Met-enkephalin Concentrations Research Articles

Neurochemical substrates of rigidity and chorea in Huntington's disease.

Huntington's disease is a progressive degenerative neurological disorder which produces a characteristic movement disorder termed chorea. Although chorea is associated with dysfunction of the basal ganglia, the underlying mechanisms by which dyskinesias such as chorea are produced, are poorly understood. Recent studies in primates have led to experimental models of chorea with postulated involvement of specific neural pathways. In the present study we attempted to determine the validity of the experimental models by measuring concentrations of gamma-aminobutyric acid (GABA), glutamate, substance P and met-enkephalin in the basal ganglia of Huntington's disease patients who manifested either chorea or rigidity/bradykinesia within 6 months of death. We also characterized changes in the Huntington's disease patients according to pathological grade, since this may be a confounding factor. We analysed post-mortem brain tissue from 12 controls, and 11 grade 3 and 12 grade 4 Huntington's disease patients. The grade 3 and 4 cases consisted of eight adult-onset choreic, nine adult-onset rigid and six juvenile-onset rigid patients. We also analysed the putamen and globus pallidus from 11 grade 2 adult onset choreic Huntington's disease patients. A model of chorea based on experimental studies in primates proposes that a loss of striatal GABAergic inhibitory projections to the globus pallidus externa leads to increased activity of the inhibitory globus pallidus externa GABAergic neurons which project to the subthalamic nucleus. It is believed that the loss of GABAergic inputs to the globus pallidus externa precedes a loss of GABAergic input to the globus pallidus interna, which occurs later in the disease and is associated with the development of rigidity and bradykinesia. In the choreic Huntington's disease patients whom we studied, there was a greater loss of GABA in the globus pallidus externa than in the globus pallidus interna, and the globus pallidus interna: globus pallidus externa GABA ratio was significantly increased compared with rigid patients. There were also increases in GABA in the subthalamic nucleus in the choreic patients, although this did not reach significance. A differential loss of met-enkephalin in the globus pallidus externa compared with substance P loss in the globus pallidus interna was not observed in either the choreic patients with advanced disease or the grade II patients. There was a significant increase in GABA concentrations in the ventroanterior nucleus of the thalamus in the choreic patients compared with rigid/bradykinetic patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Perinatal morphine exposure alters peptidergic development in the striatum

It has been reported that perinatal exposure to opiates affects mRNA synthesis, body growth and brain development in mammals, including humans. We have observed that morphine administration in drinking water during the perinatal period alters peptide development in the striatum of the rat. There is a marked increase in substance P and met-enkephalin content, the latter is maintained even at 30 days postnatally. The transient increase or earlier maturation of substance P content is correlated by a more precocious axon terminal organization as revealed by immunocytochemical staining. The increased metenkephalin content is correlated by a higher abundance of preproenkephalin A mRNA and this correlation is particularly evident at 15 days postnatally. At earlier times both northern blotting and in situ hybridization techniques fail to show any significant difference between control and morphine exposed rats, likely because the peptide content is not very different in the two groups or at least the gap is not as wide as at later times.

Regulation of NK cell activity and the level of the intracellular cAMP in human peripheral blood lymphocytes by Met-enkephalin.

The effect of Met-enkephalin on natural killer (NK) activity and on intracellular cyclic adenosine monophosphate (cAMP) level in human peripheral blood lymphocytes was measured 1/4, 1, 2, and 24 h after incubation. Concentrations of Met-enkephalin ranged from 10(-12) to 10(-8) M. Cyclic changes in NK activity and in the intracellular cAMP level after treatment with Met-enkephalin were observed. Kinetics of changes caused by high (10(-9), 10(-8) M) and low concentrations (10(-12), 10(-11), 10(-10) M) of Met-enkephalin differed from each other. Early, nearly threefold increase in the level of intracellular cAMP was found 15 min after treating peripheral blood lymphocytes with 10(-12) M Met-enkephalin. By contrast, a nearly 75% decrease of intracellular cAMP level was found 2 h after treatment with 10(-9) M Met-enkephalin. Generally, early intensive changes in the cAMP level in peripheral blood lymphocytes, induced by Met-enkephalin, were followed by delayed, subtle changes in NK cell activity in the opposite direction.

