This investigation tested the hypothesis that adenosine (A1) receptor blockade modulates the cardioprotective effects of isoflurane.
Hemodynamics and percentage segment shortening (%SS) in the left anterior descending coronary artery (LAD) perfusion territory were evaluated in barbiturate-anesthetized dogs (n = 31) at selected intervals after pretreatment with the selective A1 receptor antagonist (8-cyclopentyl-1,3,dipropyl-xanthine; DPCPX 0.8 mg/kg, intravenously) or drug vehicle in the presence or absence of 1 minimum alveolar concentration (MAC) isoflurane. Dogs were subjected to five 5-min occlusions and reperfusions of the LAD, followed by 180 min of final reperfusion. Isoflurane was administered for 30 min before and during LAD occlusions and reperfusions and was discontinued at the onset of final reperfusion. Two other groups of dogs (n = 17) were used to measure interstitial concentrations of purines in the LAD region using a microdialysis technique in the presence and absence of isoflurane.
Dogs receiving drug vehicle or DPCPX exhibited no recovery of %SS after 180 min of reperfusion (-5 +/- 7 and 5 +/- 11% of baseline, respectively, +/- SEM). In contrast, dogs receiving isoflurane alone demonstrated complete recovery of %SS at 60 min after reperfusion. DPCPX pretreatment partially attenuated isoflurane-induced enhancement of recovery of %SS (34 +/- 11% of baseline 180 min after reperfusion; P < 0.05). Interstitial purine concentrations were increased during multiple occlusions and reperfusions of the LAD in dogs not receiving isoflurane, but they were unchanged by coronary artery occlusion and reperfusion in dogs receiving isoflurane.
The results indicate that isoflurane-induced cardioprotection in stunned myocardium is partially mediated by adenosine type 1 receptor activation and is accompanied by decreases in endogenous adenosine release.
Multiple periods of brief coronary artery occlusion interspersed with reperfusion have been shown to cause reversible, postischemic contractile dysfunction known as stunned myocardium.  The precise mechanism of this phenomenon and the ability to pharmacologically modulate this process are presently under intense investigation. Adenosine has been shown to play an important cardioprotective role in postischemic reperfused myocardium. [2–5] This endogenous nucleoside has been demonstrated to decrease oxygen-derived free radical formation,  reduce adenosine triphosphate (ATP) metabolism during ischemia, increase ATP synthesis during reperfusion, [7,8] and inhibit neutrophil adherence to endothelium.  Adenosine also enhances the recovery of stunned myocardium by stimulating glycolysis and improving energy balance during ischemia,  inhibiting calcium (Ca2+) influx through L-type Ca2+ channels, [11,12] blocking norepinephrine release,  and attenuating contractile response to beta1-adrenoceptor stimulation.  Most recently, adenosine-induced cardioprotection has been linked to activation of ATP-dependent potassium (KATP) channels via adenosine type 1 (A1) receptors. 
Volatile anesthetics, including isoflurane, enhance the recovery of stunned myocardium. [16–18] Recent investigations from our laboratory [17,18] have demonstrated that the cardioprotective effects of isoflurane in postischemic, reperfused myocardium are mediated, at least in part, by KATPchannel activation. Although A sub 1 receptors are known to be coupled to KATPchannels, [4,15] the role of adenosine receptor activation during isoflurane-induced cardioprotection has not been defined. The present investigation tested the hypothesis that isoflurane-induced cardioprotection in stunned myocardium is modulated by A1receptors. In addition, because A1receptor activation during ischemic preconditioning may alter interstitial adenosine concentrations,  a second group of experiments were performed to evaluate interstitial purine concentrations during brief occlusions and reperfusions of the LAD in the presence and absence of isoflurane.
Method and Materials
All experimental procedures and protocols used in this investigation were reviewed and approved by the Animal Care and Use Committee of the Medical College of Wisconsin. Further, all conformed to the Guiding Principles in the Care and Use of Animals of the American Physiologic Society and were in accordance with the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, DC, 1996).
