Met-enkephalin and other opioid peptides in the stress response of chickens: Lessons from laboratory animals and livestock

Krystyna Pierzchała-Koziec^1*Colin Guy Scanes^2*

• ¹University of Agriculture in Krakow, Krakow, Poland
• ²University of Wisconsin-Madison, Madison, United States

Met-enkephalin is found in multiple tissues of rats including the anterior pituitary gland, neurointermediate lobe of the pituitary gland, adrenal gland, hypothalamus, heart, lung, spleen, liver, seminal vesicle, vas deferens, kidney, bladder detrusor, and duodenum with the highest concentration in the neurointermediate lobe (Kolta et al., 1992). Similarly, in chickens, Metenkephalin is synthesized in the hypothalamus, anterior pituitary gland, adrenal glands, duodenum, proventriculus and crop (Scanes and Pierzchala-Koziec, 2024a;2025). Stress (imposition of mechanical restraint) in rats is followed rapidly by increases in plasma concentrations of native (pentapeptide) Met-enkephalin (Pierzchała and Van Loon, 1990). Concentrations of Met-enkephalin are also elevated in lambs isolated from other sheep including the dams (Pierzchała-Koziec et al., 2018;2019). In chickens, both plasma concentrations of pentapeptide Met-enkephalin and PENK expression are elevated in young chickens subjected to restraint stress (Scanes et al., 2024). There are also effects of other stresses on plasma concentrations of pentapeptide Met-enkephalin and/or PENK expression. For instance, withholding water was accompanied by depressed concentrations of Met-enkephalin in both the anterior pituitary and adrenal glands together with increased PENK expression in the same organs (Scanes and Pierzchala-Koziec, 2024a). Moreover, there are decreased plasma concentrations of Met -enkephalin in chickens deprived of feed (Scanes and Pierzchala-Koziec, 2024a). There is increasing evidence that Met-enkephalin plays a role in the immune system (e.g. Zhao et al., 2016;Tian et al., 2018;2024;Wang et al., 2018). What is unclear is the relationships between Metenkephalin and immune functioning in chickens. The neurotransmitter, acetylcholine, plays an important role in the control of release and synthesis of the native pentapeptide Met-enkephalin. Release of Met-enkephalin from the adrenal glands is under cholinergic control with the nicotinic agonist, nicotine, increasing concentrations of both native Met-and Leu-enkephalin in the adrenal medulla and other tissues in rats (Van Loon et al., 1991;Pierzchała-Koziec and Van Loon, 1994). Moreover, there is in vitro Met-enkephalin release and PENK gene expression in the hypothalamus, anterior pituitary gland, adrenal glands, crop, proventriculus and duodenum in chickens (Scanes et al., 2024;2025). At least intestinal explants exhibit shifts in both expression of the PENK gene and release of Met-enkephalin in the presence of both nicotinic and muscarinic cholinergic antagonists (Scanes et al., 2025).Opioids down-regulate the Met-enkephalin system. The classical opioid, morphine, depresses plasma concentrations of Met-enkephalin and PENK expression in both the anterior pituitary and adrenal glands in young chickens (Scanes and Pierzchala-Koziec, 2024b). Moreover, the effects of restraint stress are attenuated by the administration of the opioid antagonist, naltrexone (Scanes et al., 2024). Stress in rats is followed rapidly by shifts in plasma concentrations of total Met-enkephalin (Pierzchała and Van Loon, 1990); the latter being generated by enzymatic hydrolysis of plasma proteins. There are analogous changes in plasma concentrations of total Met-enkephalin in lambs isolated from their dams (Pierzchała-Koziec et al., 2018;2019). It is not clear what total Metenkephalin signifies. Possibilities include the following:1) Proenkephalin or peptides larger than pentapeptide, Met-enkephalin that are derived from proenkephalin but lacking immuno-reactivity in the native Met-enkephalin radioimmunoassay. 