Antisense proenkephalin cDNA transfection decreases opioid binding in NG 108-15 cells

The proenkephalin A (PENK) gene codes for several opioid peptides, including Met-enkephalin, an endogenous ligand of opioid receptors. These peptides are thought to play an important role in a variety of neural processes. To study the role of the PENK gene in opioid related processes, an antisense sequence of PENK gene was subcloned into pSVL SV 40 late promoter expression vector and stably transfected into NG 108-15 cells, which contain opioid receptors. The sense orientation of the same fragment was also cloned and transfected, serving as a control. The presence and expression of transfected recombinant plasmids in NG 108 cells were confirmed by DNA and RNA PCR and by subsequent sequencing. Surprisingly, the endogeneous PENK message level was found to be 3 times higher in antisense cells than in sense or NG 108 cells. This high steady-state mRNA level seemed to be due to the increased stability of PENK mRNA rather than to an elevated transcription rate. Nevertheless, the level of total Met-enkephalin was found to be reduced in antisense-transfected cells, though free Met-enkephalin content did not differ from sense-transfected or non-transfected cells. We suggest that both the increased PENK message and the unchanged levels of free Met-enkephalin may be the result of compensatory mechanisms induced by translational inhibition by antisense, although the underlying processes remain to be determined. Binding of the opioid ligand [ 3H]diprenorphine was significantly reduced by 50–80% in the antisense-transfected cell lines, but not in the sense cells. In contrast, binding of the muscarinic ligand [ 3H]scopolamine, or α 2-adrenergic ligand [ 3H]rauwolscine were unchanged either in antisense or sense cells. Thus the presence of antisense PENK in NG 108-15 cells appeared to have a specific effect on opioid receptors, though this effect was not mediated through alterations in the receptor's endogenous ligand. While our results did not enable us to determine the effect of altering endogenous opioid ligand levels on opioid receptor disposition in NG 108-15 cells, we speculate that opioid receptors may be regulated by the level of PENK mRNA.

RAPID COMMUNICATION: PLASMA β-ENDORPHIN, BUT NOT MET-ENKEPHALIN LEVELS ARE ABNORMAL IN CHRONIC ALCOHOLICS

Plasma beta-endorphin, met-enkephalin and ACTH concentrations were measured in 20 male alcoholics (age: 32-60 yr; duration of ethanol addiction: 13.2 +/- 6.2 yr; mean +/- SE) immediately after admission to the hospital (at a time not exceeding 8 hr from the last ethanol consumption) and after 5 weeks of forced abstinence. The results were compared with those obtained in 12 age-matched normal controls. Plasma ACTH and met-enkephalin levels were normal in alcoholics on both occasions. In contrast, in samples taken at admission to the hospital, the circulating concentrations of beta-endorphin in alcoholics were half (17.1 +/- 5.3 pg/ml; mean +/- SE) of those observed in the normal controls (34.1 +/- 6.0). beta-endorphin levels rose significantly after 5 weeks of abstinence (30.1 +/- 4.9); at this time, they were not significantly different from those observed in normal controls. These data indicate that acute alcohol consumption induces significant alterations in plasma beta-endorphin, but not met-enkephalin levels, which are reversed after 5 weeks of abstinence.

Chronic MPTP treatment reduces substance P and met-enkephalin content in the basal ganglia of the marmoset

Common marmosets were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 1.25–2.5 mg/kg s.c., twice a week) for 5–10 consecutive months. The initial doses of MPTP produced a severe parkinsonian syndrome but motor activity was partially recovered at the end of treatment. Fifteen days or 6 months after the last MPTP dose, monkeys were sacrificed. In addition to a strong decrease of dopamine in the striatum, there were significant reductions in substance P and Met-enkephalin content in the substantia nigra, caudate nucleus and putamen. In the globus pallidus, the reduction in peptide levels did not reach statistical significance as compared to controls. Neurotensin levels were also decreased in the caudate nucleus. The chronic administration of MPTP for 5–10 months induces changes in substance P and Met-enkephalin systems which resemble the degeneration found in brains from parkinsonian patients.

