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PubMed1990

Detection of Met-enkephalin and Leu-enkephalin in the brain of the hagfish, Eptatretus stouti, and the lamprey, Petromyzon marinus.

Acid extracts of the brain of the pacific hagfish, Eptatretus stouti, and the marine lamprey, Petromyzon marinus, were each fractionated by gel filtration chromatography and aliquots of column fractions were screened with radioimmunoassays (RIAs) specific for pro-dynorphin-related end products and for pro-enkephalin-related end products. Only pro-enkephalin-related immunoreactive forms were detected. The enkephalin-sized immunoreactive material, isolated for each species, was separately fractionated by reverse-phase high-performance liquid chromatography (HPLC). Aliquots of column fractions were screened with RIAs specific for Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg-Phe, and Met-enkephalin-Arg-Gly-Leu. In the hagfish brain, immunoreactive forms with the same retention times as synthetic Met-enkephalin and Leu-enkephalin were detected in a ratio of approximately 2:1. In addition, an immunoreactive form was detected with the Met-enkephalin-Arg-Phe-specific RIA. This form had the same chromatographic properties as synthetic Met-enkephalin-Arg-Phe. Analyses with the Met-enkephalin-Arg-Gly-Leu RIA were negative. HPLC analysis of the lamprey enkephalin-related material revealed the presence of authentic Met-enkaphalin and Leu-enkephalin in a molar ratio of 3:1. C-terminally extended forms of Met-enkaphalin were not detected in the lamprey extracts. Collectively these observations indicate that pro-enkephalin-related opioid peptides are present in the brain of cyclostomes.

Amino Acid Sequence↗
PubMed1985

No inotropic action of enkephalins or enkephalin derivatives on electrically-stimulated atria isolated from lean and obese rats.

Inotropic actions of the endogenous enkephalins, leucine enkephalin [( Leu] enkephalin) and methionine enkephalin [( Met] enkephalin), and derivatives, [D-Ala2-methionine] enkephalinamide (DAMEA) and [D Ala2-leucine]enkephalinamide (DALEA) were tested, alone or in combination with noradrenaline (NA), (+/-)-isoprenaline or carbachol, on electrically-stimulated atria excised from Sprague-Dawley, fatty, Zucker (fa/fa) and lean, hooded heterozygous (Fa/fa) rats. [Met] enkephalin, [Leu] enkephalin, DAMEA and DALEA (4 X 10(-7)M to 4 X 10(-4)M) caused no significant changes in atrial tension in any group compared to pre-injection control values or those following the infusion of Krebs-Henseleit control solution. NA and isoprenaline (10(-7) to 10(-6)M) caused significant, dose-related increases in atrial tension in each of the three strains of rats tested with the Fa/fa group showing the greatest change and fastest rate of tension development. [Met] enkephalin, [Leu] enkephalin, DAMEA or DALEA (4 X 10(-6)M) infused concurrently with NA or isoprenaline (10(-6)M) evoked atrial tension changes within each group that were not different from those observed when NA or isoprenaline was administered alone. Carbachol (10(-9) and 10(-8)M) caused a dose-related decrease (10% and 30-40%, respectively, from pre-injection control values) in atrial tension in auricles excised from all three groups. Again, infusion of [Met] enkephalin, [Leu] enkephalin, DAMEA or DALEA (4 X 10(-6)M) together with carbachol (10(-8)M) did not affect atrial tension changes of auricles isolated from any group compared to when carbachol was given alone. The results indicate that the endogenous pentapeptides, [( Met] or [Leu] enkephalin), or derivatives (DAMEA and DALEA) do not affect atrial tension of electrically-stimulated auricles isolated from Sprague-Dawley, fa/fa or Fa/fa rats. In addition, these pentapeptides do not modify the positive inotropic actions of NA or isoprenaline or the negative inotropic effects of carbachol. It is suggested that in vivo, the enkephalins or enkephalin derivatives do not have a direct action on the heart to alter myocardial contractility.

Animals↗
PubMed1978

In vitro release of [5-methionine]enkephalin and [5-leucine]-enkephalin from the rat globus pallidus.

