Introduction

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Signal transduction mechanisms mediating pharmacologically induced cardioprotection against ischemia have been the subject of many recent investigations. It has been demonstrated that many mediators, including adenosine (Auchampach and Gross, 1993; Carr et al., 1997), acetylcholine (Yao and Gross, 1993; Yao et al., 1999), and monophosphoryl lipid A (Nelson et al., 1991; Yao et al., 1993) induce cardioprotection. Interestingly, many cardioprotective agents are thought to mediate cardioprotection via a pertussis toxin-sensitive pathway. Recently, we have demonstrated that another G-protein coupled receptor, the ₁-opioid receptor, has potent cardioprotective properties against ischemia and arrhythmias when stimulated prior to sustained coronary artery occlusion (Schultz et al., 1998a,b; Fryer et al., 2000b). More recently, we have attempted to characterize the signal transduction cascade mediating opioid-induced cardioprotection.

Opioid receptor activation is thought to be an integral component of ischemic preconditioning (IPC)-induced cardioprotection and mediated by a similar signal transduction cascade (Schultz et al., 1995). Central to the genesis of IPC is the proposed activation of protein kinase C (Ytrehus et al., 1994). More specifically, distinct isoforms of PKC are now thought to be important mediators of this cardioprotection (Ping et al., 1997; Kawamura et al., 1998). In addition to PKC activation, the involvement of a tyrosine kinase (TK) and mitogen-activated protein (MAP) kinase cascade are also likely to be involved in IPC (Fryer et al., 1998; Maulik et al., 1998). Indeed, Maulik et al. (1996) demonstrated that a tyrosine kinase-phospholipase D-sensitive pathway during IPC triggers the activation of MAP kinases and MAP kinase-activated protein kinase 2 in rat hearts. More recently, we have demonstrated that both opioids and IPC induce cardioprotection dependent on the cytosolic activation of extracellular signal-regulated kinase during myocardial reperfusion (Fryer et al., 2001a). Additionally, it has been suggested that the Src family of receptor TKs, specifically Src and Lck, are activated during IPC in conscious rabbits (Ping et al., 1999; Song et al., 2000).

In corroboration with these studies, our laboratory has previously demonstrated that both PKC (Fryer et al., 1999) and TK (Fryer et al., 1998) are important mediators of IPC in an in vivo rat model and have since demonstrated that opioid-induced cardioprotection is dependent upon the activation of similar pathways. We have also demonstrated that both IPC and opioid-induced cardioprotection are dependent upon activation of the mitochondrial ATP-sensitive potassium (K_ATP) channel but are independent of the sarcolemmal K_ATP channel (Fryer et al., 2000a,b). Furthermore, evidence from our laboratory suggests that specific PKC isoform (, _I, , and ) translocation to subcellular loci is important after opioid administration and subsequent cardioprotection and that the PKC--selective antagonist, rottlerin, could abolish opioid-induced cardioprotection (Fryer et al., 2001b). However, whether TK activation is proximal or distal to PKC activation during opioid-induced cardioprotection remains to be established.

Therefore, in the present investigation we demonstrate that tyrosine kinases are important in the genesis of opioid-induced cardioprotection and that the TKs involved are most likely not members of the Src/EGF receptor family. Additionally, we demonstrate that inhibition of TK does not inhibit PKC translocation, suggesting that these kinases exist either in a linear cascade where PKC activation is proximal to the activation of TK similar to previous data found in rabbits (Baines et al., 1998) or that, alternatively, these enzymes exist in a parallel pathway leading to cardioprotection (Vahlhaus et al., 1998)

	Experimental Procedures

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This study was performed in accordance with the guidelines of the Animal Care Committee of the Medical College of Wisconsin, which is accredited by the American Association of Laboratory Animal Care.

General Surgical Preparation. Male Wistar rats, 350 to 450 g, were anesthetized with inactin (100 mg/kg), a long-acting barbiturate. A tracheotomy was performed, and the trachea was intubated and connected to a rodent ventilator (model CIV-101, Columbus Instruments, Columbus, OH). The rats were ventilated at 60 to 65 breaths per minute. Atelectasis was prevented by maintaining a positive end-expiratory pressure of 5 to 10 mm of H₂O. Arterial pH, PCO₂, and PO₂ were monitored by a blood gas system (AVL 995 pH/blood gas analyzer; Roche Diagnostics Corp., Roswell, GA) and maintained within a normal physiological range (pH 7.35-7.45; PCO₂, 25-40 mm Hg; and PO₂, 80-110 mm Hg).

