Methionine-enkephalin Levels Research Articles

Increased myocardial methionine‐enkephalin with reduced arterial oxygenation in congenital heart disease

Cardiac opioid peptides have been identified to exert important adaptive metabolic signalling for cardioprotection against ischaemia or hypoxia-related injury. To determine myocardial methionine-enkephalin content in children with hypoxemic congenital heart defects and to correlate myocardial content of methionine-enkephalin with the extent of arterial oxygen desaturation. Children (n= 20, median age of 16 months), undergoing cardiac surgical repair (tetralogy of Fallot, 17/20), were included in this study. Arterial oxygen saturation was measured on admission. Myocardial samples obtained during surgery were assayed via radioimmunochemistry for methionine-enkephalin content. Greater methionine-enkephalin content was measured in the right ventricles of the patients suffering from recent cyanotic spells compared with those with no recent spells (cyanotic spells: 2418 ± 844 pg/g wet weight tissue, n= 6; no spells: 1175 ± 189 pg/g wet weight tissue, n= 14, P= 0.04). An inverse correlation was evident between the arterial oxygen saturation and myocardial methionine-enkephalin content. Myocardial methionine-enkephalin levels increase with the severity of hypoxic stress in congenital cardiac disease and may play an important adaptive role in countering adrenergic over-activity and related excess demand on myocardial metabolic capacity.

Proteinase-activated Receptor 1 Contributed to Up-regulation of Enkephalin in Keratinocytes of Patients with Obstructive Jaundice

Skin synthesis of endogenous opioids such as enkephalin is considered to be increased in cholestatic rodents, which may induce antinociception in cholestatic liver disease. No studies have reported yet the expression of skin enkephalin in patients with cholestasis. Electrical pain threshold, postoperative morphine consumption, and skin enkephalin expression were measured in patients with jaundice (n = 18) and control patients (n = 16). Male Sprague-Dawley rats (n = 52) and human keratinocyte cell line HaCaT were used in vivo and in vitro studies, respectively. Nociceptive thresholds and plasma and skin levels of methionine-enkephalin were compared in protease-activated receptors-1-antagonized and control bile duct-ligated rats. In in vitro study, the effect on thrombin-induced enkephalin expression was examined and the role of extracellular regulated protein kinases 1/2 and p38 was investigated. The authors found that: (1) the electrical pain threshold (mean ± SD) was 1.1 ± 0.1 mA in control patients, whereas it was significantly increased in patients with jaundice (1.7 ± 0.3 mA); 48-h postoperative morphine consumption was approximately 50% higher in the control group than that in the group with jaundice; (2) Skin keratinocytes enkephalin expression was increased in the patients with jaundice; (3) Protease-activated receptors-1 antagonist 1 μg·kg(-1)·day(-1) treatment to the bile duct-ligated rats significantly reduced plasma levels of methionine-enkephalin, nociceptive thresholds, and keratinocytes enkephalin expression; and (4) protease-activated receptors-1 activation induced enkephalin expression through phosphorylation of extracellular regulated protein kinases 1/2 and p38 in keratinocytes. Protease-activated receptors-1 activation in peripheral keratinocytes may play an important role in the local synthesis of enkephalin during cholestasis.

Augmented myocardial methionine-enkephalin in a murine model of cardiac angiotensin II-overexpression

