reperfusion injury

Life Sciences 84 (2009) 657–663

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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e

Activation of peripheral δ2 opioid receptors increases cardiac tolerance to ischemia/reperfusion injury Involvement of protein kinase C, NO-synthase, KATP channels and the autonomic nervous system Leonid N. Maslov a, Yury B. Lishmanov a, Peter R. Oeltgen b, Eva I. Barzakh a, Andrey V. Krylatov a, Meera Govindaswami b, Stephen A. Brown c,⁎ a b c

Laboratory of Experimental Cardiology, Research Institute of Cardiology, Siberian Branch, Russian Academy of Medical Sciences, Tomsk 634012, Russia Lexington Veterans Administration Medical Center and Department of Pathology, University of Kentucky, Lexington, Kentucky 40511, USA Lexington Veterans Administration Medical Center and Department of Medicine, University of Kentucky, Lexington, Kentucky 40511, USA

a r t i c l e

i n f o

Article history: Received 19 August 2008 Accepted 6 February 2009 Keywords: Delta opioid Deltorphin II PKC NOS KATP channel Tyrosine kinase Catecholamine

a b s t r a c t Aims: This study aims to investigate the role of peripheral δ2 opioid receptors in cardiac tolerance to ischemia/reperfusion injury and to examine the contribution of PKC, TK, KATP channels and the autonomic nervous system in δ2 cardioprotection. Main methods: Deltorphin II and various inhibitors were administered in vivo prior to coronary artery occlusion and reperfusion in a rat model. The animals were monitored for the development of arrhythmias, infarct development and the effects of selected inhibitors. Key findings: Pretreatment with peripheral and δ2 specific opioid receptor (OR) antagonists completely abolished the cardioprotective effects of deltorphin II. In contrast, the selective δ1 OR antagonist 7benzylidenenaltrexone (BNTX) had no effect. The protein kinase C (PKC) inhibitor chelerythrine and the NO-synthase inhibitor L-NAME (N-nitro-L-arginine methyl ester) also reversed both deltorphin II effects. The nonselective ATP-sensitive K+ (KATP) channel inhibitor glibenclamide and the selective mitochondrial KATP channel inhibitor 5-hydroxydecanoic acid only abolished the infarct-sparing effect of deltorphin II. Inhibition of tyrosine kinase (TK) with genistein, the ganglion blocker hexamethonium and the depletion of endogenous catecholamine storage with guanethidine reversed the antiarrhythmic action of deltorphin II but did not change its infarct-sparing action. Significance: The cardioprotective mechanism of deltorphin II is mediated via stimulation of peripheral δ2 opioid receptors. PKC and NOS are involved in both its infarct-sparing and antiarrhythmic effects. Infarctsparing is dependent upon mitochondrial KATP channel activation while the antiarrhythmic effect is dependent upon TK activation. Endogenous catecholamine depletion reduced antiarrhythmic effects but did not alter the infarct-sparing effect of deltorphin II. Published by Elsevier Inc.

Introduction The role of δ1 opioid receptors (ORs) in the regulation of cardiac tolerance to ischemia/reperfusion has been extensively studied (Fryer et al. 2000, 2001; Schultz et al. 1998a,b; Sigg et al. 2002). There is also a significant body of literature on the involvement of κ ORs in cardiac ischemic and pharmacological preconditioning (Cao et al. 2003; Schultz et al. 1998a). However, the role of δ2 OR in cardioprotection is less well understood. In swine and rat, pretreatment with deltorphin-D, a putative δ2 OR agonist, can decrease the infarct size/area at risk (IS/ AAR) ratio, but has no effect on coronary occlusion induced arrhythmias ⁎ Corresponding author. E-mail addresses: [email protected] (L.N. Maslov), [email protected] (S.A. Brown). 0024-3205/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.lfs.2009.02.016

(Sigg et al. 2002; Govindaswami et al. 2000). However, it is unclear whether this cardioprotective effect of deltorphin-D is mediated via δ2 OR occupancy. Moreover, it is unclear whether these receptors are located in the brain or in the peripheral organs. More recently, the compound ARD-353, which has high affinity for both δ1 and δ2 OR, has been shown to provide an infarct-limiting effect (Watson et al. 2006). These studies suggest that cardioprotection by ARD-353 is mediated via δ1 OR stimulation because the selective δ1 OR antagonist 7-benzylidenenaltrexone (BNTX) abolished this effect. However, the selective δ2 OR antagonist naltriben (NTB) was not tested. Consequently, there is a possibility that δ2 OR may be also involved in the infarctsparing effect of ARD-353. The mechanism of this infarct-reducing effect of δ2 OR stimulation remains to be determined. There is, however, extensive data on the molecular nature of the cardioprotective action of

