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Reactive oxygen species (ROS) are formed by all aerobic organisms, and are involved in the numerous physiological and pathophysiological processes. Opioid peptides belong to a class of bioactive compounds of great interest because of their opiate-like activity. We determined the influence of methionine-enkephalin (MENK) on age-associated oxidant/antioxidant status in liver of CBA mice. Lipid peroxidation (LPO), total superoxide dismutase (tSOD), catalase (CAT), and glutathione peroxidase (Gpx) activities of 1, 4, 10 and 18 months old male and female control and MENK treated (10 mg/kg bw) CBA mice were determined. MENK showed gender-related effect on both oxidant/antioxidant parameters. It stimulated LPO in males, but suppressed in females. CAT and Gpx activities were lowered upon MENK exposure in males, but in females the activities were modulated by MENK. The relative mRNA levels for the antioxidant enzymes CuZnSOD, MnSOD, CAT and Gpx-1 were determined by reverse transcriptase polymerase chain reaction (RT-PCR) in groups where differences in activities between control and treated samples were observed. Changes of mRNA level in MENK treated groups showed that transcriptional regulation is both gender- and age-related. Comparison of enzyme activities and mRNA levels in control and MENK treated groups showed that, in some cases parallel changes occurred, while in other cases nonparallel changes were found. These results suggest that transcriptional changes are in accordance with enzyme activities in some cases, while in other cases posttranscriptional regulation of antioxidant enzymes may exist.

Opioid peptides have been recognized as modulators of reactive oxygen species (ROS) in mouse macrophages and human neutrophils. Since the effect cannot be ascribed to its direct scavenger properties, in this study, we tested the hypothesis that methionine-enkephalin (MENK) modulates ROS by alteration of antioxidant enzyme activity (AOE). For this purpose superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) are measured in red blood cells of 1, 4, 10, and 18-month-old CBA mice of both sexes injected with 10 mg/kg MENK. The results indicate that MENK-affected antioxidant enzyme activity of red blood cells is age- but not sex-related. The most abundant effects were observed at the reproductive stage. Increased sensitivity to oxidative stress by opioid peptides was in both sexes mainly due to increased SOD activity followed by GPX decrease. Thus, the damage ascribed to opioid peptides might be, at least partly, ascribed to deleterious effects of accumulated hydrogen peroxide (H2O2).

The purpose of this study was to investigate the effect of opioide peptide Met-enkephalin (MENK) on resistance to oxidative stress in the brain of 4, 10 and 18 months old CBA mice of both sexes. This was done by determination of oxidant status via lipid peroxidation (LPO) and antioxidant status by determination of total superoxide dismutase (tSOD), catalase (CAT) and glutathione peroxidase (Gpx). Results showed that brain of adult male mice is less resistant to oxidative stress than brain of adult females. The difference is mainly due to higher CAT activity and lower LPO activity in female brain. MENK decreased resistance to stress in the brain of both sexes but the effect appeared earlier in males (10 months of age) than in females (18 months of age). Also, MENK could pronounce its effect on resistance to oxidative stress in a gender-related manner: in female mice via regulation of antioxidant enzyme activities and in male mice via regulation of oxidant processes respectively.

Age- and sex-associated differences in lipid peroxidation (LPO), and total sialic acid content (TSA) in response to abuse of drugs have been reported both in humans and experimental animals. However, no data on the influence of gender and age on these parameters have been reported for methionine-enkephalin (MENK). In this study we examined the influence of age and gender on MENK-induced LPO levels in the liver and TSA content in splenocytes of CBA mice. LPO production, which was age- and gender-associated was differentially regulated by MENK at a dose of 10 mg or 2.5 mg/kg body weight. At the higher dose, MENK stimulated LPO production in younger males and females but suppressed only in older male mice. At the lower dose, MENK induced strong suppression in males while being without any effect in females. In TSA levels, the age-associated increase was greater in males and much lower in females, with higher TSA levels in younger (2.5, 4.5 months) and decreased levels in older female mice (9 months) being observed. Contrary to the effect on LPO level, TSA level in MENK-treated mice was suppressed in both sexes but only in young 2.5-month-old mice. These data provide evidence that some immunomodulatory properties of MENK are age- and gender-associated which may be relevant to the potential use of MENK as adjuvant therapy in patients with immunocompromised status.

Gender- and age-related differences in nitric oxide (NO) release and in response to drugs of abuse has been reported in both humans and experimental animals. So far, we have demonstrated in vivo methionine-enkephalin- (MENK-) modulated NO release in mice. However, no data on the influence of age and gender on this immunomodulatory effect of MENK have been reported. In this study we examined the influence of age (2, 4, 8 month old mice) and gender (male and female mice) on MENK-induced NO release of mouse peritoneal macrophages (PEMs) of the CBA strain of mice. NO release was not age but was gender related in that males generally produced more NO than females. The effect of MENK on NO release was age (demonstrated only in mature 4 and 8 month old mice) and gender related in that it could be observed only in female mice. Apoptotic cells that paralleled the increase of NO in MENK-treated female mice were, however, observed also in male mice although MENK was in males without effect. These data provide evidence that some immunomodulatory properties of MENK are age and gender related which may be relevant to the potential use of MENK in adjuvant therapy for immunocompromised status., (Copyright 2001 Academic Press.)