Endogenous [5-methionine]enkephalin (Metenkephalin) and [5-leucine]enkephalin (Leu-enkephalin) are released from perfused slices of rat globus pallidus by increased K(+) in a Ca(2+)-dependent manner. Tissue perfused for 40 min contained only 26% of the Met-enkephalin and 44% of the Leu-enkephalin found in the freshly dissected tissue. After perfusion, the mean (+/-SEM) ratio (wt/wt) of Met-enkephalin to Leu-enkephalin was 3.4 +/- 0.2 compared with 5.8 +/- 0.2 in the fresh tissue. The degradation of trace amounts of synthetic [(3)H]enkephalins in the perfusing medium during stimulated release seems to reflect the accelerated degradation of enkephalin released from the tissue: 63% of the Met-enkephalin and 23% of the Leu-enkephalin were degraded in a medium containing bacitracin (30 mug/ml). The mean ratio (wt/wt) of the Met-enkephalin to the Leu-enkephalin recovered after release by exposure of slices to 50 mM K(+) was 2.7 +/- 0.3. When perfusates were corrected for degradation, this ratio increased to about 5.5 which is higher than that found in the perfused tissue. The differences in release, tissue loss, and catabolism of the two enkephalins may be reflecting differences in the metabolic systems operating on the pentapeptides, but this interpretation will have to be validated by in vivo release experiments. In any event these observations strongly suggest that both enkephalins can be considered candidate neurotransmitters in the rat globus pallidus.

Animals↗
PubMed1991

Immunohistochemical localization of [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8 in sympathetic and parasympathetic neurons and nerve fibers projecting to the rat submandibular gland.

The localization of [Met5]enkephalin, [Met5]enkephalin-Arg6-Gly7-Leu8, vasoactive intestinal polypeptide and tyrosine hydroxylase immunoreactivities was studied in the submandibular gland of adult Sprague-Dawley and Wistar rats using the indirect immunofluorescence technique. Immunoreactivities for [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8, a proenkephalin A-derived octapeptide, showed identical distributions. A large number of enkephalin-immunoreactive nerve fibers were detected around secretory acini, along intercalated ducts, convoluted granular tubules, intra- and interlobular ducts, as well as in close contact with blood vessels. The submandibular ganglia contained several enkephalin-immunoreactive neurons and nerve fibers. In the superior cervical ganglion numerous enkephalin-immunoreactive neurons and nerve fibers were also detected. Immunohistochemical co-localization studies indicated that [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8 immunoreactivities co-exist with vasoactive intestinal polypeptide in a subpopulation of neurons of the rat submandibular ganglia, in nerve trunks along the salivary ducts of the gland, and in nerve fibers around the acini. Uni- or bilateral superior cervical ganglionectomies for 1-4 weeks resulted in a complete disappearance of tyrosine hydroxylase immunoreactivity in the glandular parenchyma, while moderate tyrosine hydroxylase immunoreactivity was seen in some neurons of the submandibular ganglia. Abundant [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibers were still seen around the acini and blood vessels, as well as close to salivary ducts. These operations did not affect the [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive neurons in the submandibular ganglia. Many principal neurons in the superior cervical ganglion contained both [Met5]enkephalin-Arg6-Gly7-Leu8 and tyrosine hydroxylase immunoreactivity. Nerve ligation experiments indicated that [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive sympathetic fibers project along the external carotid nerve. Accordingly, nerve fibers were found around the acini and blood vessels as well as in nerve trunks along the salivary ducts of the submandibular gland, showing co-localization of [Met5]enkephalin-Arg6-Gly7-Leu8 and tyrosine hydroxylase. Taken together, these observations suggest that the nerve fibers of the rat submandibular gland containing proenkephalin A-derived peptides are of both sympathetic and parasympathetic origin.

Animals↗
PubMed1982

Methionine-enkephalin and leucine-enkephalin in human sympathoadrenal system and pheochromocytoma.

