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Meeting Abstracts  |   January 1997
Isoflurane and Halothane Increase Adenosine Triphosphate Preservation, but Do Not Provide Additive Recovery of Function after Ischemia, in Preconditioned Rat Hearts
Author Notes
  • (Boutros) Assistant Professor of Anesthesiology, SUNY Health Science Center at Brooklyn.
  • (Wang) Visiting Scholar, SUNY Health Science Center at Brooklyn; and Assistant Professor of Anesthesia, Xuzhou Medical College, People's Republic of China.
  • (Capuano) Research Associate, SUNY Health Science Center at Brooklyn.
  • Received from the Department of Anesthesiology, State University of New York Health Science Center at Brooklyn, and Xuzhou Medical College, People's Republic of China. Submitted for publication December 4, 1995. Accepted for publication August 18, 1996.
  • Address reprint requests to Dr. Boutros: Department of Anesthesiology-Box 6, State University of New York Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, New York 11203–2098. Address electronic mail to: aBoutros@NetMail.HSCBKLYN.edu.
Article Information
Meeting Abstracts   |   January 1997
Isoflurane and Halothane Increase Adenosine Triphosphate Preservation, but Do Not Provide Additive Recovery of Function after Ischemia, in Preconditioned Rat Hearts
Anesthesiology 1 1997, Vol.86, 109-117. doi:
Anesthesiology 1 1997, Vol.86, 109-117. doi:
Preconditioning is a phenomenon in which a brief period of ischemia renders the myocardium resistant to subsequent ischemia, reduces infarct size, and limits ultrastructural abnormality. [1] This endogenous protective mechanism has been shown to occur in various species, including humans, dogs, rabbits, pigs, and rats. [2] The mechanisms of protection, although not completely understood, involve release of adenosine and activation of A1receptors that regulate adenylate cyclase activity through guanosine triphosphate, thus binding G sub i proteins intracellularly. [3] Giproteins have several functions, one of which is to inhibit Calcium sup ++ influx during ischemia. The Giprotein pathway also may activate the KATPchannel [4] and appears to regulate cellular metabolism, particularly glycolysis. [5] Biochemical analysis indicates that preconditioning also contributes to conservation of adenosine triphosphate (ATP) content during subsequent ischemia. [6] 
Transient ischemia may occur during anesthesia and surgery. Volatile anesthetics are often used for patients with coronary artery disease (CAD) who are at risk for having intermittent ischemia during cardiac and noncardiac surgery. Previous reports indicate a beneficial effect of volatile anesthetics given before and during ischemia. [7,8] Halothane decreases the accumulation of intracellular calcium in the postischemic period, [9] improves functional recovery, and reduces the incidence and duration of dysrhythmias on reperfusion in isolated hearts. [10] It may also decrease the release of adenosine during hypoperfusion and ischemia, producing a favorable condition for reperfusion in isolated guinea pig hearts. [11] Isoflurane enhances the functional recovery of the ischemic myocardium. [12] 
We postulated that combining preconditioning with halothane or isoflurane would provide additional protection for hemodynamic recovery and ATP sparing after ischemia in normothermic rat hearts.
Materials and Methods
Male Sprague-Dawley rats (weighing 350–425 g) were fed a standard diet and acclimated in a quiet quarantine room for 1 week before the experiments. The protocol was reviewed and approved by the Animal Care and Use Committee of SUNY Health Science Center at Brooklyn. All chemicals were obtained from Sigma Chemical Company (St. Louis, MO).
Forty-two rats were injected with 10 mg ketamine and 500 U heparin intraperitoneally; when they were unresponsive to noxious stimulation, their hearts were rapidly removed through a median sternotomy incision by transsecting all the major vessels. The heart was submerged in cold perfusate (4 degrees Celsius) and then attached to perfusion apparatus via the aorta. Retrograde perfusion was initiated immediately with Krebs-Henseleit bicarbonate (K-H) buffer that had the following composition: 155 mM/l Sodium sup +, 5.6 mM/l Potassium sup +, 138 mM/l Chlorine sup -, 2.16 mM/l Calcium2+, 1.19 mM/l PO43+, 25 mM/l HCO3, 0.56 mM/l Magnesium2+, 11 mM/l glucose, and 13 mM/l sucrose. The solution was equilibrated with 95% oxygen and 5% carbon dioxide at 37 +/- 0.5 degrees Celsius, achieving a PO2of 560 mmHg, PCO2of 37 mmHg, and a pH of 7.4 +/- 0.2. The myocardial temperature was maintained at 37 degrees Celsius throughout the experiment. The perfusion set-up is based on a modification of the system described by Neely and associates. [13] 
The hearts were perfused in the Langendorff mode under constant pressure of 80 mmHg. During the first 20 min of perfusion, the vena cava was ligated, the left atrium was opened, and a fluid-filled latex balloon catheter was introduced into the left ventricle with a microtip manometer placed in the balloon. Balloon volume was adjusted to maintain an end-diastolic pressure of 6–8 mmHg. This preload volume was held constant during the entire experiment to allow continuous recording of the ventricular pressure. The pulmonary artery was cannulated with small-caliber polyethylene tubing and ligated. Coronary flow was measured using the output volume of this tubing into a graduated cylinder at 5-min intervals. Pacing wires were attached to the pulmonary outflow tract and hearts were paced at a cycle length of 200 ms (300 bpm) using a ventrix stimulator. Stimulation was stopped during ischemic periods. Cardiac function (peak) left ventricular pressure, developed pressure (end systolic minus end diastolic), and positive and negative dP/dt were continuously recorded with a pressure amplifier and direct differentiator (Astro Med, Boston MA). A three-way stopcock placed above the aortic root was positioned to stop flow and thus produce global ischemia. Pacing was resumed 3 min after reperfusion. Hearts were defibrillated when necessary.