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δ1 and κ OR agonists that demonstrate that protein kinase C (PKC) is involved in both the δ OR (Fryer et al. 2000; Schultz et al. 1998a,b; Cao et al. 2003) and κ OR (Cao et al. 2003) mediated increase in cardiac tolerance to ischemia/reperfusion injury. It has also been demonstrated that the cardioprotective action of δ1 and κ agonists is mediated via ATPsensitive potassium (KATP) channel opening (Schultz et al. 1998b; Fryer et al. 2000). Tyrosine kinase (TK) is also involved in the infarct-limiting action of δ1 agonists (Fryer et al. 2001). In addition, studies have demonstrated that the autonomic nervous system can be involved in the cardiovascular effects of enkephalins via opioid receptor stimulation (Sander et al. 1989). There is one report that these receptors are located outside the blood-brain barrier (BBB) (Giles et al. 1983). There is also a report that the n. vagus is involved in development of antifibrillatory effect of fentanyl (Saini et al. 1988). However, the role of the autonomic nervous system in the antiarrhythmic and cardioprotective effect of δ2 agonists is unknown. Thus, the purpose of these studies was twofold: first, to investigate the role of peripheral δ2 opioid receptors in the regulation of cardiac tolerance to ischemia/reperfusion in rats, and second, to examine the contribution of PKC, TK, KATP channel, and the autonomic nervous system to the mechanism of δ2 agonist-induced changes in the cardiac resistance to ischemia/reperfusion injury. Methods This study was approved by the Ethical Committee of the Institute of Cardiology of Tomsk Science Center of Russian Academy of Medical Sciences. Male Wistar rats weighing 250–300 g were housed at 23 ± 1 °C with a relative humidity of 60–70% and a light/dark cycle of 12 h with free access to water and standard rat chow. The rats were anesthetized via intraperitoneal administration of α-chloralose at a dose of 50 mg/kg. A tracheotomy was performed, and the trachea was intubated with a cannula connected to a ventilator (modified model RO-6, Kasnogvardeets, St. Petersburg, Russia). The rats were ventilated with room air supplemented with 100% O2. Atelectasis was prevented by maintaining a positive end-expiratory pressure of 5 to 10 mm H2O. Arterial pH, PCO2, and PO2 were monitored throughout the protocol with a blood gas analyzer (Stat Profile M, Nova Biomedical Corporation, Waltham, MA, USA) and maintained within a normal physiological range (pH, 7.35–7.45; PCO2, 25–40 mm Hg; PO2, 80–110 mm Hg) by adjusting the respiratory rate and/or tidal volume. Body temperature was maintained at 37 °C by the use of a heating pad. The femoral vein was cannulated for delivery of vehicle or drug infusion. Ischemia, reperfusion and myocardial staining were performed according to the method of Schultz et al. (1998b). A left thoracotomy was performed at the fifth intercostal space, followed by a pericardiotomy and adjustment of the left atrial appendage to reveal the location of the left coronary artery. A ligature (6-0 Prolene) was passed below the left descending vein and coronary artery from the area immediately below the left atrial appendage to the right portion of the left ventricle (LV). The ends of the suture were threaded through a polypropylene tube to form a snare. Occlusion of the coronary artery and subsequent regional left ventricular ischemia was produced by pulling the ends of the suture taut and clamping the snare onto the epicardial surface with a hemostat. The ST segment elevation on the electrocardiogram (ECG) verified coronary artery occlusion. The duration of occlusion was 45 min. Reperfusion of the heart was initiated via unclamping the hemostat and relieving the snare tension and was confirmed by visualizing a marked epicardial hyperemic response. The duration of reperfusion was 2 h. On completion of the experimental protocols, the coronary artery was reoccluded, and the area at risk (AAR) was determined by negative staining. Patent blue violet dye (150 μl, 10% w/v) was injected into the femoral vein to effectively stain the nonoccluded area of the LV. The duration of administration was 20–30 s. The heart was excised, and the LV was removed from the remaining tissue and subsequently cut into 5 thin cross-sectioned pieces. This allowed for the delineation of the

normal area, which stained blue, vs. the area at risk (AAR), which subsequently remained pink. The AAR was excised from the nonischemic area, and the tissues were placed in separate vials and incubated for 15 min in a 0.1% solution of nitrotetrazolium blue chloride in 100 mM phosphate buffer (pH 7.4) at 37 °C. The infarcted myocardium was dissected from the AAR. Infarct size (IS) and area at risk were determined by gravimetric analysis. IS was expressed as a percentage of the AAR (IS/AAR). The ratio of area at risk to left-ventricle mass was calculated to estimate the effect of test compounds on collateral coronary perfusion. A standard peripheral lead electrocardiogram (ECG) was continuously recorded during ischemia and the first 10 min of reperfusion. ECG recordings were performed using a potential amplifier UBF4-03 (Russia) and Pentium computer using the original applied program as elaborated by the programmer, Razenkov. Arrhythmias were quantified during the first 10 min of ischemia (phase 1a), following 35 min of ischemia (phase 1b), and the first 10 min of reperfusion. According to Russell et al. (1984), arrhythmogenesis of heart rhythm disturbance in the first 10 min of coronary artery occlusion is different from the mechanism of arrhythmia development after 10 min of ischemia. Therefore, in this study we analyzed the incidence of arrhythmias during phase 1a and phase 1b and following reperfusion. Following equilibration of heart rhythm, rats were divided into various experimental groups. Rats were pretreated with vehicle (control), pretreated with deltorphin II alone, pretreated with antagonist alone, pretreated with both deltorphin II and antagonists or pretreated with deltorphin II and inhibitors (chelerythrine, glibenclamide, 5-hydroxydecanoate). Deltorphin II was given intravenously as a bolus dose 15 min before the onset of ischemia, whereas antagonists were given 25 min before coronary artery occlusion. Following treatment, hearts were occluded for 45 min followed by 2 h of reperfusion before staining. The dose of deltorphin II was guided by results from our preliminary experiments with the nonselective peptide OR agonist dalargin (H-Tyr-D-Ala-Gly-Phe-Leu-Arg-OH) which exhibited an antifibrillatory effect during coronary artery ligation at a dose of 150 nM/kg (Maslov and Lishmanov 1991, 1993). Therefore, the selective δ2 OR agonist deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2) (Mattia et al. 1991) was injected at a dose of 150 nM/kg (0.12 mg/kg). Deltorphin II was dissolved in 0.9% NaCl solution. The nonselective opioid receptor antagonist naltrexone hydrochloride was used at a dose of 5 mg/kg (Tao et al. 2003). The nonselective peripherally acting OR inhibitor naloxone methiodide was administered at a dose of 5 mg/kg (Maslov et al. 2003). The selective δ1 OR antagonist BNTX (7-benzylidenenaltrexone maleate) was given at a dose of 0.7 mg/kg (Sofuoglu et al. 1993) and the selective δ2 OR inhibitor NTB (naltriben mesylate) was used at a dose of 0.3 mg/kg (Kamei et al.1995). The sarcolemmal and mitochondrial ATP-sensitive potassium channel (KATP channel) inhibitor glibenclamide was administered intravenously at a dose of 0.3 mg/kg 45 min before coronary artery occlusion as recommended by Schultz et al. (1997). The selective mitochondrial KATP channel (mitKATP channel) blocker 5-hydroxydecanoic acid sodium (5-hydroxydecanoate) was used at a dose of 5 mg/kg 5 min before ischemia (Fryer et al. 2000). The protein kinase C (PKC) inhibitor chelerythrine chloride was given at a dose of 5 mg/kg 25 min before coronary artery ligation (Fryer et al. 1999). The tyrosine kinase (TK) inhibitor genistein was administered at a dose of 5 mg/kg 45 min before occlusion (Fryer et al. 1999, 2001). The nitric oxide synthase (NOS) inhibitor, L-NAME hydrochloride (N-nitro-L-arginine methyl ester), was given at a dose of 10 mg/kg 25 min before ischemia (Hajnal et al. 2005) while the ganglion blocker hexamethonium chloride was administered at a dose of 10 mg/kg 25 min before coronary artery occlusion (Sander et al. 1989). Guanethidine monosulfate, a compound which depletes peripheral storage of endogenous catecholamines (Benowitz 1998), was given at a dose of 50 mg/kg subcutaneously every day during 3 days (Lishmanov et al. 1998). The last injection of guanethidine was performed 24 h before occlusion. We found that the aforementioned

