values before left anterior descending coronary artery occlusion.

Results: Myocardial infarct size was 27 +/- 2% (n = 8) of the left ventricular area at risk in control experiments. Isoflurane significantly (P < 0.05) decreased infarct size (13 +/- 2%; n = 7). Hyperglycemia alone did not alter infarct size (29 +/- 3%; n = 7) but abolished the protective effect of isoflurane (25 +/- 2%; n = 8). N-Acetylcysteine alone did not affect infarct size (28 +/- 2%; n = 8) but restored isoflurane-induced cardioprotection during hyperglycemia (10 +/- 1%; n = 7).     

Remifentanil Preconditioning Confers Cardioprotection via Cardiac κ- and δ-Opioid Receptors

Background: Remifentanil preconditioning (RPC) reduces the infarct size in anesthetized rat hearts, and this effect seems to be mediated by all three types of opioid receptors (ORs). Because there is evidence of only [kappa]- and [delta]- but not [mu]-ORs in the rat heart, the authors investigated whether RPC confers cardioprotection via cardiac [kappa]- and [delta]-OR as well as via extracardiac [mu]-OR agonist activity. The authors also investigated the involvement of signaling mechanisms, namely protein kinase C and mitochondrial adenosine triphosphate-sensitive potassium (KATP) channels.

Methods: The hearts of male Sprague-Dawley rats weighing 190-210 g were removed, mounted on a Langendorff apparatus, and perfused retrogradely at 100 cm H2O with Krebs-Ringer's solution. All hearts were subjected to 30 min of ischemia and 2 h of reperfusion. The study consisted of three series of experiments on the effect of ischemic preconditioning or RPC (10, 50, and 100 ng/ml remifentanil) after blockade of OR subtypes ([delta]-OR antagonist naltrindol, [kappa]-OR antagonist nor-binaltorphimine, and [mu]-OR antagonist CTOP). The involvement of protein kinase C or the KATP channel in the cardioprotection of RPC was also investigated using specific blockers in each group. RPC was produced by three cycles of 5-min perfusion of remifentanil in Krebs-Ringer's solution interspersed with a 5-min reperfusion with Krebs solution only. Infarct size, as a percentage of the area at risk, was determined by 2,3,5-triphenyltetrazolium staining.

Results: Infarct size as a percentage of the area at risk was significantly reduced after RPC from 51.9 +/- 5.0% (control, n = 8) to 36.2 +/- 10.0% (100 ng/ml RPC, n = 8, P < 0.01). This effect was stopped by pretreatment with naltrindol (52.3 +/- 5.2%) and nor-binaltorphimine (43.5 +/- 6.0%) but not CTOP (37.1 +/- 6.0%). Chelerythrine and GF109203X, both protein kinase C inhibitors, abolished the effects of RPC or ischemic preconditioning on infarct size as a percentage of area at risk. 5-Hydroxydecanoate (a selective mitochondrial KATP channel blocker) also abolished the cardioprotection of RPC and IPC, but HMR-1098 (a selective inhibitor of the sarcolemmal KATP channel) did not.     

Hyperglycemia prevents isoflurane-induced preconditioning against myocardial infarction.

BACKGROUND: Volatile anesthetics stimulate but hyperglycemia attenuates activity of mitochondrial adenosine triphosphate-regulated potassium channels. The authors tested the hypothesis that acute hyperglycemia interferes with isoflurane-induced preconditioning in vivo. METHODS: Barbiturate-anesthetized dogs (n = 79) were instrumented for measurement of hemodynamics. Myocardial infarct size and collateral blood flow were assessed with triphenyltetrazolium chloride staining and radioactive microspheres, respectively. All dogs were subjected to a 60-min left anterior descending coronary artery occlusion followed by 3 h of reperfusion. Dogs were randomly assigned to receive an infusion of normal saline (normoglycemic controls) or 15% dextrose in water to increase blood glucose concentrations to 300 or 600 mg/dl in the absence or presence of isoflurane (0.5 or 1.0 minimum alveolar concentration [MAC]) in separate experimental groups. Isoflurane was discontinued, and blood glucose concentrations were allowed to return to baseline values before left anterior descending coronary artery occlusion. RESULTS: Myocardial infarct size was 26 +/- 1% of the left ventricular area at risk in control experiments. Isoflurane reduced infarct size (15 +/- 2 and 13 +/- 1% during 0.5 and 1.0 MAC, respectively). Hyperglycemia alone did not alter infarct size (26 +/- 2 and 33 +/- 4% during 300 and 600 mg/dl, respectively). Moderate hyperglycemia blocked the protective effects of 0.5 MAC (25 +/- 2%) but not 1.0 MAC isoflurane (13 +/- 2%). In contrast, severe hyperglycemia prevented reductions of infarct size during both 0.5 MAC (29 +/- 3%) and 1.0 MAC isoflurane (28 +/- 4%). CONCLUSIONS: Acute hyperglycemia attenuates reductions in myocardial infarct size produced by isoflurane in dogs.     

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.     

Mechanism of Preconditioning by Isoflurane in Rabbits: A Direct Role for Reactive Oxygen Species

Background: Reactive oxygen species (ROS) contribute to myocardial protection during ischemic preconditioning, but the role of the ROS in protection against ischemic injury produced by volatile anesthetics has only recently been explored. We tested the hypothesis that ROS mediate isoflurane-induced preconditioning in vivo.