Implantation of instruments has been described in detail previously.  Briefly, mongrel dogs were anesthetized with sodium barbital (200 mg/kg) and sodium pentobarbital (15 mg/kg). The lungs of each dog were ventilated via positive pressure with an air and oxygen mixture after tracheal intubation. A double, pressure transducer-tipped catheter was inserted into the aorta and left ventricle for measurement of aortic and left ventricular pressures and the maximum rate of increase of left ventricular pressure (dP/dtmax). An ultrasonic transit time flow probe was placed around the ascending thoracic aorta for measurement of aortic blood flow. Heparin-filled catheters were inserted into the left atrial appendage and the right femoral artery for administration of radioactive microspheres and withdrawal of reference blood flow samples, respectively. A precalibrated Doppler ultrasonic flow transducer was placed around the left anterior descending coronary artery (LAD) for measurement of coronary blood flow velocity. A silk ligature was loosely placed around the LAD for subsequent coronary artery occlusion. A pair of ultrasonic segment length transducers was implanted in the LAD subendocardium for measurement of changes in regional contractile function. Segment length and coronary blood flow velocity signals were monitored by ultrasonic amplifiers. Relative diastolic coronary vascular resistance was calculated as the ratio of end-diastolic arterial pressure to peak diastolic coronary blood flow velocity. The pressure-work index, an estimate of global myocardial oxygen consumption, was determined.  End-systolic segment length (ESL) was measured at 10 ms before maximum negative left ventricular dP/dt, and end-diastolic segment length (EDL) was measured 10 ms before dP/dt first exceeded 140 mmHg/s (immediately before the onset of left ventricular isovolumic contraction). Percent segment shortening (%SS) was calculated using the formula: %SS =(EDL-ESL)[center dot] 100 [center dot] EDL sup -1. All hemodynamic data were continuously monitored on a polygraph and digitized via a computer interfaced with an analog to digital converter.
Regional Myocardial Blood Flow
Carbonized plastic microspheres (15 +/- 2 micro meter [SD] in diameter) labeled with141Ce,103Ru,51Cr, or95Nb were used to measure regional myocardial perfusion as previously described. Briefly, 2–3 x 106microspheres were administered into the left atrium as a bolus during a 10-s period and flushed in with 10 ml of warm (37 degrees Celsius) saline. A few seconds before the microsphere injection, a timed collection of reference arterial flow was started from the femoral arterial catheter and withdrawn at a constant rate of 7 ml/min for 3 min. Transmural tissue samples were selected for mapping of tissue flow in the myocardium at the conclusion of each experiment. The samples were obtained from two regions of the left ventricle: 1) normal zone (myocardium supplied by the left circumflex coronary artery [LCCA]); and 2) ischemic zone (distal to the LAD occlusion). Myocardial tissue samples were subdivided into subepicardial, midmyocardial, and subendocardial layers of approximately equal thickness and weight (0.75 g). Samples were weighed, placed in scintillation vials, and the activity of each isotope determined. Similarly, the activity of each isotope in the reference blood sample was assessed. Tissue blood flow (ml [center dot] min sup -1 [center dot] g sup -1) was calculated as Qr[center dot] Cm[center dot] Crsup -1, wherein Qr= rate of withdrawal of the reference blood flow sample (ml/min); Cm= activity (cpm/g) of the myocardial tissue sample; and Cr= activity (cpm) of the reference blood flow sample. Transmural blood flow was considered as the average of subepicardial, midmyocardial, and subendocardial blood flows.
The experimental design is illustrated in Figure 1. Dogs were randomly assigned to receive drug vehicle (50% polyethylene glycol in 0.1 N sodium hydroxide and normal saline) or the selective A1receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.8 mg/kg, intravenously) in the presence or absence of 1 minimum alveolar concentration (MAC)(1.28% end-tidal concentration) isoflurane. End-tidal concentrations of isoflurane were measured at the tip of the endotracheal tube by an infrared anesthetic analyzer that was calibrated with known standards before and during experimentation.