L.N. Maslov et al. / Life Sciences 84 (2009) 657–663

course of administration of guanethidine at a dose of 50 mg/kg completely depleted endogenous catecholamine in the heart (Lishmanov et al. 1998). Deltorphin II, α-chloralose, 5-hydroxydecanoate (5-HD), genistein, L-NAME, hexamethonium, guanethidine, chelerythrine, naltrexone, and naloxone methiodide were dissolved in 0.9% NaCl solution. Chelerythrine was poorly dissolved in cold water. Therefore, this compound was dissolved in warm water (55 °C). Glibenclamide, NTB and BNTX were preliminary dissolved in 0.1 ml DMSO then dissolved in 0.9 ml 20% hydroxypropyl-β-cyclodextrin solution that was used for intravenous administration. All solutions were prepared immediately before use. Deltorphin II was synthesized by the Multiple Peptide Systems (San Diego, California, USA). Naltrexone, naloxone methiodide, glibenclamide, 5-hydroxydecanoate, L-NAME, genistein, patent blue violet, nitrotetrazolium blue, α-chloralose, and hexamethonium were obtained from Sigma-Aldrich Corporation (St. Louis, USA). NTB, BNTX, and hydroxypropyl-β-cyclodextrin were purchased from Tocris Cookson (Bristol, UK) and chelerythrine was from LC Laboratories (Woburn, MA, USA). Guanethidine was synthesized by the International Laboratory (San Bruno, CA, USA) and purchased from Advanced Technology and Industrial Co. (Hong Kong, China). Quantitative values are expressed as the mean ± SEM. Qualitative values are expressed as percents. One-way analysis of variance with Newman–Keuls post hoc test was used to determine whether any significant differences existed in any parameter between groups in qualitative values. The Chi squared test was used to determine any difference among groups for the incidence of arrhythmias. Significant differences were determined at p b 0.05. Results Baseline function All rats equilibrated quickly after instrumentation. Baseline levels for all rats before treatment were the following: heart rate, 332 ± 9 beats/ min; systolic pressure, 119 ± 4 mm Hg; diastolic pressure, 91 ± 1 mm Hg; mean arterial blood pressure, 103 mm Hg; rate pressure product, 34 ± 1 mm Hg/s/1000. After 45 min of occlusion and 120 min of reperfusion, untreated hearts (n = 24) exhibited a reduction in rate pressure product (26 ± 2 mm Hg/s/1000) due to a decrease in mean arterial blood pressure. There were 0–1 deaths in any one group. Therefore there was no significant difference in survival between groups. Infarct size

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Table 2 Effect of chelerythrine, glibenclamide and 5-hydroxydecanoate on the cardioprotective effect of deltorphin II. Protocols

n

Control (vehicle) Deltorphin II Chelerythrine + deltorphin II Chelerythrine Glibenclamide + deltorphin II Glibenclamide 5-HD+deltorphin II 5-HD

19 508.2 ± 6.6 203.9 ± 5.6 137.0 ± 6.1 40.1 ± 3.9 67.2 ± 3.6 19 488.6 ± 5.8 209.2 ± 4.9 103.5 ± 7.4⁎ 42.8 ± 3.8 49.5 ± 3.5⁎ 13 498.3 ± 5.2 208.1 ± 6.6 132.8 ± 7.8 41.7 ± 4.6 63.8 ± 4.4