2) Met-enkephalin binding to proteins in the circulation and/or secretory granules.3) A combination of possibilities 1 and 2.Multiple peptides are derived from pro-enkephalin in the bovine and presumably chicken adrenal glands including extended Met-enkephalin, Met-enkephalin [Arg 6 , Phe 7 ] peptides B, E, F and I, and BAM 22, 20 and 12 (Stern et al., 1979;1981) with different activities (Figure 1). To the best of our knowledge there are no reports of Leu-enkephalin, dynorphins A and B together and α-and β-neoendorphins in birds. There are few reports of dynorphins even in humans. Similarly, there is only a single report of circulating concentrations of β endorphin in chickens in which the molecular forms of β endorphin were examined (Hylka and Thommes, 1991). In addition, plasma concentrations of both ACTH and β endorphin are increased by stressors, either exposure to ether or administration of lipopolysaccharide, in domestic geese (Barna et al., 1998). The physiological relevance of circulating Met-enkephalin and other endogenous opioid peptides in birds remains poorly understood. In order to better document the physiology of Met-enkephalin acting as a hormone, studies of its circulating forms and their regulation are essential. Thus, during the past few years we have determined to measure immunoreactive Met-enkephalin in plasma and tissues to characterize the large circulating forms of peptidase-derivable Met-enkephalin, and to define in hens the physiologic regulation of plasma responses to psychological stresses of free Met-enkephalin (five amino acids) and extended form of Met-enkephalin. Similarly to rats (Pierzchala and Van Loon, 1990), restraint or crowded stress, induced the biphasic responses of Met-enkephalin (Scanes et al., 2024;Scanes and Pierzchala-Koziec, 2025a).Restraint stress in rats caused plasma native Met-enkephalin increased in parallel with the increases in plasma epinephrine and norepinephrine. Thereafter, there was a divergence in the plasma concentrations of Met-enkephalin and catecholamines during the period of restraint stress. Plasma Met-enkephalin showed a biphasic response to 30 min of restraint: increase at 1 and 30 min of stress, in contrast catecholamines were increased only at 1-3 min of restraint. It seems probable that the brief duration of the initial peak of plasma Met-enkephalin induced by restraint stress is a result of a central nervous system regulatory mechanism (interaction with sympathetic nervous system) than of a limitation in Met-enkephalin pool size, since the more severe stress of immobilization produced a prolonged elevation of plasma Met-enkephalin (Pierzchała-Koziec and Van Loon, 1994). In hens, depletion of peripheral catecholamines sources, did not decreased Metenkephalin responses to restraint stress, what might indicate additional, apart from catecholamines, regulators of synthesis and release of opioid such as acetylcholine, insulin, ghrelin. This short review on the role of Met-enkephalin in modulating of stress responses showed that in spite of extended scientific research there are still open questions on opioids peptides in birds:1) Met-enkephalin is produced by multiple organs. It is unclear which, if any, are the major sources of circulating concentrations of Met-enkephalin. 2) It is also unclear the extent to which, if any, that erythrocytes, leukocytes and/or thrombocytes release or degrade Met-enkephalin. 3) Whether Met-enkephalin exerts its effects via paracrine and endocrine manners. 4) Plasma concentrations of total Met-enkephalin (generated by enzymatic hydrolysis of plasma proteins) greatly exceed those of native pentapeptide Met-enkephalin. It is not clear the extent to which total Met-enkephalin reflects larger cleavage products of Proenkephalin and/or binding of Met-enkephalin to plasma proteins. 5) There are a series of extended Met-enkephalin peptides in cattle. What is not clear is whether these are also found in chickens and other birds, whether they are secreted in response to stimuli and their actions. 6) There are no published reports on effects of stress and other physiological interventions on circulating concentrations of Leu-enkephalin, prodynorphin derived peptides or nociceptin in chickens or in other birds. 7) There are few published reports (< 5) on effects of stress and other physiological interventions on circulating concentrations of β endorphin in chickens or in other birds.Answers to above questions will clarify the role of endogenous opioids in the stress and may facilitate opioid peptides being indicators of stress/failures in welfare. Moreover, it is speculated that research on opioid peptides will provide new bases for dissecting the multiple facets of stress and the responses to these.FIGURE 1 Structure of selected opioid peptides isolated from cattle adrenal glands together with putative chicken homologues and biological activities in a presumed KOR assay.X Relative to dynorphin1-13 as 100 A Calculated from IC50s from Goldstein et al., 1979;Kilpatrick et al., 1981 NA Not available [Key: pink -dibasic amino-acid residues, green -Met-enkephalin residues, blue -Leu-enkephalin residues, red -difference with between chicken and bovine sequence)