Met- and leu-enkephalin immuno- and bio-reactivity in human stomach and pancreas

Acid extracts of human pancreas and gastric corpus and antral mucosa and muscularis were investigated for the presence of met-enkephalin and leu-enkephalin by radioimmunoassay, Sephadex chromatography and radioreceptor assay. As the assays for leu-enkephalin crossreacted with those for met-enkephalin, only cyanogen bromide-treated samples were used for the determination of leu-enkephalin. Met-enkephalin immunoreactivity was destroyed by more than 94% when treated with cyanogen bromide. Serial extract dilutions displaced ¹²⁵ I labelled met-enkephalin and ¹²⁵ I leu-enkephalin in the respective enkephalin radioimmunoassay both roughly parallel to the standard curves. Sephadex chromatography of the extracts resulted in elution of met-enkephalin and leu-enkephalin immunoreactivity similar to the size of ³H-met-enkephalin, and these eluates displaced ³H-met-enkephalin from rat brain membranes in an opioid radioreceptor assay. The highest concentration of met-enkephalin and leu-enkephalin immunoreactivity in tissue obtained at surgery was in the mucosa of the body of the stomach. Met- and leu-enkephalin receptor bioreactivity concentrations exceeded immunoreactivity concentrations. These investigations provide evidence of the presence of met-enkephalin- and leu-enkephalin-like substances in human stomach and pancreas.     

刺激迷走神经向中端对大鼠不同脑区脑啡肽含量的影响

自从1975年(Hughes et al. 1975)发现脑啡肽以来,经多方面研究已认为脑啡肽可能是一种神经递质,它具有多种生理功能(范少光、汤健,1978)。据新近文献报道,脑啡肽与催产素及加压素三者具有共同存在的并存关系(Martin and Voigt。1981)。 已经证明,刺激迷走神经向中端,可引起脑内释放乙酰胆硷,从而促使神经垂体释放压加素和催产素,肾上腺释放肾上腺素(Chang, et al. 1937;1961,1964;吕运明等1965,1977;唐正荣1981)。但是,刺激迷走神经向中端,是否也能引起脑内脑啡肽的释放,迷走神经传入纤维与脑内脑啡肽能神经原之间,是否存有机能上的联系?关于这个     

Production of enkephalins by the human and bovine corpus luteum

In search of early pregnancy factors, we detected by radioimmunoassay the presence of enkephalin in bovine and human corpus luteum. In vitro met-enkephalin release by bovine corpus luteum is about 0.5 to 1 pmole/mg of fresh tissue/24 hrs. The content of the fresh tissue is between 0.7 and 1.9 pmoles per gram of human tissue, and 0.9 pmoles for bovine tissue. Furthermore, we determined the presence of leu-enkephalin and met-enkephalin Arg-Gly-Leu to. The ratios observed confirm a pro-enkephalin A expression in the ovary. Opiates or opioid-like peptides are present in the female genitalia at the time of early embryo development. The roles of these opioid peptides is discussed in term of ovum transport, granulosa cell physiology and early pregnancy factors.     

The effect of the aminopeptidase inhibitor bestatin on the enkephalin level in the rat brain

Met- and leu-enkephalin contents in midbrain (including hypothalamus) and striatum of rats were determined by radioimmunoassay after bestatin (racemate) injection (200 g, i.c.v.). It was found that bestatin administration influenced the midbrain met-enkephalin content, values and directions of the changes observed being dependent upon the time after the injection. The data obtained confirm the participation of aminopeptidase in enkephalin inactivation and present evidence for the possibility of regional variations of enkephalin catabolism pathways in the brain.     

Differential localization of leu-enkephalin and hyperglycemic hormone in axon terminals of the sinus gland of the crabsCarcinus maenas,eriocheir sinensis,andUca pugilator

Summary Leu-enkephalin containing secretory granules were demonstrated in axon terminals of immunogoldlabeled electron-microscopic sections of the sinus gland of three brachyuran crustaceans. These granules have a diameter of 120+-15 nm and differ in electron density from those located in adjacent terminals containing hyperglycemic or molt-inhibiting hormone. These neurohormones do not show co-localization with leu-enkephalin. The cross-reactivity of leu-enkephalin antiserum with met-enkephalin is less than 1%. The sinus glands of the three species examined show no immunoreactivity for FMRF-amide. A modulatory activity of endogenous enkephalin by paracrine mechanisms is suggested.     