Volatile anesthetics were administered by placing an agent-specific vaporizer between the fresh gas supply and the perfusate. A Drager infrared gas analyzer continuously controlled the delivered vapor concentration. The concentration of halothane (0.47 +/- 0.02 mM) and isoflurane (0.84 +/- 0.03 mM) in the perfusate was measured using a gas chromatograph (Perkin-Elmer, Norwalk, CT).
Experimental Design
All experiments lasted 115 min (Figure 1) beginning with a 20-min period of stabilizing perfusion for equilibration. The time-control group received no ischemia. The untreated group underwent 60 min of perfusion followed by 25 min of ischemia. The halothane and isoflurane groups received 20 min of perfusion, after which halothane or isoflurane was administered for 40 min before ischemia. The preconditioning group had 30 min of perfusion followed by two 5-min periods of ischemia separated by 10 min of reperfusion. The halothane + preconditioning and the isoflurane + preconditioning groups had 20 min of perfusion, followed by 10 min of treatment with either halothane or isoflurane and two 5-min periods of ischemia separated by 10 min of reperfusion. Halothane or isoflurane were present throughout the experiment, except during the equilibration period, the ischemic period, and the reperfusion period. During ischemia, the hearts were enclosed in a water-jacketed container and maintained at 37 degrees Celsius. Reperfusion lasted for 30 min in all groups.
Figure 1. Experimental protocol. Isc = ischemia; Rep = reperfusion.
Figure 1. Experimental protocol. Isc = ischemia; Rep = reperfusion.
Figure 1. Experimental protocol. Isc = ischemia; Rep = reperfusion.
×
Biochemical Analyses
Ventricular transmural tissue specimens were taken at the end of each experiment. The tissues were frozen immediately in liquid nitrogen and stored at minus 80 degrees Celsius until the time of analysis. After freeze-drying, the tissues were weighed. Adenosine triphosphate was extracted from the tissues by homogenizing in 3N ice-cold perchloric acid and measured, after neutralization, using the firefly Luciferin-Luciferase assay. [14] 
Statistics
All values are expressed as mean +/- SEM. Functional parameters at identical time points were compared for the groups by analysis of variance. If the F ratios were significant, a Bonferroni-Dunn post hoc test was applied to assess the significance of individual comparisons (P < 0.05). Recovery parameters within groups were analyzed by repeated-measures (before and after ischemia), single-factor analysis of variance, and multicomparison was tested with Scheffe's F test. The incidence of ventricular fibrillation in each group was compared using chi squared analysis. We used Stat View 4 (ABACUS, Berkeley, CA) for the statistical analysis.
Results
Preischemic Hemodynamics
Control group hemodynamic values remained stable for the 115 min length of the experiment. Preischemic values of left ventricular end-diastolic pressure (LVEDP), developed pressure, +dP/dt, -dP/dt, and coronary flow for all groups were compared with baseline values and are shown in Table 1. Preconditioning caused a transient increase in coronary flow that was not significantly different from the baseline value. In the isoflurane and isoflurane + preconditioning groups, there were no significant differences in LVEDP, developed pressure, +dP/dt, and -dP/dt; however, the coronary flow increased from 18.8 +/- 0.9 to 26.3 +/- 2.4 ml/min in the isoflurane group and from 18.5 +/- 1.1 to 27.2 +/- 2.2 ml/min in the isoflurane + preconditioning group (P < 0.001). Halothane significantly depressed the contractile function, and developed pressure, +dP/dt, and -dP/dt decreased by 16%, 13%, and 16%, respectively (P <0.01). Halothane did not increase coronary flow significantly. Halothane combined with preconditioning also depressed contractile function, and developed pressure, +dP/dt, and -dP/dt decreased 22%, 33%, and 35%, respectively (P < 0.001). Halothane + preconditioning caused an increase in coronary flow from 18 +/- 2 ml/min to 25 +/- 2 ml/min (P < 0.001).
Table 1. No comment.
Image not available
Table 1. No comment.