Thirty minutes after instrumentation was completed, baseline systemic and coronary hemodynamics were recorded. All dogs were subjected to five 5-min periods of LAD occlusion separated by 5-min periods of reperfusion and followed by a final 180 min reperfusion, during which hemodynamics and contractile function were continuously monitored. Regional myocardial blood flow was measured under baseline conditions, during the fifth coronary artery occlusion, and after 60 and 180 min of reperfusion. In one group of experiments, dogs received drug vehicle (5 ml) over 10 min and underwent repetitive occlusions and reperfusions 30 min after administration of drug vehicle. In a second group of experiments conducted in an identical fashion, dogs were pretreated with DPCPX (0.8 mg/kg) to examine the effect of A1receptor blockade on recovery of function after multiple episodes of ischemia and reperfusion. In two final groups of experiments, the effects of isoflurane on recovery of stunned myocardium were assessed in dogs pretreated with drug vehicle or DPCPX. Immediately after receiving drug vehicle or DPCPX, isoflurane (1 MAC) was administered for 30 min before and during LAD occlusion and reperfusion. Isoflurane was then discontinued at the onset of the final reperfusion.
Determination of Myocardial Interstitial Purine Concentrations
The effects of multiple LAD occlusions and reperfusions on interstitial purine concentrations were measured in the presence or absence of 1 MAC isoflurane in a separate groups of experiments using a cardiac microdialysis technique as previously described. [21,22] Briefly, each microdialysis probe was constructed with silica tubing placed over each end of a single dialysis fiber, exposing a 2-cm dialysis window. The probe was inserted into the myocardial wall so that the dialysis window was completely imbedded in the midmyocardium of the ischemic region. One end of the probe was glued into a larger piece of silica tubing that was connected to a gas-tight glass syringe filled with a Krebs/Henseleit buffer consisting of 118 mmol/l NaCl, 25 mmol/l NaHCO3, 11 mmol/l glucose, 4.7 mmol/l KCl, 1.25 mmol/l CaCl2, 1.2 mmol/l MgSO4, and 1.2 mmol/l KH2PO4and equilibrated with 95% N2/5% CO2. The microdialysis fiber was implanted in myocardium perfused by the LAD, and 90 min later, a syringe pump was used to perfuse the probe at 2 micro liter/min, and the effluent was collected in glass tubes. Effluent samples were obtained at selected intervals before and during multiple occlusions and reperfusions in the presence and absence of isoflurane.
High performance liquid chromatography was used to analyze dialysate purines.  Briefly, a sample (5 micro Liter) of the raw dialysate or standard was injected onto a 1090 Series II Liquid Chromatograph (Hewlett Packard Co., Palo Alto, CA) using an autosampler. A microbore column ODS-2 C18(5 micro meter) 250 x 1.0 mm (Phenomenex, Palo Alto, CA) with a linear gradient of 10 mmol/l KH2PO4(A) and 10 mmol/l KH2PO4in 50%(v/v) methanol/water (B)(pH = 4.55 adjusted with phosphoric acid) were used for separation. The gradient started at 4–10% B in 10 min, 10–70% B in 5 min, 70–100% B in 12 min, held at 100% B for 2 min, 100–4% B in 1 min, and a 20-min postrun at 4% B. The flow rate was 50 micro liter/min. A photo diode array detector was used to simultaneously record chromatograms at 254 nm (hypoxanthine, inosine), 260 nm (adenosine), and 270 nm (xanthine) with a 4-nm bandwidth referenced to 450 nm with a 10-nm bandwidth. These are the optimal wavelengths for detection of each compound. Chromatograms were stored and analyzed on a Chem Station (Hewlett Packard Co.). Peak area was calculated and compared with that of known standards to determine the purine concentration of each sample, with a detection limit of 50 fmol. 
Statistical analysis of data within and between groups under baseline conditions, during drug and anesthetic interventions, and during LAD occlusions and reperfusions was performed with multiple analysis of variance (MANOVA) for repeated measures followed by application of Student's t test with Bonferroni's correction for multiplicity. Changes within and between groups were considered statistically significant when the P value was less than 0.05. All data are expressed as mean +/- SEM.
Hemodynamic Effects of Ischemia and Reperfusion in Dogs Receiving Drug Vehicle
There were no significant differences in baseline hemodynamics between experimental groups. Arterial blood gases were maintained within the physiologic range in each group throughout the experiment. LAD occlusion significantly (P < 0.05) decreased dP/dtmax, cardiac output, and stroke volume in drug vehicle-pretreated dogs (Table 1). No changes in heart rate, mean arterial pressure, or pressure-work index were observed. LAD occlusion caused regional dyskinesia during each 5-min occlusion (Figure 2). Persistent and progressive decreases in regional contractile function were observed during each 5-min reperfusion period and during 180 min after final reperfusion of the LAD. Impairment of regional contractile function was accompanied by increases in heart rate and diastolic coronary and systemic vascular resistances and decreases in cardiac output, stroke volume, and diastolic coronary blood flow velocity.
