Met-enk Levels Research Articles

Prenatal ethanol exposure and enkephalinergic neurotransmission.

Endogenous opioids (enkephalins, endorphins and dynorphins) are small peptides that play a main role in pain perception and analgesia, as well as in alcohol (ethanol) reinforcement and reward. Alcohol reinforcement involves the ethanol-induced activation of the endogenous opioid system, a process that may augment the hedonic value and the reinforcing properties of the drug, which in turn increases substance consumption. Changes in opioidergic transmission may contribute to alcohol intoxication and to the neuroadaptive responses produced by the long-lasting exposure to ethanol. Opioidergic transmission may be altered by ethanol at distinct levels, including the expression of precursor mRNAs, biosynthesis and release of opioid peptides, as well as ligand binding to opioid receptors. In adult rats, β-endorphinergic and enkephalinergic transmission, through activation of mu and delta opioid receptors, mediate ethanol reinforcement and high alcohol drinking behavior. Prenatal ethanol exposure (PEE) selectively modifies Methionine-enkephalin (Met-enk) content in several brain regions of infant and adolescent rats, particularly those of the reward circuits. In preweanling rats, Met-enk content is decreased in the ventral tegmental area but is increased in the prefrontal cortex and the nucleus accumbens and other brain areas, as a consequence of a short and moderate ethanol exposure during late gestation. PEE also increases Met-enk levels in the prefrontal cortex and other brain regions of 30-day-old adolescent rats. These findings suggest that mesocorticolimbic enkephalins are essential in ethanol reinforcement in offspring, as previously reported in adult rats.

Prenatal ethanol exposure alters met-enkephalin expression in brain regions related with reinforcement: Possible mechanism for ethanol consumption in offspring

The endogenous opioid system is involved in ethanol reinforcement. Ethanol-induced changes in opioidergic transmission have been extensively studied in adult organisms. However, the impact of ethanol exposure at low or moderate doses during early ontogeny has been barely explored. We investigated the effect of prenatal ethanol exposure on alcohol intake and Methionine-enkephalin (Met-enk) content in rat offspring. Met-enk content was assessed in the ventral tegmental area [VTA], nucleus accumbens [NAcc], prefrontal cortex [PFC], substantia nigra [SN], caudate-putamen [CP], amygdala, hypothalamus and hippocampus. Pregnant rats were treated with ethanol (2g/kg) or water during GDs 17–20. At PDs 14 and 15, preweanlings were evaluated in an intake test (5% and 10% ethanol, or water). Met-enk content in brain regions of infants prenatally exposed to ethanol was quantitated by radioimmunoassay. Ethanol consumption was facilitated by prenatal experience with the drug, particularly in females. Met-enk content in mesocorticolimbic regions – PFC and NAcc – was increased as a consequence of prenatal exposure to ethanol. Conversely, Met-enk levels in the VTA were reduced by prenatal ethanol manipulation. Prenatal ethanol also increased peptide levels in the medial-posterior zone of the CP, and strongly augmented Met-enk content in the hippocampus and hypothalamus. These findings show that prenatal ethanol exposure stimulates consumption of the drug in infant rats, and induces selective changes in Met-enk levels in regions of the mesocorticolimbic and nigrostriatal systems, the hypothalamus and hippocampus. Our results support the role of mesocorticolimbic enkephalins in ethanol reinforcement in offspring, as has been reported in adults.

Effects of acute ethanol administration on methionine–enkephalin expression and release in regions of the rat brain

The dopaminergic mesolimbic system plays a key role in mediating the reinforcing properties of ethanol and other drugs of abuse. Ethanol reinforcement and high alcohol drinking behaviour have been suggested to involve the ethanol-induced activation of endogenous opioid systems. Ethanol may alter opioidergic transmission at different levels, including opioid peptide biosynthesis and release, as well as binding to opioid receptors. The aim of this work was to investigate the effects of different ethanol doses on methionine-enkephalin (Met-enk) release from the rat nucleus accumbens (NAcc). Ethanol effects were also studied on Met-enk content in the NAcc, prefrontal cortex (PFC) and caudate-putamen (CP). Met-enk release was studied by microdialysis in Wistar anesthetized rats and peptide concentrations were quantitated by radioimmunoassay. Ethanol was administered by intraperitoneal injection after a 2-h basal release period. Ethanol doses of 0.5, 1 and 2.5 g/kg induced a 2.7-, 4.9- and 3.4-fold increase in Met-enk release from the NAcc. However, ethanol responses followed different kinetics, with earliest effects observed with the highest ethanol dose. In comparison, a 2.5-fold increase in peptide release was produced by 100 mM KCl. Ethanol, at a dose of 2.5 g/kg, induced a significant 66.7% decrease in Met-enk content in the NAcc, as well as a 76.4% reduction in peptide levels in the CP. Lower ethanol doses did not alter Met-enk content in these regions. On the other hand, an ethanol dose of 0.5 g/kg produced a non-significant decrease in Met-enk levels in the PFC. Our results suggest that ethanol-induced changes in enkephalin expression and release in regions of the mesocorticolimbic and nigrostriatal pathways could be involved in ethanol central effects. Released enkephalins by ethanol may modulate the dopaminergic activity of mesolimbic neurons and play a critical role in ethanol reinforcement mechanisms.

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.

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.