LV, mg

AAR, mg

IS, mg

AAR/LV, % IS/AAR, %

14 501.5 ± 5.5 212.2 ± 4.5 141.3 ± 6.8 42.3 ± 4.1 66.6 ± 3.9 16 504.4 ± 6.3 211.3 ± 6.1 127.6 ± 7.1 41.9 ± 4.4 60.4 ± 4.2 14 492.2 ± 6.2 206.4 ± 6.8 135.6 ± 6.2 41.9 ± 3.9 65.7 ± 3.8 14 498.8 ± 5.9 210.2 ± 5.1 125.0 ± 6.6 42.1 ± 4.1 59.5 ± 4.1 14 502.5 ± 6.5 212.4 ± 4.8 137.6 ± 6.1 42.2 ± 3.4 64.8 ± 3.6

Values are means ± SEM. n, no. of animals. ⁎p b 0.01 vs. the control group. LV, left ventricle; AAR, area at risk; IS, infarct size; 5-HD, 5-hydroxydecanoate. Vehicle control is 20% hydroxypropyl-beta-cyclodextrin solution.

no significant effect on blood pressure and heart rate. Pretreatment with deltorphin II markedly reduced infarct size from 65.4 ± 2.6% in vehicle treated rats to 50.6 ± 3.1% in treated rats (Table 1). Pretreatment with the nonselective OR antagonist naltrexone abrogated the cardioprotective effect of deltorphin II as did inhibition of peripheral ORs by naloxone methiodide. Pretreatment with the selective δ2 OR antagonist NTB also blocked the infarct-limiting effect of δ2 agonist (Table 1). However, pretreatment with the δ1 OR antagonist BNTX had no effect on the cardioprotective action of deltorphin II. No significant differences were noted in the ratio of left-ventricle mass to area at risk between groups. When administered alone, neither naltrexone (5 mg/kg), naloxone methiodide (5 mg/kg), NTB (0.3 mg/kg), nor BNTX (0.7 mg/kg) had any effect on the IS/AAR ratio. These studies also demonstrated that the selective PKC inhibitor chelerythrine abrogated the infarct-reducing effect of deltorphin II (Table 2). Pretreatment with the nonselective KATP channel inhibitor glibenclamide partially abolished the cardioprotective effect of deltorphin II as did the selective mitKATP channel inhibitor 5-HD (Table 2). As shown in Table 3, the NOS blocker L-NAME also reversed the infarctlimiting effect of deltorphin II. On the other hand, pretreatment with the TK inhibitor genistein did not alter the cardioprotective effect of deltorphin II. Similarly, blocking the peripheral autonomic ganglion with hexamethonium did not abolish the infarct-reducing effect of deltorphin II (Table 3). In addition, depletion of endogenous catecholamine storage with guanethidine had no effect on the cardioprotective action of deltorphin II. When administered alone, chelerythrine, glibenclamide, 5-HD, genistein, L-NAME, hexamethonium and guanethidine had no effect on the IS/AAR ratio.

Our studies demonstrate that the selective δ2 OR agonist deltorphin II (0.12 mg/kg) exhibited cardioprotective properties but it had Table 1 Effect of opioid receptor antagonist on the cardioprotective effect of deltorphin II.

Table 3 Effect of L-NAME, genistein, hexamethonium and guanethidine on the infarct-limiting effect of deltorphin II.

Protocol

n

Protocols

Control (vehicle) Deltorphin II Naltrexone + deltorphin II Naltrexone Naloxone meth + deltorphin II Naloxone meth BNTX + deltorphin II BNTX Naltriben + deltorphin II Naltriben

24 493.9 ± 5.6 207.1 ± 3.6 135.4 ± 6.9 42.0 ± 2.4 65.4 ± 2.6 19 498.7 ± 4.7 202.8 ± 4.5 102.6 ± 8.1⁎ 40.6 ± 3.2 50.6 ± 3.1⁎ 14 502.8 ± 6.3 200.9 ± 5.8 118.9 ± 7.9 39.9 ± 3.6 59.2 ± 3.6

LV, mg

AAR, mg

IS, mg

AAR/LV, % IS/AAR, %

14 486.6 ± 6.6 208.6 ± 4.5 140.1 ± 6.1 42.8 ± 3.2 67.2 ± 3.8 14 506.3 ± 5.4 202.1 ± 5.2 118.8 ± 6.9 39.9 ± 3.4 58.8 ± 3.9 14 14 14 14

489.5 ± 5.8 504.2 ± 6.9 507.6 ± 7.0 488.5 ± 6.7

209.2 ± 6.2 134.3 ± 8.8 42.7 ± 3.8 205.1 ± 5.8 97.6 ± 9.4⁎ 40.6 ± 3.6 204.2 ± 6.6 135.8 ± 8.4 40.2 ± 3.9 207.3 ± 5.7 119.2 ± 12.1 42.4 ± 3.5

14 504.6 ± 6.9 202.5 ± 6.7 133.4 ± 6.7

64.2 ± 3.7 47.6 ± 4.2⁎ 66.5 ± 4.1 57.5 ± 3.8

40.1 ± 3.9 65.9 ± 3.5

Values are means ± SEM. n, no. of animals. ⁎p b 0.01 vs. the control group. LV, left ventricle; AAR, area at risk; IS, infarct size; Naloxone meth, naloxone methiodide. Vehicle control is 20% hydroxypropyl-beta-cyclodextrin solution.

n

LV, mg

Control (vehicle) 18 494.9 ± 5.7 Deltorphin II 16 504.7 ± 6.2 L-NAME + 14 498.2 ± 5.8 deltorphin II L-NAME 13 501.3 ± 5.6 Genistein + 14 499.2 ± 4.7 deltorphin II Genistein 14 502.8 ± 6.3 Hex + deltorphin II 14 497.1 ± 8.8 Hex 14 502.3 ± 6.9 Guanethidine + 15 488.1 ± 8.9 deltorphin II Guanethidine 14 491.2 ± 6.2

