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The opioid growth factor (OGF)-OGF receptor (OGFr) pathway is present in the ocular surface and functions to maintain homeostasis of the epithelium. The OGF-OGFr pathway has been reported to be dysregulated in diabetic individuals and animal models, and is reflected in elevations of the inhibitory growth factor, OGF, chemically termed [Met 5 ]-enkephalin. Recently, our laboratory reported elevated levels of OGF and OGFr in the serum and corneal epithelium of type 1 diabetic rats, suggesting that dysregulation of the OGF-OGFr axis may lead to dry eye, abnormal corneal surface sensitivity, and delayed re-epithelialization. Blockade of OGF-OGFr pathway using naltrexone, a potent opioid receptor antagonist, reverses dry eye symptoms and restores corneal surface sensitivity in diabetic rats when used as a therapy. Based on the evidence that both OGF and OGFr are elevated in type 1 diabetic rats, this study examined whether systemic or topical naltrexone treatment initiated at the time of induction of hyperglycemia could protect against the development of diabetic ocular surface complications. Diabetic male Sprague-Dawley rats treated systemically or topically with naltrexone had a delayed onset of dry eye and altered corneal surface sensitivity, and an improved healing rate for corneal wounds, that were comparable to non-diabetic rats. Serum levels of OGF were normal for rats receiving systemic naltrexone, and OGF tissue levels were normal for type 1 diabetic rats receiving twice daily naltrexone drops. OGFr levels remained elevated. These data support the role of the OGF-OGFr axis in regulation of ocular surface complications, and suggest that naltrexone therapy may be beneficial for pre-diabetic and early diabetic individuals.

Introduction: Bariatric surgery, as the only effective treatment of obesity, has strong effects on the metabolism, and nervous and endocrine systems. Thus, based on the different opinions about the efficaciousness of morbid obesity treatments, the aim of the present study was to estimate the association of serum ghrelin and Met-enkephalin (native, five amino acids and cryptic, precursor of enkephalin) concentrations with body mass index (BMI) value in bariatric patients within 30 postoperative days., Material and Methods: The study was performed on 38 female patients divided into two groups: I - BMI lower than 40 kg/m² (n = 18) and II - BMI higher than 40 kg/m² (n = 20). Blood was taken before (-24 h), and 72 h and 30 days after the sleeve gastrectomy. Routine haematological, anthropometric, and metabolic parameters as well as thyroid-stimulating hormone (TSH), ghrelin, and Met-enkephalin values were measured in all patients., Results: There were statistically significant differences between the two groups before the surgery in terms of TSH, both forms of Metenkephalin, triglycerides concentrations, and activity of alanine transaminase (ALT), gamma glutamyltransferase (GGTP), and C-reactive protein (CRP). After 72 h, the serum levels of cryptic Met-enkephalin and CRP, and activity of enzymes varied between the two groups of patients. Thirty days after the surgery, some metabolic and immune parameters were still different in both female groups in favour of patients with lover BMI. However, significant differences were noticed in the levels of ghrelin (increase), and native (decrease) and cryptic Met-enkephalins (increase)., Conclusions: The activity of endogenous peptides in bariatric patients is connected with the degree of obesity. Ghrelin level increases are negatively correlated with native Met-enkephalin changes shortly after bariatric surgery. The interplay of ghrelin and opioids might be considered as a predictor of postoperative weight loss success.