Drugs and Materials. Inactin (thiobutabarbital sodium) was purchased from Sigma/RBI (Natick, MA) and was dissolved in distilled water. 2,3,5-Triphenyltetrazolium chloride was purchased from Sigma Chemical Co. (St. Louis, MO). TAN-67 was kindly synthesized and furnished by Dr. Hiroshi Nagase of Toray Industries (Kanagawa, Japan) and dissolved in saline. DADLE was purchased from Sigma/RBI and dissolved in saline. Genistein was purchased from Sigma/RBI and was dissolved in Alkamuls EL-620 (Aventis, Strasbourg, France), 95% ethanol, and saline. Daidzein and lavendustin A were purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). Daidzein was dissolved in polyethylene glycol, 1 N NaOH, and Dulbecco's phosphate-buffered saline. Lavendustin A was dissolved in 95% ethanol and Dulbecco's phosphate-buffered saline. PP2 was purchased from Calbiochem (San Diego, CA) and dissolved in 0.1 ml dimethyl sulfoxide.

Study Groups and Experimental Protocols. Rats were assigned to 12 experimental groups (Fig. 1). All animals were subjected to 30 min of ischemia and 2 h of reperfusion (control). The effect of opioids was assessed by administering the nonselective -opioid receptor agonist, DADLE (1 mg/kg), or via administration of the ₁-selective opioid receptor agonist, TAN-67 (10 mg/kg) 15 min prior to sustained ischemia. We have previously shown that TAN-67-induced cardioprotection is dependent upon selective stimulation of the ₁-opioid receptor (Schultz et al., 1998b). The effect of TK inhibition was examined in the absence or presence of DADLE and TAN-67 via administration of the TK antagonist, genistein (5 mg/kg), 30 min prior to the ischemic period in the absence or presence of TAN-67. To determine whether any non-TK-related effects of genistein contributed to abolishment of cardioprotection, we administered the structural analog of genistein, daidzein (5 mg/kg), which lacks TK inhibitory activity but retains many of non-TK-related effects of genistein, 30 min prior to ischemia in the absence or presence of TAN-67. Additionally, we examined the role of a Src/EGF receptor TK in TAN-67-induced cardioprotection with the selective inhibitor, lavendustin A (1.0 mg/kg), administered 30 min prior to the ischemic period in the absence or presence of TAN-67. We have previously shown that this dose and timing of genistein and lavendustin A attenuates IPC in rats and have also demonstrated in rats that this dose and timing of administration of daidzein does not attenuate IPC-induced cardioprotection (Fryer et al., 1998). Importantly, we also used the Src-selective tyrosine kinase antagonist, PP2 (0.1 mg/kg), to further characterize the importance of inhibiting Src tyrosine kinases in the presence or absence of DADLE. This dose of PP2 is sufficient to inhibit the activity of Src tyrosine kinases when taking into account the route of administration, plasma volume of the rat and IC₅₀ of PP2 (4-5 nM).

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Protocol bar of experiments used to determine the importance of a tyrosine kinase in cardioprotection induced by 1-opioids. All animals were subjected to 30 min of ischemia and 2 h of reperfusion. Cardioprotection was induced via the administration of DADLE or TAN-67 15 min prior to ischemia as an i.v. bolus or as an i.v. infusion, respectively. Genistein, daidzein, and lavendustin A were administered 30 min prior to ischemia in the absence or presence of TAN-67 or DADLE. PP2 was administered 20 min prior to ischemia in the absence or presence of DADLE. Infarct size analysis was performed after 2 h of reperfusion, and immunohistochemistry of PKC translocation was performed immediately after TAN-67 infusion for 15 min.

Determination of Infarct Size. Upon completion of the above protocols, the coronary artery was reoccluded, and the area at risk (AAR) was determined by negative staining. Patent blue dye was administered via the jugular vein to stain the nonoccluded area of the left ventricle. The rat was euthanized with a 15% KCl solution. The heart was excised, and the left ventricle was removed from the remaining tissue and subsequently cut into six thin cross-sectional pieces. The AAR was excised from the nonischemic area, and the tissues were placed in separate vials and incubated for 15 min with a 1% triphenyltetrazolium chloride stain in 100 mM phosphate buffer at 37°C. Tissues were stored in vials of 10% formaldehyde overnight, and the infarcted myocardium was dissected from the AAR under the illumination of a dissecting microscope (Cambridge Instruments, Monsey, NY). Infarct size (IS) and AAR were determined by gravimetric analysis. IS was expressed as a percentage of the AAR (IS/AAR).