AAR, mg

IS, mg

208.2 ± 4.1 139.9 ± 6.9 212.3 ± 4.4 104.8 ± 7.1⁎ 210.4 ± 5.2 133.8 ± 6.6

AAR/LV, % IS/AAR, % 42.1 ± 4.0 67.2 ± 4.2 42.0 ± 3.9 49.4 ± 4.1⁎ 42.2 ± 3.8 63.6 ± 3.9

208.9 ± 4.9 143.9 ± 6.2 41.7 ± 3.7 68.9 ± 3.8 208.3 ± 4.8 103.5 ± 10.8⁎ 41.7 ± 4.2 49.7 ± 4.8⁎ 211.1 ± 4.7 142.3 ± 6.8 205.3 ± 8.4 96.1 ± 14.1⁎ 210.3 ± 4.9 137.5 ± 6.4 209.6 ± 6.1 105.8 ± 10.6⁎

42.0 ± 4.4 41.3 ± 6.1 41.8 ± 6.5 42.9 ± 6.9

67.4 ± 4.1 46.8 ± 6.3⁎ 65.4 ± 4.3 50.5 ± 5.4⁎

207.4 ± 6.2 134.0 ± 6.8

42.2 ± 5.1 64.6 ± 4.4

Values are means ± SEM. n, no. of animals. ⁎p b 0.05 vs. the control group. LV, left ventricle; AAR, area at risk; IS, infarct size; Hex, hexamethonium. Vehicle control is saline solution (0.9% NaCl).

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L.N. Maslov et al. / Life Sciences 84 (2009) 657–663

Table 4 Effect of opioid receptor antagonists on the antiarrhythmic effect of deltorphin II during a 45-min ischemia event. Group

n

Control (vehicle) Deltorphin II Naltrexone + deltorphin II Naltrexone Naloxone methiodide + deltorphin II Naloxone methiodide BNTX + deltorphin II BNTX Naltriben + deltorphin II Naltriben

Phase 1a

24 19 14 14 14 14 14 14 14 14

Phase 1b

WVA

VPC

VT

VF

WVA

VPC

VT

VF

n (%)

n (%)

n (%)

n (%)

n (%)

n (%)

n (%)

n (%)

3(13) ⁎⁎⁎10(53) 2(14) 3(21) 1(7) 2(14) ⁎8(57) 2(14) 4(28) 3(21)

21(87) ⁎⁎⁎9(47) 12(86) 13(92) 13(93) 11(78) 9(64) 12(86) 10(71) 11(78)

21(87) ⁎⁎8(42) 12(86) 11(78) 12(86) 12(86) 9(64) 10(71) 9(64) 12(86)

10(42) ⁎⁎2(10) 6(43) 5(35) 5(36) 6(43) ⁎⁎1(7) 6(42) 6(43) 4(28)

6(25) ⁎⁎12(63) 6(43) 7(50) 3(21) 7(50) ⁎⁎9(64) 3(21) 6(43) 5(35)

18(75) ⁎⁎7(37) 8(57) 8(57) 11(79) 8(57) ⁎⁎5(36) 10(71) 8(57) 8(57)

13(54) 7(37) 5(36) 5(36) 11(79) 5(36) 5(36) 6(42) 8(57) 6(42)

8(33) 4(21) 3(21) 3(21) 2(14) 4(28) 1(7) 4(28) 3(21) 4(28)

Note. n is number of animals. WVA, without ventricular arrhythmias; VPC — ventricular premature contraction; VT, ventricular tachycardia; VF, ventricular fibrillation. ⁎p b 0.05, ⁎⁎p b 0.025, ⁎⁎⁎p b 0.01 vs. the control group. The vehicle control is 20% hydroxypropyl-beta-cyclodextrin solution.

Incidence of arrhythmias The severity and incidence of ventricular arrhythmias was determined during the first 10 min of occlusion period (phase 1a), following 35 min of ischemia (phase 1b) and the first 10 min of reperfusion. There were no incidences of ventricular tachycardia and ventricular fibrillation during reperfusion and the incidence of premature ventricular contractions fluctuated from 8% to 12% in different groups. Our studies demonstrate that 90% of rats are completely tolerant to arrhythmogenous impact of reperfusion. Consequently, we could not demonstrate any antiarrhythmic effect of deltorphin II or any of the other compounds tested during the reperfusion period. We have found that stimulation of δ2 OR with deltorphin II decreased the severity and incidence of ventricular arrhythmias both in phase 1a and phase 1b (Table 4). Pretreatment with the nonselective OR antagonist naltrexone completely abolished an antiarrhythmic effect of deltorphin II. Inhibition of peripheral OR by naloxone methiodide also reversed the antiarrhythmic effect of deltorphin II as did pretreatment with the selective δ2 OR antagonist NTB (Table 4). However, the δ1 OR inhibitor BNTX had no effect on the δ2 agonist-induced tolerance to arrhythmogenic impact of ischemia. Control animals demonstrated that neither naltrexone (5 mg/kg), naloxone methiodide (5 mg/kg), NTB (0.3 mg/kg), or BNTX (0.7 mg/kg) could effect the incidence of ventricular arrhythmias when administered alone. As shown in Table 5, inhibition of PKC with chelerythrine reversed the antiarrhythmic effect of deltorphin II. Pretreatment with either the nonselective KATP channel inhibitor glibenclamide or the selective mitKATP channel inhibitor 5-HD also did not reverse the δ2 agonistinduced tolerance to arrhythmogenic impact of ischemia (Table 5). Both KATP channel inhibitors even enhanced antifibrillatory effect of deltorphin II in phase 1b. Treatment with inhibitors alone had no significant effects.