The endogenous neuropeptide opioid growth factor, chemically termed [Met 5 ]-enkephalin, has growth inhibitory and immunomodulatory properties. Opioid growth factor is distributed widely throughout most tissues, is autocrine and paracrine produced, and interacts at the nuclear-associated receptor, OGFr. Serum levels of opioid growth factor are decreased in patients with multiple sclerosis and in animals with experimental autoimmune encephalomyelitis suggesting that the OGF-OGFr pathway becomes dysregulated in this disease. This study begins to assess other cytokines that are altered following opioid growth factor or low-dose naltrexone modulation of the OGF-OGFr axis in mice with experimental autoimmune encephalomyelitis using serum samples collected in mice treated for 10 or 20 days and assayed by a multiplex cytokine assay for inflammatory markers. Cytokines of interest were validated in mice at six days following immunization for experimental autoimmune encephalomyelitis. In addition, selected cytokines were validated with serum from MS patients treated with low-dose naltrexone alone or low-dose naltrexone in combination with glatiramer acetate (Copaxone®). Experimental autoimmune encephalomyelitis mice had elevated levels of 7 of 10 cytokines. Treatment with opioid growth factor or low-dose naltrexone resulted in elevated expression levels of the IL-6 cytokine, and significantly reduced IL-10 values, relative to saline-treated experimental autoimmune encephalomyelitis mice. TNF-γ values were increased in experimental autoimmune encephalomyelitis mice relative to normal, but were not altered by opioid growth factor or low-dose naltrexone. IFN-γ levels were reduced in opioid growth factor- or low-dose naltrexone-treated experimental autoimmune encephalomyelitis mice relative to saline-treated mice at 10 days, and elevated relative to normal values at 20 days. Validation studies revealed that within six days of immunization, opioid growth factor or low-dose naltrexone modulated IL-6 and IL-10 cytokine expression. Validation in human serum revealed markedly reduced IL-6 cytokine levels in MS patients taking low-dose naltrexone relative to standard care. In summary, modulation of the OGF-OGFr pathway regulates some inflammatory cytokines, and together with opioid growth factor serum levels, may begin to form a panel of valid biomarkers to monitor progression of multiple sclerosis and response to therapy. Impact statement Modulation of the opioid growth factor (OGF)-OGF receptor (OGFr) alters inflammatory cytokine expression in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Multiplex cytokine assays demonstrated that mice with chronic EAE and treated with either OGF or low-dose naltrexone (LDN) had decreased expression of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and the anti-inflammatory cytokine IL-10 within 10 days or treatment, as well as increased serum expression of the pro-inflammatory cytokine IL-6, relative to immunized mice receiving saline. Multiplex data were validated using ELISA kits and serum from MS patients treated with LDN and revealed decreased in IL-6 levels in patients taking LDN relative to standard care alone. These data, along with serum levels of OGF, begin to formulate a selective biomarker profile for MS that is easily measured and effective at monitoring disease progression and response to therapy.

Low-dose naltrexone is a widely used off-label therapeutic prescribed for a variety of immune-related disorders. The mechanism underlying low-dose naltrexone's efficacy for fatigue, Crohn's disease, fibromyalgia, and multiple sclerosis is, in part, intermittent blockade of opioid receptors followed by upregulation of endogenous opioids. Short, intermittent blockade by naltrexone specifically blocks the opioid growth factor receptor resulting in biofeedback events that increase production of the endogenous opioid growth factor (OGF) (chemically termed [Met 5 ]-enkephalin) facilitating interactions between opioid growth factor and opioid growth factor receptor that ultimately, result in inhibited cell proliferation. Preclinical studies have reported that enkephalin levels are deficient in animal models of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Our hypothesis is that serum enkephalin levels are diminished in humans with multiple sclerosis and experimental autoimmune encephalomyelitis mice, and that change in serum opioid growth factor levels may serve as a reasonable candidate biomarker for the onset of experimental autoimmune encephalomyelitis and response to therapy. To address this, we designed a two-part study to measure endogenous opioids in multiple sclerosis patients, and to investigate the temporal pattern of decline in serum enkephalin concentrations in mice with chronic progressive experimental autoimmune encephalomyelitis and treated with low-dose naltrexone. For comparison, we investigated whether low-dose naltrexone exposure in normal mice also resulted in altered enkephalin levels. In both animal models, we monitored tactile and heat sensitivity, as well as differential white blood cell counts as indicators of inflammation. Serum [Met 5 ]-enkephalin levels were lower in humans with multiple sclerosis relative to non-multiple sclerosis patients, and low-dose naltrexone restored their levels. In experimental autoimmune encephalomyelitis mice, [Met 5 ]-enkephalin levels were depressed prior to the appearance of clinical disease, and were restored with low-dose naltrexone treatment. Low-dose naltrexone therapy had no effect on serum [Met 5 ]-enkephalin or β-endorphin in normal mice. Thus, [Met 5 ]-enkephalin (i.e. opioid growth factor) may be a reasonable candidate biomarker for multiple sclerosis, and may signal new pathways for treatment of autoimmune disorders. Impact statement This report presents human and animal data identifying a novel biomarker for the onset and progression of multiple sclerosis (MS). Humans diagnosed with MS have reduced serum levels of OGF (i.e. [Met 5 ]-enkephalin) relative to non-MS neurologic patients, and low-dose naltrexone (LDN) therapy restored their enkephalin levels. Serum OGF levels were reduced in mice immunized with MOG 35-55 prior to any clinical behavioral sign of experimental autoimmune encephalomyelitis, and LDN therapy restored their serum OGF levels. β-endorphin concentrations were not altered by LDN in humans or mice. Thus, blood levels of OGF may serve as a new, selective biomarker for the progression of MS, as well as response to therapy.