The endogenous opioid system has been reported to interact with both the cardiac sympathetic and renin-angiotensin systems in exerting a local regulatory action on the heart. The goal of this investigation was to examine how cardiac levels of enkephalin production are altered in the development of normotensive primary hypertrophy due to elevated intra-cardiac angiotensin II (Ang II) production. Atrial and ventricular methionine-enkephalin (ME) levels were measured by quantitative radioimmunoassay in 14 and 28-week-old male transgenic mice (TG1306/1R) and control mice. The TG1306/1R exhibit cardiac specific Ang II overexpression and cardiac hypertrophy, but not hypertension. TG1306/1R mice had significantly higher heart/body weight ratios (15-20%) than control littermates at both 14 (p=0.02) and 28 weeks (p=0.04). Relative to controls, ME content was significantly elevated (approximately two-fold) in atria and ventricles in the older 28-week TG1306/1R mice only. A significant inverse correlation between heart size and ME level was observed for 28-week TG1306/1R only. We have provided evidence that a marked elevation of myocardial enkephalin level is observed in the established (but not early) phase of cardiac hypertrophy associated with cardiac-specific Ang II-overexpression. This study identifies a potentially important relationship between two endogenous peptidergic signalling systems involved in the regulation of growth and function of the hypertrophic heart.

Levels of Methionine Enkephalin and Substance P are Elevated in Inflamed Human Tooth Pulp

Purpose of the study: Endogenous opioid peptides such as methionine enkephalin (ME) and neuropeptides such as substance P (SP) are involved in modulating pain sensitive neurons. Recent studies have used human peripheral tissue such as dental pulp to investigate the effects whether inflammation and decay affect neuropeptide levels, this study investigates whether ME and SP levels are altered in inflamed human tooth pulp. Methods: After Ethics Committee approval, ME and SP were extracted in 250 µl of 0.5M acetic acid from the tooth pulp of 8 patients: 4 with healthy pulps and 4 with inflamed pulps. We used liquid chromatography and mass spectrometry (LC–MS) to detect changes in peptide concentration in the extracts. Peptide levels were compared to standard curves for each peptide, each sample was run in triplicate. Results: Both ME (1.20 ng/µl) and SP (1.86 ng/µl) were present in healthy pulp, and the levels of both peptides were significantly increased in inflamed tissue (4.37 ng/µl) for ME and 6.90 ng/µl for SP (P = 0.029 and P < 0.001 for ME and SP respectively). [Figure 1][1] illustrates the changes in peptide levels in inflammation compared to a healthy status. ![Figure 1][2] Figure 1 Graph shows the concentration of ME and SP in both experimental groups (n = 8). *Significant difference compared inflamed with control group (P = 0.029 for ME and P < 0.001 for SP). Conclusions: Inflammation in teeth can lead to severe pain in patients. We have, for the first time, identified an elevation of an endogenous opioid in inflamed human teeth, providing further evidence for endogenous analgesic mechanisms that may be therapeutically manipulated. [1]: #F1 [2]: pending:yes

Effects of chronic ethanol administration on brain interstitial fluid levels of Methionine-enkephalin as measured by microdialysis in vivo

The level of Met-enkephalin in the brain is inversely correlated with ethanol consumption and is controlled partially through efflux activity of peptide transport system-1 (PTS-1) at the blood–brain barrier (BBB). Prolonged alcohol drinking can perturb aspects of this system, including a loss of control of Met-enkephalin levels at the transcriptional and translational levels, and impaired release of Met-enkephalin from tissue sources. Met-enkephalin levels in whole brain homogenates often first paradoxically increase after a few days of ethanol drinking and then decrease with the development of physical dependence. Which of those various changes drives the others is unclear. To clarify these interactions, we here determined the levels of Met-enkephalin in striatal interstitial fluid (ISF) by microdialysis, striatal tissue homogenates, and serum after chronic ethanol treatment and alcohol withdrawal. Mice received ethanol (5%) in liquid diet for 7 days (ethanol-treated) and others withdrawn for a day following 7-day treatment (withdrawal). There was a significant ( P < 0.05) difference in the levels of Met-enkephalin in striatal microdialysate between the control (79.1 ± 5.9 pg/ml) and ethanol-treated group (94.9 ± 4.3 pg/ml), which was lost by withdrawing ethanol (83.9 ± 3.8 pg/ml). In contrast, ethanol treatment did not affect Met-enkephalin levels in the striatal tissue. In the ethanol-treated group, there was a significant ( P < 0.05) reduction of the levels of Met-enkephalin in serum to 70.5% of control levels. This decrease was restored to the level of control by withdrawing ethanol. These reversible changes in ISF and serum are readily explained by the known changes in the efflux activity of PTS-1 at the BBB.