The NOS blocker L-NAME reversed the antiarrhythmic effect of deltorphin II (Table 6). In addition, pretreatment with the TK inhibitor genistein, the ganglion blocker hexamethonium or depletion of endogenous catecholamines with guanethidine also reversed the antiarrhythmic effect of deltorphin II (Table 6). It should be noted that, when administered alone, chelerythrine, genistein, L-NAME, hexamethonium, guanethidine, glibenclamide and 5-HD had no effect on the incidence of ischemia-induced arrhythmias. Discussion Our studies demonstrate that pretreatment with the selective δ2 OR agonist deltorphin II not only decreased the IS/AAR ratio but also reduced the incidence of ischemia-induced arrhythmias. The infarctsparing effect of deltorphin II was independent of collateral coronary blood flow because it did not alter the ratio of area at risk to left ventricle. The infarct-reducing and antiarrhythmic of deltorphin II was mediated via peripheral δ2 OR activation because both effects were abolished by pretreatment with the nonselective OR antagonist naltrexone, the selective δ2 OR antagonist NTB and the peripherally acting OR inhibitor naloxone methiodide. However, the selective δ1 OR antagonist BNTX did not alter both protective effects δ2 OR agonist. Our results also indicate that endogenous agonists of opioid receptors probably are not involved in the regulation of cardiac tolerance to the arrhythmogenous impact of ischemia/reperfusion in naïve rats because OR antagonists alone had no effect on the IS/AAR and the incidence of ischemia-induced arrhythmias. The cardioprotective effect of deltorphin II is reversed by pretreatment with the PKC inhibitor chelerythrine, the nonselective KATP channel blocker glibenclamide and the selective mitKATP channel inhibitor 5-HD. These findings indicate that PKC and mitKATP channel play an important role in the mechanism of the infarct-reducing effect of

Table 5 Effect of chelerythrine, glibenclamide, 5-hydroxydecanoate on the antiarrhythmic effect of deltorphin II during a 45-min ischemia event. Groups

Control (vehicle) Deltorphin II Chelerythrine + deltorphin II Chelerythrine Glibenclamide + deltorphin II Glibenclamide 5-HD + deltorphin II 5-HD

n

19 19 13 14 16 14 14 14

Phase 1a

Phase 1b

WVA

VPC

VT

VF

WVA

VPC

VT

VF

n (%)

n (%)

n (%)

n (%)

n (%)

n (%)

n (%)

n (%)

2(11) ⁎⁎⁎10(53) 1(8) 1(8) 5(31) 3(21) ⁎⁎7(50) 3(21)

17(89) 11(57) 12(92) 11(78) 11(69) 12(92) ⁎8(57) 9(64)

16(84) ⁎10(53) 11(85) 11(85) ⁎9(56) 9(64) ⁎7(50) 9(64)

7(37) ⁎⁎2(10) 3(23) 4(28) ⁎2(13) 4(28) 4(29) 3(21)

4(21) ⁎⁎12(63) 2(15) 2(15) ⁎⁎⁎11(69) 4(28) ⁎⁎9(64) 4(28)

15(79) ⁎⁎7(37) 11(85) 11(85) ⁎⁎⁎5(31) 10(71) ⁎⁎5(36) 9(64)

10(53) 7(37) 9(69) 10(71) 4(25) 6(43) 4(29) 5(35)

6(32) 4(21) 2(15) 4(28) ⁎⁎⁎0 3(21) ⁎⁎0 3(21)

Note. n is number of animals. WVA, without ventricular arrhythmias; VPC — ventricular premature contraction; VT, ventricular tachycardia; VF, ventricular fibrillation; 5-HD, 5-hydroxydecanoate. ⁎p b 0.05, ⁎⁎p b 0.025, ⁎⁎⁎p b 0.01 vs. the control group. The vehicle control is 20% hydroxypropyl-beta-cyclodextrin solution.

L.N. Maslov et al. / Life Sciences 84 (2009) 657–663

661

Table 6 Effect of L-NAME, genistein, hexamethonium, guanethidine on the antiarrhythmic effect of deltorphin II during a 45-min ischemia event. Groups

Control (vehicle) Deltorphin II L-NAME + deltorphin II L-NAME Genistein + deltorphin II Genistein Hex + deltorphin II Hex Guanethidine + deltorphin II Guanethidine

n

18 16 14 13 14 14 14 14 15 14

Phase 1a

Phase 1b

WVA

VPC

VT

VF

WVA

VPC

VT

VF

n (%)

n (%)

n (%)

n (%)

n (%)

n (%)

n (%)

n (%)

2(11) ⁎⁎9(56) 2(14) 1(7) 3(21) 3(21) 2(14) 3(21) 2(13) 3(21)

16(89) ⁎⁎7(44) 12(86) 12(92) 13(92) 11(78) 12(86) 9(64) 13(87) 9(64)

15(83) ⁎⁎6(38) 10(71) 11(84) 11(78) 11(78) 12(86) 10(71) 11(73) 9(64)

6(33) 4(25) 4(29) 5(38) 5(36) 4(29) 6(43) 4(28) 5(33) 4(28)

4(22) ⁎⁎11(69) 3(21) 3(2) 5(36) 3(21) 6(43) 6(43) 3(20) 5(35)

13(72) ⁎6(31) 11(79) 11(84) 9(64) 11(79) 8(57) 7(50) 12(80) 7(50)

10(56) 6(31) 9(64) 8(61) 6(42) 9(64) 5(36) 7(50) 10(67) 8(57)

6(33) 4(25) 5(36) 5(38) 4(28) 4(28) 3(21) 3(21) 5(33) 3(21)