To elucidate the physiological and pathophysiological significance of methionine- and leucine-enkephalin (Met-and Leu-enkephalin, respectively) in human sympathoadrenal system, the contents of these peptides in normal human sympathetic nervous system, adrenal medulla, and pheochromocytomas were determined by specific radioimmunoassays combined with reverse-phase high-performance liquid chromatography. Met-enkephalin-LI and Leu-enkephalin-LI, respectively) were detected by radioimmunoassay in adrenal glands, adrenal medulla, stellate ganglia, sympathetic trunks, and celiac ganglia, and their contents in adrenal medulla were highest. Existence of authentic Met- and Leu-enkephalin was confirmed by reverse-phase high-performance liquid chromatography. Met-enkephalin was approximately 74% of Met-enkephalin-LI, whereas Leu-enkephalin was approximately 30% of Leu-enkephalin-LI in human adrenal medulla. The ratio of Met- to Leu-enkephalin was 2.6 in human adrenal medulla, whereas it was higher in sympathetic ganglia or trunks. In four cases of pheochromocytoma marked difference in Met- and Leu-enkephalin contents was found between medullary and extramedullary tumors. The contents were about three orders higher and the Met- to Leu-enkephalin ratio was lower in medullary than in extramedullary pheochromocytomas, reflecting the tissues where the tumors arose. These results suggest the physiological roles of Met- and Leu-enkephalin in sympathetic nervous system and adrenal glands and their pathophysiological significances in pheochromocytomas.

Adrenal Gland Neoplasms↗
PubMed1981

Immunohistochemical localization of leucine-enkephalin in the spinal cord of the cat: enkephalin-containing marginal neurons and pain modulation.

This study examined the spinal cord distribution of the endogenous opioid peptide leucine-enkephalin in the cat using immunohistochemical techniques. The distribution of nerve processes was studied in untreated cats; colchicine was administered to study the distribution and morphology of spinal enkephalin-containing perikarya. Enkephalin immunoreactive processes were greatest in laminae I and II (marginal layer and substantia gelatinosa) of the superficial dorsal horn. In many sections, the outer substantia gelatinosa (SG), lamina IIa, was discernibly less immunoreactive than I or IIb. Laminae III and IV were relatively devoid of staining. Laminae V and VII had moderate enkephalin-immunoreactivity, lamina VI somewhat less. Enkephalin immunoreactivity in lamina X, around the central canal, was very dense. Enkephalin-containing beaded varicosities coursed throughout the ventral horn. Although previous studies in the rat emphasized the enkephalin-somata of the SG, we found that in the cat the majority of superficial dorsal horn enkephalin-somata are in the marginal layer. These enkephalin-containing marginal cells were morphologically similar to a population of marginal neurons which project to the brainstem and/or the thalamus. Some light staining small SG neurons were also identified; many were located at the lamina I-II border. Considerably more cells were found ventral to the SG, in lamina III, and at the IV-V border. These latter cells had dendrites coursing dorsally, toward the SG. Numerous immunoreactive cells were found in lamina VIII, in a band across the intermediate gray. These cells fused medially with cells of lamina X. Enkephalin cells were also found in the sacral autonomic nucleus and encircling the central cervical nucleus, Clarke's column, and stilling's nucleus. Although surrounded by labeled cells, the latter regions were devoid of enkephalin-immunoreactive processes. Many of these spinal enkephalin neurons are morphologically similar to and distributed in regions known to contain projection neurons. Thus it is suggested that many spinal enkephalin neurons, generally thought to be local circuit neurons, project rostrally, to other spinal levels and perhaps to brainstem and/or thalamus.

Animals↗
PubMed1994

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 D2/D3 antagonist. From a functional point of view, the dopaminergic D2 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 met-enkephalin release, presumably potentiated by 6-hydroxydopamine treatment, act via a functional modification of the nigrostriatal dopaminergic pathway.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1983

Purification and characterization of enkephalin convertase, an enkephalin-synthesizing carboxypeptidase.

