Endomorphin 1 activates nitric oxide synthase 2 activity and downregulates nitric oxide synthase 2 mRNA expression.

Endomorphins 1 and 2 are newly discovered opioid tetrapeptides whose structure is more resistant to enzymatic degradation than that of other opioid peptides. Endomorphins 1 and 2 are considered as endogenous ligands with a high affinity for mu receptors. A number of studies have shown that opioid peptides per se can induce release of nitric oxide from rodent and human immune cells. Endomorphins seemed to be involved in the process of vasodilatation by stimulating release of nitric oxide. In our study we stimulated in vitro J774 macrophages with different concentrations of endomorphin 1 or 2 for measuring nitric oxide release and nitric oxide synthase 2 (NOS 2) mRNA expression. Results showed that 48 h incubation did not enhance nitric oxide release when measured with the Griess method. On the other hand, using real-time amperometric detection of nitric oxide release shortly after challenge with endomorphins, we showed that only 10(-6) M endomorphin 1 was able to stimulate nitric oxide release from a J774 macrophage cell line by activation of NOS 2 isoenzyme. The peak release was 1000-1500 s after stimulation and was in the range of nitric oxide release stimulated with 10 microg/ml lipopolysaccharide. In contrast to this, endomorphin 2 failed to induce nitric oxide release in all tested concentrations. Using a specific inhibitor of nitric oxide synthase 2 (N-(3-[aminomethyl]benzyl)acetamidine, 1400W) we eliminated the stimulatory effect of endomorphin 1 on nitric oxide release. The expression of mRNA for NOS 2 in J774 macrophages, after 30 min incubation with either lipopolysaccharide or 10(-6) M endomorphin 1 was not upregulated. As expected, lipopolysaccharide induced de novo NOS 2 transcription within 4 h. At the same time, in contrast to lipopolysaccharide, mRNA expression of cells treated with endomorphin 1 was downregulated. Since a mu-opioid receptor specific antagonist beta-funaltrexamine hydrochloride inhibited nitric oxide release from endomorphin 1-treated cells, the effect seemed to be mu-opioid receptor mediated.

The influence of season on oxidant-antioxidant status in trained and sedentary subjects.

The association between oxidative stress and cardiovascular diseases is a widely accepted fact today. Generally, men have a higher risk of cardiovascular incidents and mortality from acute myocardial infarction and strokes. We have examined sport-associated circannual rhythms of oxidant and antioxidant processes by measuring plasma LPO, erythrocyte SOD, CAT, Gpx activity and plasma hormonal status in both sedentary and long-term trained men and women. We have shown seasonal variations in both oxidant and antioxidant status in all examined groups. The largest difference was observed in the oxidant status between sedentary men and women during autumn and winter, which is considered a period of high coronary risk for men. Sport decreased LPO in trained men in autumn, while the same effect in trained women was shifted towards summer. These data state that regular, long-term physical exercise training induces adaptive responses that confer protection against oxidative stress, as well as the beneficial effect of exercise with regard to season, particularly in men during a period of high coronary risk (autumn and winter, respectively) and in women during summer.

Met-enkephalin modulation of age-related changes in red cell antioxidant status.

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).

Immunostimulatory effect of natural clinoptilolite as a possible mechanism of its antimetastatic ability.

PURPOSE: Many biochemical processes are closely related to ion exchange, adsorption, and catalysis. Zeolites reversibly bind small molecules such as oxygen or nitric oxide; they possess size and shape selectivity, the possibility of metalloenzyme mimicry, and immunomodulatory activity. These properties make them interesting for pharmaceutical industry and medicine. METHODS: The experiments were performed on mice. Different biochemical and molecular methods were used. RESULTS: Micronized zeolite (MZ) administered by gastric intubation to mice injected with melanoma cells significantly reduced the number of melanoma metastases. In mice fed MZ for 28 days, concentration of lipid-bound sialic acid (LSA) in serum increased, but lipid peroxidation in liver decreased. The lymphocytes from lymph nodes of these mice provoked a significantly higher alogeneic graft-versus-host (GVH) reaction than cells of control mice. After i.p. application of MZ, the number of peritoneal macrophages, as well as their production of superoxide anion, increased. However, NO generation was totally abolished. At the same time, translocation of p65 (NFkappaB subunit) to the nucleus of splenic cells was observed. CONCLUSION: Here we report antimetastatic and immunostimulatory effect of MZ and we propose a possible mechanism of its action.

The effect of methionine-enkephalin on nitric oxide release in mice is age and gender related.

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.