Preproenkephalin targeted antisenses cross the blood–brain barrier to reduce brain methionine enkephalin levels and increase voluntary ethanol drinking

Antisense potentially can manipulate target gene expression in the brain if it can cross the blood–brain barrier (BBB). We designed three (10mer, 17mer, and 19mer) phosphorothioated antisenses (PS-ODNs) directed against the precursor molecule of methionine enkephalin (Met-Enk), an opiate peptide which suppresses voluntary ethanol drinking. We measured the ability of the antisenses to cross the BBB, accumulate in the brain and CSF, decrease levels of Met-Enk in brain and blood, and affect voluntary ethanol drinking. Each antisense readily crossed the BBB, with 0.07–0.16% of the i.v. dose accumulating per gram of brain. Capillary depletion and CSF sampling each confirmed that the antisenses entered the CNS. Gel electrophoresis of radioactivity recovered from brain and serum showed intact antisense and a higher molecular weight form likely representing antisense bound to protein, but no degradation products. Each antisense molecule and a cocktail of all three reduced Met-Enk levels in brain and serum. Met-Enk levels in the brain were reduced more rapidly and for a longer duration than Met-Enk levels in the serum, indicating a degree of selective targeting to the CNS. Additionally, administration of the cocktail was more effective in reducing Met-Enk levels than any of the individual antisenses. Each antisense increased voluntary ethanol drinking by about 20% and the cocktail increased it by about 80%. Taken together, these results used pharmacokinetic, immunochemical, and behavioral methods to show that PS-ODN antisenses that readily cross the BBB can decrease brain levels of Met-Enk and increase voluntary ethanol drinking.

Influence of Ethanol Dependence and Methionine Enkephalin Antisense on Serum Endomorphin‐1 and Methionine Enkephalin Levels

Opiate peptides are involved in the physical dependence on ethanol. Levels of methionine enkephalin (MEnk), for example, are affected by ethanol. No study on the effect of ethanol on endomorphin, the endogenous ligand for the mu-opiate receptor, has yet been conducted. We examined the effect of ethanol ingestion on serum endomorphin (EM)-1 and MEnk levels. We also determined the effect of antisense directed at MEnk on serum levels of EM-1 and MEnk. Serum EM-1 levels steadily decreased about 20% during 56 days of ethanol ingestion in liquid feed, whereas a similar decrease in serum MEnk levels was not statistically significant. Serum MEnk levels decreased about 20% by 48 hr after antisense injection and then returned to baseline, whereas serum EM-1 levels increased by about 80% and remained elevated for about 2 weeks. In mice not treated with antisense or alcohol, there was no correlation between the serum levels of EM-1 and MEnk. These results show that serum levels of EM-1 are decreased by physical dependence on ethanol and that this effect is not directly mediated through MEnk.

Effects of chronic ethanol on brain and serum level of methionine enkephalin

Most evidence agrees that levels of methionine enkephalin (Met-Enk) in brain are inversely correlated with ethanol drinking and withdrawal seizures. One area of discrepancy is the effect of chronic ethanol administration on the level of immunoactive Met-Enk in brain, with some authors reporting increased and others reporting decreased levels. These reports differed greatly in terms of method of ethanol administration, species used, length of time ethanol was administered, and the region of brain examined. We found that all studies could be resolved by considering only length of time ethanol was administered, with Met-Enk levels first increasing and then decreasing. We tested this finding by determining the effect of 4–56 days of ethanol delivered in liquid feed on levels of brain Met-Enk. We found that brain levels of Met-Enk peaked after 7 days of ethanol ingestion and declined to levels lower than control by 28 days. Exposure to ethanol abolished a correlation between brain and serum levels of Met-Enk which occurred in controls. HPLC showed that whereas 100% of immunoactivity eluted in the position of Met-Enk in controls, only about 50% eluted as Met-Enk in mice exposed to ethanol. These results support the hypothesis that exposure to ethanol alters brain Met-Enk in a way consistent with the reinforcement of physical dependence.