Enkephalin convertase, an enkephalin-synthesizing carboxypeptidase present in adrenal medulla chromaffin granules, has also been detected in brain and pituitary. To determine whether these three carboxypeptidase activities represent the same enzyme, we purified and characterized enkephalin convertase from adrenal medulla, whole brain, and whole pituitary. Enzyme from all three tissues co-purifies on DEAE-cellulose, gel filtration, concanavalin A, and L-arginine affinity columns, resulting in a 135,000-fold, 110,000-fold, and 2,800-fold purification for bovine adrenal medulla, brain, and pituitary, respectively. Purified enkephalin convertase appears homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showing a single band with an apparent molecular weight of 50,000 for enzyme isolated from all three tissues. Adrenal, brain, and pituitary enkephalin convertase are similarly inhibited by hexapeptide enkephalin precursors and active site-directed inhibitors. Both [Met]-and [Leu]enkephalin-Arg6 inhibit enkephalin convertase with Ki values between 50 and 80 microM, while [Met]-and [Leu]enkephalin-Lys6 are 3-fold less potent. Two active site-directed inhibitors, guanidinopropylsuccinic acid and guanidinoethylmercaptosuccinic acid, are potent inhibitors of all three enzymes with Ki values of 8-9 nM. A series of dansylated di-, tri-, and tetrapeptide substrates are hydrolyzed by enkephalin convertase with similar kinetic properties (Km, Vmax, and Kcat/Km) for the three enzymes. This evidence suggests that enkephalin convertase activity represents the same enzyme in adrenal medulla, brain, and pituitary. Enkephalin convertase may be involved in the production of other peptide neurotransmitters and hormones besides enkephalin.

Adrenal Glands↗
PubMedDate not supplied

Enkephalin-containing polypeptides are potent inhibitors of enkephalin degradation.

Enkephalin-containing polypeptides derived from pro-enkephalin A, pro-enkephalin B, or pro-opiomelanocortin were inhibitors of enkephalin degradation by aminoenkephalinases purified from cytosol or membranes. Of the peptides, Argo-Met-enkephalin was the most potent inhibitor for the aminoenkephalinases, with an IC50 of about 0.6 microM, it was more effective than bestatin (IC50 = 0.8-1.0 microM). This inhibition was partly due to substrate competition. Argo-Met-enkephalin was hydrolyzed by aminoenkephalinases to form Arg, Tyr, and Gly-Gly-Phe-Met in a substrate-inhibited manner. The hexapeptide also inhibited the breakdown of Arg- and Tyr-beta-naphthylamide by the membrane aminoenkephalinase. Since Argo-Met-enkephalin did not inhibit leucine aminopeptidase, it was a more selective inhibitor than bestatin of Met-enkephalin breakdown by aminopeptidases. Argo-Met-enkephalin inhibited enkephalin breakdown by synaptosomal plasma membranes but not by brain slices. Our data suggest that in addition to their possible role as opioids, the enkephalin-containing polypeptides may be regulators of enkephalin levels.

Aminopeptidases↗
PubMedDate not supplied

Detection of Met-enkephalin and Leu-enkephalin in the posterior pituitary of the holostean fish, Amia calva.

Immunohistochemical analysis of the pituitary of the holostean fish, Amia calva, indicated that enkephalin-related immunoreactivity was restricted to the pars nervosa, and was not detected in other regions of the pituitary. Fractionation of acid extracts of posterior pituitaries by reverse phase HPLC followed by RIA analysis indicated the presence of immunoreactive Met-enkephalin and Leu-enkephalin. No immunoreactive forms were detected with RIAs specific for either Met-enkephalin-RF or Met-enkephalin-RGL. The molar ratio of Met- to Leu-enkephalin in this terminal field was 3:1 (n = 4). HPLC fractions were also digested with trypsin and carboxypeptidase B to test for C-terminally extended forms of Met-enkephalin. A novel modified form of Met-enkephalin was detected. Extracts of the posterior pituitary, forebrain, midbrain, hypothalamus and hindbrain were screened with RIAs specific for the Pro-dynorphin end products, alpha-neo-endorphin, dynorphin A(1-17), dynorphin A(1-8) and dynorphin B(1-13). The results of these analyses were negative. Collectively, these data suggest that a Pro-enkephalin-like molecule is present in holostean fish. The holostean enkephalin precursor contains at least Met-enkephalin and Leu-enkephalin. However, Pro-dynorphin-related end products with antigenic determinants similar to mammalian dynorphin A(1-17), dynorphin A(1-8), dynorphin B(1-13) and alpha-neo-endorphin could not be detected in the brain or pituitary of this species.

Animals↗
PubMed1995

Comparison of immunohistochemical localization of [Met5]enkephalin-Arg6-Gly7-Leu8, [Met5]enkephalin, neuropeptide Y and vasoactive intestinal polypeptide in the superior cervical ganglion of the rat.