Neutrophil neutral endopeptidase variation and its regulation by opioid peptides.

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.

The link between met-enkephalin-induced down-regulation of APN activity and the release of superoxide anion.

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.

Gender-related differences in murine T- and B-lymphocyte proliferative ability in response to in vivo [Met(5)]enkephalin administration.

Gender-related differences in response to drugs of abuse, such as cocaine and morphine, have been reported both in humans and in experimental animals. Besides causing analgesia, morphine has recently been shown to exert strong immunosuppressive activity. However, no data on the influence of gender on the immunomodulatory effects of morphine or opioid peptides have been reported yet. The aim of this study was to test the influence of gender on the immunomodulatory ability of the endogenous opioid peptide [Met(5)]enkephalin (MENK) in mice. This was done by comparing the proliferative ability of splenic T- and B-lymphocytes 14 h after systemic (intraperitoneal; i.p.) administration of [Met(5)]enkephalin (2.5, 5 or 10 mg/kg body weight). The proliferative ability of T- and B-lymphocytes was assessed by testing their in vitro response to graded concentrations of the T- and B-cell mitogens, concanavalin-A (Con-A) and lipopolysaccharide (LPS), respectively. The results obtained showed that [Met(5)]enkephalin, at a dose of 2.5 mg/kg, enhanced the proliferative ability of T-lymphocytes in male mice, but not in female mice. Similarly, [Met(5)]enkephalin, at doses of 2.5 and 5 mg/kg, enhanced the proliferative ability of splenic B-lymphocytes in male mice, whereas in female mice a decrease was observed ([Met(5)]enkephalin 2.5 mg/kg, LPS 10 microg/ml). [Met(5)]enkephalin, at a dose of 10 mg/kg, did not affect the proliferative ability of either lymphocyte population, regardless of gender. The [Met(5)]enkephalin-induced stimulatory effect on both T- and B-lymphocyte proliferation was reversed by naloxone (10 mg/kg body weight), injected prior to [Met(5)]enkephalin, suggesting an involvement of opioid receptors. Thus, the data presented provide evidence for the gender-related response of murine splenic lymphocytes to immunomodulation by [Met(5)]enkephalin, administered in vivo. This finding may be relevant to the potential use of [Met(5)]enkephalin in adjuvant therapy for immunocompromised states, such as acquired immunodeficiency syndrome (AIDS) or malignancies.

The role of aminopeptidase N in Met-enkephalin modulated superoxide anion release.

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.

Calcitonin gene-related peptide receptors in pig spleen and the involvement of the CGRP(1)receptor in the splenocyte function.

Calcitonin gene-related peptide (CGRP)-positive nerve fibers have been found in the trabecula and parenchyme area of pig spleen. Receptor studies have demonstrated that the CGRP binding site in pig spleen membranes has an average K(d)2.24 +/- 0.48 nM and B(max)78 +/- 4.09 fmol/mg of protein. In the K(d)range demonstrated in the binding studies, the dose-dependent suppressive effect of CGRP on spleen T lymphocyte proliferation was found with the maximal effect in 10(-9)M concentration. The same effect, but in a different concentration, was found on peripheral blood T lymphocytes with the maximum in 10(-6)M concentration. Contrary to the results obtained through the simultaneous presence of CGRP and mitogen, preincubation with CGRP led to a stimulation of peripheral blood lymphocytes in response to ConA and had no effect on spleen T lymphocytes. These results illustrate the difference in CGRP effect between lymphocytes of different origins. Using CGRP(1)receptor antagonist CGRP(8-37), we established that the CGRP suppressive effect on spleen T lymphocyte proliferation is CGRP(1)-receptor mediated.

The role of cytokines in MET-enkephalin-modulated nitric oxide release.

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.

Hydrogen peroxide modulation of the superoxide anion production by stimulated neutrophils.

Hydrogen peroxide (H2O2) pretreatment of human neutrophils results in a suppression of the superoxide anion (O2) production in response to surface-acting stimulants such as lipopolysaccharide (LPS) and opsonized zymosan. This effect was not observed when phorbol myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (fMLP) or tumor necrosis factor alpha (TNF alpha) were used as a stimuli. Since the response to PMA and other stimuli was unimpaired by preincubation with H2O2, we assume that the H2O2 modulated O2 production is probably due to alteration of the LPS receptor conformation rather than effecting directly NADPH-oxidase. The balance of reactive oxygen species (ROS) produced by neutrophils in the state of sepsis may thus be autoregulated by negative feedback phenomena of locally produced H202.