×
Postischemic Hemodynamics
Hemodynamic recovery was severely impaired in rats in the untreated group. Figure 2shows the time course of changes in LVEDP during reperfusion for all groups. Ischemia produced a significant increase in LVEDP in all groups when compared with baseline values (P <0.001). The treated groups had a lower LVEDP than did the untreated group (P < 0.001). There was no significant difference in the LVEDP among any of the treated groups at the end of 25 min of ischemia. In the untreated group, LVEDP increased further to reached a maximum value at 5 min of reperfusion and remained high during the rest of the experiment. The treated groups also showed an increase in LVEDP in the first 5 min of reperfusion, but this decreased subsequently and was significantly lower when compared with the untreated group at the end of reperfusion (P <0.001).
Figure 2. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on left ventricular end-diastolic pressure, which is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for untreated groups compared with preconditioning, halothane, and halothane + preconditioning groups. (dagger) P < 0.01 for the preconditioned group compared with the halothane group. No significant difference was found among the preconditioned group and the halothane + preconditioning groups. (B) Effects of preconditioning (preconditioning), isoflurane (Iso), and isoflurane + preconditioning (Iso + preconditioning) on left ventricular end-diastolic pressure, which is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with the preconditioning, isoflurane, and isoflurane + preconditioning groups. No significant difference was found among the treated groups.
Figure 2. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on left ventricular end-diastolic pressure, which is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for untreated groups compared with preconditioning, halothane, and halothane + preconditioning groups. (dagger) P < 0.01 for the preconditioned group compared with the halothane group. No significant difference was found among the preconditioned group and the halothane + preconditioning groups. (B) Effects of preconditioning (preconditioning), isoflurane (Iso), and isoflurane + preconditioning (Iso + preconditioning) on left ventricular end-diastolic pressure, which is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with the preconditioning, isoflurane, and isoflurane + preconditioning groups. No significant difference was found among the treated groups.
Figure 2. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on left ventricular end-diastolic pressure, which is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for untreated groups compared with preconditioning, halothane, and halothane + preconditioning groups. (dagger) P < 0.01 for the preconditioned group compared with the halothane group. No significant difference was found among the preconditioned group and the halothane + preconditioning groups. (B) Effects of preconditioning (preconditioning), isoflurane (Iso), and isoflurane + preconditioning (Iso + preconditioning) on left ventricular end-diastolic pressure, which is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with the preconditioning, isoflurane, and isoflurane + preconditioning groups. No significant difference was found among the treated groups.
×
(Figure 3) shows the recovery of developed pressure during 30 min of reperfusion. In the untreated group, developed pressure recovered only 30.4% and was significantly different from the preconditioning, isoflurane, and isoflurane + preconditioning groups (85%, 84%, and 90%, respectively; P < 0.001). The halothane and halothane + preconditioning groups recovered 83% and 92%, respectively (P < 0.001). No significant difference in developed pressure was found among the treated groups.
Figure 3. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on the recovery of developed pressure in the reperfusion phase. Developed pressure is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for untreated groups compared with preconditioning, halothane, and halothane + preconditioning. No significant difference was found among treated groups. (B) Effects of preconditioning (Pc), isoflurane (Iso), and isoflurane + preconditioning on the recovery of developed pressure in the reperfusion phase. Developed pressure is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with preconditioning, isoflurane, and isoflurane + preconditioning. No significant difference was found among treated groups.
Figure 3. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on the recovery of developed pressure in the reperfusion phase. Developed pressure is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for untreated groups compared with preconditioning, halothane, and halothane + preconditioning. No significant difference was found among treated groups. (B) Effects of preconditioning (Pc), isoflurane (Iso), and isoflurane + preconditioning on the recovery of developed pressure in the reperfusion phase. Developed pressure is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with preconditioning, isoflurane, and isoflurane + preconditioning. No significant difference was found among treated groups.
Figure 3. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on the recovery of developed pressure in the reperfusion phase. Developed pressure is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for untreated groups compared with preconditioning, halothane, and halothane + preconditioning. No significant difference was found among treated groups. (B) Effects of preconditioning (Pc), isoflurane (Iso), and isoflurane + preconditioning on the recovery of developed pressure in the reperfusion phase. Developed pressure is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with preconditioning, isoflurane, and isoflurane + preconditioning. No significant difference was found among treated groups.
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(Figure 4) shows the recovery of +dP/dt during reperfusion. In the untreated group, +dP/dt was 900 +/- 108 mmHg/s at the end of reperfusion. In the preconditioning, isoflurane, and isoflurane + preconditioning groups, +dP/dt was 2,766 +/- 205, 2,650 +/- 219, and 2,775 +/- 251 mmHg/s, respectively. In the halothane and halothane + preconditioning groups, +dP/dt was 2,616 +/- 216 and 2,816 +/- 186 mmHg/s, respectively, which was significantly different from the +dP/dt of the untreated group at the end of reperfusion (P < 0.001). There was no significant difference in +dP/dt among treated groups. Similar findings were obtained for -dP/dt (Figure 5). At 30 min of reperfusion, the value for the untreated group was 433 +/- 74 mmHg/s, and there was a significant difference between the untreated and treated groups at the end of reperfusion (P < 0.001). There was no significant difference in -dP/dt among treated groups.