The neurotoxic actions of ibotenic acid on cholinergic and opioid peptidergic systems in the central nervous system of the rat.

The neurotoxic effects produced by ibotenic acid (IA) induced chemical lesions of the central nervous system (CNS) cholinergic system were examined on the opioid peptidergic system in adult rats. Forebrain cholinergic systems were bilaterally lesioned by the infusion of IA (1 or 5 micrograms/site) into the nucleus basalis magnocellularis (NBM). One week after the injections, the animals were sacrificed, and activities of acetylcholinesterase (AChE), choline acetyltransferase (ChAT) and concentrations of beta-endorphin (beta-End) and Met-enkephalin (Met-Enk) were measured in different brain regions. Animals treated with IA showed a decrease in the activity of ChAT (-24%), AChE (-36%) and beta-End level (-33%) in the frontoparietal cortex (FC). For the first time we report that these changes were associated with a compensatory increase in the activity of ChAT (+27%), AChE (+25%), beta-End level (+66%) in the remaining part of the cortex, i.e. cortex devoid of frontal cortex (C-FC). Met-enkephalin level increased by 59% in the frontoparietal cortex and did not change in the cortex devoid of frontal cortex upon IA treatment. These results suggest that IA treatment results in changes in the activity of cortical ChAT and AChE, and beta-End level in the same direction. Injection of IA in the NBM did not cause a change in the activity of ChAT or AChE in other brain regions such as hippocampus, striatum or midbrain. In addition to cortex devoid of frontal cortex, midbrain also showed a significant increase in the beta-End level in the IA treated animals. However, pituitary beta-End decreased in the neurotoxin treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

β-Endorphin and met-enkephalin in cattle during late gestation and parturition — release from uteroplacental tissues

Plasma concentrations of β-endorphin and met-enkephalin during term and pre-term caesarean sections, and the release of these peptides by bovine uteroplacental tissues, were investigated. Blood samples were taken from the jugular vein and from a branch of the uterine vein. A release of metenkephalin into the maternal circulation by the uterus or placenta could be demonstrated.

Stress-induced changes in brain Met-enkephalin, Leu-enkephalin and dynorphin concentrations

Methionine-enkephalin (Met-enkephalin), leucine-enkephalin (Leu-enkephalin) and dynorphin A (1–17) (dynorphin A) concentrations in discrete brain areas were determined in the mice showing behavioral changes induced by stress using radioimmunoassay (RIA). In the present experiment, we used environment-induced conditioned suppression of motility and forced swimming-induced immobility. In the environment-induced conditioned suppression of motility, Met-enkephalin concentration in the striatum and hypothalamus significantly decreased. Leu-enkephalin concentration in the hypothalamus also decreased. Dynorphin A concentraion in the striatum decreased, but significantly increased in the hypothalamus and pituitary. In the forced swimming-induced immobility, Met-enkephalin concentration in the striatum significantly decreased. Leu-enkephalin concentration in the hypothalamus and pituitary significantly decreased. Dynorphin A concentration in the pituitary decreased, but significantly increased in the hypothalamus. Our results indicated that the concentrations of Met-enkephalin, Leu-enkephalin and dynorphin A in the discrete brain areas changed in two different stressful situations. These findings suggested that these peptides might modulate the behavioral changes induced by stressors.

Effect of diabetes on the levels of two forms of Met-enkephalin in plasma and peripheral tissues of the rat

Levels of native and cryptic or peptidase-derivable (after being digested with trypsin and carboxypeptidase) Met-enkephalin were measured by a specific radioimmunoassay method in plasma, anterior and neurointermediate lobes of pituitary and various peripheral tissues of streptozotocin (STZ) diabetic rats. The results show that the highest concentration of native and cryptic Met-enkephalin were found in the neurointermediate lobe of pituitary. Streptozotocine-induced diabetes alters the concentration of either or both forms of Met-enkephalin in plasma, the anterior and neurointermediate lobes of the pituitary, heart, lung, spleen, liver, seminal vesicle, vas deferens, kidney, bladder detrusor, and duodenum. One of the most pronounced effects of diabetes observed in this study is seen in the seminal vesicles where native Met-enkephalin was depleted to less than 10% of the control value. The uneven distribution of Met-enkephalin in peripheral tissues may suggest that these tissues process and/or metabolize Met-enkephalin to different degrees. Our data also suggest that STZ-induced diabetes alters the enkephalinergic activity in some of these tissues. It is suggested that some of the peripheral pathophysiological symptoms associated with diabetes may be attributed, in part, to altered activity of enkephalinergic systems.

