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1Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland; 2Institut Universitaire de Technologie, Université d'Auvergne, Aubière Cedex, France; 3Department of Surgery, Monash University, Alfred Hospital, and Wynn Domain, Baker Heart Research Institute, Melbourne, Australia; and 4University of North Texas Health Science Center, Fort Worth, Texas
Submitted 3 November 2004 ; accepted in final form 18 May 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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opioid peptides; heart; opioid peptide processing
. Thus opiate receptor signaling is an important facet of the myocardial adaptation to stress, although the source of the opioids and the location of their receptors are less well defined. The concentration of circulating opioid peptides (9) is presumed to be too low to explain their direct action on heart tissues (23, 34). However, bioactive opioid peptides synthesized within the myocardium could produce local concentrations much higher than those achieved in plasma. Moreover, the widespread and rapid enzymatic inactivation of endogenous opioids suggests that their influence is primarily paracrine. Further evidence for the local opioid production derives from the observation that opiate receptor antagonists prevent the beneficial effects of IPC in isolated hearts (13). Despite the abundance of myocardial proenkephalin (PE) mRNA and its translation by cardiac cells (15, 24, 28), the myocardial synthesis and processing of PE during IPC remain largely unknown.
Although isolated cardiomyocytes actively translated and processed PE, the fully processed enkephalins, e.g., methionine-enkephalin (ME), was much lower than that reported for the brain, despite comparable transcript contents in both tissues (11, 28). Because there is no evidence for granular storage of these bioactive peptides, the ventricular myocardium may translate preproenkephalin mRNA continually and then hydrolyze the resulting precursor when needed into an array of intermediate and fully processed enkephalins (1, 37).
ME and ME-arginine-phenylalanine (MEAP) represent >90% of the fully processed bioactive enkephalins in the heart (37). In the absence of vesicular storage, acute changes in ME and MEAP during cellular stress would thus require changes in the synthesis and processing of the larger peptides. The present study provides evidence for a putative scheme of enkephalin peptide processing within the myocardium and tests a three-part hypothesis: 1) that a 30-min period of ischemia and reflow (I/R) reduces postischemic ventricular function and depletes large PE precursors (PEP) and small bioactive enkephalins (MEAP and ME), 2) that brief periods of IPC before I/R will preserve ventricular function and increase those same pools of enkephalin, and 3) that preventing enkephalin degradation will substitute for IPC, raise myocardial enkephalin content, and preserve postischemic ventricular function.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Experimental groups. Hearts were subjected to 30 min of global ischemia and I/R in the presence or absence of IPC. IPC consisted of three episodes of 2 min of ischemia followed by 5 min of perfusion. Another group of hearts, similarly perfused, were pretreated with a cocktail of peptidase inhibitors (0.1 µmol/ml doses of amastatin, apstatin, bestatin, thiorphan, phosphoramidon, and des-Tyr-Leu-enkephalin). Rats received one dose of this cocktail intraperitoneally 24 h before each perfusion and then again 1 h before the heart was collected for isolated perfusion. A third dose was slowly infused into the perfusate during the first 5 min of perfusion of the heart. The experimental protocols are described in detail in Fig. 1. A separate substudy was conducted with hearts prepared as described above, without inhibitor, to evaluate the postischemic recovery of function after the addition of selected enkephalins to the coronary inflow. ME, MEAP, or leucine-enkephalin (LE) was added to the perfusate (10–8 M) for 20 min before initiation of global ischemia in lieu of the IPC. After 30 min of ischemia, recovery of ventricular function was evaluated during 20 min of reflow with the continued inclusion of the enkephalin. These protocols differed in two other respects: the amino acid complement was omitted, and the hearts were paced at 300 beats/min to reduce functional variation.
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-mercaptoethanol was added, the tissue was homogenized (Polytron, Brinkman), and the supernatant was collected after centrifugation at 25,000 g for 30 min. The pellet was rehomogenized in another 2.5 vol of 1 N acetic acid-0.2 N HCl, and centrifugation was repeated. The supernatants were combined and stored at –80°C. The extracts were thawed, neutralized with 10 N NaOH, and filtered through 0.45-µm syringe filters. Peptides in 2 ml of filtered extract were separated by size-exclusion chromatography on Bio-Gel P-10 columns (1.5 x 10 cm, 15 ml). The samples were eluted with 0.01 M phosphate-buffered saline (PBS), pH 7.0, containing 0.1% gelatin. Fractions corresponding to small (e.g., ME and MEAP), intermediate (e.g., peptide B), and large (e.g., PE) enkephalin sequences were collected (37) and frozen in 0.5-ml aliquots for later radioimmunoassay.