Background: Postoperative pain management is a critical aspect of patient care. The inflammatory state of the post-sternotomy surgical wound sensitizes nerve endings, causing pain. Unrelieved or improperly managed pain compromises wound healing. Peripheral opioid receptors play a major role in analgesia, particularly under inflammatory conditions where both opioid receptor expression and efficacy are increased. Leukocytic opioid peptides include β-endorphin (END), met-enkephalin (ENK), and dynorphin-A (DYN), with END and ENK being predominant., Methodology/principal Findings: This work represents the first study of inflammatory cells collected from post-sternotomy wounds of patients undergoing cardiac surgery including coronary artery bypass grafting (CABG). Wound fluid (WF) and cells were collected from sternal wounds using a JP Blake drain at 24, 48, and 72 hours post sternum closure. Anti-CD15 staining and flow cytometry revealed that polymorphonuclear neutrophils (PMN) are the predominant cells present in wound fluid collected post-surgery. Compared to peripheral blood (PB) derived PMN, significant increases in CD177+/CD66b+ PMN were observed suggesting activation of wound-site PMN. Such activation was associated with higher levels of opioid peptide expression in PMN derived from WF. Indeed, increased level of opioid peptides in sternal wound environment was noted 72 h post-surgery. We demonstrate that WF contains factors that can significantly induce POMC transcription in human PMNs. IL-10 and IL-4 were abundant in WF and both cytokines significantly induced POMC gene expression suggesting that WF factors such as IL-10 and IL-4 contribute towards increased opioid peptide expression in wound-site PMN., Conclusions/significance: This approach provided a unique opportunity to study the cross-talk between inflammation and opioid peptides in PMN at a sternotomy wound-site. Wound-site PMN exhibited induction of END and ENK. In addition, sternal wound fluid significantly induced END expression in PMN. Taken together, these data constitute first clinical evidence that human wound-site PMNs are direct contributors of opioids at the sternal wound-site.

Objective: Met- and leu-enkephalins are endogenous opioid neuropeptides with potent analgesic, vasoactive, immunomodulatory and anti-apoptotic properties. We hypothesised that clinical or immunological variables of early systemic sclerosis (SSc) might be correlated to plasma enkephalin levels., Methods: Plasma samples were collected at study entry of the Genetics versus Environment in Scleroderma Outcomes Study (GENISOS) cohort (early SSc, n=116). Plasma met-enkephalin and leu-enkephalin levels (microg/ml) were measured by high performance liquid chromatography (HPLC) and correlated to clinical and laboratory parameters in the GENISOS database. Statistical analyses were performed by nonparametric Wilcoxon rank sum tests and Pearson correlation coefficients., Results: Significantly lower plasma met-enkephalin levels were associated with anti-topoisomerase-I seropositivity (6+8.3 vs. 14.9+22.8 microg/ml, p=0.02). Plasma leu-enkephalin levels were significantly higher in SSc patients with digital pulp loss (95.6+130 vs. 64.9+101 microg/ml, p=0.02). Lower mean plasma met-enkephalin levels and inversely higher leu-enkephalin levels were noted in SSc patients with Raynaud's phenomena (p=NS)., Conclusion: The associations of plasma enkephalin levels to immunologic or clinical pathologies may underscore their vasogenic or fibrogenic significance and potential as therapeutic targets in early SSc.

To address the role of the opioid system in the pathogenesis of hepatic encephalopathy (HE) we measured plasma met-enkephalin, beta-endorphin and leu-enkephalin in patients with different grades of HE compared to control subjects and patients with cirrhosis. Plasma met-enkephalin levels were significantly higher in patients with cirrhosis and all grades of HE than controls. Plasma beta-endorphin levels were similar in the 3 groups. Plasma leu-enkephalin levels were significantly higher in HE grades II, III and IV than in controls, patients with cirrhosis and HE grade I patients. Our results support data on the involvement of met-enkephalin and leu-enkephalin in the pathogenesis of HE and provide a rationale for the use of opioid receptor antagonists in the treatment of HE.