Hemodynamic Effects of Ischemia and Reperfusion in Dogs Receiving DPCPX
LAD occlusion in DPCPX-pretreated dogs (Table 2) caused hemodynamic effects that were similar to those observed in dogs receiving drug vehicle. Regional dyskinesia was observed during each LAD occlusion (Figure 2), and decreases in %SS were demonstrated during each 5-min reperfusion period and throughout 180 min of final reperfusion. Contractile dysfunction during reperfusion was accompanied by increases in mean arterial pressure and systemic vascular resistance and by decreases in dP/dtmax, cardiac output, and stroke volume at 180 min after reperfusion. No significant differences in systemic and coronary hemodynamics and %SS were observed between drug vehicle- and DPCPX-pretreated dogs (Table 1and Table 2).
Hemodynamic Effects of Ischemia and Reperfusion in Dogs Receiving Isoflurane Alone
Isoflurane decreased heart rate, mean arterial and left ventricular systolic pressures, dP/dtmax, diastolic coronary vascular resistance, cardiac output, and pressure-work index (Table 3). Diastolic coronary blood flow velocity, systemic vascular resistance, and stroke volume were unchanged. LAD occlusion caused no additional hemodynamic effects in isoflurane-anesthetized dogs. Dogs receiving isoflurane demonstrated significantly lower heart rate, mean arterial and left ventricular systolic pressures, dP/dtmax, systemic vascular resistance, and pressure-work index than dogs receiving drug vehicle or DPCPX alone. Equivalent degrees of systolic dyskinesia occurred during the first LAD occlusion in each group (Figure 2). Dogs receiving isoflurane alone demonstrated complete recovery of %SS to control values during the first reperfusion period and during final reperfusion. Isoflurane-pretreated dogs also demonstrated significantly greater regional contractile function than drug vehicle- or DPCPX-pretreated dogs during the second and fourth 5-min reperfusions and throughout 180 min of final reperfusion (Figure 2). Heart rate and pressure-work index were lower in dogs receiving isoflurane alone compared with those receiving drug vehicle or DPCPX during the 180 min of final reperfusion.
Hemodynamic Effects of Ischemia and Reperfusion in Dogs Receiving Isoflurane and DPCPX
Isoflurane decreased heart rate, mean arterial and left ventricular systolic pressures, dP/dtmax, cardiac output and pressure-work index in dogs pretreated with DPCPX (Table 4). In contrast to dogs receiving isoflurane alone, diastolic coronary vascular resistance was unchanged in DPCPX-pretreated dogs during administration of isoflurane. No differences in hemodynamics were observed in dogs receiving DPCPX and isoflurane compared with those receiving isoflurane alone during coronary artery occlusions or after final reperfusion. Dogs pretreated with DPCPX and isoflurane demonstrated equivalent degrees of systolic dyskinesia during each LAD occlusion compared with dogs receiving isoflurane alone. However, %SS failed to recover to control values during final reperfusion of the LAD, and %SS was decreased in isoflurane-anesthetized dogs receiving DPCPX compared with dogs receiving isoflurane alone 180 min after final reperfusion (Figure 2).
Effects of Myocardial Stunning on Regional Myocardial Perfusion
No differences in myocardial perfusion to the ischemic or normal regions (Table 5) at baseline or during coronary artery occlusion and reperfusion were observed between groups. Multiple LAD occlusions and reperfusions decreased transmural blood flow in the ischemic zone to equivalent degrees in all four experimental groups.
Effects of Myocardial Stunning on Interstitial Purine Concentrations
Multiple episodes of ischemia and reperfusion caused increases in interstitial adenosine, inosine, hypoxanthine, and xanthine in control dogs (Figure 3). Administration of isoflurane caused initial decreases in adenosine (Figure 3(A)) and xanthine (Figure 3(B)) concentrations that returned to baseline values after 30 min equilibration at 1 MAC. Isoflurane abolished increases in interstitial purine concentrations during multiple occlusions and reperfusion of the LAD in contrast to the increases observed in the absence of this volatile anesthetic.