Corrigenda

CORRIGENDACorrigendaPublished Online:01 Apr 1998https://doi.org/10.1152/jappl.1998.84.4.1/aOriginal articleMoreSectionsPDF (14 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Volume 83, August 1997 Page 522, Abstract: In line 20 of the Abstract, “arterial Po 2” was mistakenly printed instead of “arterial Pco 2.” The corrected Abstract is reprinted below. Waters, Karen A., André Laferrière, Julie Paquette, Cynthia Goodyer, and Immanuela R. Moss. Curtailed respiration by repeated vs. isolated hypoxia in maturing piglets is unrelated to NTS ME or SP levels. J. Appl. Physiol. 83(2): 522–529, 1997.—In early development, respiratory disorders can produce recurring hypoxic episodes during sleep. To examine possible effects of daily repeated vs. isolated hypoxic hypoxia, cardiorespiratory functions and central, respiratory-related neuromodulator levels in 21- to 32-day-old, chronically instrumented, unsedated piglets were compared between a fifth sequential daily hypoxia and an isolated hypoxia (10% O2-90% N2 for 30 min). Diaphragmatic electromyographic activity, heart rate and arterial pressure, and pH and gas tensions were measured. In vivo microdialysis, via chronically implanted guides, served to sample interstitial substance P (SP) and methionine-enkephalin (ME) at the level of the respiratory-related nucleus tractus solitarii (NTS). Compared with an isolated hypoxia, repeated hypoxia resulted in 1) lower respiratory frequency (f), ventilation equivalent, and arterial pH, higher arterial Pco 2 during hypoxia, and lower f in recovery from hypoxia; and 2) increased SP concentrations but no change in ME concentrations. We conclude that, in these maturing swine, repeated vs. isolated hypoxic exposure curtails respiratory responses to hypoxia by a mechanism(s) unrelated to SP or ME levels at the NTS.This article has no references to display. Download PDF Back to Top Next FiguresReferencesRelatedInformationRelated articlesCurtailed respiration by repeated vs. isolated hypoxia in maturing piglets is unrelated to NTS ME or SP levels 01 Aug 1997Journal of Applied Physiology More from this issue > Volume 84Issue 4April 1998Pages 1a-1a Copyright & PermissionsCopyright © 1998 the American Physiological Societyhttps://doi.org/10.1152/jappl.1998.84.4.1/aHistory Published online 1 April 1998 Published in print 1 April 1998 Metrics Downloaded 56 times

Curtailed respiration by repeated vs. isolated hypoxia in maturing piglets is unrelated to NTS ME or SP levels.

In early development, respiratory disorders can produce recurring hypoxic episodes during sleep. To examine possible effects of daily repeated vs. isolated hypoxic hypoxia, cardiorespiratory functions and central, respiratory-related neuromodulator levels in 21- to 32-day-old, chronically instrumented, unsedated piglets were compared between a fifth sequential daily hypoxia and an isolated hypoxia (10% O2-90% N2 for 30 min). Diaphragmatic electromyographic activity, heart rate and arterial pressure, and pH and gas tensions were measured. In vivo microdialysis, via chronically implanted guides, served to sample interstitial substance P (SP) and methionine-enkephalin (ME) at the level of the respiratory-related nucleus tractus solitarii (NTS). Compared with an isolated hypoxia, repeated hypoxia resulted in 1) lower respiratory frequency (f), ventilation equivalent, and arterial pH, higher arterial PO2 during hypoxia, and lower f in recovery from hypoxia; and 2) increased SP concentrations but no change in ME concentrations. We conclude that, in these maturing swine, repeated vs. isolated hypoxic exposure curtails respiratory responses to hypoxia by a mechanism(s) unrelated to SP or ME levels at the NTS.

