Synthesis Of Enkephalins Research Articles

Efficient Synthesis of Norbuprenorphines Coupled with Enkephalins and Investigation of Their Permeability.

An efficient approach for the synthesis of norbuprenorphin derivatives through coupling of enkephalins and norbuprenorphine intermediates is described. Norbuprenorphine derivative was synthesized from thebaine and then, its reaction with succinic acid and phthalic acid was also studied. Meanwhile, the synthesis of enkephalins was done using solid phase peptide synthesis approach. Furthermore, after cleavage of the peptide from the surface of the resin, the coupling of enkephalins with norbuprenorphine derivative was done using TBTU as a coupling reagent then the derivatives were purified using preparative high-pressure liquid chromatography and their structures were confirmed using high-resolution mass spectrometry data. Later, their permeability across membranes was investigated. After PAMPA studies, it was found that the permeability of all norbuprenorphin-enkephalin derivatives was increased; however, succinic and phthalic acid derivatives showed higher permeability than norbuprenorphine-Leu-enkephalin.

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.

Role of acupuncture in stress management

ABSTRACT: Stress factors impair physiological and psychological balance state of the individuals. This situation can cause various diseases. Today, diseases caused by stress are treated with methods like educational program for coping with stress, psychotherapy, drug therapy. Recently, acupuncture treatment is added to these methods. Acupuncture treatment effects to modulate various neurophysiologic mechanisms in during resistance period of stress. This mechanism provides support for the person’s psychological state. Acupuncture treatment increases the secretion of hormones such as endomorphine, beta endorphine, enkephalin, serotonin and cortisol from central nervous system. Stress factors increase endomorphin, beta endorphin, enkephalin, serotonin and oxytocin levels in the central nervous system and plasma in the resistance period to stress. Acupuncture applications increase the synthesis and release of endomorphin, beta endorphin, enkephalin, serotonin and oxytocin from the central nervous system which strengthens the individual’s resistance period to cope with stress. Therefore, method of acupuncture is also used in the treatment of diseases caused by stress. KEY WORDS: stress, acupuncture, beta endorphin, enkephalin, serotonin, oxytocin

Morphine-induced conditioned place preference in rats is inhibited by electroacupuncture at 2 Hz: Role of enkephalin in the nucleus accumbens

Our previous studies have demonstrated that morphine-induced conditioned place preference (CPP) can be inhibited by 2 Hz electroacupuncture (EA). This inhibition can be blocked by either the opioid receptor antagonist naloxone (i.p.) or lesion in the nucleus accumbens (NAc), providing evidence that endogenous opioid system in the NAc mediates the effects of EA. Here we report that 1) A single session of 2 Hz EA produced a significant increase of the content of enkephalin in the NAc of morphine-induced CPP rats, and this effect was stronger in three consecutive sessions of EA; 2) Intracerebroventricular injection of the μ-opioid receptor antagonist CTAP or δ-opioid receptor antagonist NTI, but not κ-opioid receptor antagonist nor-BNI, dose-dependently reversed the inhibitory effects of 2 Hz EA on the expression of morphine-induced CPP; 3) Three consecutive sessions of 2 Hz EA up-regulated the mRNA level of preproenkephalin in the NAc of morphine-induced CPP rats. The results suggest that the inhibitory effects of 2 Hz EA on the expression of the morphine CPP is mediated by μ- and δ-, but not κ-opioid receptor, possibly via accelerating both the release and synthesis of enkephalin in the NAc. These findings support the possibility of using 2 Hz EA for the treatment of opiate addiction.