The cardioprotective role of adenosine during myocardial ischemia and reperfusion injury has received considerable recent attention. Adenosine reduced myocardial damage after a 15-min coronary artery occlusion and reperfusion [2,3,5] or after multiple brief coronary artery occlusions and reperfusion.  Functional recovery of stunned myocardium was enhanced in dogs receiving intracoronary adenosine before and during ischemia but not during reperfusion alone.  Increases in endogenous adenosine attained through blockade of adenosine deaminase [2,5] or inhibition of nucleoside transport  also enhanced contractile function of postischemic, reperfused myocardium.
The mechanisms by which adenosine exerts its cardioprotective effects are not fully understood. Stimulation of A1receptors by adenosine has been firmly linked to activation of KATPchannels in ventricular myocardium. [4,15,23–26] KATPchannel activation has been shown to enhance functional recovery of stunned myocardium,  decrease myocardial infarct size,  and mimic the effects of ischemic preconditioning  in vivo, actions which are blocked by the selective KATPchannel antagonist, glyburide (glibenclamide). [27,29,30] It has been hypothesized that KATPchannel activation represents the end-effector in an endogenous cardioprotective signal transduction system that becomes activated during myocardial ischemia. [25,26] Stimulation of A1receptors by adenosine may play an important role in this process because these receptors are coupled to K sub ATP channels via inhibitory G proteins in ventricular myocytes. 
A recent investigation  from our laboratory demonstrated that isoflurane-induced cardioprotection in stunned myocardium is abolished by glyburide pretreatment. This finding suggests that isoflurane activates KATPchannels in myocardium. The role of adenosine in the modulation of cardioprotective signal transduction during isoflurane anesthesia has not been previously investigated. The present results show that the cardioprotective effects of isoflurane are attenuated by pretreatment with DPCPX. DPCPX, a xanthine derivative, previously has been shown to be 700 times more selective for A1versus A2receptors using radioligand binding studies and in vitro functional assays.  Although isoflurane enhanced recovery of function of stunned myocardium to baseline values, DPCPX pretreatment in dogs receiving isoflurane attenuated recovery of segment shortening after 180 min of reperfusion. These results indicate that isoflurane-induced cardioprotection is partially mediated by A1receptor activation. Interestingly, Yao et al.  demonstrated that cardioprotection induced by cyclopentyladenosine, a selective A1receptor agonist, was blocked by glyburide, indicating that an important interaction exists between A1receptors and myocardial KATPchannels. The results of the present and previous [17,18] investigations demonstrate that the myocardial protective effect of isoflurane involves activation of A1receptors and KATPchannels, although it is unclear at present whether the effect is sequential or mediated by independent effects on the receptor and the channel. The findings also indicate that DPCPX partially attenuates, whereas glyburide completely abolishes,  the beneficial effects of isoflurane on functional recovery of stunned myocardium, results which support the hypothesis that KATPchannel activation represents the end-effector in isoflurane-induced cardioprotection. The mechanism of KATPchannel-induced cardioprotection is incompletely understood, but it may result from decreases in action potential duration, decreased intracellular calcium accumulation, and preservation of cellular energy stores. [32–34] Whether isoflurane modulates other signaling pathways or other elements in the adenosine/KATPpathway of cardioprotective signal transduction has not been examined during this investigation and is unknown.
The actions of DPCPX to attenuate rather than completely abolish the enhanced recovery of stunned myocardium by isoflurane were probably not related to the use of an insufficient dose of DPCPX. During preliminary studies,  a higher dose of DPCPX (1 mg/kg. intravenously) exacerbated myocardial stunning and caused significantly greater reductions in %SS during reperfusion when compared with dogs receiving drug vehicle alone. The dosage of DPCPX (0.8 mg/kg) used during the present investigation did not adversely affect the recovery of stunned myocardium compared with control dogs. Similarly, the dosage of glyburide used in our previous investigation  had no effect on recovery of stunned myocardium. Nevertheless, glyburide abolished the beneficial effects of isoflurane on functional recovery of stunned myocardium, demonstrating that isoflurane-induced cardioprotection critically depends on KATPchannel activation. 