Met-enkephalin levels during PTCA-induced myocardial ischemia

Met-enkephalin (Met-enk) has been demonstrated to modulate myocardial-ischemia mechanisms via the opioid receptors, but no studies are now available on Met-enk levels in the coronary circulation. In this experience Met-enk levels were evaluated in aortic root and in coronary sinus at baseline (T0), during PTCA induced transient ischemia (T1) and during reperfusion (T2). No significant differences were found at any time. Thus, it appears that there is no Met-enk extraction from the coronary circulation during provoked myocardial ischemia and no Met-enk release from the ischemic heart.

Plasma met-enkephalin levels in diabetic patients: Influence of autonomic neuropathy

The presence of opioid peptides within pancreatic islets in several animal species and in humans suggests that these peptides could play a role in pancreatic endocrine secretion, influencing glucose metabolism. We measured plasma met-enkephalin (met-Enk) levels in eight neuropathic (four with insulin-dependent diabetes mellitus [IDDM] and four with non—insulin-dependent diabetes mellitus [NIDDM]) and eight nonneuropathic (four IDDM and four NIDDM) diabetic patients to study met-Enk secretion in diabetic patients with asymptomatic autonomic neuropathy. Plasma met-Enk levels were significantly lower in neuropathic compared with nonneuropathic patients both in the IDDM group (28.7 ± 4.8 v 61.6 ± 4.1 pg/mL, P < .0025) and in the NIDDM group (26.5 ± 3.6 v 44.3 ± 4.6 pg/mL, P < .0125). This study suggests that the presence of neuropathy in diabetic patients, even if asymptomatic, is associated with a significant decrease of plasma met-Enk levels, thus contributing to a worsening of metabolic control under stress conditions.

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.

Striatal neuropeptide levels in Parkinson's disease patients

Substance P (SP), Met-enkephalin (Met-enk) and cholecystokinin-8-S (CCK-8-S) were measured by a combined HPLC/RIA method in the caudate nucleus and anterior putamen from controls and from Parkinson's disease (PD) patients. SP levels were reduced in caudate in PD, but unchanged in putamen. No differences in Met-enk content were found in parkinsonians compared to controls. However, a significant correlation between DA and Met-enk levels in caudate nucleus from PD was observed. The concentration of CCK-8-S was unaltered in caudate nucleus or putamen in PD. The decrease in caudate nucleus SP levels might be related to the decrease in nigral SP levels in PD, while the reduction in Met-enk levels appears to be a feature of a subgroup of parkinsonian patients.

Plasma met-enkephalin in type I diabetes

Physiological and pathological evidence suggests that opioid peptides may play a role in glucose homeostasis. We measured plasma levels of β-endorphin (β-END) and met-enkephalin (met-ENK) in 22 type I diabetic patients and 15 healthy women (control group). No differences were observed in plasma β-END levels, whereas plasma met-ENK levels were significantly higher (Student's t test, P < .005) in diabetics than in controls before (68 ± 3 pg/mL v 32 ± 7 pg/mL) and 1 hour after, a standard meal and administration of insulin therapy (81 ± 9 pg/mL v 32 ± 7 pg/mL). This is the first report of met-ENK levels in insulin-dependent diabetes mellitus (IDDM), and an impaired feedback of insulin/met-ENK is suggested.

Dexamethasone increases both catecholamines and methionine-enkephalin in cultured bovine adrenal chromaffin cells and human extramedullary pheochromocytoma cells

The effect of dexamethasone on dispersed cells in primary monolayer culture from bovine adrenal medulla and human extramedullary pheochromocytoma was examined by estimating the level of catecholamines (CAs) and Methionine-enkephalin (Met-enk) in the medium and cells. In cultured bovine adrenal chromaffin cells, dexamethasone caused significant increase in Met-enk levels 18 hours after administration. There was no release of Met-enk and CAs in the medium 10 min after administration, although nicotine did cause a significant release of Met-enk and CAs. A dose response increase in the level of CAs and Met-enk in bovine adrenal chromaffin cells was obtained with doses varying between 0 and 10 −6M dexamethasone 18 hours after administration. In cultured human extramedullary pheochromocytoma cells, dexamehtasone significantly increased the levels of norepinephrine and Met-enk in a dose dependent manner 24 hours after administration. These results suggest that dexamethasone does not act as a secretagogue but may be related to the synthesis of Met-enk and CAs.

Diurnal fluctuations in methionine-enkephalin levels in the hypothalamus and preoptic area of the male rat: effects of pinealectomy.

Methionine-enkephalin (met-enk) levels were measured at 09.30-10.30, 16.30-17.30 and 23.30-00.30 h in the anterior hypothalamus-preoptic area (AHPOA), medial basal hypothalamus (MBH), anterior, intermediate and posterior lobe of the pituitary gland of male rats. Met-enk levels in the AHPOA and MBH fluctuated significantly through a 24-hour period; from low levels at 09.30-10.30 h, met-enk in the AHPOA and MBH rose slowly in the afternoon to reach peak concentrations at 23.30-00.30 h. Pinealectomy appears to depress or shift the normally occurring increments in met-enk levels at 23.30-00.30 h in the AHPOA only. Serum testosterone (T) levels displayed an inverse relationship with the met-enk rhythm; high concentrations during the day were associated with low met-enk levels and as T levels decreased to a nadir at 23.30-00.30 h, the met-enk in the MBH and AHPOA attained peak concentrations. Daily changes in serum T levels were not affected by pinealectomy.