Arginine vasopressin enhances periaqueductal gray synthesis and secretion of enkephalin and endorphin in the rat

Previous study has proven that arginine vasopressin (AVP) enhances periaqueductal gray (PAG) secreting enkephalin and endorphin in vivo in the rat. Present work investigated that AVP effect on PAG secretion and synthesis of enkephalin and endorphin in vitro. Radioimmunoassy results showed that AVP increased leucine-enkephalin (L-Ek), methionine-enkephalin (M-Ek), β-endorphin (β-Ep) rather than dynorphin A 1–13 (DynA 1–13) concentrations in PAG slice culture liquid, and V 2 receptor antagonist: d(CH 2) 5[D-Ile 2, Ile 4, Ala 9-NH 2]AVP decreased L-Ek, M-Ek and β-Ep, not DynA 1–13 concentrations in PAG slice culture liquid in a dose-dependent manner, but V 1 receptor antagonist: d(CH 2) 5Tyr(Me)AVP did not change these peptide concentrations in PAG slice culture liquid. RT-PCR data displayed that AVP enhanced proenkephalin and proopiomelanocortin (Pro-β-Ep) rather than prodynorphin mRNA expressions in culture PAG tissues, and V 2 receptor antagonist weakened proenkephalin and proopiomelanocortin (Pro-β-Ep), not prodynorphin mRNA expressions in culture PAG tissues, but V 1 receptor antagonist did not influence these mRNA expressions in culture PAG tissues. The data suggest that AVP enhances PAG synthesizing and secreting enkephalin and endorphin rather than dynorphin through V 2 receptors.

To the descending pain-control system in rats, inflammation-induced primary and secondary hyperalgesia are two different things*1

The periaqueductal gray matter and the rostral ventromedial medulla (RVM), with its projections to the spinal dorsal horn, constitute the efferent channel of the ‘descending pain-control system’. Noxious stimulation of a peripheral tissue causes more pain if this tissue is inflamed (primary hyperalgesia). In such cases, stimulation of neighboring but uninflamed tissues also becomes more painful (secondary hyperalgesia). In animal models of inflammation, the descending pain-control system sends down, simultaneously, inhibitory and facilitatory influences, but inhibition predominates for primary hyperalgesia while facilitation predominates for secondary hyperalgesia. Descending inhibition and facilitation during peripheral inflammation are due not only to previously existing descending modulation, but also to inflammation-induced changes in RVM which involve receptors for NMDA, AMPA, cholecystokinin and neurotensin, as well as synthesis of enkephalins and nitric oxide.

To the descending pain-control system in rats, inflammation-induced primary and secondary hyperalgesia are two different things

The periaqueductal gray matter and the rostral ventromedial medulla (RVM), with its projections to the spinal dorsal horn, constitute the efferent channel of the ‘descending pain-control system’. Noxious stimulation of a peripheral tissue causes more pain if this tissue is inflamed (primary hyperalgesia). In such cases, stimulation of neighboring but uninflamed tissues also becomes more painful (secondary hyperalgesia). In animal models of inflammation, the descending pain-control system sends down, simultaneously, inhibitory and facilitatory influences, but inhibition predominates for primary hyperalgesia while facilitation predominates for secondary hyperalgesia. Descending inhibition and facilitation during peripheral inflammation are due not only to previously existing descending modulation, but also to inflammation-induced changes in RVM which involve receptors for NMDA, AMPA, cholecystokinin and neurotensin, as well as synthesis of enkephalins and nitric oxide.

Effects of c-fos antisense oligodeoxynucleotide on 5-HT-induced upregulation of preprodynorphin, preproenkephalin, and glutamic acid decarboxylase mRNA expression in cultured rat spinal dorsal horn neurons

Effects of c-fos antisense oligodeoxynucleotide (ASO) on serotonin (5-HT)-induced upregulation of preprodynorphin (ppDyn), preproenkephalin (ppEnk), and glutamic acid decarboxylase (GAD), a special chemical marker for γ-aminobutyric acid (GABA) neurons, mRNAs in cultured spinal dorsal horn neurons were investigated in order to extend our understanding of expressions of opioid peptides and GABA in spinal cord regulated by the descending serotonergic efferents. Reverse transcription-polymerase chain reaction analysis revealed a time-course increase in the expression of mRNAs encoding c-fos, ppDyn, ppEnk, and GAD after administration of 5-HT (100 nM). Administration of c-fos ASO (0.02 nM) 30 min prior to 5-HT application markedly blocked the expression of c-fos gene. Moreover, c-fos ASO pretreatment significantly decreased the 5-HT-induced upregulation of ppDyn and ppEnk mRNAs, but failed to affect the expression level of GAD mRNA. These results suggest that the serotoningic raphe-spinal efferents might play an important role in regulating the synthesis of enkephalin, dynorphin, and GABA in the spinal dorsal horn neurons. The immediate early oncogene c-fos might be involved in the 5-HT-induced increase in ppDyn and ppEnk expression. However, under the present experimental conditions, c-fos does not seem to be associated with the upregulation of GAD mRNA induced by 5-HT.