Perfusate enkephalin extraction.
Perfusate from the isolated hearts was collected in concentrated acetic acid and HCl, producing final concentrations of 1 and 0.2 N, respectively. -Mercaptoethanol was added to achieve a 0.1% final concentration. Peptides were extracted on prewetted Sep-Pak C-18 cartridges (Millipore/Waters, Bedford, MA). The cartridges were washed with 10 ml of 0.1% trifluoroacetic acid, the peptides were eluted with 6 ml of 60% acetonitrile in 0.1% trifluoroacetic acid, and the eluent was evaporated under vacuum. The freeze-dried extracts were reconstituted in 2 ml of PBS and chromatographically processed as described above.
Radioimmunoassays. Samples were assayed with COOH-terminal-directed antisera specific for ME and MEAP. 125I-labeled ME and 125I-labeled MEAP were prepared after oxidation of the iodine with chloramine-T, and the products of the reaction were separated by a combination of size-exclusion and anion-exchange chromatography with Sephadex QAE-A25, as previously described (1, 37).
Enkephalin-hydrolyzing activity.
Enkephalin-hydrolyzing activity (EHA) was determined radiometrically using 125I-labeled ME as substrate (5). After isolated heart perfusion, 
100 mg of left ventricle were excised near the apex and homogenized in 1 ml of 8% glycerol in PBS. The homogenates were stored at –80°C until enzyme assays were performed. Preliminary assays were conducted to determine the optimum assay conditions needed to maintain zero-order kinetics. The enzyme reaction mixture consisted of 50 nmol of ME, 5 x 105 disintegrations/min of 125I-labeled ME in 250 µl of 0.05 M Tris·HCl buffer, pH 7.4, 50 µl of diluted homogenate containing 50 µg of the original tissue, and an additional 200 µl of the Tris buffer. After 10 min of incubation at 30°C in a shaking incubator, the tubes were placed in ice water, and 33% glacial acetic acid (100 µl) was added to stop the reaction. All determinations included appropriate time 0 controls. The reaction products were separated by selective adsorption of the intact substrate on 0.5-ml Poropak Q columns, which were prepared and stored in 100% ethanol. Before being used, the columns were washed with 10 ml of 5% ethanol. The enzyme extracts (100 µl) were then loaded onto the columns to adsorb the intact substrate. The free 125I-labeled products of the hydrolysis were eluted with 10 ml of 5% ethanol and quantified by gamma spectrometry.
Statistics. Student's t-tests were used to evaluate differences in peptide content due to the cocktail of peptidase inhibitors. The effects of ischemia, I/R, and IPC + I/R were evaluated by ANOVA, with multiple comparisons tested post hoc by Tukey's test. Comparisons with P < 0.05 were deemed significantly different.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Effects of ischemia and reflow. Figure 4, A and B, illustrates enkephalin distribution after 30 min of global ischemia and after ischemia followed by 40 min of reflow with and without prior IPC. Ischemia alone reduced the PEP pool by 34%, MEAP by 54%, and ME by 60%; thus the total content of extracted peptides declined by 42%. I/R did not restore PEP or ME, and both remained below that observed in control hearts. However, in contrast to ischemia alone, reflow increased MEAP by 53% compared with the normoxic control and by 230% compared with ischemia alone. Accordingly, the decline in total peptide content was less severe after reflow than after ischemia alone. The decline in the PEP pool suggested that ischemia shifts the normal balance between synthesis and processing to one characterized by reduced synthesis and/or increased processing. The failure of the smaller peptides, ME and MEAP, to accumulate during ischemia in the absence of flow would be consistent with their rapid degradation. This presumes that the elevated EHA (Table 2) remained functionally effective during ischemia. The dramatic restoration of MEAP during reflow without a parallel increase in ME suggests that I/R differentially alters the conversion, secretion, and/or degradation of the two peptides (steps 3, 4, and 5, respectively, in Fig. 2).
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Preconditioning before prolonged ischemia followed by reflow. IPC + I/R depressed the PEP pool by 45% compared with normoxia (Fig. 4A) and, again, appeared to accelerate the processing from the PEP pool compared with its parallel treatment, I/R alone. IPC + I/R reduced the PEP pool by an additional 30% compared with the reduction after I/R alone. Although both protocols raised MEAP well above control values, IPC + I/R increased ME by 100% compared with normoxic time-matched control and by 500% compared with I/R alone (Fig. 4B). The IPC-mediated increase in peptide content coincided with a greater recovery of postischemic contractile function than I/R alone.