Note. n is number of animals. WVA, without ventricular arrhythmias; VPC — ventricular premature contraction; VT, ventricular tachycardia; VF, ventricular fibrillation; Hex, hexamethonium. ⁎p b 0.05, ⁎⁎p b 0.025, ⁎⁎⁎p b 0.01 vs. the control group. The vehicle control is saline solution (0.9% NaCl).

deltorphin II. Moreover, these results agree with the findings of other investigators indicating that the cardioprotective effect of opioids are mediated via PKC activation and mitKATP channel opening (Cao et al. 2003; Fryer et al. 2000; 2001; Schultz et al.1996,1998b). The selective TK inhibitor genistein also did not alter deltorphin II induced cardioprotection. This fact contrasts with Fryer's data (Fryer et al. 2001), who found that tyrosine kinase is involved in δ1 opioid receptor-mediated cardioprotection. The possibility exists that infarct-limiting signaling pathways that mediated cardioprotection via δ1 and δ2 OR stimulation are different. Tyrosine kinase is involved in δ1 OR mediated increase in cardiac tolerance to ischemia but may not play a substantial role in infarct-reducing action of deltorphin II. We have also demonstrated that pretreatment with the NOS inhibitor L-NAME abolished the cardioprotective action of deltorphin II. This result was surprising since a number of studies indicate the involvement of NOS in the delayed cardioprotective effect of opioids (Patel et al. 2004; Jiang et al. 2006; Guo et al. 2005), but none has demonstrated the participation of this enzyme in the acute cardioprotective effect of opioids. However, it should be noted that NOS can be involved in vasodilator responses to opioid peptides (Champion et al. 2002). Morphine can induce NO release from endothelium in vitro (Bilfinger et al. 2002). There are also data that acetylcholine and bradykinin trigger preconditioning through a pathway that includes NOS (Krieg et al. 2004). It, therefore, seems likely that NO can also be involved in part in the mechanism of action for the cardioprotective effects of deltorphin II. The role of NO in ischemia/reperfusion injury of the heart remains controversial. There are also data indicating that endothelial NOS overexpression in transgenic mice attenuates myocardial reperfusion injury (Jones et al. 2004). It has also been demonstrated that endothelial NOS-deficient transgenic mice may be more sensitive to myocardial ischemia/reperfusion (Sharp et al. 2002). Other studies indicate that NOS inhibition can exacerbate ischemia/reperfusion injury of isolated perfused rat heart (Kobara et al. 2003). Based on our studies, we hypothesize that NO plays an important role in providing deltorphin II induced cardioprotection. Our data indicated that the autonomic nervous system is not involved in the infarct-reducing effect of deltorphin II because pretreatment with the ganglion blocker hexamethonium or a depletion of endogenous catecholamines' storage with guanethidine did not alter cardioprotective effect of this δ2 OR agonist. We found that pretreatment with PKC inhibitor chelerythrine abrogated the antiarrhythmic effect of deltorphin II. This indicates that PKC is involved in deltorphin II induced increase in cardiac tolerance to the arrhythmogenic impact of ischemia. This is in agreement with the widely held hypothesis that PKC plays a key role in the signaling pathway underlining the opioid induced resistance of heart tissue to ischemia/ reperfusion injury. We also demonstrated that pretreatment with NOS inhibitor L-NAME reversed the antiarrhythmic effect of δ2 OR agonist. Hence, NOS may play a significant role in the deltorphin II induced increase in cardiac electrical stability. Our hypothesis is supported by the data that NOS inhibition can increase cardiac vulnerability to arrhyth-

mogenous impact of reperfusion (Kawahara et al. 2003). In contrast, the NO-donors almost completely abolished reperfusion induced ventricular fibrillation (Bilinska et al. 1996). Based on Fryer's data (Fryer et al. 2000), we anticipated that KATP channel inhibition would abolish the antiarrhythmic effect of deltorphin II. However, glibenclamide and 5-HD not only reversed the δ2 agonist-induced tolerance to the arrhythmogenous impact of ischemia but also enhanced the antifibrillatory action of this opioid during phase 1b. It should be noted that Fryer et al. used the selective δ1 OR agonist TAN-67 while we used the δ2 agonist deltorphin II. Consequently, we hypothesize that the signaling pathway mediating the antiarrhythmic effect of δ1 OR stimulation is different from the signaling pathway underlying the antiarrhythmic effect of δ2 OR activation. Since the TK inhibitor genistein abolished the antiarrhythmic effect of deltorphin II, TK also plays an important role in deltorphin II induced cardiac electrical stability. This study also demonstrated that pretreatment with the ganglion blocker hexamethonium abrogated the antiarrhythmic effect of deltorphin II. Such evidence leads us to propose that the autonomic nervous system is involved in the antiarrhythmic effect of this peptide. Our data are in agreement with the widely held hypothesis that the autonomic nervous system plays a significant role in arrhythmogenesis. Furthermore, our studies indicate that depletion of stored endogenous catecholamines by guanethidine pretreatment reversed the antiarrhythmic action of deltorphin II while the guanethidine treatment alone had no effect on the incidence of ischemia-induced arrhythmias. The aforementioned findings were unexpected since it is generally held that adrenergic system activation is involved in onset of ischemia/reperfusion induced arrhythmias. However, a number of studies indicate that if adrenergic receptor stimulation occurred in the pre-ischemic period, the heart became tolerant to ischemia/reperfusion injury (Frances et al. 2003; Imani et al. 2008). It has also been established that treatment with exogenous agonists of α and β adrenergic receptors can result in infarctreducing effects (Frances et al. 2003) and antiarrhythmic effect (Imani et al. 2008; Vegh and Parratt 2002) if they are administered before ischemia. Recently, it was shown that antiarrhythmic effect of noradrenaline is mediated via mitKATP channel opening (Imani et al. 2008). Therefore, we hypothesize that deltorphin II can induce a release of endogenous catecholamines which in turn increases cardiac tolerance to arrhythmogenic impact of ischemia/reperfusion. Although the foregoing evidence supports our hypothesis, there are reports that morphine, fentanyl and the selective κ-OR agonist U-62066E can elevate norepinephrine and epinephrine levels in blood plasma of animals and man (Hoehe and Duka 1993; Rimoy et al. 1994). The intrathecallyadministered deltorphin II induces norepinephrine released in the spinal cord (Grabow et al. 1999). However, there is a report that deltorphin failed to modify basal plasma levels of both norepinephrine and epinephrine in healthy man (degli Uberti et al. 1993). It should be noted that authors of this report used a deltorphin with a chemical structure (H-Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) different from deltorphin II