Background: Remote preconditioning is known to be cardioprotective, but the exact mechanism has not been fully elucidated. The objective of the current study was to investigate the role of kappa-opioid receptors in cardioprotection by remote preconditioning and reveal possible underlying mechanisms., Methods: Remote preconditioning was induced in anesthetized male Sprague-Dawley rats by three cycles of 5 min of right femoral artery occlusion followed by 5 min of reperfusion. Myocardial ischemia-reperfusion was achieved by ligation of the left anterior descending coronary artery for 30 min and then reperfusion for 120 min. Infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining. Levels of lactate dehydrogenase, dynorphin, and met-enkephalin in plasma were measured. The opening of the mitochondrial permeability transition pore was monitored with fluorescent calcein in isolated ventricular myocytes., Results: Both remote preconditioning and U-50,488H (10 mg/kg intravenous), a kappa-opioid receptor agonist, significantly decreased the infarct size and plasma lactate dehydrogenase level induced by ischemia-reperfusion, and these effects were attenuated by nor-binaltorphimine (10 mg/kg intravenous), a kappa-opioid receptor antagonist, and atractyloside (5 mg/kg intravenous), a mitochondrial permeability transition pore activator. However, administration of naltrindole (5 mg/kg), a delta-opioid receptor antagonist, had no effect on the cardioprotection by remote preconditioning. The dynorphin plasma level was increased after remote preconditioning treatment, but the met-enkephalin level did not change. In isolated ventricular myocytes loaded with calcein, U-50,488H (300 microM) decreased the mitochondrial permeability transition pore opening induced by calcium (200 microM), and this effect was attenuated by cotreatment with nor-binaltorphimine (5 microM) or atractyloside (20 microM)., Conclusion: Activation of cardiac kappa-opioid receptors is involved in the cardioprotection induced by remote preconditioning, and the mitochondrial permeability transition pore may participate in the postreceptor pathway.

The local increase in Met-enkephalin level during zymosan-induced peritonitis may be related to its local production by exudatory leukocytes (as evidenced by our previous experiments) and influx from distal centers, as its level is significantly decreased in blood, inguinal lymph nodes, and some brain areas (striatum, hypothalamus, and pituitary gland).

Weight-trained men [OT; n = 11; age = 22.0 +/- 0.9 (SE) yr] resistance trained daily at 100% one-repetition maximum (1-RM) intensity for 2 wk, resulting in 1-RM strength decrements and in an overtrained state. A control group (Con; n = 6; age = 23.7 +/- 2.4 yr) trained 1 day/wk at a low relative intensity (50% 1 RM). After 2 wk, the OT group exhibited slightly increased exercise-induced testosterone (preexercise = 26.5 +/- 1.3 nmol/l, postexercise = 29.1 +/- 5.9 nmol/l) and testosterone-to-cortisol ratio (preexercise = 0. 049 +/- 0.007 nmol/l, postexercise = 0.061 +/- 0.006 nmol/l) and decreased exercise-induced cortisol (preexercise = 656.1 +/- 98.1 nmol/l, postexercise = 503.1 +/- 39.7 nmol/l). Serum concentrations for growth hormone and plasma peptide F [preproenkephalin (107-140)] were similar for both groups throughout the overtraining period. This hormonal profile is distinctly different from what has been previously reported for other types of overtraining, indicating that high-relative-intensity resistance exercise overtraining may not be successfully monitered via circulating testosterone and cortisol. Unlike overtraining conditions with endurance athletes, altered resting concentrations of pituitary, adrenal, or gonadal hormones were not evident, and exercise-induced concentrations were only modestly affected.