The localization of [Met5]enkephalin-Arg6-Gly7-Leu8 and [Met5]enkephalin immunoreactivities was studied in the rat superior cervical ganglion. The distribution of these enkephalin-containing peptides in the ganglion was correlated to that of neuropeptide Y and vasoactive intestinal polypeptide. Three different populations of peptide-containing postganglionic neurons were demonstrated. (1) A minor population (10-20%) of principal neurons was immunoreactive for [Met5]enkephalin-Arg6-Gly7-Leu8 but not immunoreactive for neuropeptide Y nor vasoactive intestinal polypeptide. (2) The major population (about 50-70%) was immunoreactive for neuropeptide Y but not for [Met5]enkephalin-Arg6-Gly7-Leu8. (3) Few vasoactive intestinal polypeptide-immunoreactive principal neurons (less than 2% of all principal neurons) were observed in the ganglion. All vasoactive intestinal polypeptide-immunoreactive neurons were also immunoreactive for neuropeptide Y but not for [Met5]enkephalin-Arg6-Gly7-Leu8. [Met5]enkephalin-Arg6-Gly7-Leu8- and [Met5]enkephalin-immunoreactive nerve fibers had a similar distribution. These enkephalin immunoreactive nerve fibers were seen to enclose both neuropeptide Y-containing principal neurons and neurons devoid of neuropeptide Y immunoreactivity. Furthermore, there were enkephalin-immunoreactive fiber baskets around vasoactive intestinal polypeptide-immunoreactive neurons and sometimes also around solitary enkephalin-immunoreactive neurons. Previously reported diverse role of enkephalins in the rat superior cervical ganglion is supported by this study.

Animals↗
PubMed1977

The effect of enkephalin and enkephalin analogs on the guinea-pig ileum and rat brain opilate receptor.

We have synthesized the two naturally occurring enkephalins (Methionine-Enkephalin and Leucine-Enkephalin) as well as four other analogs (Methinonine-Enkephalin amide, S-Benzyl sulphonium analog of Methionine-Enkephalin, 3-Benzyl-tyrosine analog of Methionine-Enkephalin and 3-Benzyl-tyrosine analog of Leucine-Enkephalin) in order to study their relative potency and their inactivation of different tissues. Morphine-like activity of the peptides was determined by measuring their inhibitory effect in the guinea-pig ileum and their interaction with the brain opiate receptor; the order of affinity of the three most active peptides (Methionine-Enkephalin amide greater than Methionine-Enkephalin greater than Leucine-Enkephalin) for the rat brain opiate receptor was found to be the same in the guinea-pig ileum. The rate of inactivation of the peptides was also studied in the guinea-pig ileum and in the rat brain membrane preparation. Methionine-Enkephalin amide was relatively more resistant to the ileum peptidases than the other peptides, but was destroyed at the same rate by brain membranes; this variation in inactivation of Methionine-Enkephalin amide probably indicates the presence of differnet peptidases in each tissue preparation.

Animals↗
PubMed1991

The phylogeny of Met-enkephalin and Leu-enkephalin: studies on the holostean fish Lepisosteus platyrhincus and the Australian lungfish, Neoceratodus forsteri.

Acid extracts of the brain of the holostean fish Lepisosteus platyrhincus and the forebrain of the dipnoan fish Neoceratodus forsteri were separately fractionated by Sephadex G-50 column chromatography. For both species, Met-enkephalin-related immunoreactivity was detected coeluting with the total volume internal standard. Higher-molecular-weight Met-enkephalin-containing immunoreactive peaks were not detected in these chromatographs. Furthermore, immunoreactive forms with antigenic determinants identical to mammalian dynorphin A(1-17), dynorphin A(1-8), alpha-neo-endorphin, or dynorphin B(1-13) were not detected in either species. Reverse-phase HPLC analysis of enkephalin-sized immunoreactive material indicated the presence of authentic Met-enkephalin and Leu-enkephalin in the extracts of both species. In the brain of L. platyrhincus the molar ratio of Met-enkephalin to Leu-enkephalin was approximately 3:1, whereas, the molar ratio of these enkephalins in the forebrain of N. forsteri was approximately 5:1 [corrected]. C-terminally extended forms of Met-enkephalin were also detected in the extracts of both species. These results suggest that the ancestral proenkephalin gene of both actinopterygian and sarcopterygian fish contained both the Met-enkephalin and Leu-enkephalin sequences.