Immunofluorescent Staining of PKC Isoforms. Subcellular localization of PKC isoforms were performed and compared by immunofluorescence staining after various interventions with control hearts as previously described (Wang and Ashraf, 1999; Wang et al., 1999; Fryer et al., 2001b). Control and experimental specimens were harvested immediately prior to ischemia. Left ventricular tissue was embedded in OCT compound, rapidly frozen in liquid nitrogen, and stored at 70°C until use. Transverse cryosections (5 µm) were prepared with a cryostat (Jung Friocut 2800E; Leica, Wetzlar, Germany) and collected on poly-L-lysine-coated slides. Sections were fixed for 10 min in a 70% acetone-30% methanol mixture at 20°C, rinsed in PBS, and incubated in 10% normal goat serum in PBS for 30 min to block nonspecific binding. Primary antibodies (rabbit polyclonal antibodies against PKC-, -_I, -, and -) were diluted with PBS containing 0.1% bovine serum albumin. Sections were then incubated for 1 h at room temperature with diluted primary antibodies and subsequently washed three times in PBS. Sections were then incubated for 45 min with indocarbocyanine-conjugated goat anti-rabbit IgG, followed by washing once with 0.1% Triton X-100 in PBS and twice with PBS. Nuclear staining was achieved with bis-benzamide (10 mg/ml in PBS) for 30 s and washed three times with PBS. Sections were examined and photographed with a microscope equipped with fluorescence optics (BH-2 with a PM-CBSP camera; Olympus, Tokyo, Japan).

Exclusion Criteria. A total of 73 rats successfully completed the above protocols for infarct size analysis. Rats were excluded from data analysis if they exhibited severe hypotension (<30 mm Hg systolic blood pressure) or if we were unable to maintain adequate blood gas values within a normal physiological range.

Statistical Analysis of Data. All values are expressed as mean ± S.E.M. Analysis of variance (ANOVA) with Newman-Keuls post hoc test was used to determine whether any significant differences existed among groups for left ventricular (LV) weight, IS, and AAR. A two-way ANOVA with repeated measures for time and treatment was performed on the hemodynamic data. Significant differences were determined at p < 0.05.

	Results

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Hemodynamics. Table 1 summarizes the hemodynamic data obtained for the following experiments. No significant differences were seen between control and treatment groups for mean blood pressure (MBP), and only animals administered both PP2 and DADLE had a reduced rate pressure product (RPP) at 2 h of reperfusion. Heart rate was significantly decreased (p < 0.05 versus control) in the lavendustin A + TAN-67 group at baseline and 2 h of reperfusion and in animals administered genistein in the presence of TAN-67 at 15 min of ischemia. This effect was also seen in animals administered DADLE at 2 h of reperfusion or administered DADLE in the presence of PP2; however, PP2 alone only reduced heart rate at 2 h of reperfusion.

Infarct Size Following Various Interventions. LV weight and AAR expressed as a percentage of the LV (AAR/LV) were not significantly different in any of the groups (data not shown). IS/AAR (%) for animals untreated or treated with opioids in the presence or absence of genistein, daidzein, or lavendustin is shown in Figs. 2 to 5. IS/AAR in control animals averaged 58.2 ± 0.6. ₁/₂-opioid receptor stimulation with DADLE, 1 mg/kg, significantly (p < 0.05) reduced IS/AAR (34.8 ± 3.8) versus control. Similarly, the ₁-selective opioid receptor agonist, TAN-67 (10 mg/kg), also reduced IS/AAR (28.8 ± 3.6). Genistein, daidzein, and lavendustin A did not affect IS/AAR versus control in nonopioid-treated animals (52.3 ± 1.2, 47.6 ± 6.5, and 56.2 ± 3.0, respectively). Genistein, administered in the presence of 10 mg/kg TAN-67 or 1 mg/kg DADLE, completely abolished cardioprotection (59.1 ± 3.2 and 61.5 ± 3.4, respectively). Conversely, daidzein did not attenuate TAN-67-induced cardioprotection (30.9 ± 5.7). In contrast to the effects of genistein, the Src/EGF TK inhibitor, lavendustin A, did not attenuate TAN-67-induced reduction in infarct size (22.1 ± 6.8) at a dose previously shown to attenuate cardioprotection from IPC. Additionally, the Src-selective TK inhibitor, PP2, had no effect on DADLE-induced cardioprotection (31.1 ± 7.3) and had no effect on infarct size compared with control when administered in the absence of the opioid agonist (50.8 ± 2.6).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   IS/AAR in animals administered TAN-67 or DADLE in the presence or absence of the tyrosine kinase antagonist, genistein. *, p < 0.05 versus control; , p < 0.05 versus TAN-67; ¥, p < 0.05 versus DADLE.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   IS/AAR in animals administered TAN-67 in the presence or absence of the inactive analog of genistein, lacking tyrosine kinase inhibitory activity, daidzein. *, p < 0.05 versus control.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   IS/AAR in animals administered TAN-67 in the presence or absence of the Src/EGF receptor tyrosine kinase antagonist, lavendustin A (Lav A). *, p < 0.05 versus control.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   IS/AAR in animals administered DADLE in the presence or absence of the Src-selective receptor tyrosine kinase antagonist, PP2. *, p < 0.05 versus control.