Effect of low and high doses of nitrous oxide on preproenkephalin mRNA and its peptide methionine enkephalin levels in the hypothalamus

Effect of low and high doses of nitrous oxide on preproenkephalin mRNA and its peptide methionine enkephalin levels in the hypothalamus

Effect of low and high doses of nitrous oxide on preproenkephalin mRNA and its peptide methionine enkephalin levels in the hypothalamus

The effect of exposure to nitrous oxide (N 2O) on the levels of preproenkephalin mRNA in the hypothalamus of rats was examined. In the first experiment, rats were exposed to 1000 ppm N 2O for 8 h a day over 4 days. Compared with controls (which were exposed to air over the same duration), the N 2O exposed animals exhibited significant elevations in preproenkephalin mRNA levels in the hypothalamus. In a second experiment, rats were exposed to 60% N 2O or air for 12, 24 and 48 h duration, and hypothalamic levels of preproenkephalin mRNA as well as methionine enkephalin were analyzed. Compared with controls, N 2O exposed rats exhibited significant elevations in preproenkephalin mRNA levels. The levels on preproenkephalin mRNA were significantly higher after 48 h of N 2O exposure than after 12 h of N 2O exposure. Similarly, the concentration of methionine enkephalin was significantly higher after 24 and 48 h of exposure to N 2O than after exposure to 12 h of N 2O or air. These results indicate that (a) exposure to N 2O results in significant elevations in preproenkephalin mRNA levels, (b) the increased preproenkephalin mRNA levels appear to be proportional to the concentration of N 2O exposure as well as the duration of N 2O exposure, and (c) N 2O-induced elevation in preproenkephalin mRNA levels is associated with corresponding increase in tissue concentrations of methionine enkephalin. In total, these results suggest that N 2O selectively stimulates synthesis of methionine enkephalin in the diencephalic region of the brain.

Changes in methionine-enkephalin levels in specific rat brain regions following repeated treatment with selective dopaminergic agonists and antagonists

The present study was conducted to investigate the effects of repeated treatment with selective dopaminergic agents on the level of methionine-enkephalin (Met-Enk) in rat brain cortex (CTX), hypothalamus, (HYPO), hippocampus (HIPP) and midbrain (MID). Male Sprague-Dawley rats were kept under controlled conditions for at least one week. After adaptation period, rats were randomly assigned into nine groups (7–9 rats per group) for intraperitoneal treatment with dopaminergic agents. Group 1 served as control, while, groups 2, 3 and 4 were treated with either SKF-81297, SCH 23390 or their combination, respectively. Groups 5, 6 and 7 received either LY 171555, (-)-sulpiride or their combination, whereas, groups 8 and 9 were treated with nomifensine or selegiline, respectively. One hour after the last injection, rats were sacrificed, brains were removed and dissected into different regions, then extracted and their Met-Enk levels determined by radioimmunoassay (RIA). Administration of SKF-81297 or SCH 23390 significantly elevated Met-Enk levels in all brain regions examined, while their combination elevated Met-Enk levels in HYPO and HIPP only. On the other hand, treatment with LY 171555 or (-)-sulpiride, but not their combination, markedly increased Met-Enk content in all brain regions examined. Treatment with nomifensine increased Met-Enk levels in all brain regions investigated, whereas, treatment with selegiline significantly elevated Met-Enk in HYPO, HIPP and MID but not in CTX. These findings clearly indicate that dopaminergic agonists and antagonists alter Met-Enk levels in specific rat brain regions.

Opioid and tachykinin neuropeptides in prolactin-secreting human pituitary adenomas.