Chronic intrathecal cannulation affects hypothalamic opioids depending on the technique employed

Previous studies have shown that chronic intrathecal cannulation can interfere with the homeostasis of central opioid peptides. These results show that beta-endorphin and Met-enkephalin concentrations did not change in the hypothalamus of rats bearing a chronic cannula inserted between C 8-T 1 up to L 3 and fixed to the processus transversus T 1. These results suggest that chronically cannulated rats can be considered as normal when studying hypothalamic beta-endorphin or Met-enkephalin concentrations, and used in physiological studies, depending on the technique employed.

CD10 (CALLA)/neutral endopeptidase 24.11 modulates inflammatory peptide- induced changes in neutrophil morphology, migration, and adhesion proteins and is itself regulated by neutrophil activation

The common acute lymphoblastic leukemia antigen (CALLA, CD10), which is expressed on early lymphoid progenitors and neutrophils, is the zinc metalloprotease, neutral endopeptidase 24.11 (NEP, “enkephalinase”). The CD10 cell surface enzyme is known to hydrolyze a variety of biologically active peptides including met-enkephalin, formyl-met-leu- phe (f-MLP), and substance P. These three CD10/NEP substrates induce the migration and aggregation of neutrophils, suggesting that each of the peptides can function as a mediator of neutrophil inflammatory responses. Recently, inhibition of CD10/NEP was found to reduce the concentration of metenkephalin needed to activate human and invertebrate granulocytes by several orders of magnitude. Herein we show that f-MLP and substance P induce rapid changes in neutrophil morphology, migration, and adhesion molecule expression, including upregulation of Mo1 (CD11b/CD18) and shedding of LAM-1 (also known as LECAM-1, Leu8, or TQ-1, the human homologue of murine gp100MEL14). Importantly, these coordinated changes are potentiated by inhibition of cell surface CD10/NEP enzymatic activity. Neutrophil cell surface CD10/NEP enzymatic activity is also shown to be regulated by the activation state of the cell during the time period in which the enzyme has its most pronounced effects. These results suggest that in neutrophils, CD10/NEP functions to control responsiveness to multiple inflammatory peptides.

CD10 (CALLA)/ Neutral Endopeptidase 24.11 Modulates Inflammatory Peptide-Induced Changes in Neutrophil Morphology, Migration, and Adhesion Proteins and Is Itself Regulated by Neutrophil Activation

AbstractThe common acute lymphoblastic leukemia antigen (CALLA, CD10), which is expressed on early lymphoid progenitors and neutrophils, is the zinc metalloprotease, neutral endopeptidase 24.11 (NEP, “enkephalinase”). The CD10 cell surface enzyme is known to hydrolyze a variety of biologically active peptides including metenkephalin, formyl-met-leu-phe (f-MLP), and substance P. These three CD10/NEP substrates induce the migration and aggregation of neutrophils, suggesting that each of the peptides can function as a mediator of neutrophil inflammatory responses. Recently, inhibition of CD10/NEP was found to reduce the concentration of metenkephalin needed to activate human and invertebrate granulocytes by several orders of magnitude. Herein we show that f-MLP and substance P induce rapid changes in neutrophil morphology, migration, and adhesion molecule expression, including upregulation of Mo1 (CD11b/CD18) and shedding of LAM-1 (also known as LECAM-1, Leu8, or TQ-1, the human homologue of murine gp100MEL14). Importantly, these coordinated changes are potentiated by inhibition of cell surface CD10/NEP enzymatic activity. Neutrophil cell surface CD10/NEP enzymatic activity is also shown to be regulated by the activation state of the cell during the time period in which the enzyme has its most pronounced effects. These results suggest that in neutrophils, CD10/NEP functions to control responsiveness to multiple inflammatory peptides.