Figure 4. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on the recovery of the left ventricular positive value of +dP/dt during reperfusion. +dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with preconditioning, halothane, and halothane + preconditioning. No significant difference was found among treated groups. (B) Effects of preconditioning (Pc), Isoflurane (Iso), and isoflurane + preconditioning on the recovery of the left ventricular positive value of +dP/dt during reperfusion. +dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group compared with preconditioning, isoflurane, and isoflurane + preconditioning. No significant difference was found among treated groups.
Figure 4. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on the recovery of the left ventricular positive value of +dP/dt during reperfusion. +dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with preconditioning, halothane, and halothane + preconditioning. No significant difference was found among treated groups. (B) Effects of preconditioning (Pc), Isoflurane (Iso), and isoflurane + preconditioning on the recovery of the left ventricular positive value of +dP/dt during reperfusion. +dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group compared with preconditioning, isoflurane, and isoflurane + preconditioning. No significant difference was found among treated groups.
Figure 4. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on the recovery of the left ventricular positive value of +dP/dt during reperfusion. +dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with preconditioning, halothane, and halothane + preconditioning. No significant difference was found among treated groups. (B) Effects of preconditioning (Pc), Isoflurane (Iso), and isoflurane + preconditioning on the recovery of the left ventricular positive value of +dP/dt during reperfusion. +dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group compared with preconditioning, isoflurane, and isoflurane + preconditioning. No significant difference was found among treated groups.
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Figure 5. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on the recovery of the left ventricular negative value of -dP/dt during reperfusion. -dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with preconditioning, halothane, and halothane + preconditioning. No significant difference was found among treated groups. (B) Effects of preconditioning (Pc), isoflurane (Iso), and isoflurane + preconditioning on the recovery of the left ventricular negative value of -dP/dt during reperfusion. -dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group compared with preconditioning, isoflurane, and isoflurane + preconditioning. No significant difference was found among treated groups.
Figure 5. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on the recovery of the left ventricular negative value of -dP/dt during reperfusion. -dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with preconditioning, halothane, and halothane + preconditioning. No significant difference was found among treated groups. (B) Effects of preconditioning (Pc), isoflurane (Iso), and isoflurane + preconditioning on the recovery of the left ventricular negative value of -dP/dt during reperfusion. -dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group compared with preconditioning, isoflurane, and isoflurane + preconditioning. No significant difference was found among treated groups.
Figure 5. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on the recovery of the left ventricular negative value of -dP/dt during reperfusion. -dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with preconditioning, halothane, and halothane + preconditioning. No significant difference was found among treated groups. (B) Effects of preconditioning (Pc), isoflurane (Iso), and isoflurane + preconditioning on the recovery of the left ventricular negative value of -dP/dt during reperfusion. -dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group compared with preconditioning, isoflurane, and isoflurane + preconditioning. No significant difference was found among treated groups.
×
In the untreated group, coronary flow recovered 76% at 30 min of reperfusion, which was significantly lower than the preconditioning, isoflurane, and isoflurane + preconditioning groups (109%, 111%, and 122%, respectively; P < 0.001). The halothane and halothane + preconditioning groups recovered 101% and 120%, respectively (P < 0.001). The recovery of coronary flow for halothane-treated hearts was lower than other treated groups (P < 0.05), and both anesthetics + preconditioning-treated groups had significantly increased coronary flow recovery compared with preconditioning alone (P < 0.05).
Adenosine Triphosphate Content
In the time-control group, ATP concentration was 18.3 +/- 1.7 micro Meter/g dry weight at the end of 115 min of perfusion without ischemia. Adenosine triphosphate content was better preserved in the preconditioning group (10.8 +/- 0.9 micro Meter/g), isoflurane group (11.2 +/- 1.2 micro Meter/g), halothane group (10.2 +/- 1 micro Meter/g), isoflurane + preconditioning group (13.1 +/- 0.9 micro Meter/g), and halothane + preconditioning group (12.4 +/- 0.9 micro Meter/g) than in the untreated group (5.4 +/- 1.2 micro Meter/g)(P < 0.001) at the end of the 30-min reperfusion period. The isoflurane + preconditioning group showed greater preservation of ATP content than did the preconditioning and isoflurane groups (P < 0.001 and P < 0.01). The halothane + preconditioning group also showed better preservation of ATP than did the preconditioning and halothane groups (P < 0.05 and P < 0.001). There was no significant difference in ATP sparing among the preconditioning, isoflurane, and halothane groups.
Reperfusion-induced Ventricular Fibrillation
Ventricular fibrillation occurred immediately or within 3 min of reperfusion in all hearts in the untreated group (100%). The incidence of reperfusion-induced ventricular fibrillation was significantly decreased (P < 0.05) in the six hearts of the treated groups compared with those in the untreated group: three hearts in the isoflurane group; two hearts in the preconditioning, isoflurane + preconditioning, and halothane groups; and only one heart in the halothane + preconditioning group (P <0.01). There was no significant difference among treated groups.
Discussion
Our findings indicate that exposure to isoflurane or halothane before an ischemic insult improves recovery of hemodynamic function and has a favorable effect on ATP preservation. This corresponds to previous findings of Kashimoto and coworkers [15] and Warltier and associates. [7] Isoflurane and halothane also decreased the incidence of ventricular fibrillation during reperfusion. Isoflurane produced a better recovery of coronary flow than did halothane.