Two opioid neuropeptides, methionine enkephalin (ME) and beta-endorphin (BE), and one tachykinin neuropeptide, substance P (SP), were quantified in 10 prolactin (PRL)-secreting human pituitary adenomas and in 10 control human pituitaries. Immunohistochemical techniques provided appropriate staining for PRL. Reversed-phase high performance liquid chromatography (RP-HPLC) was used to purify these three neuropeptides before their analysis, radioimmunoassay (RIA) was used for the quantification of SP-like immunoreactivity (SP-LI), and liquid secondary-ion mass spectrometry (LSIMS) was used for the qualitative and quantitative analysis of ME and a tryptic peptide of BE. This study shows that, for 90% of the cases studied here (excluding one hypothyroidism case), the tachykinin A neuropeptide SP-LI level is decreased, the POMC peptide BE level is not altered, and the proenkephalin A neuropeptide ME level is increased in these PRL-secreting tumors.

Neuropeptide levels early after trauma: immunomodulatory effects?

The levels of endogenous opioids, beta-endorphin and methionine-enkephalin, were analyzed in 21 severely traumatized patients (ISS 32, mortality 42.8%) from a first blood sample drawn at the scene of the injury before resuscitation within 32 +/- 16 minutes after the injury and for 8 days after trauma. Additionally, the respiratory burst function of polymorphonuclear neutrophils (PMNs) was assessed and the results were compared with those obtained from 5 healthy control patients undergoing elective surgery with the same analgesic regimen as the multiple trauma patients. Compared with elective surgery anesthesia (controls 3.3, surgery 3.2 fmol/L), the beta-endorphin levels on-scene were markedly elevated (survivors 10.1 fmol/L, non-survivors 15.0 fmol/L) (p < 0.05). Methionine-enkephalin levels after trauma were not different from those of the controls. The stimulation of PMNs with different concentrations of the opioids at the first day after trauma gave results comparable with those of the controls. On the third day after trauma the reactivity of PMNs to low opioid concentrations was markedly suppressed to 79.6% of the baseline value (p < 0.05). Endogenous opioids seem to be able to modulate the nonspecific immune-response after trauma.

Alterations in Hypothalamic μ–Opiate Receptor—Mediated Responses But Not Methionine Enkephalin or Proenkephalin Messenger Rna Levels in Rats With Acute Cholestasis

Endogenous opioids, including methionine enkephalin, have been implicated in the control of adrenocorticotrophic hormone release by acting through mu-opiate receptors in the hypothalamus. Recently, alterations in the central opioid system have been postulated to occur in cholestasis. In addition, alterations in hypothalamic corticotropin-releasing hormone content and messenger RNA levels, as well as basal release, have been described in bile duct-resected rats, and hypothalamic methionine enkephalin is colocalized with corticotropin-releasing hormone in hypothalamic neurons. Therefore hypothalamic and pituitary methionine enkephalin content and hypothalamic proenkephalin messenger RNA levels, as well as hypothalamic mu-opiate receptor-mediated responses in vitro and in vivo, were studied in rats with acute cholestasis caused by bile duct resection and in respective controls. Hypothalamic and pituitary methionine enkephalin levels were similar in bile duct-resected, sham-resected and unoperated control rats. In addition, hypothalamic proenkephalin steady state messenger RNA levels were similar in the three groups of animals. mu-Opiate receptor stimulation of hypothalamic explants in vitro with the specific mu-opiate receptor agonist ligand [D-Ala2,N-Me-Phe4,Gly-ol]-Enkephalin resulted in 8.2% and 16.9% inhibition of corticotropin-releasing hormone release in sham-resected and unoperated control rats, respectively. In contrast, treatment of hypothalamic explants from bile duct-resected rats with [D-Ala2,N-Me-Phe4,Gly-ol]-Enkephalin resulted in a significant 22.5% increase in corticotropin-releasing hormone release. Systemic administration of the mu-opiate receptor agonist morphine to rats in vivo resulted in significantly higher incremental rises in plasma adrenocorticotropic hormone levels in sham-resected and unoperated control animals than in bile duct-resected rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Methionine enkephalin concentration and enkephalin-degrading activity are elevated in blood in children with cholestasis

