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Delta opioid agonists and volatile anesthetics facilitate cardioprotection via potentiation of K_ATP channel opening¹

HEMAL H. PATEL^2,*, LYNDA M. LUDWIG^2,*,, RYAN M. FRYER^2,, ANNA K. HSU^*, DAVID C. WARLTIER^*, and GARRETT J. GROSS^*3

^* Medical College of Wisconsin, Department of Pharmacology and Toxicology, Milwaukee, Wisconsin, USA;
Harvard Institutes of Medicine, Center for Neurological Disease, Boston, Massachusetts, USA; and
Medical College of Wisconsin, Department of Anesthesiology, Milwaukee, Wisconsin, USA

³Correspondence: Medical College of Wisconsin, Department of Pharmacology and Toxicology, 8701 Watertown Plank Road, Milwaukee, WI 53226. E-mail: ggross{at}mcw.edu

SPECIFIC AIMS

This study used an in vivo rat model of ischemia-reperfusion injury to 1) examine the potentiation of cardiac K_ATP channel opening by opioids and volatile anesthetics to confer protection against myocardial infarction and 2) determine whether combination therapy using opioids and volatile anesthetics could potentially enhance the cardioprotective effect beyond that of either stimulus alone.

PRINCIPAL FINDINGS

1. Potentiation of K_ATP channel activity by opioids
To study the effect of opioids on K_ATP channel activity, the delta opioid agonist TAN-67 was given alone or before the K_ATP channel opener diazoxide. Given individually, only high doses of TAN-67 or diazoxide produced a cardioprotective effect. In combination, however, high and low doses of TAN-67 and diazoxide produced a significant reduction in myocardial infarct size (9±3% and 17±7%, respectively) compared to the control group (58±2%). The protection due to combined drug therapy was better than protection produced by either drug given alone. These data suggest that pretreatment with an opioid agonist potentiates the opening of cardiac K_ATP channels produced by a K_ATP channel opener to produce an additive cardioprotective effect.

2. Potentiation of K_ATP channel activity by volatile anesthetics
To study the effect of volatile anesthetics on K_ATP channel activity, isoflurane was administered before diazoxide. Individual treatment produced a modest cardioprotective effect, but when combined with high and low doses of the volatile anesthetic isoflurane and diazoxide, the K_ATP channel opener, produced a significant reduction in myocardial infarct size (14±3% and 31±7%, respectively) compared to the control group. These data suggest that volatile anesthetics produce a cardioprotective effect through potentiation of cardiac K_ATP channel opening.

3. Combined effects of opioids and volatile anesthetics
Since opioids and volatile anesthetics appear to potentiate the opening of cardiac K_ATP channels, we hypothesized that combining an opioid with a volatile anesthetic should produce a cardioprotective effect similar to that seen with the opioid or isoflurane and diazoxide combination. Delta opioid agonists TAN-67 or BW373 U86 were given with isoflurane to determine whether this is the case. Treatment with either TAN-67 or BW373 U86 and isoflurane produced a significant reduction in myocardial infarct size (16±7% and 7±2%, respectively) compared to the control group (Fig. 1 ). The nonselective K_ATP channel blocker glibenclamide attenuated the marked cardioprotective effect conferred by combined treatment with BW373 U86 and isoflurane, with a resultant infarct size (52±4%) similar to that of the control group (Fig. 1) . These data suggest that opioids and volatile anesthetics mediate their protective effects via opening of cardiac K_ATP channels, and combining the two produces a potentiation of K_ATP channel opening and cardioprotection.

View larger version (20K): [in this window] [in a new window]   Figure 1. Opioids, volatile anesthetics, and KATP channel blockade. Rats were given isoflurane (1 MAC), an infusion of TAN-67 (10 mg/kg) or BW373 U86 (5 mg/kg), or a combination of isoflurane and TAN-67 or BW373 U86. Rats were also administered isoflurane and BW373 U86 in the presence of a glibenclamide (3 mg/kg) bolus. After drug intervention, the rats underwent 30 min of ischemia, followed by 2 h of reperfusion. Isoflurane was discontinued at the start of reperfusion. AAR was determined by injection of patent blue dye and separated from the normal tissue. Infarct size was determined by TTC staining. Percent infarct is expressed as a percentage of the AAR (IS/AAR). Values represent mean ± SE. *Significant differences from the control group (P<0.05). +Significant difference from isoflurane (1MAC) and BW373 U86 (5 mg/kg) groups (P<0.05). Represents significant difference from the ISO group (1MAC). Significant difference from the isoflurane and BW373 U86 combination group.