Amino Acid Sequence↗
PubMed1994

Immunocytochemical evidence for the presence of Met-enkephalin and Leu-enkephalin in distinct neurons in the brain of the elasmobranch fish Scyliorhinus canicula.

Immunohistochemical methods have been used to investigate the distribution of various opioid peptides derived from mammalian proenkephalin in the central nervous system of Scyliorhinus canicula. The results indicate that both Leu- and Met-enkephalin-immunoreactive peptides are present in the dogfish brain. In contrast, enkephalin forms similar to Met-enkephalin-Arg-Phe or Met-enkephalin-Arg-Gly-Leu, and mammalian alpha-neo-endorphin, dynorphin A (1-8), dynorphin A (1-13), and dynorphin A (1-17) were not detected. Met- and Leu-enkephalin immunoreactivities were found in distinct neurons of the telencephalon and hypothalamus. In particular, cell bodies reacting only with the Met-enkephalin antiserum were localized in the preoptic nucleus and in the suprachiasmatic region of the hypothalamus. Conversely, cell bodies reacting only with the Leu-enkephalin antiserum were localized in the pallium and the nucleus lobi lateralis hypothalami. Several areas of the telencephalon and diencephalon exhibited both Met- and Leu-enkephalin-like immunoreactivity, but the two immunoreactive peptides were clearly contained in distinct perikarya. The overall distribution of Met-enkephalin-immunoreactive elements in the dogfish exhibited similarities to the distribution of proenkephalin-derived peptides previously reported for the brain of tetrapods. The fact that Met- and Leu-enkephalin-like peptides were detected in distinct neurons, together with the absence of dynorphin-related peptides, suggests the existence of a novel Leu-enkephalin-containing precursor in the dogfish brain.

Amino Acid Sequence↗
PubMed1988

Differential release of enkephalin and enkephalin-containing peptides from perfused cat adrenal glands.

We have compared the enkephalin-like material derived from proenkephalin released from perfused cat adrenal glands stimulated with pilocarpine (5 X 10(-4)M) and nicotine (5 X 10(-6) M). In addition, two doses of acetylcholine (10(-5) and 10(-4) M) and 50 mM K+ were tested. Free Met-enkephalin immunoreactivity and total Met-enkephalin immunoreactivity, as determined by enzymatic digestion of large enkephalin-containing fragments, were coreleased with catecholamines. Free Met-enkephalin immunoreactivity represented 13% of total immunoreactivity for nicotinic stimulation, 46% for pilocarpine, 33% for 10(-5) M acetylcholine, 22% for 10(-4) M acetylcholine, and 16% for 50 mM K+. Analysis of the perfusate by gel filtration showed that 80% of the total Met-enkephalin immunoreactivity whose release was induced by pilocarpine was eluted in fractions corresponding to fragments of low molecular weight, whereas these fractions accounted only for 10% of the total Met-enkephalin immunoreactivity whose release was induced by nicotine. HPLC analysis of low-molecular-weight peptide fractions revealed that Met-enkephalin, Met-enkephalin-Arg-Gly-Leu, and Met-enkephalin-Arg-Phe represented 69% of total Met-enkephalin immunoreactivity whose release was induced by pilocarpine. These results indicate that selective activation of muscarinic receptors is followed by release of low-molecular-weight material, whereas nicotine application also yielded high-molecular-weight peptides. Furthermore, increasing the acetylcholine concentration from 10(-5) to 10(-4) M and using 50 mM K+ increased proportionally the high-molecular-weight peptide secretion. Results are discussed in relation to the existence of a heterogeneous population of granules either in the same cell or in different cells, containing proenkephalin-derived peptides. (ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMedDate not supplied

Effects of Met-enkephalin on the mechanical activity and distribution of Met-enkephalin-like immunoreactivity in the cat large intestine.