Pharmacokinetics and pharmacodynamics of famotidine in infants.

The pharmacokinetics and pharmacodynamics of intravenous famotidine were evaluated in 10 infants ranging from 5 to 19 days of age who had a therapeutic indication for the prophylactic treatment of stress ulceration. After a 0.5-mg/kg infusion of famotidine, timed serum (n = 6), urine (24-hour collection), and repeated measurements of gastric pH were obtained. The mean +/- standard deviation maximum plasma concentration (Cmax) was 640.66 +/- 250.66 ng/mL, the elimination half-life (t1/2 beta) was 10.51 +/- 5.43 hours, and the apparent volume of distribution at steady state (Vdss) was 0.82 +/- 0.29 L/kg. Plasma clearance (Cl) and renal clearance (ClR) were 0.132 +/- 0.061 L/hr/kg and 0.093 +/- 0.056 L/hr/kg, respectively. No significant correlations were found between t1/2 beta, Vdss, Cl, and ClR and age. Six of the nine infants who had intragastric pH monitoring maintained a gastric pH > 4 until the final 24-hour sampling point. In this study, the t1/2 beta of famotidine was prolonged and the Vdss, Cl, ClR were reduced compared with corresponding parameters in previously reported studies of children older than one year of age and adults.

Methionine-enkephalin primes human neutrophils for enhanced superoxide anion production.

Methionine-enkephalin (MENK) is not an effective stimulus for inducing the superoxide (O2-) generation of human neutrophils, but it enhanced the O2- generation stimulated by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) or human recombinant interferon gamma (hrIFN gamma) when the cells had been preincubated with MENK for 30 min at 37 degrees C. The priming effect of MENK was not observed with stimulus such as lipopolysaccharide (LPS) or phorbol-12-myristate-13-acetate (PMA). The enhancing effect of MENK was abrogated if cells were treated with protein kinase C (PKC) inhibitor H7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) before fMLP or IFN gamma. This finding indicates that MENK is a potent modulator of human neutrophils and can contribute to inflammatory process.

Enkephalins in hematopoiesis.

Recent data support the view that neuropeptide mediators, in particular opioid peptides, participate in the control of hematopoiesis. The main arguments are: neuropeptides modulate the functions of lymphoid cells, macrophages and mature granulocytes; they control cell proliferation and differentiation in many tissues, particularly during embryogenesis; lymphoid cells, macrophages, polymorphonuclear granulocytes and bone marrow stromal elements express neuropeptide receptors; bone marrow cells produce opioid-like neuropeptides; the CD10/CALLA marker of lymphoid, myeloid and marrow stromal cells is an enzyme, endopeptidase, which cleaves- and thus activates/inactivates-opioid and other neuropeptides. We have shown that opioid peptides enkephalins, opioid antagonist naloxone, and the inhibitor of enkephalin-degrading endopeptidase, thiorphan, modulate the proliferation and differentiation of hematopoietic cells in clonal and long-term cultures of mouse bone marrow. The effects partly depended on the presence of the accessory hematopoietic elements, and followed a circadian pattern. The dose-responses were irregular, showed strain-dependent and individual variations, and apparently reflected the state of the activity of target cells, cellular interactions and simultaneous signals by other mediators. The enkephalins were shown to bind to specific (opioid) receptors on the target cells, and their signals to be transmitted to the cell interior by a cascade of secondary messengers including diacyl-glycerol (DAG), protein-kinase C (PKC) and Ca++ ions. Neuropeptide regulation of hematopoiesis might belong to a complex immuno-neuroendocrine network including melatonin.

Influence of methionine-enkephalin on stress-induced parameters.

This study examines the influence of methionine-enkephalin (MENK) on stress-induced oxidative damage (lipid peroxidation; LPO) in mice liver homogenate, plasma corticosterone concentration (CS) and phagocytic activity of mouse splenocytes. The LPO value increased in the mice subjected to restraint stress and had no correlation to stress duration, while MENK had no effect. The CS concentration was enhanced after 6 h of stress and 6 h after injection of a low (2.5 mg/kg bw) dose of MENK. However, MENK and stress are adjunct modulators of LPO and corticosterone in vivo. LPO was additionally elevated when MENK (10 mg/kg bw) proceeded for 2 h after the onset of stress. However, corticosterone concentration seems to be regulated differently by the same dose of MENK depending on the duration of stress i.e. elevated in cases involving short periods of stress (2 h) and decreased in cases involving prolonged periods of stress (6 h). This modulation of LPO and corticosterone by 10 mg/kg bw of MENK and 2 h of restraint stress was paralleled with elevated phagocytosis.