Immunohistochemical Distribution of PKC Isoforms after Various Interventions. All samples analyzed for the immunohistochemical study demonstrated consistent PKC localization within each group, and representative results are shown in Fig. 5. We have previously demonstrated that TAN-67 alone induces the translocation of numerous PKC isoforms. Genistein did not affect this pattern of localization since in animals administered TAN-67 in the presence of genistein, PKC- was distinctly localized in the sarcolemmal membrane, and PKC-_I positively stained the nucleus. PKC- and - were translocated to the mitochondria and mitochondria/intercalated disks, respectively. Mitochondrial localization of PKC- and - has been previously verified by our group via confocal microscopy (Fryer et al., 2001b). These data suggest that PKC translocation and activation occur in parallel or proximal to TK activation (Fig. 6).

View larger version (183K): [in this window] [in a new window]   Fig. 6.   Immunofluorescent staining of PKC isoforms in TAN-67 pretreated hearts. A, negative control (no PKC antibody); B, PKC- in saline-treated control hearts. Diffuse cytoplasmic distribution of the -isoform is observed. PKC-I, -, and -, in control hearts were similar to that of panel B (photos not shown). C, PKC- in a TAN-67-treated heart after pretreatment with genistein. Immunofluorescence is observed in the sarcolemma (arrow). D, PKC-I in TAN-67-treated heart after pretreatment with genistein. PKC-I staining was observed in the nuclear region (arrow). E, PKC- in a TAN-67-treated heart after pretreatment with genistein. PKC is positively localized in the mitochondrial sites between myofibrils (arrow). F, PKC- in a TAN-67-treated heart after pretreatment with genistein. PKC- is prominently distributed in the intercalated disc (arrow) and mitochondrial sites (arrowhead). All original magnifications 400×.

	Discussion

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The results of the present investigation are the first to suggest the importance of a tyrosine kinase-mediated signaling pathway in opioid-induced cardioprotection. Additionally, we suggest that this cardioprotection is independent of activation of Src/EGF receptor tyrosine kinases. These conclusions are based on the finding that the nonselective TK inhibitor, genistein, could abolish cardioprotection from the ₁-opioid receptor agonist, TAN-67; however, the Src/EGF receptor tyrosine kinase selective antagonist, lavendustin A, and the Src-selective tyrosine kinase antagonist, PP2, could not attenuate TAN-67- or DADLE-induced reduction in infarct size, respectively. Furthermore, although it has been demonstrated that genistein has nontyrosine kinase inhibitory activity (Huang et al., 1992; Okajima et al., 1994; Chiang et al., 1997; French et al., 1997; Paillart et al., 1997; Weinreich et al., 1997), we demonstrated with daidzein, a structural analog of genistein that lacks tyrosine kinase inhibitory activity but shares the nontyrosine kinase activities, that the effect of genistein to antagonize TAN-67-induced cardioprotection is independent of any nontyrosine kinase-related effects.

Both IPC and opioid-induced cardioprotection are mediated by complex signal transduction cascades probably involving multiple kinases. We have demonstrated that TK and PKC are important in cardioprotection induced during IPC, and the extent of involvement of these two enzymes may be directly related to the magnitude of the IPC stimulus (Fryer et al., 1998, 1999). We have shown that IPC induced by one cycle of ischemia/reperfusion can be completely abolished by genistein or chelerythrine administered alone. However, IPC induced by multiple cycles of ischemia/reperfusion could be completely abolished only by dual inhibition of both enzymes. We speculate that a single IPC stimulus and a subsequent lesser reduction in IS/AAR versus multiple cycle-induced IPC may be related directly to the amount of respective enzyme activation. This agrees with the present investigation where TAN-67 induces cardioprotection to a lesser extent than we have previously demonstrated with IPC (Fryer et al., 1999) and the finding that genistein could completely abolish opioid-induced cardioprotection.