The effects of Met-enkephalin on the spontaneous and electrically evoked activity were investigated in longitudinal and circular strips isolated from different regions of the large intestine, i.e., proximal colon, distal colon and rectum. Met-enkephalin induced dose-dependent contractile responses which were reversibly blocked by naloxone (10(-6) M). In all longitudinal strips and in the circular strips of the rectum, the effects of Met-enkephalin were prevented by TTX (10(-7) M), demonstrating their neurogenic nature. In the circular strips from the colon, Met-enkephalin induced contractile responses after TTX, proving the existence of smooth muscle opioid receptors. The comparison between the EC50 values of Met-enkephalin showed that the opioid receptors in the different regions have different sensitivity to Met-enkephalin, while the opioid receptors in the longitudinal and circular layers of the same region have equal affinity. Atropine (10(-6) M) and guanethidine (10(-6) M) did not alter significantly the EC50 values, showing that the neurogenic effects of Met-enkephalin on the spontaneous activity involve mainly nonadrenergic, noncholinergic (NANC) neurotransmitter mechanisms. When the preparations were stimulated electrically, Met-enkephalin (10(-9) M) suppressed the cholinergic components of the responses. Met-enkephalin-containing nerve fibers were found in the myenteric plexus of the three intestinal regions. In the colon, where direct smooth muscle effects were observed, fibers containing Met-enkephalin-like immunoreactivity were found to go deep into the circular layer, suggesting that they could supply Met-enkephalin input to the smooth muscle cells.

Animals↗
PubMed1977

The distribution of methionine-enkephalin and leucine-enkephalin in the brain and peripheral tissues.

1 A method is described for the rapid extraction of opioid peptides from the brain and other tissues. The method is based on acid extraction of tissues followed by adsorption of the extract onto Amberlite XAD-2 resin. Elution with methanol separates the enkephalins and alpha-endorphin from beta-endorphin.2 Over 90% of the opioid peptide activity isolated from brain and gut of several species by our method was due to methionine- and leucine-enkephalin. In contrast, the major opioid peptide activity recovered from the pituitary was due to peptides of much greater mol. wt. than the enkephalins.3 An opioid peptide with properties unlike those of the known endorphins or enkephalins was present in brain extracts. This peptide, termed epsilon-endorphin, has an apparent mol. wt. of 700 to 1200; it constituted between 5 to 10% of the total opioid activity in our extracts.4 A differential assay of methionine- and leucine-enkephalin was made either by destroying methionine-enkephalin activity with cyanogen bromide or by separating the peptides by thin layer chromatography.5 The ratio of methionine-enkephalin to leucine-enkephalin varied greatly in different brain regions. The highest proportions of leucine-enkephalin were found in the cerebral cortex and hippocampus.6 Formaldehyde perfusion and fixation of the brain in vivo had no significant effect on the brain content of enkephalin, indicating that proteolytic breakdown is not a major problem in the extraction of these peptides.7 It is suggested that the enkephalins may have a neurotransmitter role in both brain and peripheral tissues and that methionine- and leucine-enkephalin may subserve separate neuronal functions.

Animals↗
PubMed2000

Deciphering the origin of Met-enkephalin and Leu-enkephalin in Lobe-finned fish: cloning of australian lungfish proenkephalin.

The previous detection of Met-enkephalin and Leu-enkephalin in the CNS of the Australian lungfish, Neoceratodus forsteri, in a molar ratio comparable to mammals suggested that the lungfish proenkephalin precursor should contain the sequences of both Met-enkephalin and Leu-enkephalin as seen for mammalian proenkephalin. However, the cloning of a full-length proenkephalin cDNA from the CNS of the Australian lungfish indicates that the organization of this precursor is more similar to amphibian proenkephalin than mammalian proenkephalin. The Australian lungfish cDNA is 1284 nucleotides in length and the open reading frame (267 amino acids) contains seven opioid sequences (GenBank #AF232671). There are five copies of the Met-enkephalin sequence flanked by sets of paired basic amino acid proteolytic cleavage sites and two C-terminally extended forms of Met-enkephalin: YGGFMRSL and YGGFMGY. As seen for amphibians, no Leu-enkephalin sequence was detected in the Australian lungfish proenkephalin cDNA. The fact that Leu-enkephalin has been identified by radioimmunoassay and HPLC analysis in the CNS of the Australian lungfish indicates that a Leu-enkephalin-coding gene, distinct from proenkephalin, must be expressed in lungfish. Potential candidates may include a prodynorphin- or other opioid-like gene. Furthermore, the absence of a Leu-enkephalin sequence in lungfish and amphibian proenkephalin would suggest that the mutations that yielded this opioid sequence in tetrapod proenkephalin occurred at some point in the radiation of the amniote vertebrates.

Amino Acid Sequence↗