Met-enkephalin modulates stress-induced alterations of the immune response in mice.

Overnight restraint stress of mice decreased ConA-driven lymphocyte proliferation, plaque-forming cell response to sheep red blood cells (SRBC), and NK activity in the spleen, but the phagocytic activity was enhanced. Injection of methionine-enkephalin (MENK), 10 mg/kg, i.p., 30 min before restraint, abolished these changes (except for the NK activity) and attenuated the stress-induced elevation of glucocorticoids. However, MENK itself affected the immune responses like stress: It decreased NK activity and the PFC response and enhanced phagocytic activity. Contrary to results with stress, MENK had no effect on cell proliferation. The opioid-receptor antagonist naloxone given before restraint reversed the stress-induced enhancement of phagocytosis and the decrease of T-cell proliferation. Alterations of the immune responses induced by restraint stress seem to be mediated by at least two mechanisms: activation of the hypothalamus-pituitary-adrenal (HPA) axis and the secretion of opioid peptides. MENK injected before stress may interfere with either or both mechanisms. T or B lymphocytes seem to be affected by the activation of the HPA axis, and phagocytes by a direct opioid action, whereas NK cells seem to be under the influence of another control mechanism.

Neutrophil signal transduction in Met-enkephalin modulated superoxide anion release.

The present study explored the involvement of signal transduction system(s) in Met-enkephalin (MENK) modulated superoxide anion (O2-) release from human neutrophils. This opioid pentapeptide stimulated the O2- release in all samples if present at 10(-8) M concentration while in lower concentrations the stimulatory concentration was donor-dependent. The most abundant product of MENK degradation, Tyr-Gly-Gly (TGG), suppressed O2- release over a wide range of concentrations (10(-12)-10(-8) M). MENK induced O2- release was associated with a dose-dependent increase of diacylglycerol (DAG) concentration and protein-kinase C (PKC) translocation to the neutrophil membranes, with an increase of cytosolic Ca++, and could be abolished by H7, a PKC inhibitor. On the contrary, the suppressive effect of TGG was not associated with alteration of DAG concentration in neutrophil membranes. Superoxide anion release induced by low concentrations of MENK (10(12)-10(-10) M), could be blocked by NDGA, an inhibitor of the lipoxygenase pathway. We concluded that MENK-induced O2- release results mainly due to DAG/PKC pathway activation, although other secondary messengers might be involved.

The relevance of intact enkephalin molecule in predominantly delta opioid receptor mediated superoxide anion release.

The effect of intact enkephalin (MENK) molecule or its metabolite Tyr-Gly-Gly (TGG) as well as the effect of synthetic agonist for opioid receptor subtypes (DADLE and DAGO) on superoxide anion release from human neutrophils has been investigated. In lower MENK concentrations, where MENK alone had no effect on O2- release, inhibition of enkephalinase by thiorphan significantly increased O2- production, while in higher concentrations, where MENK alone was effective, inhibition of enkephalinase had no effect. Aminopeptidase inhibition by bestatin did not influence O2- release from MENK treated PMNs. While MENK predominantly stimulated, TGG suppressed O2- release. Opioid antagonist naloxone (10(-5) M) abrogated the effect of MENK on O2- release. DADLE (delta receptor agonist) increased O2- release in 10(-11) M concentration, while DAGO (mu receptor agonist) had no effect in any concentration examined. Enkephalinase inhibition increased O2- production from DADLE but not from DAGO treated PMNs. It seems, therefore, that free radical production is mainly associated with the delta subtype of the opioid receptor. Also, our observations support the hypothesis that enkephalinase might be the enzyme selectively responsible for regulating effects of enkephalins.

Side-effects of peptidoglycan monomer (PGM) treatment in mice.

The influence of single (4 mg/mouse) and multiple (1 mg/mouse per day for 5 consecutive days) injections of PGM on some hepatic enzymes, lipid peroxide generation in serum and liver, sialic acid concentration in serum and spleen and hepatic lysosomal membrane permeability was investigated. The studies performed showed that a single injection of PGM in vivo changed temporarily the permeability of lysosomal membranes, lipid peroxidation products and sialic acid concentration, and when administered in vitro modulated superoxide anion production and did not affect the activity of lysosomal membrane enzymes. Multiple injections of PGM did not cause significant changes in the examined parameters. Although the metabolic changes were time-limited and from the toxicological point of view, provoked transient effects, the results obtained may be of importance when using PGM in combined chemo-immunotherapy.