The FASEB Journal express article 10.1096/fj.02-0170fje. Published online July 1, 2002.
Delta opioid agonists and volatile anesthetics facilitate cardioprotection via potentiation of KATP channel opening Hemal H. Patel *,§, Lynda M. Ludwig *,‡,§, Ryan M. Fryer †,§, Anna K. Hsu *, David C. Warltier *,‡, Garrett J. Gross* *Medical College of Wisconsin, Department of Pharmacology and Toxicology, Milwaukee, Wisconsin; †Harvard Institutes of Medicine, Center for Neurological Disease, Boston, Massachusetts; and ‡Medical College of Wisconsin, Department of Anesthesiology, Milwaukee, Wisconsin. §These authors contributed equally to the data represented in the paper. Corresponding author: Garrett J. Gross, Medical College of Wisconsin, Department of Pharmacology and Toxicology, 8701 Watertown Plank Road, Milwaukee, WI 53226. E-mail:
[email protected] ABSTRACT Opioids and volatile anesthetics produce marked cardioprotective effects against myocardial infarction via the activation of ATP-sensitive potassium (KATP) channels, however, the effect of combined treatment with both drugs is unknown. We examined the hypothesis that opioids and volatile anesthetics potentiate cardiac KATP channel opening, thereby enhancing cardioprotection. Rats were treated with the delta opioid agonists, TAN-67 or BW373U86, or isoflurane, together or alone with and without diazoxide, a mitochondrial KATP channel opener. Glibenclamide, a non-selective KATP channel blocker, was used to further characterize the signaling mechanism involved. Myocardial infarct size (IS) was determined by tetrazolium staining and was expressed as a percent of the area at risk (AAR). High doses of TAN-67 (10 mg/kg), diazoxide (10 mg/kg), and isoflurane (1 MAC) produced a significant reduction in IS compared with the control group (30±3%, 36±5%, and 42±2 vs. 58±2%, respectively), whereas lower doses of the drugs had no effect except for the low dose of isoflurane (0.5 MAC). The combination of TAN-67 and diazoxide or isoflurane and diazoxide resulted in a marked reduction in IS compared with controls in the presence of high (9±3% and 14±3%) and low (17±7% and 31±7%) dose combinations, respectively. The combination of TAN-67 or BW373U86 and isoflurane also caused a striking reduction in IS/AAR (16±7% and 7±2%, respectively). To date, this is the first demonstration that opioids and volatile anesthetics work in conjunction to confer protection against myocardial infarction through potentiation of cardiac KATP channel opening. Key words: isoflurane • infarction • ischemia • preconditioning
I
schemic preconditioning (IPC) occurs when the myocardium is exposed to brief periods of ischemia that result in protection from subsequent prolonged ischemic insults (1). Since this original observation, numerous pharmacological interventions have been shown to mimic IPC. The signaling pathways that are induced by ischemia and various pharmacological agents converge on the mitochondrial KATP channel, which appears to function as the end effector.
Protection from stresses, such as ischemia and hypoxia, has been elicited by activation of opioid receptors in numerous organ systems, and the δ1-opioid receptor appears to be the predominant form found in the heart leading to cardioprotection during acute or delayed IPC (2). Treatment with agonists (3–5) and antagonists (2, 5) of δ1-opioid receptors results in cardioprotection and blockade of protection, respectively. It also appears that the protection induced by opioid receptor stimulation is sensitive to blockers of the KATP channel (3, 6, 7). Similar observations hold true for treatment with volatile anesthetics. It has been shown in both canine and rabbit models that isoflurane produces a marked protective effect on the myocardium, and that this effect is attenuated by treatment with inhibitors of the sarcolemmal and mitochondrial KATP channel (8–12). Not only does blockade of the mitochondrial KATP channel inhibit the protection induced by numerous stimuli, but direct stimulation of the channel via diazoxide, a mitochondrial KATP channel opener has also been beneficial. This channel appears to be the key target in establishing the mechanistic link between IPC and its cardioprotective effect. Though it is known that protection induced by opioids or anesthetics can be abolished by KATP channel blockade, it is not clear how these stimuli lead to cardioprotection via KATP channel opening. The goal of the present investigation