Figure 4C summarizes the effects of ischemia and I/R in the presence and absence of IPC from another perspective, i.e., relative changes in peptide content compared with normoxic controls. Ischemia appears to reduce the PEP pool, regardless of prior preconditioning or subsequent reflow. IPC may have increased the PEP depletion further compared with ischemia alone and, thus, may have increased PEP processing (step 2 in Fig. 2). In each case, reflow seemed to moderate the degree to which the PEP pool was depleted. Ischemia decreased the myocardial content of the processed peptides, ME and MEAP, and IPC reduced the decline in each case, suggesting again that preconditioning increases PEP processing (step 2). However, reflow produced divergent responses when MEAP and ME were compared. Reflow alone increased MEAP well above that in normoxic controls but had little effect on the depressed ME content. In contrast, when combined with preconditioning, reflow increased ME 500% from its low level to exceed the normoxic control by 100%. The parallel increase in MEAP was not different from that observed after reflow alone, but MEAP + ME content was clearly much higher. Finally, when the percent changes in the PEP pool and ME + MEAP were compared, it was apparent that preconditioning preserved or increased the processed smaller enkephalins at the expense of the existing PEP pool. Thus the improvement of postischemic mechanical function was coincident with the IPC-mediated increase in ME and/or MEAP + ME.
I/R with inhibitor cocktail. The third part of the study was designed to test whether inhibiting enkephalin degradation would increase endogenous enkephalin and, thus, represent a strategic substitute for IPC. The animals were treated with peptidase inhibitors in vivo, and the inhibitors were also added to the coronary inflow before I/R. The distribution of myocardial enkephalins was then determined after I/R with and without prior IPC. Figure 5 illustrates the results of adding the inhibitor cocktail in time-matched controls, during I/R, and during IPC + I/R. Peptidase inhibition increased MEAP and decreased ME in time-matched controls to produce a net increase in total enkephalins. Despite the higher net enkephalin content in hearts from time-matched controls, the enkephalin content was much lower after the combination of cocktail and I/R than in time-matched controls and was nearly identical to that in untreated I/R animals. Mechanical function (Table 1) was similarly depressed when cocktail and I/R were combined. In contrast, the combination of IPC and inhibitors restored enkephalins to levels almost matching those of controls treated with inhibitors. Similar to the time-matched controls, much of the restored content was attributed to higher MEAP. In contrast to the inhibitor-free protocols, the higher enkephalin content was associated with a much less robust improvement in mechanical function. The peptidase inhibitors did not alter the PEP pool during I/R. The overall PEP pool declined to a similar degree during I/R whether the inhibitors were present or not.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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The initial contents of various opioid peptides in control hearts shown in Fig. 3 represent a "snapshot" of net balance among synthesis, secretion, and relative activity of a collection of tissue peptidases. These peptidases are responsible for processing the PEP pool to MEAP and ME (step 2 in Fig. 2), for converting MEAP to ME (step 3), and for degrading ME and MEAP (step 5). Thus changes in the distribution of enkephalins resulting from ischemia, reflow, IPC, or introduction of a cocktail of peptidase inhibitors into the coronary perfusate could not be a priori specified. The myocardial MEAP content was very sensitive to peptidase blockade and nearly tripled when the cocktail was applied during control conditions. MEAP must normally have been processed and/or degraded by endogenous peptidase activity. In direct contrast, myocardial ME declined by 50% under the same control conditions. Because MEAP includes an ME sequence, the reciprocal relation between MEAP and ME suggests that some ME was derived from the conversion of MEAP to ME (step 3). Previous work indicated that, when added to myocardial homogenates, ME and MEAP were aggressively degraded by NH2-terminal aminopeptidase activities, with little evidence of interconversion (19). However, when the aminopeptidase activity was blocked, conversion of MEAP to ME was easily demonstrated (20). ME and MEAP may also be processed independently from the PEP pool at different rates, as suggested by the processing arrows in Fig. 2 (step 2). Furthermore, the preferential accumulation of MEAP and decline in ME during peptidase inhibition might also be explained if ME is more aggressively degraded than MEAP by residual myocardial peptidases (19, 20). Thus the relative importance of conversion, degradation, and secretion in determining the relative fates of ME and MEAP remains to be elucidated.