Estrogen alters proenkephalin RNAs in the paraventricular nucleus of the hypothalamus following stress

Gonadal steroids modulate activity of the hypothalamo-pituitary-adrenal axis (HPA) following stress, but the regulatory pathways of this modulation are unknown. A possible site of action is the synthesis of CRH and/or enkephalin in cells of the paraventricular nucleus of the hypothalamus (PVN). To investigate this possibility, we utilized two stressors, i.p. hypertonic saline injection (HSI) or exposure to novel environment, and examined the response of CRH or c- fos mRNAs and proenkephalin (PPE) mRNA and heteronuclear RNA (hnRNA, primary transcript). Male rats were gonadectomized and treated with estrogen or dihydrotestosterone propionate (DHTP) for 2 weeks. In situ hybridization revealed that novelty or HSI elevated levels of PPE hnRNA and c- fos mRNA in the PVN. Estrogen attenuated the elevation of PPE hnRNA in the PVN following HSI, and enhanced the c- fos mRNA response to novelty. In contrast, DHTP did not affect PPE hnRNA, but inhibited the c- fos mRNA response to novelty. These data indicate that in male rats estrogen receptor but not androgen receptor may modulate the endocrine stress response by altering PPE transcription in the PVN and that this effect depends on the type of stressor.

ChemInform Abstract: A Fast Procedure for the Preparation of Amides/Peptides from Carboxylic Acids and Azides via Two Redox Reactions: Application to the Synthesis of Methionine Enkephalin.

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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.

A Fast Procedure for the Preparation of Amides/Peptides from Carboxylic Acids and Azides via Two Redox Reactions: Application to the Synthesis of Methionine Enkephalin

Abstract A one-pot self regulated approach for the synthesis of amides/peptides based on two reduction–oxidation (redox) reactions has been described. The primary and secondary amides/peptides are made by using azidotrimethylsilane and alkyl azides/α-azido acid derivatives respectively as the direct source of amine components. Benzeneselenol, generated in the reaction medium during carboxyl activation, has been found to be an effective reducing agent for the conversion of azides to primary amines. The methodology has been applied to the synthesis of methionine enkephalin.

Neurochemistry, connectivity and plasticity of small intensely fluorescent (SIF) cells in the rat superior cervical ganglion

Applying double-labelling immunofluorescence, the peptide content of solitary and clustered small intensely fluorescent (SIF) cells, identified by an antiserum to a selective membrane glycoprotein marker, synaptophysin, was correlated with the presence of catecholamines in the rat superior cervical ganglion. Most of synaptophysin-immunoreactive solitary and clustered SIF cells apparently contained dopamine (indicated by tyrosine hydroxylase-TH) but not noradrenaline (indicated by dopamine-beta-hydroxylase-DBH). Frequently, immunoreactivities for substance P or rarely, neuropeptide Y were colocalized in TH-immunolabeled cells of both types. Immunostaining for vasoactive intestinal polypeptide was found only in solitary SIF cells and was visible in TH-immunoreactive, as well as in TH-nonreactive cells. Very few solitary SIF cells were TH- and DBH-immunoreactive. Solitary and clustered SIF cells, as a rule, were encircled by leu-enkephalin-positive fibres which were also met-enkephalin-arg6-phe7-immunoreactive, indicating proenkephalin as precursor. SIF cells were additionally approached by varicose fibres which contained immunoreactivity for calcitonin gene-related peptide (CGRP) but not for enkephalins. As observed by immuno-electronmicroscopy, fibres that were immunostained for leu-enkephalin or CGRP, deeply invaginated into SIF cell somata. In addition to close membrane appositions, CGRP-immunolabeled fibres exhibited efferent synaptic contacts wih elements of SIF cell clusters. SIF cells were non-reactive to enkephalin-antisera in control ganglia and after transection of the postganglionic nerves (axotomy); but both types exhibited leu-enkephalin in preganglionically transected ganglia (decentralization) in which enkephalin-immunoreactive fibre baskets were absent. Synthesis of enkephalin in SIF cells after decentralization was confirmed by in situ hybridization demonstrating intracytoplasmic proenkephalin messenger-RNA. The findings are indicative for a differential neurochemical equipment of SIF cells in the rat superior cervical ganglion, which mainly is independent to a topographical classification. Moreover, they demonstrate the involvement of two neuropeptides in preganglionic SIF cell innervation. Finally, the observations indicate the capacity of SIF cells for proenkephalin-expression in response to preganglionic denervation.

