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

1 Introduction

Endogenous opioid peptides and their receptors are involved in the regulation of many physiological processes, including nociception, analgesia, respiration, cardiovascular and gastrointestinal system activity, as well as nervous, endocrine, and immune functions. Opioid peptides, forming the family of enkephalins, dynorphins, and endorphins, are synthesized as large peptides (precursors) named preproenkephalin, preprodynorphin, and proopiomelanocortin, respectively.

1.1 Genes encoding opioid peptides

1.2 Opioid receptors

Opioid properties were broadly searched as important modulators of hypothalamic–pituitary–adrenal (HPA) axis activity, particularly during stress responses. Endorphin- and enkephalin-producing neurons are present in the paraventricular nucleus and the median eminence and modulate adrenocorticotropic hormone (ACTH)-controlling neurons (Van’T Veer et al., 2012).

The present communication focuses on Met-enkephalin in chickens, the effect of stress on Met-enkephalin physiology, insights gained from laboratory animals and livestock, and open questions on opioid peptides.

2 Loci for Met-enkephalin synthesis and release

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, Met-enkephalin is synthesized in the hypothalamus, anterior pituitary gland, adrenal glands, duodenum, proventriculus, and crop (Scanes and Pierzchala-Koziec, 2024a; Scanes and Pierzchala-Koziec, 2024b).

3 Met-enkephalin and stress

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 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 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 (Zhao et al., 2016; Tian et al., 2018; 2024; Wang et al., 2018). The relationships between Met-enkephalin and immune functioning in chickens remain unclear.

4 Control of pentapeptide Met-enkephalin release

The neurotransmitter acetylcholine plays an important role in regulating the release and synthesis of the native pentapeptide Met-enkephalin. The release of Met-enkephalin from the adrenal glands is under cholinergic control, as evidenced by with the nicotinic agonist nicotine, which increases 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, in vitro Met-enkephalin release and PENK gene expression have been observed in the hypothalamus, anterior pituitary gland, adrenal glands, crop, proventriculus, and duodenum in chickens (Scanes et al., 2024; 2025). Intestinal explants, at least, exhibit shifts in both PENK gene expression and Met-enkephalin release in the presence of both nicotinic and muscarinic cholinergic antagonists (Scanes et al., 2025).

Opioids downregulate 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).

5 Total immuno-reactive Met-enkephalin in plasma and tissues

Multiple peptides are derived from proenkephalin in the bovine and presumably chicken adrenal glands, including extended Met-enkephalin, Met-enkephalin [Arg⁶ and Phe⁷] peptides B, E, F, and I, and BAM 22, 20, and 12 (Stern et al., 1979; 1981), with different activities (Figure 1).

6 Other opioid peptides and stress

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 increase in response to stressors, such as exposure to ether or administration of lipopolysaccharide, in domestic geese (Barna et al., 1998).

7 Discussion and conclusions

The physiological relevance of circulating Met-enkephalin and other endogenous opioid peptides in birds remains poorly understood. To better document the physiology of Met-enkephalin acting as a hormone, studies on its circulating forms and their regulation are essential. Thus, during the past few years, we have measured immunoreactive Met-enkephalin in plasma and tissues to characterize the large circulating forms of peptidase-derivable Met-enkephalin and define, in hens, the physiological regulation of plasma responses of free Met-enkephalin (five amino acids) and the extended form of Met-enkephalin to psychological stress. Similar to rats (Pierzchala and Van Loon, 1990), restraint- or crowding-induced stress elicited biphasic responses of Met-enkephalin (Scanes et al., 2024; Scanes and Pierzchala-Koziec, 2024a).

Restraint stress in rats increased plasma native Met-enkephalin, which is 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: increasing at 1 and 30 min of stress; in contrast, catecholamines 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 results from a central nervous system regulatory mechanism (interaction with the sympathetic nervous system) rather than from 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 catecholamine sources did not decrease Met-enkephalin responses to restraint stress but may indicate the involvement of additional regulators of opioid synthesis and release, apart from catecholamines, such as acetylcholine, insulin, and ghrelin.

Answers to the abovementioned questions will clarify the role of endogenous opioids in 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.

References

• BarnaI.KoenigJ. I.PéczelyP. (1998). Characteristics of the proopiomelanocortin system in the outdoor-bred domestic gander. II. Seasonal and circadian rhythmicity; effect of ether stress and lipopolysaccharide administration. Gen. Comp. Endocrinol.109, 52–59. 10.1006/gcen.1997.7002

• GoldsteinA.TachibanaS.LowneyL. I.HunkapillerM.HoodL. (1979). Dynorphin-(1-13), an extraordinarily potent opioid peptide. Proc. Natl. Acad. Sci. USA.76 (12), 6666–6670. 10.1073/pnas.76.12.6666

• HylkaV. W.ThommesR. C. (1991). Avian beta-endorphin: alterations in immunoreactive forms in plasma and pituitary of embryonic and adult chickens. Comp. Biochem. Physiol. C100, 643–648. 10.1016/0742-8413(91)90054-W

• KilpatrickD. L.TaniguchiT.JonesB. N.SternA. S.ShivelyJ. E.HullihanJ.et al (1981). A highly potent 3200-dalton adrenal opioid peptide that contains both a [Met]- and [Leu]enkephalin sequence. Proc. Natl. Acad. Sci. U. S. A.78, 3265–3268. 10.1073/pnas.78.5.3265