We also found that ischemic preconditioning can protect the myocardium against injury induced by ischemia-reperfusion, but we failed to show an additive effect, in terms of hemodynamic recovery of pretreatment with halothane or isoflurane combined with preconditioning. We did find a small but statistically significant improvement in the ATP content of hearts that received preconditioning combined with halothane or isoflurane.
Halothane and isoflurane, like ischemic preconditioning, have been shown to activate the KATPchannels. [16–18] Studies suggest that although KATPchannels may protect the myocardium during ischemia, how this occurs is still unclear. This mechanism of protection may not be as important in rats as in other species, such as humans, swine, and dogs, because blockade of the KATPchannel in rats does not alter the mechanism of preconditioning. [19] 
Preconditioned hearts show evidence of ultrastructural damage more slowly than do hearts that have not been preconditioned, and the rate of ATP use is less. [6] Other studies have postulated that the preservation of ATP is responsible for preconditioning as a result of decreased anaerobic glycolysis and glycogen breakdown. Thus intracellular calcium concentrations could decrease as well as ATP use. [18] In this model, the hemodynamic effect of ischemia-reperfusion was severe in the untreated group. The developed pressure was significantly depressed and left ventricular diastolic pressure was elevated, reflecting an increase in left ventricular wall stiffness or contracture. This increase in pressure could be due to an increase in intracellular Calcium2+ during ischemia. The development of diastolic contracture during ischemia is probably due to persistent, calcium-activated, diastolic contraction secondary to impaired calcium sequestration by the sarcoplasmic reticulum. An increase in intracellular calcium concentration may activate membrane-located phospholipases, and other enzyme systems involved in energy production, to cause energy depletion with a resultant decrease in contractile function. [20] 
Halothane-decreased myoplasmic calcium concentration, as measured by beat-to-beat changes in bioluminescence of aequorin, is an indicator of free intracellular calcium. [21] Using a single ventricular cell voltage clamping technique, it was shown that the decrease in intracellular free calcium, with exposure to halothane and isoflurane, is at least due in part to a decrease in calcium flux through voltage-dependent calcium channels. [22] Isoflurane and halothane decreased the calcium to a similar degree and have at least one effect similar to calcium channel blockers; that is, attenuation of the slow calcium current through voltage-dependent calcium channels. This is meaningful because changes in calcium ion homeostasis play an important role in the events associated with irreversible myocardial injury. In our experiment, this decrease in the intracellular calcium influx was most evident in the lower LVEDP in the treated groups.
Our data show that preconditioning maintained a high concentration of ATP at the end of reperfusion. Richardt and colleagues [23] have shown that preconditioning with short periods of ischemia, separated by intermittent reperfusion, completely prevented the reperfusion-induced endothelial dysfunction. Adenosine, which accumulates rapidly after the onset of myocardial ischemia, plays a part as a mediator of preconditioning. [24] Adenosine may hasten repletion of endogenous vasodilator and anti-inflammatory compounds produced by endothelial cells by restoring the metabolic machinery of these cells by replenishing ATP stores or by enhancing oxygen delivery through arteriolar vasodilatation. [25] 
Volatile anesthetics are potent negative chronotropic, dromotropic, and inotropic agents. [21] Increasing evidence suggests that volatile anesthetics produce negative inotropic effects through mechanisms that affect many steps in the excitation-contraction process. Using the paced isolated rat heart model, we found that 40-min exposure of halothane, but not isoflurane, produced a significant decrease in DP, +dP/dt, and -dP/dt before the onset of ischemia. However, the recovery of DP, +dP/dt, and -dP/dt at the reperfusion period was not significantly different between these two treated groups. Using a guinea pig heart model, Marijic and associates [10] showed that, before the onset of hypoxia, left ventricular systolic pressure decreased in the halothane and isoflurane groups and reduced the oxygen demand. Volatile anesthetics might act to protect against early hypoxia-induced injury by decreasing the cardiac energy expenditure. Left ventricular diastolic pressure was significantly greater in the halothane group at the end of the 30-min reperfusion period. A greater recovery of left ventricular diastolic pressure in the halothane group compared with the isoflurane group could reflect better protection by an improved oxygen supply-to-demand ratio with halothane than with isoflurane, [21] because heart rate and left ventricular diastolic pressure, the dynamic determinants of oxygen demand and supply, decreased more with halothane than with isoflurane. It has been shown in the isolated perfused guinea pig model that halothane improves the oxygen supply-versus-demand ratio during hypoxia more by decreasing oxygen extraction than by increasing oxygen supply, and isoflurane improves this ratio more by increasing oxygen delivery and less by decreasing oxygen extraction. [10] 