Increased preproenkephalin A gene expression in the rat heart after induction of a myocardial infarction.

The expression of preproenkephalin A (ppENK) gene was investigated in the rat heart, following the onset of myocardial infarction induced by ligation of the left anterior descending coronary artery. The relative abundance of ppENK mRNA and the level of enkephalins were measured by Northern blot analysis and radioimmunoassay, respectively, in the ventricles from control-unoperated, sham-operated, and operated rats. Three hours after the surgery, a comparison between rats with infarction and sham-operated rats revealed that the relative abundance of ppENK mRNA and the level of enkephalins were increased three- to four- and two- to three-fold, respectively, in the ventricles of rats with infarction. No difference was observed between rats with infarction and sham-operated rats 24 h after the surgery, or between rats with infarction compared at time intervals of 3 and 24 h following the surgery. The abundance of the ppENK mRNA in the polysomal fraction of the ventricular septum was also measured 3 h after the surgery and found to be threefold higher in rats with infarction as compared with sham-operated rats. These results indicate that the level of enkephalins rapidly increases in the ventricles of rats following myocardial infarction, and that this higher level may be ascribed to a stimulation of the local synthesis of enkephalins.

A rostrocaudal gradient in the synthesis of enkephalin in nucleus accumbens.

A subpopulation of projection neurons in the nucleus accumbens uses the neuropeptide enkephalin as a neurotransmitter. The synthesis of enkephalin in striatal neurons is regulated by dopaminergic inputs. In the present study quantitative in situ hybridization histochemistry was used to examine the effects of unilateral 6-hydroxydopamine lesions of the ascending dopaminergic fibres on levels of enkephalin mRNA in subregions of the nucleus accumbens. It was found that the levels of enkephalin mRNA were increased throughout the nucleus after the lesion. However, the increase appeared to be much higher in rostral parts of the nucleus than in caudal parts, indicating regional differences in the effects of blockade of the dopaminergic neurotransmission. Possible causative factors for these differences are discussed.

Chronic treatment with chlorpromazine, thioridazine or haloperidol increases striatal enkephalins and their release from rat brain

The aim of this paper is to study the effect of 1, 2 or 3 months' administration of chlorpromazine (CPZ), thioridazine (TDZ) (2 or 6 mg/kg) or haloperidol (HAL) (0.25 or 1 mg/kg) IP on the level of leu- and met-enkephalin (ENK) in striatum. A dose- and time-dependent increase of striatal ENK level was observed after chronic administration of the neuroleptics (NL), but 8 days after withdrawal of chronically administered NL striatal ENK was decreased. Apomorphine pretreatment significantly attenuated the elevation in ENK produced by chronic injections of NL. In perfusion fluid obtained from the lateral ventricle of animals treated 1 month with HAL a dose-dependent increase of ENK levels was observed, which was augmented by potassium ions. It is concluded that: 1) Chronic administration of neuroleptic drugs that block dopamine receptors increases the level and the release of striatal enkephalins; 2) The results support the hypothesis that activation of dopaminergic neurons tonically inhibits the synthesis of enkephalins in the striatum.

Enkephalin Expression in Spinal Cord Neurons is Modulated by Drugs Related to Classical and Peptidergic Transmitters.