To investigate the involvement of endogenous opioids in acute increases in blood pressure and their functional relationship with atrial natriuretic factor and endothelin-1, we assessed plasma levels of beta-endorphin, met-enkephalin, dynorphin B, catecholamines, atrial natriuretic factor, and endothelin-1 before and after administration of the opioid antagonist naloxone hydrochloride (8 mg i.v.) in 28 hypertensive patients with a stress-induced acute increase in blood pressure. Ten patients with established mild or moderate essential hypertension and 10 normotensive subjects served as control groups. Opioids, atrial natriuretic factor, and endothelin-I were radioimmunoassayed after chromatographic preextraction; catecholamines were determined by high-performance liquid chromatography with electrochemical detection. Patients with an acute increase in blood pressure (systolic, 203.2 +/- 2.2 mm Hg; diastolic, 108.4 +/- 1.3) had plasma opioid, catecholamine, and atrial natriuretic factor levels significantly (P < .01) higher than hypertensive control patients (systolic pressure, 176.4 +/- 1.0 mm Hg; diastolic, 100.0 +/- 1.4), who had a hormonal pattern similar to that of normotensive subjects (systolic pressure, 123.2 +/- 1.5 mm Hg; diastolic, 75.0 +/- 2.0). Endothelin-1 did not differ in any group. In patients with an acute increase in blood pressure, naloxone significantly (P < .01) reduced blood pressure, heart rate, opioids, catecholamines, and atrial natriuretic factor 10 minutes after administration. Naloxone effects on blood pressure, heart rate, opioids, and catecholamines wore off within 20 minutes. In control groups, naloxone failed to modify any of the considered parameters. Our findings suggest that pressor effects of opioid peptides mediated by the autonomic nervous system during stress-induced acute episodes of blood pressure increase in hypertensive patients.

Release of met-enkephalin-like immunoreactivity (MLI) into the circulation was investigated during feline intestinal ischemia-reperfusion in relation to the mass of ischemic tissue and in correlation to the sympathoadrenergic, response as gauged by plasma norepinephrine (NE) and epinephrine (E) levels. Chloralose anesthetized cats were randomized to a control group (C, n = 7), 20 cm segmental (S, n = 7), or complete intestinal ischemia (l, n = 7) for 90 min followed by reperfusion for 180 min. MLI and NE/E concentrations were assayed by radioimmunoassay and chromatography, respectively. Severe mucosal lesions and cardiovascular decompensation were observed in l animals in association with increased MLI and NE levels in femoral venous and portal venous plasma, whereas no such responses were observed in the S or C groups. MLI and NE concentrations were consistently higher in portal venous than in femoral venous plasma in 1 animals and correlations between MLI and NE levels were found during reperfusion in the portal, but not the systemic, circulation. Concentrations of E remained unchanged in all groups and did not correlate to MLI release. We conclude that: 1) intestinal MLI release and sympathetic activation occur only when the ischemic insult is sufficient to cause circulatory collapse, characteristic of an "on-off" rather than a "dose-response" pattern; and 2) intestinal MLI and NE release at reperfusion are correlated whereas MLI and E release do not appear to be related events.

Objective: To test the hypothesis that met-enkephalin has a role in human ovulation and that plasma levels may differ between ovulatory and anovulatory ovarian cycles., Design: This is a descriptive study comparing levels of plasma met-enkephalin, gonadotropins, and ovarian steroids in 12 ultrasonically confirmed ovulatory cycles and 12 anovulatory cycles., Setting: The study took place in the infertility clinic of a large teaching hospital receiving primary and tertiary referrals of both private and public sector patients., Patients: All patients (n = 16) had infertility greater than or equal to 3 years and normal findings on previous investigation including evidence of ovulation., Interventions: Ovarian cycles were defined using transabdominal ultrasound scanning. Biochemical analyses were by radioimmunoassay., Main Outcome Measures: The differences between plasma met-enkephalin levels in the two groups of cycles were compared., Results: Met-enkephalin levels are significantly higher in ovulatory cycles with a significant peak in the 2 postovulatory days (Duncan's multiple range test; P less than 0.05)., Conclusion: Human ovulation is associated with cyclic elevation of plasma met-enkephalin. Further studies are required to elucidate causality.

To obtain data on the degree to which the opioid system is changed in cholestasis, endogenous opioid activity in plasma of rats with acute cholestasis was determined 5 days after bile duct resection. Total plasma opioid activity was determined using a radioreceptor technique that measured the displacement of the opiate receptor ligand [3H]-DAMGO from lysed synaptosomal fractions of normal rat brain. Plasma total opioid activity was threefold greater in bile duct-resected rats than in sham-operated and unoperated controls (P less than or equal to 0.05). Plasma levels of the individual endogenous opioid, methionine-enkephalin, were determined using a sensitive radioimmunoassay, and the specificity of the assay was confirmed using high-performance liquid chromatography. In cholestatic rats, plasma methionine-enkephalin levels were more than six-fold greater than in sham-operated controls (P less than or equal to 0.001) and more than 17-fold greater than in unoperated controls (P less than or equal to 0.001). However, plasma methionine-enkephalin levels accounted for less than 5% of total plasma opioid activity after bile duct resection. Plasma methionine-enkephalin levels in both cholestatic plasma and plasma from sham-operated animals were stable when incubated in vitro despite the presence of undiminished activity of the major enkephalin-degrading enzymes. Thus, protection of methionine-enkephalin from degradation may be a factor contributing to the elevated plasma levels of methionine-enkephalin found in cholestasis. The magnitude of the increase in plasma endogenous opioid activity in bile duct-resected rats provides support for the hypothesis that endogenous opioids contribute to the pathophysiology of cholestasis.