The incidence of ventricular fibrillation was significantly decreased in the halothane, isoflurane, and the preconditioning groups. Despite extensive investigation, the mechanisms underlying volatile anesthetic-antiarrhythmic effects are not completely understood. Halothane may have both pro- and antiarrhythmic effects. The accumulation of [Ca2+]iduring ischemia and reperfusion may be an important contributing factor to arrhythmogenesis. [26] At the cellular level, volatile anesthetics block the slow inward Calcium2+ current, [22] abolish Calcium2+-dependent action potentials, [27] decrease intracellular Calcium2+ transients, [28] and decrease the amount of Calcium2+ available for release from the sarcoplasmic reticulum. [29] Each of these actions can antagonize either abnormal automaticity or triggered activity. The mechanism by which preconditioning acts on arrhythmogenesis is still unknown. Washout or diminished production of hydrogen ion [30] or other glycolytic products have been proposed as possible mechanisms of the preconditioning effect. Preconditioning has a well-documented capacity to limit proton accumulation during ischemia that would result in a reduction in the trans-sarcolemmal proton gradient after reperfusion. This appears to be very important in the genesis of reperfusion-induced arrhythmias, possibly as a consequence of limiting reperfusion-induced calcium loading. [31] Oxygen free-radical formation by xanthine oxidase could be decreased if preconditioning allowed washout of hypoxanthine. Preconditioning with low-flow ischemia followed by total occlusion reduces ischemia-related arrhythmias by hastening the increase of extracellular potassium. [32] 
Halothane, isoflurane, and preconditioning independently provide substantial protection against ischemia-reperfusion injury. This protective effect may have a common mechanism via the KATPreceptors, but this hypothesis requires further investigation. Our premise that inhalational anesthetics combined with ischemic preconditioning may provide greater protection than either one alone could not be proved. However, we did find better preservation of ATP content compared with preconditioning alone or treatment with halothane or isoflurane alone.
REFERENCES
Murry CE, Jennings RB, Reimer KA: Preconditioning with ischemia: A delay of lethal cell injury in ischemic myocardium. Circulation 1986; 74:1124-36.
Downey JM: Ischemic preconditioning: Nature's own cardioprotective intervention. Trends Cardiovasc Med 1992; 2:170-6.
Liu GS, Thornton J, Van Winkle DM, Stanley AW, Olsson RA, Downey JM: Protection against infarction afforded by preconditioning is mediated by A1 adenosine receptors in rabbit heart. Circulation 1991; 84:350-6.
Kirsch GE, Codina J, Birnbaumer L, Brown AM: Coupling of ATP-sensitive Potassium sup + channels to A sub 1 receptors by G proteins in rat ventricular myocytes. Am J Physiol 1990; 28:H820-6.
Finegan BA, Lopaschuk GD, Gandhi M, Clanachan AS: Ischemic preconditioning inhibits glycolysis and proton production in isolated working rat hearts. Am J Physiol 1995; 38:H1767-75.
Murry CE, Richard VJ, Reimer KA, Jennings RB: Ischemic preconditioning slows energy metabolism and delays ultrastructural damage during a sustained ischemic episodes. Circ Res 1990; 66:913-31.
Warltier DC, al-Wathiqui MH, Kampine JP, Schmeling WT: Recovery of contractile function of stunned myocardium in chronically instrumented dogs is enhanced by halothane or isoflurane. Anesthesiology 1988; 69:552-65.
Mattheussen M, Rusy BF, Van Aken H, Flameng W: Recovery of function and adenosine triphosphate metabolism following myocardial ischemia induced in the presence of volatile anesthetics. Anesth Analg 1993; 76:69-75.
Hoka S, Bosnjak ZJ, Kampine JP: Halothane inhibits calcium accumulation following myocardial ischemia and calcium paradox in guinea pig hearts. Anesthesiology 1987; 67:197-202.
Marijic J, Stowe DF, Turner LA, Kampine JP, Bosnjak ZJ: Differential protective effects of halothane and isoflurane against hypoxic and reoxygenation injury in the isolated guinea pig heart. Anesthesiology 1990; 73:976-83.
Buljubasic N, Stowe DF, Marijic J, Roerig DL, Kampine JP, Bosnjak ZJ: Halothane reduces release of adenosine, inosine, and lactate with ischemia and reperfusion in isolated hearts. Anesth Analg 1993; 76:54-62.
Kashimoto S: Effects of isoflurane on myocardial metabolism during postischemic reperfusion in the rat. Acta Anaesthesiol Scand 1988; 32:199-202.
Neely JR, Liebermeister H, Battersby EJ, Morgan HE: Effect of pressure development on oxygen consumption by isolated rat heart. Am J Physiol 1967; 212:804-14.
Kass IS, Abramowicz AE, Cottrell JE, Chambers G: The barbiturate thiopental reduces ATP levels during anoxia but improves electrophysiological recovery and ionic homeostasis in the rat hippocampal slice. Neuroscience 1992; 49:537-43.
Kashimoto S, Tsuji Y, Kumazawa T: Effects of halothane and enflurane on myocardial metabolism during postischaemic reperfusion in the rat. Acta Anaesthesiol Scand 1987; 31:44-7.
Larach DR, Schuler HG: Potassium channel blockade and halothane vasodilation in conducting and resistance coronary arteries. J Pharmacol Exp Ther 1993; 267:72-81.