The effects of various neurotransmitter agonists and antagonists on the synthesis and release of methionine enkephalin (mENK) in neuronal cultures of mouse spinal cord and dorsal root ganglia have been measured. Blockade of electrical activity with tetrodotoxin between days 9 and 13 in culture caused a > 95% decrease in the number of mENK-immunoreactive neurons. This effect was also seen upon the blockade of glycine and beta-adrenergic receptors with strychnine and propranolol, respectively, and stimulation of GABA receptors with muscimol. Stimulation of beta-adrenergic receptors with isoproterenol, or blockade of glutamate and GABA receptors with 2-aminophosphonovalerate and strychnine, respectively, had a qualitatively opposite action on both the number of mENK-immunoreactive neurons and enkephalin peptide levels measured by radioimmunoassay. Application of substance P also enhanced the mENK cell number. These data suggest that, at least in the spinal cord, characteristics other than the average level of impulse activity in the afferent input may be critical to the regulation of expression of mENK.

Chronic naltrexone treatment increases expression of preproenkephalin and preprotachykinin mRNA in discrete brain regions

Long-term blockade of brain opioid receptors by the opiate antagonist naltrexone increases methionine-enkephalin content in the striatum and nucleus accumbens (Tempel et al., 1984). To determine whether these changes in peptide levels reflect increased peptide synthesis, we examined preproenkephalin mRNA content in discrete brain regions of control (placebo-treated) and chronic naltrexone-treated animals by Northern analysis. Chronic naltrexone treatment (8 d) led to an approximately 12-fold increase in the striatal content of preproenkephalin mRNA relative to that of control animals. In contrast, no statistically significant change was observed in striatal mRNA for cyclophilin (1B15) or actin. Small increases in preproenkephalin mRNA content occurred in the hippocampus (+40%) and hypothalamus (+19%). No significant changes occurred in the frontal cortex. Increases in levels of the mRNA were seen as early as 24 hr after antagonist treatment. In contrast, changes in opioid receptor density required 3-4 d to reach half-maximal up-regulation after chronic antagonist treatment. Recent evidence has suggested that substance P is regulated by opioid peptides. To determine whether substance P synthesis is altered by chronic antagonist treatment, the mRNA corresponding to the precursor for substance P was examined using a probe for exon-7 of the preprotachykinin gene. Preprotachykinin mRNA content in the striatum was increased 6-fold after chronic antagonist treatment relative to that of control animals. Substance P content was increased 3-fold after chronic antagonist treatment. These data suggest that chronic blockade of brain opioid receptors leads to the increased synthesis of both enkephalin and substance P in the striatum and that these changes are relatively specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of enkephalins by the method of polymeric activated esters based on 4-hydroxy-3-nitrobenzophenone

Leucine- and methionine-enkephalins have been synthesized by the successive growth of the peptide chain from the C-end by the method of polymeric activated esters based on 4-hydroxy-3-nitrobenzophenone with yields of 90 and 70%, respectively, calculated on the initial C-terminal amino acid. Polystyrene with 2% of divinylbenzene was used as the polymeric matrix. Using the synthesis of methionine-enkephalin as an example, the possibility has been shown of using polymeric activated esters for the synthesis of peptides with a free carboxy group.

Enkephalin convertase in the heart: similar disposition to atrial natriuretic factor.

Enkephalin convertase (EC; carboxypeptidase-E or -H) is a carboxypeptidase-B-like enzyme proposed to be involved in the synthesis of enkephalins and other neuropeptides. In this study we have characterized and localized EC in the rat heart and examined its correspondence with atrial natriuretic factor. Heart homogenates bind [3H]guanidinoethylmercaptosuccinic acid ([ 3H]GEMSA), a selective ligand for enkephalin convertase, with specificity and high affinity (Kd = 5-10 nM). The pharmacology of these sites matches that of convertase catalytic activity and of [3H]GEMSA binding in other tissues. The binding sites in the heart can be purified 4000-fold using p-aminobenzoylarginine affinity chromatography, and the purified sites have Co2+ stimulated carboxypeptidase activity identical to EC. By subcellular fractionation studies [3H]GEMSA-binding sites are localized to the granule fraction with ANF immunoreactivity. [3H]GEMSA autoradiography localizes EC in the heart to the left and right atria, with very low levels in mature and ventricles. The distribution, biochemical properties, and developmental course of EC suggest that it may be involved in the synthesis of atrial natriuretic factor.