Objective To examine the concentration and degradation of methionine enkephalin in children with cholestasis of differing aetiologies. Patients The study included 41 children aged from 3 months to 14 years, with Alagille syndrome, Byler's disease, extrahepatic biliary duct atresia, or idiopathic cholestasis. Methods Plasma methionine enkephalin was measured using a specific radioimmunoassay. Liver extracts were prepared from biopsy homogenates. Enkephalin-degrading activity was evaluated by an aluminium silicate binding assay for quantification of the degradation of ‘short’ radiolabelled peptide hormones, and with high-performance liquid chromatography. Results The patients showed statistically significant (P<0.005) elevation of blood enkephalin concentrations (mean ± SD): 190±195 versus 50±35 in healthy controls and 60±15pg/ml in disease controls. Serum enkephalin-degrading activity was also markedly elevated: 1149±269 (Alagille syndrome), 400±146 (Byler's disease), 1038±616 (extrahepatic biliary duct atresia), 549±180 (idiopathic cholestasis) versus 234±44 (disease controls), and 290±24 pg/min/ml (healthy controls; P<0.05). Enkephalin-degrading activity in the liver extracts was virtually the same in patients with extrahepatic biliary duct atresia and chronic active hepatitis: 42±18 versus 36±14pg/min/mg, respectively (P>0.05). Endogenous methionine enkephalin was stable in plasma extracted in EDTA but it was degraded in the serum samples. The inhibitory and substrate specificity data suggest that aminopeptidase-M is the major enkephalin-degrading enzyme in both the serum and liver extracts, and that its activity is elevated in children with cholestasis. Conclusion Children with cholestasis of differing aetiologies exhibit significant elevation of both circulating methionine enkephalin levels and activity of serum aminopeptidase-M. Hepatic enkephalin degradation was retained in patients with extrahepatic biliary duct atresia. The physiological and pathophysiological significance of these observations requires further study.

Characterization of methionine-enkephalin release in the rat striatum by in vivo dialysis: Effects of gamma-hydroxybutyrate on cellular and extracellular methionine-enkephalin levels

The opioïd system is implicated in mediating the effects produced upon administration of gamma-hydroxybutyrate. Gamma-hydroxybutyrate occurs endogenously in the mammalian brain, and is most probably involved in the regulation of some basic brain functions, particularly those concerning the dopaminergic nigrostriatal pathway, which is closely linked to the expression of enkephalins in the striatum. In the present study, in vivo microdialysis was used to examine the basic characteristics of methionine-enkephalin (met-enkephalin) release in the striatum of Wistar rats, using a high performance radioimmunoassay. Administration of gamma-hydroxybutyrate to the rats induced a dose-dependent decrease in the extracellular release of met-enkephalin. In parallel, a dose- and time-dependent gamma-hydroxybutyrate-induced accumulation of met-enkephalin in striatum was observed. These two phenomena (tissue accumulation and inhibition of release) were blocked by NCS-382, a gamma-hydroxybutyrate receptor antagonist. The striatal met-enkephalin accumulation does not seem to be exclusively due to the inhibition of its release. Thus, a gamma-hydroxybutyrate mediating effect on met-enkephalin synthesis is suggested, most probably occurring via functional modulation of striatal dopamine synthesis and release. To understand the role of this dopaminergic mechanism, unilateral lesions of the nigrostriatal dopaminergic pathway were carried out. In gamma-hydroxybutyrate-treated rats, striata exhibited a similar increase in met-enkephalin content. In untreated rats, only the lesioned striatum showed an identical increase in met-enkephalin levels. Thus, striatal met-enkephalin accumulation could be attributed to the suppression of the dopaminergic impulse flow, due to gamma-hydroxybutyrate or to the action of 6-hydroxydopamine. In the extracellular spaces (microdialysis experiments), gamma-hydroxybutyrate administration induced identical modifications of met-enkephalin release in lesioned or non-lesioned striata. These modifications could be reproduced by peripheral or striatal administration of sulpiride, a D 2/D 3 antagonist. From a functional point of view, the dopaminergic D 2 receptor blockade or the gamma-hydroxybutyrate-induced inhibition of dopamine release could be considered to induce similar results, with identical consequences on striatal met-enkephalin accumulation and release. These results suggest that gamma-hydroxybutyrate-induced modifications in metenkephalin release, presumably potentiated by 6-hydroxydopamine treatment, act via a functional modification of the nigrostriatal dopaminergic pathway.