Plasma concentrations of beta-endorphin and met-enkephalin were measured, with appropriate radioimmunoassays, in cows during gestation and at parturition and in newborn calves. During pregnancy beta-endorphin immunoreactivity (IR) concentration increased, but values during the last month of gestation were not different from those at parturition. Highest met-enkephalin IR levels were obtained in cows during calving. A term Caesarean section caused an increase in plasma beta-endorphin and met-enkephalin IR concentrations, but no such increase occurred in cases of a preterm Caesarean section. In calves beta-endorphin IR values were lower before umbilical cord rupture than immediately after birth. Values decreased continuously thereafter. This was also the case for met-enkephalin IR concentrations in calves born at term. In preterm calves met-enkephalin IR values were low immediately after delivery and increased during the first hour of life. A significant correlation existed between the degree of acidosis and plasma levels of both opioid peptides in the calves. We conclude that a direct stimulation of peripheral beta-endorphin release by the pain or stress associated with calving does not seem to exist in cattle, whereas met-enkephalin seems to be more directly related to parturition. In calves the change to the extrauterine environment causes an immediate, increased release of both opioids.

Met-enkephalin and related proenkephalin A-derived peptides circulate in plasma at picomolar concentration as free, native pentapeptide and at nanomolar concentration in cryptic forms. We have optimized conditions for measurement of immunoreactive Met-enkephalin in plasma and for generation by trypsin and carboxypeptidase B of much greater amounts of total peptidase-derivable Met-enkephalin in plasma of rats, dogs, and humans. Free Met-enkephalin (11 pM) is constituted by native pentapeptide and its sulfoxide. Characterization of plasma total Met-enkephalin derived by peptidic hydrolysis revealed a small amount (38 pM) of Met-enkephalin associated with peptides of molecular mass less than 30,000 D, and probably derived from proenkephalin A, but much larger amounts of Met-enkephalin associated with albumin (1.2 nM) and with a globulin-sized protein (2.8 nM). Thus, plasma protein precursors for peptidase-derivable Met-enkephalin differ structurally and chemically from proenkephalin A. Met-enkephalin generated from plasma by peptidic hydrolysis showed naloxone-reversible bioactivity comparable to synthetic Met-enkephalin. Prolonged exposure of adult, male rats to restraint stress produced biphasic plasma responses, with peaks occurring at 30 s and 30 min in both free native and total peptidase-derivable Met-enkephalin. Repeated daily exposure to this 30-min stress resulted in adaptive loss of responses of both forms to acute restraint. Initial plasma responses of Met-enkephalin paralleled those of epinephrine and norepinephrine, but subsequently showed divergence of response. In conclusion, Met-enkephalin circulates in several forms, some of which may be derived from proteins other than proenkephalin A, and plasma levels of both free native, and peptidase-derivable Met-enkephalin are modulated physiologically.

Right medullary and various cardiovascular responses to stimulation of the peripheral end of the splanchnic nerve have been investigated in the presence and absence of exogenous adrenocorticotrophin, ACTH1-24, (5 ng min-1 kg-1). The adrenal-clamp technique was employed in conscious calves, after the pituitary stalk had been cauterized and they had recovered from anaesthesia. The intravenous infusion of ACTH1-24 increased the plasma ACTH concentration by about 1100 pg ml-1 and right adrenal venous output of cortisol by about 400 ng min-1 kg body weight-1. Stimulation of the splanchnic nerve at 4 Hz for 10 min had no effect on either arterial plasma ACTH concentration or the adrenal output of cortisol. Closely similar amounts of both adrenaline and noradrenaline were released in response to nerve stimulation in the presence and absence of exogenous ACTH. In contrast, the fall in adrenal vascular resistance of about 40%, which normally occurred in response to splanchnic nerve stimulation, was completely abolished by ACTH. The adrenal produced relatively large quantities of met-enkephalin-containing peptides. During splanchnic nerve stimulation the output of these increased 2-100-fold, at which time free met5-enkephalin accounted for only 10-20% of total. During ACTH infusion the output of free met5-enkephalin was reduced at rest and during nerve stimulation, but that of total met-enkephalin-containing peptides was unaffected. These results indicate that ACTH or an adrenal steroid may alter the processing of proenkephalin in the adrenal medulla acutely but not total opiate secretion. Alternatively, the presence of ACTH could act by influencing the population of chromaffin cells activated by splanchnic nerve stimulation.