Failure of Met-Enkephalin to Enhance Natural Killer Cell Activity

Several papers have reported the enhancing effects of opiate peptides, like Met-enkephalin, on natural killer (NK) cell activity. We examined the actions of Met-enkephalin on NK activity in blood obtained from 18 different donors, of different ages, many of them on several occasions, at several ratios of effector to target cells, several concentrations of peptide, in several types of flasks, with the purity and identity of the pentapeptide verified by chromatography, in a system responsive to interferon, and with results calculated in different ways. No significant increase was found for the peptide for any ratio of cells, any concentration of peptide, or any single subject, even when the subjects with the lowest baseline NK cell activity were used or when the subjects were more than 60 years old. Instead of an increase, the combined results for all subjects at all ratios at all concentrations of Met-enkephalin showed an overall decrease of 31.3 % specific cytotoxicity. These results fail to support the reports of an enhancing effect of Met-enkephalin on NK cell activity.

The regional distribution of thyrotropin releasing hormone, leu-enkephalin, met-enkephalin, substance P, somatostatin and cholecystokinin in the rat brain and pituitary

There was no apparent difference in the regional distribution of neuropeptides in the brain of male and female rats. The highest levels of immunoreactive leu-enkephalin, TRH, substance P and somatostatin were found in the hypothalamus, while the striatum and the cerebral cortex had the highest concentrations of met-enkephalin and cholecystokinin respectively. The lowest concentrations of these were found in the cerebellum. Enkephalins (cerebral cortex), substance P (cerebral cortex and brain stem), and somatostatin (brain stem and striatum) showed higher level in the female while enkephalin and substance P contents in the anterior pituitary were higher in the male.

EFFECT OF RESERPINE ON CATECHOLAMINE CONTENTS AND MET-ENKEPHALIN AND ?-ENDORPHIN LEVELS IN THE HYPOTHALAMUS AND THE PITUITARY

1. The effects of reserpine treatment on the contents of catecholamines and opioid peptides have been studied in the rat hypothalamus and pituitary. 2. Hypothalamic and pituitary catecholamines were drastically depleted following acute reserpine treatment. 3. Reserpine treatment also resulted in a significant decrease in immunoactive met-enkephalin content in both the hypothalamus (25%) and the anterior lobe (50%), but not in the neurointermediate lobe of the pituitary. 4. No changes were observed in immunoactive beta-endorphin levels. 5. These findings suggest that the met-enkephalin contents in the hypothalamus and the anterior pituitary may be under catecholaminergic control. 6. The lack of effect of acute reserpine treatment on immunoactive beta-endorphin contents might be due to the opposing effects of adrenergic and dopaminergic mechanisms.

Nicotine-induced alterations in peripheral tissue concentrations of native and cryptic Met- and Leu-enkephalin

This study examined the peripheral tissue distribution of native and cryptic Met- and Leu-enkephalin, and regulation of tissue enkephalins by nicotine. Met- and Leu-enkephalin concentrations showed widespread variation in tissue concentration and degree of processing. HPLC characterization of homogenate of spleen revealed that both native and cryptic immunoreactive Met-enkephalin are comprised of two peaks, one representing authentic Met-enkephalin pentapeptide and the other its sulfoxide. Subacute repeated administration of nicotine 0.1 mg/kg ip, six times at 30 min intervals, increased native Met- and Leu-enkephalin in adrenal medulla without affecting cryptic Met- and Leu-enkephalin concentrations, consistent with increased processing of larger peptides to met- and Leu-enkephalin. Subacute nicotine decreased splenic concentrations of native and crypic Met-enkephalin and native Leu-enkephalin, consistent with increased release of Met- and Leu-enkephalin from spleen and decreased synthesis of proenkephalin A or inadequate processing of larger peptides to enkephalin pentapeptides in spleen to compensate for the increased release during this period. HPLC characterization revealed that nicotine-induced decrease in native Met-enkephalin in spleen resulted from reductions in both pentapeptide and its sulfoxide. Nicotine also increased native Met-enkephalin in jejunum, decreased cryptic Met-enkephalin in heart atrium, increased native Leu-enkephalin in anterior pituitary and decreased cryptic Leu-enkephalin in jejunum. Nicotine may produce some of its effects through alterations in release of enkephalins from peripheral tissues.