was to characterize the mechanistic link between the stimulus and end effect of cardioprotection and to determine whether the combination of opioids and volatile anesthetics could enhance the cardioprotective effect beyond that of either agent alone. We hypothesized that stimulation via opioids or anesthetics potentiates the opening of KATP channels, and it is this initial priming of the channel that results in a more robust protective effect. MATERIALS AND METHODS Drugs (±)-[1(S*),2α,5β]-4-[[2,5-Dimethyl-4-(2-propenyl)-1-piperazinyl](3-hydroxyphenyl)methyl]N,N-diethyl-benzamide hydrochloride (BW373U86), diazoxide, and glibenclamide were purchased from Sigma (St. Louis, MO). TAN-67 was kindly supplied by Hiroshi Nagase, of Toray Industries (Kanagawa, Japan). Study groups and experimental protocols Male Wistar rats (300–350 g) were divided randomly into groups (Fig. 1). The rats were fed standard rodent food and had access to water ad libitum. Control groups consisted of rats undergoing no drug intervention. All drugs were administered at the time points noted (Fig. 1). Opioids [high (10 mg/kg) and low (5 mg/kg) dose for TAN-67; high dose (5 mg/kg) for BW373U86], diazoxide [high (10 mg/kg) and low (3 mg/kg) dose], and glibenclamide (3 mg/kg) were administered via the jugular vein. Isoflurane [high (1 MAC) and low (0.5 MAC) dose] was administered via an Ohmeda, Isotec 3 vaporizer.
General surgical procedure Rats were anesthetized by using 120–150 mg/kg Inactin intraperitoneally. The right jugular vein was cannulated for the delivery of saline and drugs. The right carotid artery was cannulated for the measurement of blood pressure and heart rate by using a Gould PE50 or PE23 pressure transducer and a Grass Model 7 polygraph. A tracheotomy was performed, and the trachea was intubated with a cannula connected to a rodent artificial ventilator (model 683, Harvard Apparatus, South Natick, MA). The rats were ventilated with room air at 38–45 breaths/min supplemented with O2. Atelectasis was prevented by maintaining a positive end-expiratory pressure of 5–10 mm H2O. Arterial pH, pCO2, and pO2 were monitored at control, 15 min postcoronary occlusion, and at 60 and 120 min after reperfusion (AVL 995 pH/Blood Gas Analyzer). Normal values were maintained by adjusting the respiratory rate and/or tidal volume. Body temperature was maintained at 36ûC by using a heating pad. Once heart rate and blood pressure stabilized, a left thoracotomy was performed at the fifth intercostal space. A pericardiotomy was completed followed by adjustment of the left atrial appendage to expose the left coronary artery. A ligature (6–0 prolene) was passed below the left descending coronary artery and vein in the area immediately below the left atrial appendage to the right portion of the left ventricle. The ends of the suture were threaded through a propylene tube to form a snare. Occlusion for a period of 30 min was elicited by pulling on the snare. This resulted in left ventricular ischemia confirmed by epicardial cyanosis and a decrease in arterial pressure. Reperfusion, for a period of 2 h, was achieved by unclamping the hemostat and loosening the snare. Determination of infarct size After the 2-h period of reperfusion, the coronary artery was re-occluded by using the snare. The area at risk (AAR) was determined by negative staining. Patent blue dye was administered via the jugular vein to stain the non-occluded area of the left ventricle. The heart was excised, and the left ventricle was separated from the remaining tissue and cut into thin cross sections. The normal areas were stained blue, while the AAR remained pink. The normal area and AAR were separated and placed in different vials containing 1% 2,3,5-triphenyltetrazolium chloride in 100 mM phosphate buffer (pH 7.4), a histochemical stain for viable tissue. These vials were incubated at 37ûC for 15 min. Tissues were fixed overnight in 10% formaldehyde, and the infarcted tissue was dissected from the AAR by using a dissecting microscope (Cambridge Instruments). Infarct size (IS) and AAR size were determined by gravimetric analysis. IS was expressed as a percentage of the AAR (IS/AAR). Statistical analysis All values are expressed as mean ± SEM. For the hemodynamic data, left ventricular mass, area at risk, and infarct size, statistical significance was determined by performing a one-way ANOVA with Bonferroni’s Multiple Comparison Test as the post-hoc test. A P value