Cason BA, Shubayev I, Hickey RF: Blockade of adenosine triphosphate-sensitive potassium channels eliminates isoflurane-induced coronary artery vasodilation. Anesthesiology 1994; 81:1245-55
Gross GJ, Yao Z, Pieper GM, Auchampach JA: The ATP-regulated potassium channel in ischemia-reperfusion injury. Ann N Y Acad Sci 1994; 723:71-81.
Grover GJ, Dzwonczyk S, Sleph PG, Sargent CA: The ATP-sensitive potassium channel blocker glibenclamide (Glyburide) does not abolish preconditioning in isolated ischemic rat hearts. J Pharmacol Exp Ther 1993; 265:559-64.
Nayler WG, Yepez CE, Poole-Wilson PA: The effect of beta-adrenoreceptor and Calcium sup 2+ antagonist drugs on the hypoxia-induced increase in resting tension. Cardiovasc Res 1978; 12:666-74.
Stowe DF, Marijic J, Bosnjak ZJ, Kampine JP: Direct comparative effects of halothane, enflurane, and isoflurane on oxygen supply and demand in isolated hearts. Anesthesiology 1991; 74:1087-95.
Eskinder H, Rusch NJ, Supan FD, Kampine JP, Bosnjak ZJ: The effects of volatile anesthetics on L- and T-type calcium channel currents in canine cardiac Purkinje cells. Anesthesiology 1991; 74:919-26.
Richardt G, Waas W, Kranzhofer R, Mayer E, Schomig A: Adenosine inhibits exocytotic release of endogenous noradrenalin in rat heart: A protective mechanism in early myocardial ischemia. Circ Res 1987;61:117-23.
Downey JM, Liu GS, Thornton JD: Adenosine and the anti-infarct effects of preconditioning. Cardvasc Res 1993; 27:3-8.
Kaplan LJ, Bellows CF, Haywood-Blum ALM, Mitchell M, Whitman GJ: Ischemic preconditioning preserves end ischemic ATP, enhancing functional recovery and coronary flow during reperfusion. J Surg Res 1994;57:179-84.
Opie LH, Coetzee WA: Role of calcium ions in reperfusion arrhythmias: Relevance to pharmacologic intervention. Cardiovasc Drug Ther 1988; 2:623-36.
Lynch C III, Vogel S, Sperelakis N: Halothane depression of myocardial slow action potentials. Anesthesiology 1981; 55:360-8.
Bosnjak ZJ, Kampine JP: Effects of halothane on transmembrane potentials, Calcium sup 2+ transients, and papillary muscle tension in the cat. Am J Physiol 1986; 251:H374-81.
Komai H, Rusy BF: Direct effect of halothane and isoflurane on the function of the sarcoplasmic reticulum in intact rabbit atria. Anesthesiology 1990; 72:694-8.
Tani M, Neely JR: Role of intracellular Sodium sup + in Calcium sup 2+ overload and depressed recovery of ventricular function of reperfused ischemic rat hearts. Possible involvement of Hydrogen sup + -Sodium sup + and Sodium sup + -Calcium sup 2+ exchange. Circ Res 1989; 65:1045-56.
Lawson CS, Hearse DJ: Ischemic preconditioning against arrhythmias: An anti-arrhythmic or an anti-ischemic phenomenon? Ann N Y Acad Sci 1994; 723:138-57.
Fleet WF, Johnson TA, Graebner CA, Gettes LS: Effect of serial brief ischemic episodes on extracellular Potassium, pH and activation in the pig. Circulation 1985; 2:922-32.
Figure 1. Experimental protocol. Isc = ischemia; Rep = reperfusion.
Figure 1. Experimental protocol. Isc = ischemia; Rep = reperfusion.
Figure 1. Experimental protocol. Isc = ischemia; Rep = reperfusion.
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Figure 2. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on left ventricular end-diastolic pressure, which is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for untreated groups compared with preconditioning, halothane, and halothane + preconditioning groups. (dagger) P < 0.01 for the preconditioned group compared with the halothane group. No significant difference was found among the preconditioned group and the halothane + preconditioning groups. (B) Effects of preconditioning (preconditioning), isoflurane (Iso), and isoflurane + preconditioning (Iso + preconditioning) on left ventricular end-diastolic pressure, which is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with the preconditioning, isoflurane, and isoflurane + preconditioning groups. No significant difference was found among the treated groups.
Figure 2. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on left ventricular end-diastolic pressure, which is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for untreated groups compared with preconditioning, halothane, and halothane + preconditioning groups. (dagger) P < 0.01 for the preconditioned group compared with the halothane group. No significant difference was found among the preconditioned group and the halothane + preconditioning groups. (B) Effects of preconditioning (preconditioning), isoflurane (Iso), and isoflurane + preconditioning (Iso + preconditioning) on left ventricular end-diastolic pressure, which is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with the preconditioning, isoflurane, and isoflurane + preconditioning groups. No significant difference was found among the treated groups.