1. The extent to which the adrenal gland contributes to neuroendocrine responses to electrical stimulation of the peripheral end of the splanchnic nerve has been investigated in conscious calves in which the right nerve was stimulated either at 4 Hz continuously for 10 min or at 40 Hz in 1 s bursts at 10 s intervals for the same period. 2. It was confirmed that the release of neuropeptide Y (NPY) and of gastrin-releasing peptide (GRP) is potentiated by stimulation in bursts at a relatively high frequency and shown that the adrenal gland made a negligible contribution to these responses. 3. There was no detectable change in the concentration of vasoactive intestinal peptide (VIP) in the arterial plasma but the existence of a very small but highly significant rise in the output of VIP from the adrenal provided evidence that it was released within the gland in response to splanchnic nerve stimulation. 4. The concentration of calcitonin gene-related peptide (CGRP) in the arterial and adrenal venous effluent plasma was consistently below the level of detection of the assay. 5. Splanchnic nerve stimulation resulted in an abrupt rise in the output of both free and total met5-enkephalin-like immunoreactivity from the adrenal gland which was substantially potentiated by stimulating in bursts. This pattern of stimulation also increased the proportion released in a high-molecular-weight form. 6. Stimulation in bursts significantly enhanced the output of both adrenaline and noradrenaline from the adrenal and resulted in the release of proportionately more noradrenaline. Small amounts of dopamine and DOPAC were also released during splanchnic nerve stimulation and the output of dopamine was significantly increased by stimulating in bursts. 7. Both patterns of stimulation elicited an abrupt rise in mean plasma adrenocorticotrophic hormone (ACTH) concentration, which was associated with an increase in mean adrenal cortisol output and the former effect was significantly enhanced by stimulating in bursts. 8. It is concluded that certain responses to splanchnic nerve stimulation are significantly potentiated by an intermittent high-frequency pattern of stimulation, including all those that are attributable to adrenal medullary activity, whereas others are apparently unaffected by changes in stimulus pattern.

Concentrations of endogenous opioid peptides in the plasma are increased during exercise and these substances have been implicated in the pathogenesis of asthma induced by chloropropramide and alcohol in diabetic patients. This work was undertaken to determine whether exercise induced asthma might be mediated by endogenous opioids. Plasma beta endorphin, met-enkephalin, and adrenocorticotrophic hormone (ACTH) concentrations were measured in five asthmatic patients and five normal volunteers breathing cold air during exercise. In four of the patients the effect of an infusion of naloxone on FEV1 was also measured during exercise induced asthma. Exercise produced acute bronchoconstriction in all asthmatics, characterised by a fall in FEV1; whereas no change occurred in normal subjects. There was no difference in plasma met-enkephalin, beta endorphin, and ACTH concentration between the two groups. Infusion of naloxone neither prevented nor worsened exercise induced asthma. These data suggest that endogenous opioids probably do not play a part in the development of exercise induced asthma.

Plasma beta endorphin and met-enkephalin concentrations were measured in response to treadmill exercises in 15 normal women before, during, and after an intensive programme of exercise training. Significant release of beta endorphin occurred in all three test runs, and the pattern and amount of release were not altered by training. Before training dramatic release of met-enkephalin was observed in seven subjects and smaller rises observed in a further four, and this response was almost abolished by training. This represents the first observed "physiological" stimulus to met-enkephalin release. Endogenous opioid peptides play a part in adaptive changes to exercise training and probably contribute to the menstrual disturbances of women athletes.

Methionine enkephalin and catecholamines were measured in carefully collected plasma samples from 25 patients with cirrhosis and ascites, and 25 with cirrhosis without ascites, 15 disease and 15 healthy controls. Methionine enkephalin was invariably raised in the ascites group, the median value being 4.6-6.9 times that of the other three groups. Similarly, in the ascites group, median noradrenaline was increased 2.5-4.2 and median adrenaline 1.8-2.5 times that of the other groups. Plasma methionine enkephalin is considerably raised in patients with cirrhotic ascites and has actions which could enable it to be an initiating factor of ascites formation.