Nicotinic- and muscarinic-evoked release of canine adrenal catecholamines and peptides

The tissue content and overflow of norepinephrine (NE), epinephrine (Epi), dopamine (DA), Met-enkephalin (Met-Enk), and neuropeptide Y (NPY) from isolated, retrogradely perfused dog adrenal glands were studied. Under resting conditions, approximately 25% of the overflow of autocoids from the glands was Ca2+ dependent; the cholinergic antagonists hexamethonium and atropine had no effects on basal efflux. Stimulation with the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP; 3 or 50 microM) or with the muscarinic agonist pilocarpine (50 microM or 1 mM) evoked releases of autocoids. These releases were blocked or dramatically reduced by appropriate antagonists or by the removal of Ca2+ from the perfusate. Expressed as percentages of tissue stores, the rank order of overflow of autocoids was E approximately DA much greater than NE during resting conditions, DA much greater than E approximately NE during stimulation with 50 microM DMPP, and DA greater than Epi greater than NE during stimulation with 1 mM pilocarpine. These data are consistent with different mechanisms of release for the catecholamines, perhaps from different cell populations. The data support corelease of peptides and catecholamines, although clear pairing of autocoids could not be confirmed.

Association between circadian rhythms of endogenous hypothalamic opioid peptides and of natural killer cell activity

To explore in man the hypothesis that natural killer cell activity and hypothalamic - hypophyseal hormones constitute a mutally coupled multioscillatory system, we analysed and compared, in 11 healthy volunteers, the circadian variations in plasma concentrations of beta-endorphin, met-enkephalin and alpha-MSH, and of natural killer activity of peripheral blood lymphocytes. Natural killer cell activity and plasmas beta-endorphin levels showed a similar circadian rhythm with the peak in the morning (acrophases at 06.14 and 08.25, respectively), whereas the circadian rhythm of met-enkephalin was approximately in antiphase to the natural killer rhythm (acrophase close to 17.00 hours). Although daily variation of alpha-MSH showed greater inter-individual variability, a circadian rhythm was statistically validated. Analysis of correlation between rhythmometric parameters (mesor,amplitude, peak and nadir) of natural killer cell activity vs neuro-endocrine hormones revealed that the minimum and medium daily concentrations of beta-endorphin correlated directly with the corresponding parameters of natural killer activity, while the maximum and medium concentrations of met-enkephalin were inversely correlated with the peak and the mesor of natural killer activity. The amplitude of natural killer cell activity oscillations correlated directly with the peak, mesor and nadir concentrations of alpha-MSH. We show here that circadian rhythms of some neuro-endocrine hormones of the hypothalamic-hypophyseal axis, i.e. beta-endorphin, met-enkephalin and alpha-MSH, are significantly coupled to daily oscillations of NK cell activity.

Endocrine Control of Hypothalamic and Pituitary Met-Enkephalin and Beta-Endorphin Contents

This review summarizes the recent findings on the effects of endocrine manipulation on the hypothalamic and pituitary contents of met-enkephalin and beta-endorphin. In the pituitary, gonadectomy decreases beta-endorphin content in both the anterior lobe and neuro-intermediate lobe. Orchidectomy results in a decrease while ovariectomy leads to an increase in anterior lobe met-enkephalin contents. Adrenalectomy only lead to an increase in beta-endorphin contents in the anterior pituitary lobe. Hypothyroidism induced by propylthiouracil treatment is accompanied by a decrease of beta-endorphin in the neuro-intermediate lobe and a decrease in met-enkephalin in the anterior lobe while thyroidectomy entails a decrease in met-enkephalin in the anterior lobe only. Chemically induced diabetes mellitus results in a decrease in beta-endorphin content in the hypothalamus and the neuro-intermediate lobe, and a reduction in met-enkephalin level in the anterior and neuro-intermediate lobes. All these changes are reversible with appropriate hormone treatments. These results indicate the importance of hormones in the regulation of the synthesis and/or release of the opioid peptides in the hypothalamus and the pituitary.