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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]

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]

Abstract: 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. [Copyright &y& Elsevier]