The effects of either hypotension induced by sodium nitroprusside or hexamethonium or hypertension produced by angiotensin II or noradrenaline on the circulating levels of methionine enkephalin ([Met]enkephalin)-like immunoreactivity (MLI), adrenaline and noradrenaline in anaesthetized greyhounds were examined. Nitroprusside infusions (200 and 400 micrograms min-1) induced a fall in blood pressure accompanied by significant rises in plasma MLI and catecholamine concentrations. Concomitant administration of a high dose of naloxone did not alter the fall in blood pressure produced by nitroprusside but was associated with greater rises in circulating MLI and catecholamines when compared to nitroprusside alone, suggesting that [Met]enkephalin is not involved in the hypotensive action of nitroprusside. Intravenous hexamethonium (2.5 mg kg-1) provoked a fall in blood pressure which was not associated with any changes in plasma MLI. However, it produced a fall in plasma noradrenaline and a rise in plasma adrenaline. Thus it appears that neural mechanisms are required, at least in part, for the release of MLI. Angiotensin II (1.25 micrograms kg-1 min-1) and noradrenaline (8 micrograms kg-1 min-1) infusions produced an elevation in blood pressure without altering the circulating MLI levels. Study of the molecular forms of circulating MLI, before and during hypotension, revealed that the large molecular weight enkephalin-containing peptides with approximate molecular sizes of 18kD and 8kD were the predominant forms both in the basal and stimulated states. It is concluded that circulating [Met]enkephalin is not involved in the tonic control of blood pressure but it may modulate catecholamine release following hypotension as part of the stress response.

Endogenous opioid peptides rose to prominence with the discovery of the enkephalins in 1975. Since then, a vast amount of research has been directed toward understanding their role in normal and pathophysiological situations. Although the place of endogenous opioids in psychiatry remains uncertain, there is good evidence that a variety of tumors may secrete endorphins or enkephalins, and these may contribute to the non-metastatic complications of malignant disease. In addition, changes in cerebrospinal fluid met-enkephalin and beta-endorphin after acupuncture may be involved in the effectiveness of this therapy in the treatment of heroin withdrawal and severe pain. The hormonal effects of opiate agonists and antagonists are now well characterized; exercise-induced changes in circulating catecholamines are markedly enhanced by the opiate antagonist naloxone. It is possible that the opiate inhibition of catecholamine release during exercise is a reflection of endogenous opioid modulation of effort perception.

The possibility that nitrous oxide releases endogenous opioid peptides into the circulation has been tested in 10 pain-free, unstressed volunteers breathing 30% nitrous oxide in oxygen. Despite achieving plateau concentrations in venous blood, accompanied by subjective effects, there were no significant changes in plasma concentrations of immunoreactive beta-endorphin, methionine-enkephalin or ACTH. These results indicate that, in the absence of nociceptive input, the effects of the inhalation of nitrous oxide are unrelated to alterations in peripheral concentrations of these endogenous opioid peptides.

Inhibition of enkephalin hydrolysis by catecholamines in vitro suggested that local and/or humoral factors released during exercise might facilitate opiate responses by reducing the rate of opiate peptide inactivation. Several measures of enkephalin hydrolysis were determined in blood samples obtained from subjects designated as trained (VO2max, 64.3 +/- 1.6 ml.min-1.kg-1) and un-trained (VO2max, 37.4 +/- ml.min-1.kg-1) both at rest and after maximal exercise stress tests. Enkephalin hydrolyzing activity assessed under optimal conditions was equally distributed between plasma and intact red cells; however, hydrolysis by red cells increased dramatically following osmotic release of red cell contents. There were no apparent differences in enzyme concentration or its distribution between cells and plasma when comparing trained and untrained subjects; P greater than 0.05. There was also no statistical effect of maximal exercise on these measures in either group. However, when the sequential disappearance of enkephalin added to whole blood in vitro was evaluated, blood from trained subjects degraded the enkephalin more slowly than blood from untrained subjects and had half-lives in vitro 30-50% longer both before and after the exercise test; P less than 0.05. Since enzyme concentrations between the groups were similar, the longer half-lives suggest that circulating factors were responsible for moderating the rate of enkephalin metabolism in vivo and that these factors were more concentrated in trained subjects. This would facilitate opiate responses in trained subjects and perhaps provide them with added tolerance for the effort associated with elite performance levels.