Ping et al. (1999) have previously demonstrated that Src and Lck tyrosine kinase, both members of the Src tyrosine kinase family, are important in the genesis of IPC in rabbit hearts. They demonstrated that in conscious rabbits, Src activation in the particulate fraction was apparent 30 min post IPC; however, Lck activation in the particulate fraction was observed at both 5- and 30-min post IPC. We also suggest the importance of an Src/EGF receptor tyrosine kinase-mediated mechanism during IPC, since we could attenuate IPC-induced cardioprotection with both genistein and lavendustin A in an in vivo rat model (Fryer et al., 1998). Ping et al. (1999) also suggested that these Src/Lck-receptor TKs are activated by the  isoform of PKC since lavendustin had no effect on PKC- activation, but chelerythrine could abolish Src/Lck activation. Additionally, preliminary evidence from their laboratory suggests and has shown that Lck tyrosine kinase is a direct substrate of PKC- in the heart and that PKC- and Lck can physically interact (Song et al., 2000).

We suggest that TK activation following -opioid receptor activation is not mediated by an Src or EGF receptor TK. These differences in the findings of this investigation versus the findings of Ping et al. (1999) are probably explained by the stimulus used to initiate cardioprotection (ischemia versus opioid receptor stimulation) or to species differences. Additionally, these data may be explained by differential PKC isoform activation, which may regulate downstream TK activation. Ping et al. (1997) suggest that PKC- is the major isoform involved in cardioprotection in rabbits, whereas we have recently shown that PKC- plays an integral role in cardioprotection following opioid agonist administration in rats (Fryer et al., 2001b).

The use of the inactive analog of genistein, daidzein, was an important component of the present investigation. Genistein was originally thought to be a selective tyrosine kinase inhibitor (Akiyama et al., 1987). This idea, however, has been refuted since genistein has been shown to exhibit extensive nonselective effects (Akiyama and Ogawara, 1991; Huang et al., 1992; Chiang et al., 1997); however, these effects have not been associated with lavendustin A (Onada et al., 1989).

We also demonstrate that inhibition of TK with genistein does not abolish isoform-specific PKC translocation. We have previously demonstrated that PKC-, -_I, -, and - translocation follow stimulation of the ₁-opioid receptor (Fryer et al., 2001b). In the same investigation, we demonstrated that translocation of these PKC isoforms could be completely abolished by the PKC antagonist, chelerythrine, and the ₁-opioid receptor antagonist, 7-benzylidene naltrexamine. Additionally, we demonstrated that the PKC- inhibitor, rottlerin, could abolish TAN-67-induced cardioprotection with the subsequent blockade of PKC- translocation. However, rottlerin did not affect the subsequent translocation of PKC-, -_I, or -, suggesting the importance of PKC- in opioid-induced infarct size reduction (Fryer et al., 2001b). We demonstrate here that opioid-induced PKC translocation could not be inhibited by the TK antagonist, genistein. These conclusions are based on the observation that TAN-67 in the presence of genistein induced the translocation of PKC- to the sarcolemma, PKC-_I to the nucleus, PKC- to the mitochondria, and PKC- to the mitochondria and intercalated disk. Translocation to the mitochondria of PKC- and - has been previously demonstrated and confirmed by our laboratory via confocal microscopy (Fryer et al., 2001b). This suggests that TK activation is not proximal to PKC activation. Rather, TK may exist downstream from PKC as demonstrated by Baines et al. (1998) in a linear cascade or as a parallel cascade (Vahlhaus et al., 1998) where both enzymes converge on a final common signaling pathway. We did not assess the effects of lavendustin A on isoform-specific PKC translocation; however, evidence from our laboratory suggests that this drug, at the dose shown to attenuate cardioprotection from IPC, did not affect PKC translocation following IPC (unpublished observation).

In conclusion, we have demonstrated that a tyrosine kinase-sensitive mechanism mediates cardioprotection induced by ₁-opioid receptor activation. However, we clearly demonstrate that this is likely to be dependent on a soluble tyrosine kinase, as opposed to a receptor tyrosine kinase-mediated mechanism since both the Src-selective and Src/EGF receptor TK inhibitor could not abolish cardioprotection produced by a ₁-opioid agonist. Finally, these data suggest that PKC translocation to specific cellular loci is not abolished by TK inhibition, suggesting that these two key kinases in cardioprotection likely confer cardioprotection via activation of parallel signaling cascades or that PKC activation is proximal to TK activation in the in vivo rat myocardium.