I/R. The first part of the hypothesis proposed to test whether the ventricular dysfunction after I/R was associated with depletion of the PEP pool and/or the smaller bioactive peptides. In support of this thesis, the available bioactive peptides, ME + MEAP, declined sharply during ischemia and were collectively restored to near-control values during reflow. Because ME and MEAP are very similar and both were capable of restoring postischemic function when administered to isolated hearts, the sum of their contents may be the more critical determinant of opioid-mediated cardioprotection. If cardioprotection requires that collective enkephalins exceed some threshold during reflow, the continued poor mechanical performance during I/R, despite increased MEAP, may reflect the fact that, collectively, MEAP + ME had barely returned to control values.
The PEP pool and the processed enkephalins fell during ischemia, suggesting that synthesis (step 1 in Fig. 2) remained static while processing of precursor (step 2) and degradation of product (step 5) continued. After 30 min of global ischemia, resumption of reperfusion also returned the precursor pool toward control, presumably by reactivating its synthesis (step 1). This presumption is based on the tandem increase in precursors and processed enkephalins (MEAP + ME). The accumulation of the smaller peptides during reflow appears to occur by increasing precursor processing relative to synthesis, as suggested by the incomplete restoration of the PEP pool.
IPC + I/R. The second part of the hypothesis proposed to test whether the improved ventricular function after IPC was associated with increased precursor processing and/or an increased supply of ME and MEAP. In contrast to I/R alone, IPC increased MEAP and ME well above the control content at the apparent expense of increased processing from the PEP pool, which declined below the content observed with I/R alone. Thus the improved ventricular function after IPC was, as predicted, associated with a substantive increase in ME + MEAP compared with control or I/R. Compared with I/R, the exaggerated increase in the smaller peptides in the IPC + I/R hearts was accompanied by a less robust recovery in the PEP pool. The accumulation of the smaller peptides after IPC + I/R appears again to occur by a further increase in processing relative to synthesis.
IPC may increase the ME + MEAP content by protecting key processing enzymes from reperfusion injury, perhaps by increasing the cellular content of heat shock proteins, which then serve as protective chaperones.
Inhibition of peptidase activity. The third part of the hypothesis proposed to test whether the administered protease inhibitors would inhibit myocardial enkephalin degradation, raise endogenous enkephalins, and restore postischemic ventricular function to a level very similar to that expected after IPC. If effective, this strategy might provide a less invasive approach to preconditioning. Because several proteases are likely to be responsible for enkephalin degradation in heart, a cocktail of peptidase inhibitors with a spectrum of specificities was employed. Furthermore, because little is known about the pharmacokinetics of the protease inhibitors in heart, multiple doses were administered to maximize the efficacy. Pretreatment with peptidase inhibitors increased the initial enkephalin content without altering resting function. Despite the higher initial enkephalin content in the peptidase inhibitor-treated group, the combined enkephalin (ME + MEAP) content fell sharply during I/R to values very similar to those observed after I/R alone. Consistent with this observation, ventricular function was similarly not restored in the inhibitor-treated I/R hearts. When protease inhibitor cocktail treatment and IPC + I/R were combined, ME + MEAP content was quantitatively similar to that in untreated IPC + I/R hearts and protease inhibitor-treated time-matched controls. Qualitatively, the mixture was different, in that MEAP predominated when protease inhibitors were present. Ventricular function, although restored in part by protease inhibitor cocktail + IPC, was clearly reduced compared with the cocktail-treated control or IPC without cocktail treatment. The relative failure to improve function, despite the higher enkephalin content, is unexplained. An acute change in content may be required. The extended exposure to protease inhibitors may have provided sufficient time for the development of competing compensatory responses, including the potential downregulation of the participating opioid receptors. Furthermore, the broad specificity of the inhibitors employed may have reduced the availability of other nonopioid cardioprotective peptides such as bradykinin. Thus the less-than-optimal functional result might be improved by the development of more specific inhibitors and/or more refined timing in their administration.