The opioid peptide methionine-enkephalin (MENK) has significant immunomodulatory ability in addition to its neurotransmitter function. Since neutral endopeptidase (NEP, CD10, enkephalinase EC 3.4.24.11) cleaves opioid peptides, the presence and activity of NEP in neutrophils from different persons might be responsible for the diverse, neuropeptide-induced, responses of neutrophils from different donors [Ann. N. Y. Acad. Sci. 650 (1992) 146]. The results obtained showed statistically significant differences in NEP activity among donors (high, medium and low). A 10-fold higher NEP activity in neutrophils (160-280 nmol/10(6) cells/h) and in their corresponding membrane preparations (550 nmol/mg protein/min) in our study, as compared to literature data, was a result of high specificity and affinity of Suc-Ala-Ala-Phe-pNA as substrate. In control nontreated neutrophils, the number of CD10 positive cells were not correlated with NEP activity. However, in neutrophils treated with a physiological (10(-10) M) concentration of MENK, two main events occurred; not only did the number of CD10 positive cells correlate with NEP activity, but contrary to control samples, MENK upregulated the expression of CD10 marker as demonstrated by an increase of mean florescence intensity (F-mean) in donors with low NEP activity. Taken together, these data add some clarity to the diverse activity of enkephalins in association with enzyme cleavage of those molecules.

We have previously shown that methionine-enkephalin (MENK) differentially alters the production of superoxide anion (O(2)(-)) from neutrophils of different donors. This effect could be due to variable activity of proteolytic enzymes involved in the degradation of this neuropeptide. In this study, we investigated the possible association between the effect of MENK on O(2)(-)release and the two neutrophil associated hydrolytic enzymes that participate in enkephalin degradation; aminopeptidase N (APN) and neutral endopeptidase (NEP). We have demonstrated that APN but not NEP activity was down-regulated by MENK. This might be due to internalization, since APN down-regulation was observed only with intact neutrophils and not with the respective membranes. Preincubation of neutrophils with inhibitory anti CD13 MoAb (WM15) abbrogated the suppressive effect of MENK (10(-12), 10(-10)and 10(-8)M). These facts, show that in the periphery (as well as the brain) the dominant role in MENK hydrolysis can be attributed to APN. Also, they further support the idea of the link between the membrane associated CD13 and binding of the ligand to the opioid receptor., (Copyright 2000 Harcourt Publishers Ltd.)

We have previously shown that methionine-enkephalin (MENK) alters in dose-dependent fashion the capacity of human neutrophils to produce superoxide anion. The response of neutrophils from different donors was diverse and this effect could be due to variable activity of proteolytic enzymes involved in the degradation of the neuropeptide. In this study, we have demonstrated a highly individual aminopeptidase N (APN) activity of neutrophils from different donors. Preincubation of neutrophils with MENK, but not with the synthetic agonist of the mu (DAGO) or the delta (DPDPE) opioid receptor, down-regulated the APN activity. This was paralleled by a loss in cell surface expression of APN at physiological (10(-10) M) concentrations of MENK. The level of APN activity from different donors correlated with the effect of MENK on superoxide anion release. Neutrophils with low APN activity, if preincubated with MENK, released reduced amounts of superoxide anion. In contrast, neutrophils with high APN activity released increased amounts of superoxide anion after preincubation with MENK. Thus, the highly individual APN activity on the surface of neutrophils from different donors seems to be altered by MENK and to be related to the respiratory burst.

In the present study the in vitro and in vivo effect of Met-enkephalin (MENK) on nitric oxide (NO) release by mouse peritoneal macrophages was evaluated. While in vitro MENK was ineffective unless combined with suboptimal concentrations of recombinant murine interferon gamma, in vivo all the doses (2.5, 5 or 10 mg/kg bw) bimodaly modulated NO release. Only the stimulative (2.5 and 10 mg/kg bw) and not the suppressive (5 mg/kg bw) dose of MENK was opioid receptor-mediated as demonstrated by abolishing the effect by naloxone. The stimulative effect of the low (2.5 mg/kg bw) dose, that was observed only if MENK was injected p.m., was associated with the IL production and IFN gamma as demonstrated by abolishing the effect by specific antibodies. The data additionally support the idea that opioid-mediated responses might be to a large degree mediated by the release of cytokines.

In the present study, we have examined the effect of opioid peptide methionine enkephalin (MENK) on production of factors with interleukin-1 (IL-1) and tumor necrosis factor (TNF) activity by mouse peritoneal macrophages and assessed whether modification in the production of those cytokines could be related to alteration of phagocytosis by MENK. None of the MENK concentrations examined altered IL-1 or TNF activity alone. However, peritoneal macrophages co-stimulated with 1 microgram of lipopolysaccharide (LPS) and 10(-10) M MENK potentiated IL-1 activity, compared to LPS alone, but abrogated TNF activity induced by LPS. While MENK alone slightly decreased phagocytosis of sheep red blood cells (SRBC) by mouse peritoneal macrophages, cells simultaneously incubated with 1 microgram of LPS and 10(-10) M MENK had increased phagocytosis compared to LPS alone. Moreover, phagocytosis of SRBC by cells incubated overnight with the supernatant of the respective cell culture was significantly augmented. These results provide additional evidence for the immunoregulatory role of neuropeptides and suggest that the modulatory action of MENK could be mediated, at least in part, through the up-regulation of cytokines, most probably IL-1 and TNF.