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Enkephalins are opioid peptides that are found at high levels in the brain and endocrine tissues. Studies have shown that enkephalins play an important role in behavior, pain, cardiac function, cellular growth, immunity, and ischemic tolerance. Our global hypothesis is that enkephalins are released from non-neuronal tissues in response to brief ischemia or exercise, and that this release contributes to cardioprotection. To identify tissues that could serve as potential sources of enkephalins, we used real-time PCR, Western blot analysis, ELISA, immunofluorescence microscopy, and ex vivo models of enkephalin release. We found widespread expression of preproenkephalin (pPENK) mRNA and production of the enkephalin precursor protein proenkephalin (PENK) in rat and mouse tissues, as well as in tissues and cells from humans and pigs. Immunofluorescence microscopy with anti-enkephalin antisera demonstrated immunoreactivity in rat tissues, including heart and skeletal muscle myocytes, intestinal and kidney epithelium, and intestinal smooth muscle cells. Finally, isolated tissue studies showed that heart, skeletal muscle, and intestine released enkephalins ex vivo. Together our studies indicate that multiple non-neuronal tissues produce PENK and release enkephalins. These data support the hypothesis that non-neuronal tissues could play a role in both local and systemic enkephalin-mediated effects.

Exercise increases serum opioid levels and improves cardiovascular health. Here we tested the hypothesis that opioids contribute to the acute cardioprotective effects of exercise using a rat model of exercise-induced cardioprotection. For the standard protocol, rats were randomized to 4 days of treadmill training and 1 day of vigorous exercise (day 5), or to a sham exercise control group. On day 6, animals were killed, and global myocardial ischemic tolerance was assessed on a modified Langendorff apparatus. Twenty minutes of ischemia followed by 3 h of reperfusion resulted in a mean infarct size of 42 ± 4% in hearts from sham exercise controls and 21 ± 3% (P < 0.001) in the exercised group. The cardioprotective effects of exercise were gone by 5 days after the final exercise period. To determine the role of opioid receptors in exercise-induced cardioprotection, rats were exercised according to the standard protocol; however, just before exercise on days 4 and 5, rats were injected subcutaneously with 10 mg/kg of the opioid receptor antagonist naltrexone. Similar injections were performed in the sham exercise control group. Naltrexone had no significant effect on baseline myocardial ischemic tolerance in controls (infarct size 43 ± 4%). In contrast, naltrexone treatment completely blocked the cardioprotective effect of exercise (infarct size 40 ± 5%). Exercise was also associated with an early increase in myocardial mRNA levels for several opioid system genes and with sustained changes in a number of genes that regulate inflammation and apoptosis. These findings demonstrate that the acute cardioprotective effects of exercise are mediated, at least in part, through opioid receptor-dependent mechanisms that may include changes in gene expression. [ABSTRACT FROM AUTHOR]

Ischemic heart disease is a major contributor to death worldwide. Ischemic preconditioning (IPC) is a natural process that protects the heart from ischemic injury. The goal of our laboratory is to determine the mechanisms by which mediators of IPC exert their protective effects. Among the mediators of IPC are small opioid peptides called enkephalins. We found that numerous tissues, including skeletal muscle, express the precursor protein proenkephalin (PENK), and release enkephalins in a tissue-specific manner. We also found that exercise, which is potently cardioprotective, alters PENK levels in both heart and skeletal muscle, but not in intestine. Moreover, ischemia-induced enkephalin release is higher in hearts from exercised animals, as compared to control animals. Together, our data support a model wherein numerous non-neuronal tissues release enkephalins in response to ischemia and ischemic-like stresses (e.g., exercise). These enkephalins may then act locally or systemically to increase ischemic tolerance of the heart and other tissues. Our findings further suggest that enhancing or mimicking the protective effects of enkephalins may benefit patients with ischemic disease. [ABSTRACT FROM AUTHOR]

