Elsevier

Peptides

Volume 29, Issue 1, January 2008, Pages 83-92
Peptides

Proenkephalin expression and enkephalin release are widely observed in non-neuronal tissues

https://doi.org/10.1016/j.peptides.2007.11.004Get rights and content

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.

Introduction

Enkephalins and other peptide products from the precursor protein proenkephalin (PENK) act as neurotransmitters, autocrine and paracrine factors, and hormones that modulate pain, behavior, cardiac function, organogenesis, and immunity [2], [13], [18], [19], [31], [35]. Our most comprehensive knowledge of the expression and release of enkephalins come from studies of the brain and neuroendocrine systems [5], [12], [13], whereas relatively fewer studies have been done on other tissues and cells. During development, preproenkephalin (pPENK) mRNA is abundant in many tissues, including kidney, liver, skin, skeletal muscle, bone, and lung [20], [21], [30], [33]. Previous studies reported that levels of pPENK mRNA in the majority of these tissues subsequently decrease as animals approach maturity, with the exception of the brain and heart, where levels increase [20], [21].

In addition to the functions listed above, studies from our laboratory and others demonstrate that in vivo administration of exogenous enkephalins is cardioprotective [8], [10], and that enkephalins added in vitro increase ischemic tolerance of isolated cardiac myocytes [38]. To understand further the potential role of endogenous enkephalins in protecting the heart and other organs from ischemic injury, we designed experiments to identify potential sources for local and systemic enkephalin release. To this end, we measured pPENK mRNA expression, as well as PENK protein production and enkephalin release using non-neuronal tissues, including skeletal muscle, kidney, and intestine.

Section snippets

Animals

Animals were maintained and used in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the requirements of the Animal Care and Use Committee (ACUC) at the University of Iowa. All animal protocols used in these studies were approved by the University of Iowa ACUC. Studies were performed using Sprague–Dawley rats (male, 300–350 g) from Harlan (Indianapolis, IN), C57/BL6 mice (male, 25–27 g) from Jackson Laboratories (Bar Harbor, ME), and pigs

Expression of preproenkephalin (pPENK) mRNA

Earlier studies demonstrate expression of pPENK mRNA in fetal and neonatal tissues [21], [33], [34], as well as in adult brain, adrenals, heart and lung [14], [15], [22], [28], [39], [41]. To expand upon these studies, with a particular focus on adult non-neuronal tissues, we performed real-time PCR as described in Section 2: Table 1 lists the primers used for these studies. For all studies, the specificity of the PCR product was illustrated by a single peak in disassociation curves (data not

Discussion

Enkephalins play an important role in a wide range of physiological functions, including preconditioning [10], [42], a process whereby brief stress protects against subsequent ischemia-reperfusion injury [26]. Preconditioning can occur both locally and at a distance (remote preconditioning) following brief ischemia of the heart, kidney, and skeletal muscle [1], [4], [11], [16], [32]. Two studies of particular interest to our group are the one performed by Patel et al. [27], who showed that the

Acknowledgements

Funding for these studies was provided by the Department of Emergency Medicine. We would also like to thank Chantal Allamargot and Kenneth Moore (Director) of the University of Iowa Central Microscopy Core Facility for their technical and scientific help.

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