Figure 2. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on left ventricular end-diastolic pressure, which is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for untreated groups compared with preconditioning, halothane, and halothane + preconditioning groups. (dagger) P < 0.01 for the preconditioned group compared with the halothane group. No significant difference was found among the preconditioned group and the halothane + preconditioning groups. (B) Effects of preconditioning (preconditioning), isoflurane (Iso), and isoflurane + preconditioning (Iso + preconditioning) on left ventricular end-diastolic pressure, which is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with the preconditioning, isoflurane, and isoflurane + preconditioning groups. No significant difference was found among the treated groups.
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Figure 3. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on the recovery of developed pressure in the reperfusion phase. Developed pressure is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for untreated groups compared with preconditioning, halothane, and halothane + preconditioning. No significant difference was found among treated groups. (B) Effects of preconditioning (Pc), isoflurane (Iso), and isoflurane + preconditioning on the recovery of developed pressure in the reperfusion phase. Developed pressure is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with preconditioning, isoflurane, and isoflurane + preconditioning. No significant difference was found among treated groups.
Figure 3. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on the recovery of developed pressure in the reperfusion phase. Developed pressure is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for untreated groups compared with preconditioning, halothane, and halothane + preconditioning. No significant difference was found among treated groups. (B) Effects of preconditioning (Pc), isoflurane (Iso), and isoflurane + preconditioning on the recovery of developed pressure in the reperfusion phase. Developed pressure is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with preconditioning, isoflurane, and isoflurane + preconditioning. No significant difference was found among treated groups.
Figure 3. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on the recovery of developed pressure in the reperfusion phase. Developed pressure is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for untreated groups compared with preconditioning, halothane, and halothane + preconditioning. No significant difference was found among treated groups. (B) Effects of preconditioning (Pc), isoflurane (Iso), and isoflurane + preconditioning on the recovery of developed pressure in the reperfusion phase. Developed pressure is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with preconditioning, isoflurane, and isoflurane + preconditioning. No significant difference was found among treated groups.
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Figure 4. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on the recovery of the left ventricular positive value of +dP/dt during reperfusion. +dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with preconditioning, halothane, and halothane + preconditioning. No significant difference was found among treated groups. (B) Effects of preconditioning (Pc), Isoflurane (Iso), and isoflurane + preconditioning on the recovery of the left ventricular positive value of +dP/dt during reperfusion. +dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group compared with preconditioning, isoflurane, and isoflurane + preconditioning. No significant difference was found among treated groups.
Figure 4. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on the recovery of the left ventricular positive value of +dP/dt during reperfusion. +dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with preconditioning, halothane, and halothane + preconditioning. No significant difference was found among treated groups. (B) Effects of preconditioning (Pc), Isoflurane (Iso), and isoflurane + preconditioning on the recovery of the left ventricular positive value of +dP/dt during reperfusion. +dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group compared with preconditioning, isoflurane, and isoflurane + preconditioning. No significant difference was found among treated groups.
Figure 4. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on the recovery of the left ventricular positive value of +dP/dt during reperfusion. +dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with preconditioning, halothane, and halothane + preconditioning. No significant difference was found among treated groups. (B) Effects of preconditioning (Pc), Isoflurane (Iso), and isoflurane + preconditioning on the recovery of the left ventricular positive value of +dP/dt during reperfusion. +dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group compared with preconditioning, isoflurane, and isoflurane + preconditioning. No significant difference was found among treated groups.
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Figure 5. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on the recovery of the left ventricular negative value of -dP/dt during reperfusion. -dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with preconditioning, halothane, and halothane + preconditioning. No significant difference was found among treated groups. (B) Effects of preconditioning (Pc), isoflurane (Iso), and isoflurane + preconditioning on the recovery of the left ventricular negative value of -dP/dt during reperfusion. -dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group compared with preconditioning, isoflurane, and isoflurane + preconditioning. No significant difference was found among treated groups.
Figure 5. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on the recovery of the left ventricular negative value of -dP/dt during reperfusion. -dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with preconditioning, halothane, and halothane + preconditioning. No significant difference was found among treated groups. (B) Effects of preconditioning (Pc), isoflurane (Iso), and isoflurane + preconditioning on the recovery of the left ventricular negative value of -dP/dt during reperfusion. -dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group compared with preconditioning, isoflurane, and isoflurane + preconditioning. No significant difference was found among treated groups.
Figure 5. (A) Effects of preconditioning (Pc), halothane (Hal), and halothane + preconditioning on the recovery of the left ventricular negative value of -dP/dt during reperfusion. -dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group (U) compared with preconditioning, halothane, and halothane + preconditioning. No significant difference was found among treated groups. (B) Effects of preconditioning (Pc), isoflurane (Iso), and isoflurane + preconditioning on the recovery of the left ventricular negative value of -dP/dt during reperfusion. -dP/dt is plotted as a function of time. Zero time is the beginning of reperfusion. Bars represent SEM. *P < 0.001 for the untreated group compared with preconditioning, isoflurane, and isoflurane + preconditioning. No significant difference was found among treated groups.
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Table 1. No comment.
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Table 1. No comment.
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