Myocardial ischemia results in ATP depletion and the degradation of adenine nucleotides to adenosine and inosine. During reperfusion, adenosine and inosine are released into the interstitial space, taken up by endothelial cells, and further metabolized to hypoxanthine and xanthine.  It has been suggested that during a brief period of ischemia, adenosine release, acting through activation of A1receptors and possibly via KATPchannels,  results in reductions in high-energy phosphate use during a subsequent prolonged period of ischemia.  Whereas A1receptor activation may trigger ischemic preconditioning,  interstitial and venous adenosine concentrations are paradoxically reduced in preconditioned myocardium  and in response to KATPagonists, respectively.  The results of the present investigation demonstrate that isoflurane abolishes increases in interstitial adenosine during multiple occlusions and reperfusions of the LAD, extending the findings of previous investigations during which isoflurane was shown to preserve high-energy phosphate content of postischemic, reperfused myocardium. [39,40] These anesthetic actions to attenuate ATP breakdown during ischemia are similar to those observed during ischemic preconditioning and in response to other KATPchannel agonists. These collective findings indicate that isoflurane either activates A1receptors directly or indirectly enhances the sensitivity of A1receptors to decreased amounts of endogenously released adenosine. Interstitial purine concentrations measured during this investigation provide an index of ATP depletion during ischemia, with a microdialysis efficiency of 59 +/- 1%.  A phase lag may exist between changes in tissue purine concentration and their subsequent collection and measurement. Such a phase delay would be expected to be similar among groups and thus not alter the results. In addition, tissue trauma caused by microdialysis fiber implantation was not likely to have altered the results because interstitial purine concentrations were stable 90 min after fiber implantation. 
The cardioprotective effects of isoflurane demonstrated in the present investigation were probably not related to differential alterations in myocardial oxygen consumption. The pressure-work index, a global estimate of myocardial oxygen consumption,  was similar in dogs receiving isoflurane in the presence or absence of DPCPX. There also were no differences in systemic hemodynamics between dogs receiving isoflurane with or without DPCPX pretreatment. Only dogs receiving isoflurane alone without DPCPX demonstrated complete recovery of segment shortening after 180 min of reperfusion. These results indicate that isoflurane-induced alterations in the primary determinants of myocardial oxygen supply and demand relations can, at best, account for only a fraction of the beneficial actions of this volatile anesthetic during ischemia. Coronary sinus oxygen tension was not determined, and direct measurements of myocardial oxygen consumption were not made in the present investigation.
Isoflurane previously has been shown to produce coronary vasodilation, and recently this action has been attributed to KATPchannel activation.  In the present investigation, isoflurane decreased diastolic coronary vascular resistance in drug vehicle- but not DPCPX-pretreated dogs. Although direct activation of A2receptors causes coronary vasodilation, these results suggest that stimulation of A1receptors by isoflurane indirectly contributes to anesthetic-induced coronary vasodilation. The results support the contention that isoflurane may act via a similar pathway involving stimulation of A1receptors coupled to KATPchannels in coronary vascular smooth muscle cells and ventricular myocytes to cause coronary vasodilation and antiischemic effects, respectively. Although isoflurane alone reduced diastolic coronary vascular resistance, alterations in myocardial perfusion do not account for the cardioprotective effects of isoflurane observed in the present investigation. Myocardial perfusion was similar in dogs receiving isoflurane with or without DPCPX. In addition, transmural collateral blood flow was very low (less or equal to 0.10 ml [center dot] min1[center dot] g sup -1), and no differences were observed between experimental groups.
In summary, the present results demonstrate that the cardioprotective effects of isoflurane in stunned myocardium are mediated, in part, by A1receptor activation. These effects are accompanied by decreases in endogenous adenosine release from the ischemic zone, findings similar to those found during ischemic preconditioning. The results provide evidence to support the hypothesis that isoflurane-induced cardioprotection occurs via direct activation or enhanced sensitivity of A1receptors coupled to KATPchannels in canine myocardium.
The authors thank Todd Schmeling and David Schwabe for technical assistance and Angela Barnes for assistance in preparation of this manuscript.