• KoltaM. G.PierzchałaK.HoudiA. A.Van LoonG. R. (1992). Effect of diabetes on the levels of two forms of Met-enkephalin in plasma and peripheral tissues of the rat. Neuropeptides21 (1), 55–63. 10.1016/0143-4179(92)90152-m

• PierzchałaK.Van LoonG. R. (1990). Plasma native and peptidase-derivable Met-enkephalin responses to restraint stress in rats. Adaptation to repeated restraint. J. Clin. Invest.85 (3), 861–873. 10.1172/JCI114513

• Pierzchała-KoziecK.Van LoonG. R. (1994). Effects of nicotine on the concentration of native and cryptic Met- and Leu-enkephalin in peripheral tissues. J. Physiol. Pharmacol.45 (2), 319–330.

• Pierzchała-KoziecK.KępysB.OeltgenP.ScanesC. G. (2018). Developmental changes in the pituitary-adrenocortical axis and plasma enkephalin concentration in response to isolation stress in growing lambs. Folia Biol.66, 53–61.

• Pierzchała-KoziecK.Dziedzicka-WasylewskaM.ScanesC. G. (2019). Isolation stress impacts Met-enkephalin in the hypothalamo-pituitary-adrenocortical axis in growing Polish Mountain sheep: a possible role of the opioids in modulation of HPA axis. Stress14, 1–9. 10.1080/10253890.2018.1553947

• ScanesC. G.Pierzchala-KoziecK. (2024a). Disparate effects of stressors on Met-enkephalin system parameters and on plasma concentrations of corticosterone in young female chickens. Anim. (Basel)14 (15), 2201. 10.3390/ani14152201

• ScanesC. G.Pierzchała-KoziecK. (2024b). Morphine influences circulating and tissue concentrations of met-enkephalin and proenkephalin (PENK) expression and plasma concentrations of corticosterone in chickens. Poult. Sci.103, 103712. 10.1016/j.psj.2024.103712

• ScanesC. G.Pierzchała-KoziecK.GajewskaA. (2024). Effects of restraint stress on circulating corticosterone and met enkephalin in chickens: induction of shifts in insulin secretion and carbohydrate metabolism. Anim. (Basel)14, 752. 10.3390/ani14050752

• ScanesC. G.JaszczaK.GajewskaA.Pierzchala-KoziecK. (2025). Effects of cholinergic and opioid antagonists on in vitro release of Met-enkephalin, somatostatin and insulin-like growth factor-1 by and PENK expression in crop, proventriculus and duodenum of newly hatched chickens. Anim. (Basel)15 (12), 1702. 10.3390/ani15121702

• SternA. S.LewisR. V.KimuraS.RossierJ.GerberL. D.BrinkL.et al (1979). Isolation of the opioid heptapeptide Met-enkephalin [arg6, Phe7] from bovine adrenal medullary granules and striatum. Proc. Natl. Acad. Sci. U. S. A.76, 6680–6683. 10.1073/pnas.76.12.6680

• SternA. S.JonesB. N.ShivelyJ. E.SteinS.UdenfriendS. (1981). Two adrenal opioid polypeptides: proposed intermediates in the processing of proenkephalin. Proc. Natl. Acad. Sci. U. S. A.78, 1962–1966. 10.1073/pnas.78.3.1962

• TianJ.JiaoX.WangX.GengJ.WangR.LiuN.et al (2018). Novel effect of methionine enkephalin against influenza A virus infection through inhibiting TLR7-MyD88-TRAF6-NF-κB p65 signaling pathway. Int. Immunopharmacol.55, 38–48. 10.1016/j.intimp.2017.12.001

• TianJ.FuW.XieZ.ZhaoY.YangH.ZhaoJ. (2024). Methionine enkephalin (MENK) protected macrophages from ferroptosis by downregulating HMOX1 and ferritin. Proteome Sci.22, 2. 10.1186/s12953-024-00228-x

• Van LoonG. R.PierzchalaK.HoudiA. A. (1991). Nicotine-induced alterations in peripheral tissue concentrations of native and cryptic Met- and Leu-enkephalin. Neuropeptides19(1):35–41. 10.1016/0143-4179(91)90071

• Van’T VeerA.YanoJ. M.CarrollF. I.CohenB. M.CarlezonW. A.Jr. (2012). Corticotropin-releasing factor (CRF)-induced disruption of attention in rats is blocked by the κ-opioid receptor antagonist JDTic. Neuropsychopharmacology37, 2809–2816. 10.1038/npp.2012.151

• WangX.JiaoX.MengY.ChenH.GriffinN.GaoX.et al (2018). Methionine enkephalin (MENK) inhibits human gastric cancer through regulating tumor associated macrophages (TAMs) and PI3K/AKT/mTOR signaling pathway inside cancer cells. Int. Immunopharmacol.65, 312–322. 10.1016/j.intimp.2018.10.023

• ZhaoD.PlotnikoffN.GriffinN.SongT.ShanF. (2016). Methionine enkephalin, its role in immunoregulation and cancer therapy. Int. Immunopharmacol.37, 59–64. 10.1016/j.intimp.2016.02.015