Enkephalin hydrolysis. Cardiac EHA was measured to evaluate potential I/R- and IPC-mediated changes in enzyme activity and to verify the efficacy of the added peptidase inhibitors. EHA was significant, confirming the myocardial capacity for the aggressive local degradation of enkephalin (step 5 in Fig. 2). The near restoration of ME + MEAP during I/R and the accumulation of ME + MEAP during IPC + I/R are not likely the result of acute changes in the overall capacity for enkephalin hydrolysis, because EHA was similar in both protocols and was not different from that in time-matched controls (Table 2). The peptidase inhibitors suppressed EHA by >50%; however, there were no differences when EHA from ischemic and preconditioned hearts was compared with EHA from cocktail-treated controls. The enzyme activity and the effect of the inhibitors were, however, assessed under optimal substrate conditions and at a significant dilution of tissue and inhibitor. Thus the enzyme content was likely unchanged by I/R or IPC + I/R, although subtle changes in effective enzyme activity in vivo, secondary to factors such as activation and available substrate, were not assessed and could have contributed to differences in the peptide distributions. These data confirm that myocardial EHA is prodigious in heart (4) and, thus, represents a significant barrier to effective diffusion of myocardial enkephalins. This hydrolytic barrier thus reinforces the paracrine character of myocardial enkephalin and the importance of its local synthesis.
Conversion of MEAP to ME.
The idea that myocardial ME may arise from the hydrolysis of MEAP [conversion (step 3 in Fig. 2)] is empirically supported by the rise in MEAP and the reciprocal decline in ME under control conditions when peptidase inhibitors were added. A similar rise in MEAP and decline in ME were observed after I/R when peptidase activity may have been impaired by hostile conditions during reflow. Reduced conversion of MEAP to ME during peptidase inhibition would not be surprising, however, in the absence of added inhibitor; a similar reduction in conversion during reflow suggests again that hostile conditions in the interstitium during I/R (e.g., pH, oxyradicals, and Ca2+) have modified the putative converting enzyme activity. Consistent with the thesis that I/R suppressed the conversion (step 3), IPC + I/R, which presumably improved interstitial conditions, eliminated the inverse relation between ME and MEAP. Furthermore, adding the peptidase cocktail to the IPC + I/R protocol restored the reciprocal relation by increasing MEAP and decreasing ME. The relative importance of this conversion is unclear, and some of the changes might also be attributed to differential changes in secretion. The greater release of ME than of MEAP (Table 3) in the coronary effluent observed throughout the study provides some logical support for that alternative. In addition, the decline in ME secretion after IPC + I/R may have contributed to the robust increase in tissue ME after IPC + I/R. The relative significance of individual endogenous enkephalins is also unclear. Although all the endogenous enkephalins are qualitatively 
-agonists, MEAP may be more potent in some respects, perhaps in part because of a slower rate of degradation (6, 7, 19). Despite potential differences, however, all three enkephalins evaluated in the present study were cardioprotective when added exogenously.
Peptidase inhibitors. Despite the multiple enzyme targets, comparison of the differential responses to the addition of peptidase inhibitors provides some insight into enkephalin processing in the heart. The synthesis of precursor and its processing to enkephalin were apparently impaired by I/R. This is based on the observation that the peptide pattern after I/R was unaltered by the addition of the peptidase cocktail. In direct contrast, the cocktail produced very similar shifts in the distribution pattern in time-matched control and IPC + I/R hearts. Thus it would appear that IPC may have reactivated or preserved key components of the synthetic and processing apparatus during reflow. These observations suggest a more complex interaction between IPC and I/R, which may include synthesis, differential PEP processing, and conversion of MEAP to ME (steps 1, 2, and 3, respectively, in Fig. 2). The net effect of IPC appears to be an increase in the local availability of bioactive, cardioprotective enkephalins as a result of an increase in their processing from existing precursor pools.
Enkephalin secretion. Changes in enkephalin secretion (step 4 in Fig. 2) could contribute to changes in the tissue content. Because ME secretion during I/R was not different from that in time-matched controls, an increase in secretion could not provide a clear explanation for the low myocardial ME content after I/R. In contrast, however, the clear decline in ME secretion during IPC + I/R might explain in part the dramatic increase in myocardial ME content observed when I/R was preceded by IPC. In contrast, the reciprocal increase in MEAP in the coronary effluent during IPC + I/R may reflect the parallel rise in MEAP in the tissue available for secretion. The combined increase in MEAP in tissue and effluent reinforces the interpretation that IPC increased the generation of MEAP.
Limitations. The inherent limitations associated with tissue peptide measurements suggested that the broad changes in constituent peptides were more reliable than the individual details. Immunoassays of hormones are always relative determinations, and the assay of rapidly degraded peptides in partially purified tissue extracts should, by their very nature, be viewed as estimates. Because ME and MEAP have identical NH2-terminal sequences, the assay for each is directed at the COOH-terminal epitope, which distinguishes them from each other. Consequently, the assays are less sensitive to alterations at the other end (NH2 terminus) of the molecule. Because NH2-terminal extensions or deletions are generally inactive, their contribution to the resulting measurements would overestimate the biologically active opioid content of the sample. Size-exclusion chromatography before assay eliminates many, but not all, potential contaminants in each size range. For instance, the initial step in the hydrolysis of MEAP reduces its size by one amino acid, its cross-reactivity by 70%, and its biological activity by >99% (37). Thus incompletely degraded MEAP would constitute a potential contaminant in the assay. Although most intermediates cross-react poorly, absolute purification is impractical, and their potential contribution to individual estimates of specific peptides should always be considered.