Methods: Pentobarbital-anesthetized rabbits were instrumented for measurement of hemodynamics and were subjected to a 30 min coronary artery occlusion followed by 3 h reperfusion. Rabbits were randomly assigned to receive vehicle (0.9% saline), or the ROS scavengers N-acetylcysteine (NAC; 150 mg/kg) or N-2-mercaptopropionyl glycine (2-MPG; 1 mg [middle dot] kg-1[middle dot] min-1), in the presence or absence of 1.0 minimum alveolar concentration (MAC) isoflurane. Isoflurane was administered for 30 min and then discontinued 15 min before coronary artery occlusion. A fluorescent probe for superoxide anion production (dihydroethidium, 2 mg) was administered in the absence of the volatile anesthetic or 5 min before exposure to isoflurane in 2 additional groups (n = 8). Myocardial infarct size and superoxide anion production were assessed using triphenyltetrazolium staining and confocal fluorescence microscopy, respectively.

Results: Isoflurane (P < 0.05) decreased infarct size to 24 +/- 4% (mean +/- SEM; n = 10) of the left ventricular area at risk compared with control experiments (43 +/- 3%; n = 8). NAC (43 +/- 3%; n = 7) and 2-MPG (42 +/- 5%; n = 8) abolished this beneficial effect, but had no effect on myocardial infarct size (47 +/- 3%; n = 8 and 46 +/- 3; n = 7, respectively) when administered alone. Isoflurane increased superoxide anion production as compared with control experiments (28 +/- 12 vs. -6 +/- 9 fluorescence units;P < 0.05).     

Isoflurane does not produce a second window of preconditioning against myocardial infarction in vivo

The administration of a volatile anesthetic shortly before a prolonged ischemic episode exerts protective effects against myocardial infarction similar to those of ischemic preconditioning. A second window of preconditioning (SWOP) against myocardial infarction can also be elicited by brief episodes of ischemia when this occurs 24 h before prolonged coronary artery occlusion. Whether remote exposure to a volatile anesthetic also causes delayed myocardial protection is unknown. We tested the hypothesis that the administration of isoflurane 24 h before ischemia produces a SWOP against infarction. Barbiturate-anesthetized dogs (n = 25) were instrumented for measurement of hemodynamics, including aortic and left ventricular (LV) pressures and LV +dP/dt(max), and subjected to a 60-min left anterior descending coronary artery occlusion followed by 3 h of reperfusion. Myocardial infarct size and coronary collateral blood flow were assessed with triphenyltetrazolium chloride staining and radioactive microspheres, respectively. Two groups of dogs received 1.0 minimum alveolar anesthetic concentration isoflurane for 30 min or 6 h that was discontinued 30 min (acute) or 24 h (delayed) before ischemia and reperfusion, respectively. A control group of dogs did not receive isoflurane. Infarct size was 27% +/- 3% of the LV area at risk in the absence of pretreatment with isoflurane. Acute, but not remote, administration of isoflurane reduced infarct size (12% +/- 1% and 31% +/- 3%, respectively). No differences in hemodynamics or transmural myocardial perfusion during or after occlusion were observed between groups. The results indicate that isoflurane does not produce a SWOP when administered 24 h before prolonged myocardial ischemia in vivo. IMPLICATIONS: Isoflurane mimics the beneficial effects of ischemic preconditioning by protecting myocardium against infarction when it is administered shortly before a prolonged ischemic episode. However, unlike ischemic preconditioning, isoflurane does not produce a second window of protection 24 h after administration in dogs.     

Mechanism of Myocardial Protection by Isoflurane: Role of Adenosine Triphosphate-regulated Potassium (K sub ATP) Channels

Background: The mechanism of the protective actions of volatile anesthetics in ischemic myocardium has not been clearly elucidated. The role of myocardial adenosine triphosphate-regulated potassium (KATP) channels in isoflurane-induced enhancement of recovery of regional contractile function after multiple brief occlusions and reperfusion of the left anterior descending coronary artery (LAD) was studied in dogs anesthetized with barbiturates.

Methods: Dogs (n = 32) were instrumented to measure left ventricular and aortic blood pressure, cardiac output, LAD coronary blood flow velocity, and subendocardial segment length. Regional myocardial perfusion was measured using radioactive microspheres. Hemodynamics and percentage segment shortening (%SS) in the LAD perfusion territory were evaluated after instrumentation was complete; after pretreatment with the KATP channel antagonist, glyburide (0.05 mg/kg sup -1) or drug vehicle (polyethylene glycol in ethyl alcohol; control experiments); and in the presence or absence of 1 MAC isoflurane administered for 30 min before and during five 5-min occlusions and reperfusion of the LAD in four experimental groups. Isoflurane was discontinued at the onset of the final reperfusion period. Measurements of hemodynamics, %SS, and myocardial perfusion were repeated at several intervals during 180 min after reperfusion of the LAD.

Results: Left anterior descending coronary artery occlusion caused regional dyskinesia during each 5-min occlusion in each dog. Control and glyburide-pretreated dogs demonstrated poor recovery of %SS by 180 min after reperfusion (2 +/- 10 and 7 +/- 6% of baseline, respectively). In contrast, dogs anesthetized with isoflurane exhibited complete recovery of function (%SS) by 180 min after reperfusion (82 +/- 8% of baseline). Enhanced recovery of regional contractile function by isoflurane was abolished by pretreatment with glyburide 180 min after reperfusion (16 +/- 10% of baseline). Improvement of functional recovery of stunned myocardium by isoflurane, and the blockade of this action by glyburide, was not associated with changes in hemodynamics or regional myocardial perfusion.     

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).