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(Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2). Therefore, the effects of these peptides may be also different. It has been shown that intravenous administration of enkephalins to conscious dogs can induce a shortterm tachycardia (approximately, 2 min) (Sander et al. 1989; Giles et al. 1983). This tachycardia was mediated via peripheral OR stimulation (Giles et al. 1983) and abolished by pretreatment with hexamethonium or propranolol (Sander et al. 1989). However, other studies indicate that opioids can inhibit norepinephrine release from peripheral sympathetic terminals via presynaptic OR stimulation (Szabo et al. 1986; Cadet et al. 1998). This anti-adrenergic effect is exhibited only under conditions of an evoked norepinephrine release (Szabo et al. 1986; Cadet et al. 1998). We hypothesize that exogenous opioids inhibit stimulated norepinephrine leakage, but may in fact induce a short-term liberation of catecholamines. Perhaps, this catecholamine release in the pre-ischemic period enhances cardiac tolerance to the arrhythmogenic impact of ischemia. However, some data are not consistent with this hypothesis. Thus, it is unclear why endogenous catecholamines are not involved in the infarct-reducing effect of deltorphin II. It is also unknown why the mitKATP channel is not involved in the antiarrhythmic effect of the δ2 agonist. Therefore, this working hypothesis of the involvement of endogenous catecholamines in the antiarrhythmic effect of deltorphin II requires further experimental examination. In summary, our data indicated that infarct-reducing and antiarrhythmic effects of deltorphin II are mediated via stimulation of peripheral δ2 opioid receptors. Protein kinase C and NO-synthase are involved in the cardioprotective and antiarrhythmic effects of deltorphin II. The infarct-sparing mechanisms of action of deltorphin II also relies in part on mitochondrial KATP channel activation. Our studies indicate that tyrosine kinase is involved in the mechanism of action resulting in the antiarrhythmic effect of deltorphin II. Our data also indicate that endogenous catecholamines are involved in the antiarrhythmic effect of deltorphin II. However, depletion of endogenous catecholamine storage does not alter cardioprotective effect of deltorphin II. Our results also demonstrate that mitochondrial KATP channel is not involved in the antiarrhythmic effect in response to δ2 opioid receptor stimulation. Acknowledgements The authors are grateful to Dr. Kevin J. Gormley (National Institute on Drug Abuse, Division of Basic Research, Bethesda, MD, USA) for peptide ligands of the opioid receptors. This research was supported in part by the Russian Foundation of Basic Research. References Benowitz NL. Compounds affected cardiovascular system and kidney. Antihypertensive drugs. (Chapter 11). In: Katzung B (Ed) Basic and Clinical Pharmacology, Sixth edition. Prentice-Hall International Inc. 1995; Zvzrtau EE (Ed) Translation from English to Russian, Moscow, Petersburg. Binom, Nevskyi Dialect; Vol 1. pp 192–221, 1998 Bilfinger TV, Vosswinkel JA, Cadet P, Rialas CM, Magazine HI, Stefano GB. Direct assessment and diminished production of morphine stimulated NO by diabetic endothelium from saphenous vein. Acta Pharmacologica Sinica 23 (2), 97–102, 2002. Bilinska M, Maczewski M, Beresewicz A. Donors of nitric oxide mimic effects of ischaemic preconditioning on reperfusion induced arrhythmias in isolated rat heart. Molecular and Cellular Biochemistry 160–161 (1), 265–271, 1996. Cadet P, Weeks BS, Bilfinger TV, Mantione KJ, Casares F, Stefano GB, Rialas CM, Fimiani C, Bilfinger TV, Salzet M, Stefano GB. Endomorphin-1 and -2 inhibit human vascular sympathetic norepinephrine release: lack of interaction with mu 3 opiate receptor subtype. Acta Pharmacologica Sinica 19 (5), 403–407, 1998. Cao Z, Liu L, Van Winkle DM. Activation of δ- and κ-opioid receptors by opioid peptides protects cardiomyocytes via KATP channels. American Journal of Physiology Heart and Circulatory Physiology 285 (3), H1032–H1039, 2003. Champion HC, Bivalacqua TJ, Zadina JE, Kastin AJ, Hyman AL, Kadowitz PJ. Role of nitric oxide in mediating vasodilator responses to opioid peptides in the rat. Clinical and Experimental Pharmacology and Physiology 29 (3), 229–232, 2002. degli Uberti EC, Ambrosio MR, Vergnani L, Portaluppi F, Bondanelli M, Trasforini G, Margutti A, Salvadori S. Stress-induced activation of sympathetic nervous system is attenuated by the δ-opioid receptor agonist deltorphin in healthy man. Journal of Clinical Endocrinology and Metabolism 77 (6), 1490–1494, 1993.

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