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.

Carcinomas of the cervix and corpus uteri in humans: stage-dependent blood levels of substance(s) immunologically cross-reactive with insulin.

Inasmuch as the elevated levels of substance(s) immunologically cross-reactive with insulin (SICRI) in a diabetic woman with carcinoma of the corpus uteri decreased following the surgical removal of the uterus and ovaries, 80 women with cervical carcinomas of various stages and 70 women with carcinomas of the corpus uteri of various stages were screened for the levels of SICRI and C-peptide. The levels of SICRI in the second, third, and fourth stages of the cancers were elevated (up to six times above the normal levels of immunoreactive insulin) and stage-dependent. The levels of C-peptide, which are related to the insulin-secreting activity of pancreatic beta-cells, were normal and independent of the stage of cancer.

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).

Thymic influence of proliferation and interaction of fractionated spleen cells of mice.

Cells from the spleens of mice were separated into seven subpopulations by centrifugation on a discontinuous density gradient of dextran. The lightest fraction (containing stem cells), the three heaviest fractions (containing small lymphocytes), or a mixture of these, were injected into thymectomized or sham-thymectomized irradiated mice together with sheep red blood cells as antigen. After 8 days, the spleens of treated mice were tested for their content of cells lysing sheep red blood cells in agar (PFC) or cells mounting graft-versus-host reaction in newborn mice (GVHR). Heavy fractions were depleted of progenitors of PFC, but contained progenitors of cells mounting GVHR. The light fraction was depleted of both. If light cells were given together with heavy ones, the potential to generate PFC was restored, but only if the recipients were without their thymus; by contrast, the potential to generate GVH-reactive cells was strongly suppressed in the absence of a thymus. It is concluded that, in this model, stem cells probably interact with immunocompetent cells, and that the interaction depends on the thymus.

Modulation of lipopolysaccharide-induced production of cytokines by methionine-enkephalin.

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.

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.

Suppression of immune response in rats by stress and drugs interfering with metabolism of serotonin.

Rats immunized with sheep erythrocytes were stressed by repeated restraint and/or treated with a precursor of serotonin (5-hydroxytryptophan, 5-HTP) or with an inhibitor of serotonin synthesis (parachlorophenylalanine, PCPA). As expected, repeated stress reduced the plaque-forming cell (PFC) response. Treatment with 5-HTP also reduced the PFC response, and potentiated the immunosuppressive effect of stress. This was accompanied by increased metabolism of serotonin in the brain, as indicated by increased concentration of its metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in cerebral tissue. Treatment with PCPA also suppressed the PFC response, but this suppression was accompanied by decreased levels of brain serotonin and of 5-HIAA. Plasma corticosterone levels were elevated, reaching statistical significance, in rats treated with PCPA. Both drugs suppressed the in vitro PFC response of peripheral blood lymphocytes. Thus, although 5-HTP and PCPA altered serotonin metabolism in the brain in a diametrically opposite manner, their effects on the immune response were the same. Putative central effects of the drugs on serotoninergic regulation of the immune response were apparently obscured by their effects on corticosterone secretion as well as by their direct effects on immunocompetent cells.

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.

Met-enkephalin induced escape from dexamethasone immunosuppression.

The present study was undertaken to examine whether and what type of interaction occurs between a synthetic glucocorticoid, dexamethasone (DEX) and an opioid peptide, met-enkephalin (MENK) upon superoxide anion (O2-) release from human polymorphonuclear cells (PMN). MENK (10(-8) M) abolished suppressed O2- release from PMNs treated with 10(-7) M DEX. This was the case in unstimulated but not in PMNs stimulated with PMA. The effect of MENK was mediated through pertussis-toxin (PTX) sensitive G-protein and since it was abolished by H7 probably involves protein kinase C (PKC) as a second messenger system. Thus, MENK can abolish DEX induced suppression of O2- release from human PMNs possibly through the interaction of second messenger pathways.

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.

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 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.

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.

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.

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.

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 effect of Met-enkephalin on mice liver lysosomes.

The unsedimentable activities of acid phosphatase (AP) and beta-glucosidase (BG), from mice liver lysosomes significantly increased 6 h after a single i/p injection of Met-enkephalin (MENK). The activity of AP in the serum at the same time remained unchanged. Multiple injections of MENK (8 x 10 mg/kg) induced a significant decrease in AP activity in the serum and no change in the unsedimentable activities of AP or BG. MENK did not elicit any significant extracellular release of lactate dehydrogenase (LDH) either, indicating that, under the experimental conditions described, the cells remained intact. Other parameters, such as the activities of AP and BG in the liver and total sialic acid content in the serum and spleen remained unaltered. Moreover, MENK in concentrations of 10(-12) M, 10(-8) M, 10(-6) M or 10(-4) M did not change the activities of the lysosomal enzyme markers AP or BG in vitro. These data indicate far less pronounced transient effects of MENK on lysosomal membranes and enzymes compared to Leu-enkephalin which may be relevant for the use of MENK in combined chemo-immunotherapy.

Effect of enkephalins on bone marrow cells.

Mouse bone marrow cells were incubated with methionine- or leucine-enkephalin (10(-15)-10(-6) M) before seeding into soft agar cultures. In marrow samples harvested at different times, enkephalins decreased GM colony count on average by 30-40%. In individual experiments, however, the same concentration of enkephalins caused even stimulation, or at other times had effect. In view of the circadian periodicity of neuroendocrine functions and hematopoietic activity, the enkephalin effect on bone marrow cells was tested on marrow samples harvested at fixed time points (6 am, 6 pm), using enkephalin concentrations in the physiological range (10(-12)-10(-9) M). The seeding efficiency of the 6-pm cell population was on average 50% above that of the 6-am population. The 6-pm cell population was also more susceptible to the inhibitory effect of the enkephalins (35% inhibition) than the 6-am population (15% inhibition), and the variability in response was considerably reduced. With progenitor cell-enriched population, obtained by fluorescence-activated cell sorting (FACS) of 6-am bone marrow samples, in 3 out of 6 experiments Met- and Leu-enkephalin showed 30-35% inhibition of GM colony formation over a wide range of concentrations (10(-15)-10(-6)). In the other 3 experiments, suppression as well as stimulation or no alteration in colony count were observed. This variability probably reflected quality (purity) of the progenitor cell population, and may indicate that the enkephalins affected hematopoietic cells via a population of accessory cells.

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.