CONCLUSIONS AND SIGNIFICANCE

Our results demonstrate that opioids and volatile anesthetics work cooperatively with diazoxide to produce a potent cardioprotective effect in anesthetized rats. When administered alone, high doses of opioids, diazoxide, and isoflurane produce a modest cardioprotective effect. At low doses, the protective effects of these agents were not apparent except for the low dose of isoflurane. When given in combination, a higher dose of TAN-67 or isoflurane plus diazoxide produced a reduction in infarct size that was significantly greater than the high or low dose of each drug given alone. We demonstrate that combining opioid receptor agonists (TAN-67 or BW373 U86) with isoflurane produced a marked cardioprotective effect substantially greater than that observed when the drugs were administered alone. These cardioprotective effects could be antagonized by glibenclamide, a nonselective K_ATP channel blocker, implying that the effect of these drugs is mediated by modulation of K_ATP channel activity. These results suggest that opioids and volatile anesthetics work by priming the opening of cardiac K_ATP channels, possibly bringing the threshold for protection lower, which can then be augmented by these drugs in combination.

Priming of K_ATP channels in relation to decreasing the latency to channel opening and enhancing the effect of openers such as diazoxide and pinacidil has been reported before. It was shown that adenosine primed the opening of the mitochondrial K_ATP channel and used protein kinase C (PKC) as a potential mediator for this action. It has been shown that the activity of openers of the K_ATP channel such as diazoxide and pinacidil in isolated myocytes is enhanced by pretreatment with adenosine or a PKC activator. It seems likely that G-protein-coupled receptors such as the adenosine or opioid receptor result in activation of PKC, which then functions not only to open the K_ATP channel but also to decrease the latency of opening. This activity of PKC provides a primed channel for direct K_ATP openers to further enhance a cardioprotective signal. Studies in multiple species show that a variety of stimuli, including opioids and volatile anesthetics, produce a cardioprotective effect that is sensitive to K_ATP channel blockade.

We show in our model that delta opioids agonists and isoflurane act in a fashion in the intact animal similar to that observed with adenosine in isolated cardiomyocytes. Various G-protein-coupled receptors may use similar pathways to facilitate cardioprotection. Our laboratory has recently shown not only the involvement of K_ATP channels in opioid-mediated signaling that leads to protection, but that an essential component in the mechanism is PKC-. Delta opioid receptor stimulation by selective agonists results in translocation of PKC- to the mitochondria, where it potentially functions to modulate channel activity. The translocation of this particular isoform, in contrast to other isoforms of PKC, was blocked by selective PKC inhibitors and by BNTX, a selective ₁-opioid receptor antagonist. By inhibiting translocation of PKC, the cardioprotective effect of TAN-67 (a delta receptor agonist) was abolished.

The signaling mechanisms activated by volatile anesthetics have only recently been studied. It is not clear whether these agents act on specific receptors or directly on effector molecules. It is likely these compounds activate mediators such as PKC to modulate K_ATP channel activity. We have previously shown that isoflurane is capable of activating adenosine receptors and possibly signals through PKC activation. In fact, we have demonstrated that blockade of G-proteins abolishes cardioprotection produced by isoflurane. To definitively establish this link, additional studies are needed. However, our investigations relating to the priming effects on K_ATP channels show striking similarities between opioids and volatile anesthetics.

Previous studies suggest that opioids and volatile anesthetics use the K_ATP channel for cardioprotection, so it would seem likely that giving one drug first should prime channel opening for the subsequent drug. We demonstrated that by combining delta opioid agonists and isoflurane, we enhanced the cardioprotective effect produced by either drug alone. Giving a K_ATP channel blocker before both stimuli attenuated the protective effects of combined drug treatment, suggesting that the channel was prevented from being primed and/or opened. Our observations suggest that stimuli that modify K_ATP channel activity can be combined to enhance a cardioprotective signal.

Numerous studies have focused on the potential involvement of reactive oxygen species (ROS) in the mechanism of protective signals and involvement of the mitochondrial K_ATP channel as the source of this ROS burst. It does not seem likely that the channel contributes directly to the generation of ROS, but may in fact be susceptible to modulation by ROS. This could represent a potential mechanism of K_ATP channel priming independent of PKC.

In summary, it appears that the K_ATP channel is an end effector of cardioprotection after administration of opioids or isoflurane. The activity of this channel, whether primed or unprimed, leads to the infarct-sparing effects of various stimuli. This is an important concept and may provide a possible therapeutic target. Finally, opioids and volatile anesthetics, two compounds already used clinically in combination, have the potential to produce a dramatic beneficial effect in patients suffering an acute myocardial infarction.

View larger version (45K): [in this window] [in a new window]   Figure 2. Schematic diagram. Opioids and volatile anesthetics may confer cardioprotection against myocardial infarction via similar signaling pathways. Activation of G-protein-coupled receptors may stimulate PKC to enhance KATP channel activity. Opioids and volatile anesthetics may also have direct effects on mitochondria to generate reactive oxygen species (ROS), which could directly modulate KATP channels.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0170fje; to cite this article, use FASEB J. (July 1, 2002) 10.1096/fj.02-0170fje

² These authors contributed equally to the data represented in the paper.

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