Methionine-enkephalin concentrations in discrete brain regions, spinal cord, pituitary gland and peripheral tissues of U-50,488H-tolerant and abstinent rats.

Effects were determined of chronic administration and withdrawal of a highly selective kappa-opioid receptor agonist, U-50,488H, on methionine-enkephalin levels in central and peripheral tissues of male Sprague-Dawley rats. Rats were rendered tolerant to and physically dependent on U-50,488H by twice daily injections of 25 mg/kg of this compound for 5 days. Rats deemed abstinent were injected with this drug for 4 days and sacrificed on 5th day. Methionine-enkephalin concentration increased in the hippocampus of U-50,488H-tolerant-dependent rats, whereas in abstinent rats, its level was elevated only in the hypothalamus. Levels of methionine-enkephalin in the pituitary gland of U-50,488H-tolerant-dependent or abstinent rats were unchanged. Among peripheral tissues, methionine-enkephalin concentration decreased in the adrenal gland of U-50,488H-tolerant-dependent rats. In the U-50,488H-abstinent rats, methionine-enkephalin concentration was elevated in the heart. In tissues of morphine- and U-50,488H-tolerant-dependent and abstinent rats methionine-enkephalin concentrations were affected differentially, suggesting inherent differences in mu- and kappa-opiate-mediated tolerance-dependence and abstinence processes.

Deletions of the synenkephalin domain which do not alter cell-specific proteolytic processing or secretory targeting of human proenkephalin.

To identify signals that direct the proteolytic processing and regulated secretion of human proenkephalin (hPE), we have transfected the hPE gene or minigene constructs into pituitary tumor cells, either rat GH4Cl cells or mouse AtT-20 cells. Cells transfected with either the hPE gene or minigene contained similar levels of methionine-enkephalin (ME)-containing peptides and hPE mRNA. In the GH4Cl clones, ME was present predominantly in high-molecular-mass forms (5-25 kDa). In contrast, the AtT-20 clones contained almost exclusively free ME and low-molecular-mass forms (< 5 kDa), with very little high-molecular-mass species present. Thus, among pituitary cells, corticotroph-derived cells appear better equipped to process hPE than lactotroph-derived cells. Despite limited proteolytic processing, GH4Cl clones secreted large amounts of unprocessed (> 20 kDa) hPE into the medium, making up to 10% of endogenous rat prolactin secretion. Both precursor and processed forms of ME were cosecreted acutely (< 1 h) with rat prolactin, and release of both polypeptides was stimulated up to 12-fold by secretagogues. Thus, complete proteolytic processing was not required for accurate targeting of hPE to the regulated secretory pathway. When transfected with constructs bearing deletions of amino-terminal amino acids 2-43 or 2-67, i.e., part or nearly all of the synenkephalin moiety, GH4Cl cells handled the modified protein much like cells expressing the complete protein. They did not process the modified hPE extensively, but the protein was correctly targeted to the regulated secretory pathway. AtT-20 cells transfected with truncated hPE cDNA constructs expressed and processed the protein as efficiently as cells expressing unmodified hPE and expressed predominantly low-molecular-mass forms of ME. Therefore, the structural features required for correct targeting and processing are not present in the cysteine-rich amino-terminal third of the prohormone. It is interesting that the deletions did not include the SHLL peptide motif in synenkephalin, a motif that has been proposed as a sorting signal.