Significance.
Opioid influences are often mediated by reducing Ca2+ influx and subsequent neurotransmitter release. Cardioprotective enkephalins may stabilize the heart by adjusting neurotransmitter release to ensure an appropriate balance between fuel supply and metabolic demand. Catecholamines are energetically inefficient, presumably because they increase cAMP, deplete fuel supplies, and increase intracellular Ca2+. Reduced energy and excess Ca2+ facilitate myocardial necrosis (25), and chronic sympathetic stimulation facilitates cardiocyte apoptosis (for review see Ref. 10). Protection may also be afforded by limiting Ca2+ overload and the resulting Ca2+-dependent arrhythmias (25, 36). Enkephalin inhibits the chronotropic effect of sympathetic stimulation in vivo (29) and reverses the inotropic effect of -adrenergic stimulation in isolated cardiac myocytes and perfused hearts in vitro (23, 36). The interactions in vitro reduced L-type Ca2+ currents (36) and inhibited cAMP formation (23). Sympatholytic opioid influences in vivo are therefore potentially cardioprotective during metabolic stresses such as ischemia and intense exercise. Ganglionic blockade and chemical sympathectomy increased total myocardial opioids (1, 37), suggesting that the normal processing and release of myocardial opioid peptides depend on continued autonomic input. Continued opioid processing might therefore constitute a form of local feedback that normally moderates or restrains sympathetic stimulation.
Enkephalins may also stabilize the heart by improving vagal transmission (12). As indicated above and elsewhere (12, 16, 22), cardiac enkephalins are elevated during ischemia, and low enkephalin concentrations are vagotonic (12, 16). Thus improved vagal transmission may help protect the heart by reducing myocardial oxygen demand directly and/or indirectly by opposing excitatory sympathetic input.
In summary, myocardial enkephalins were distributed between large precursors and small bioactive products. We have provided evidence for a schematic framework for peptide processing within the heart. The processing was responsive to local myocardial conditions. I/R reduced processing and differentially altered the distribution of bioactive products in favor of MEAP. IPC increased bioactive cardioprotective enkephalins (ME + MEAP), probably by activating, preserving, or restoring the synthesis and processing of existing precursor pools.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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-opioid receptors regulate vagal bradycardia in canine sinoatrial node. Am J Physiol Heart Circ Physiol 285: H1332–H1339, 2003. inhibition during reperfusion in intact rat hearts. Circ Res 94: 960–966, 2004. mediates convergence of protection signaling to inhibit the mitochondrial permeability pore. J Clin Invest 113: 1535–1549, 2004.[CrossRef][ISI][Medline]
- and -opioid receptor stimulation affects cardiac myocyte function and Ca2+ release from an intracellular pool in myocytes and neurons. Circ Res 70: 66–81, 1992.This article has been cited by other articles:
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  S. H. Deo, M. A. Barlow, L. Gonzalez, D. Yoshishige, and J. L. Caffrey Repeated Arterial Occlusion, Delta-Opioid Receptor (DOR) Plasticity and Vagal Transmission Within the Sinoatrial Node of the Anesthetized Dog Experimental Biology and Medicine, January 1, 2009; 234(1): 84 - 94. [Abstract] [Full Text] [PDF]
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 A. J. Zatta, H. Kin, D. Yoshishige, R. Jiang, N. Wang, J. G. Reeves, J. Mykytenko, R. A. Guyton, Z.-Q. Zhao, J. L. Caffrey, et al. Evidence that cardioprotection by postconditioning involves preservation of myocardial opioid content and selective opioid receptor activation Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1444 - H1451. [Abstract] [Full Text] [PDF]
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M. A. Barlow, S. Deo, S. Johnson, and J. L. Caffrey Vagotonic effects of enkephalin are not mediated by sympatholytic mechanisms. Experimental Biology and Medicine, April 1, 2006; 231(4): 387 - 395. [Abstract] [Full Text] [PDF]
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Significant inhibitor effect (2-tailed unpaired t-test).
), and I/R(
: P < 0.05.