Enkephalins are opioid peptides that act as neurotransmitters and modulators of cardiac function. Plasma enkephalin levels increase in response to ischemia and exercise. The level of bioactive peptides in blood is regulated at many levels, including inactivation by plasma or cell-associated peptidases. Previous studies showed that Met-enkephalin (ME) and Leu-enkephalin (LE) bound to plasma proteins, and that binding to these proteins protected LE from proteolysis. We found that Met-enkephalin-Arg-Phe (MEAP), and Met-enkephalin-Arg-Gly-Leu (MEAGL) also bound to human serum proteins; however, this binding was of significantly higher affinity that that of ME and LE. In addition, the enkephalin binding capacity of plasma was considerably greater than that of serum from the same donor, suggesting that enkephalins avidly bind to proteins that are present in plasma but not in serum. Finally, we observed a unique degradation pattern when MEAP was hydrolyzed by plasma carboxypeptidases, as compared to ME, LE and MEAGL. Together, our data suggest complex mechanisms by which enkephalin-plasma interactions may regulate systemic enkephalin bioactivity. [ABSTRACT FROM AUTHOR]

Ischemia-reperfusion injury contributes to the pathophysiology of numerous diseases. Ischemic preconditioning increases tissue resistance to ischemia-reperfusion injury. Among the mediators of ischemic preconditioning are opioid peptides called enkephalins. We found that numerous non-neuronal tissues express the enkephalin precursor protein proenkephalin. Additionally, using isolated heart, intestine, and skeletal muscle, we found a tissue-specific pattern of enkephalin release. We also found that ischemia increased cardiac enkephalin release, and that exercise altered the efficiency of enkephalin processing and release. In addition to acting as potential sources for enkephalin release, heart, intestine, and skeletal muscle all express mRNAs for opioid receptors, suggesting that these tissues are also targets for opioid-mediated effects. Consistent with this conclusion, purified enkephalins induced opioid-dependent decreases in intestinal contractility. Taken together, our data suggest that skeletal muscle, heart, and intestine may release enkephalins that may in turn mediate autocrine, paracrine, and systemic effects, including ischemic preconditiong. [ABSTRACT FROM AUTHOR]

Adipocytes are previously unrecognized sources of proinflammatory mediators that contribute to metabolic syndrome, diabetes, and atherosclerosis. Epicardial fat overlying coronary arteries produces higher levels of inflammatory mediators than subcutaneous fat. Because of their unique properties and location, we hypothesize that epicardial adipocytes (EpAd) may play an important role in transducing inflammatory responses to coronary arteries. Enkephalins are naturally occurring endogenous delta opioids that improve myocardial ischemic tolerance. We found that EpAd express CD14, which plays a crucial role in transducing signaling by endotoxin and other inflammatory mediators, and release significant levels of IL-8 in response to both endotoxin and TNF-alpha. Met5-enkephalin-Arg6-Phe7 (MEAP; 1 µM) inhibited this IL-8 release by 50% or more, as did PCP-enkephalin. Microarray analysis revealed that MEAP blocked LPS-induced expression of a number of immunomodulatory genes. Together, these data suggest that EpAd may contribute to proinflammatory responses in coronary arteries, and that endogenous enkephalins may blunt these effects, thus playing a protective role in coronary artery disease. [ABSTRACT FROM AUTHOR]

Exercise is associated with both increased serum opioid levels and improvements in cardiovascular health. We hypothesize that one class of opioids, the enkephalins, contributes to the acute cardioprotective effects of exercise. We further hypothesize that enkephalins are released from skeletal muscle in response to exercise, and act hormonally to evoke a cardioprotective effect. To test these hypotheses, we assessed the effect of exercise on skeletal and cardiac muscle proenkephalin (PENK) expression, as well as on myocardial ischemic tolerance. We found that exercise was profoundly cardioprotective in the rat. Additionally, exercise was associated with a change in PENK levels in skeletal and cardiac muscle, but not in intestine. Finally, hearts from exercised animals appeared to have an enhanced ischemia-induced enkephalin release relative to control animals. These findings, coupled with our recent report that enkephalins can be absorbed from skeletal muscle and can induce a cardioprotective effect, strongly support our hypothesis that exercise-induced enkephalin release from skeletal muscle, acting hormonally, contributes to acute cardioprotection. [ABSTRACT FROM AUTHOR]