Multiple Molecular Forms of Enkephalins in the Guinea Pig Hippocampus

Abstract: The combined techniques of HPLC and radioimmunoassay were used to identify and quantitate enkephalin-related peptides in the guinea pig hippocampus. Both met- and leu-enkephalin were identified, in approximately a 2:1 ratio, as well as a third enkephalin-like molecule that is neither met- nor leu-enkephalin. The third enkephalin elutes earlier than met- or leu-enkephalin from a reversed-phase column, has a molecular weight similar to the other enkephalins, and is as active as these enkephalins are in inhibiting binding of labeled opiates to rat brain membranes. All regions of the hippocampus (dentate gyrus, CA1–2, CA3–4, and subiculum) contain all three immunoreactive peptides. Immunocytochemical techniques, using antisera raised against met-enkephalin, show with one antiserum immunoreactivity in the granule cell-mossy fiber system, and with the other scattered immunoreactive cells mostly in the CA2 region. Enkephalins are not confined to the mossy fiber system, as previously suggested, but may be a component of another hippocampal innervation.     

Pupillary effects of leucine and methionine enkephalin in rats after intraperitoneal administration

Changes in pupil size after peripheral administration of met-enkephalin, leu-enkephalin, or morphine were studied in the rat. With a simple pupillographic technique, the pupil diameter of male, S.D. rats (250–300 g) was measured by a series of photographs taken every 60 sec for at least 45 min after the last drug injection. Morphine (8 mg/kg, SC) caused mydriasis characterized by rapid and marked fluctuations of pupil size. Mydriasis also occurred after leu-enkephalin (5 and 10 mg/kg, IP) and met-enkephalin (20 mg/kg, IP). Both peptides induced morphine-like fluctuations. When given 15 min after morphine, leu-enkephalin (5 and 10 mg/kg) increased the mydriatic effect of morphine from 172 percent of control to 224 and 272 percent, respectively. Met-enkephalin (20 mg/kg, but not 10 mg/kg) also enhanced the mydriatic response of morphine, to 244 percent of control. These interactions appear to represent simple addition rather than potentiation. The effects of both peptides were reversed by naloxone (1 mg/kg, SC), suggesting an opiate receptor interaction for the pupillary effects of the enkephalins. The rat pupil thus provides one of the few in vivo models permitting quantification of enkephalin action after parenteral administration.     

Enkephalin and cyclic nucleotide content in the brain structures of rats at different stages of the formation and development of alcoholic dependence

Rats with increased alcohol motivation have been found to have a rise in enkephalin levels in limbic cortex and a decrease in met-enkephalin levels in the brain basal ganglia. Reduction of met-enkephalin to leu-enkephalin ratio in basal ganglia, limbic cortex and hypothalamus may serve as an index of increased inclination to ethanol in these animals. Alcohol dependence is characterized by reduced cAMP content in the majority of brain structures studied, sharply decreased met-enkephalin levels in limbic cortex and hypothalamus, and diminished cAMP and cGMP content in hypothalamus. In the third stage of experimental alcoholism the partial normalization of met-enkephalin and cAMP levels is observed in brain structures, with cGMP content increased in hypothalamus and considerably reduced in basal ganglia.     

Met-enkephalin: Rapid separation from brain extracts using high-pressure liquid chromatography,and quantitation by binding assay

Met-enkephalin can be rapidly separated with high recovery from leu-enkephalin, -endorphin, and enkephalin metabolites by high-pressure liquid chromatography on a Dupont SCX column. The technique permits separation of endogenous enkephalins from brain extracts for measurement by opiate-receptor binding assay, immunoassay, or for study of the incorporation of radiolabeled amino acids into enkephalin. Using this technique, met-enkephalin was separated from striatal extracts of rats killed by focused microwave irradiation and quantitated by the opiate-receptor binding assay. The concentration of met-enkephalin in the column eluate was 1.3 g/g tissue. This value is comparable to that obtained by radioimmunoassay without purification of the extracts.Pharmacology Research Associate, Pharmacology Research Associate Program, National Institute of General Medical Sciences.     

Characteristics of a monoclonal beta-endorphin antibody recognizing the N-terminus of opioid peptides

The properties of a mouse monoclonal antibody to beta-endorphin secreted by a clone of hybrid myelomas (3-E7) are described. The antibody displays virtually complete cross-reactivity to met-enkephalin and leu-enkephalin, but no cross-reactivity to beta-lipotropin, alpha-N-acetyl-beta-endorphin and des-Tyr1-beta-endorphin. Substantial cross-reactivity is seen with some other naturally occurring opioid peptides bearing the enkephalin sequence, such as dynorphin, alpha-neo-endorphin and BAM 22, but cross-reactivity is lacking in the case of certain synthetic enkephalin derivatives possessing a D-amino acid in position 2. The data indicate that for the binding of an antigen to the antibody the N-terminal tyrosine moiety is essential. The antibody recognizes, thus, a site which is of functional significance for the interaction of many naturally occurring opioid peptides with the opiate receptor.     

Monoclonal antibody to enkephalins with binding characteristics similar to opiate receptor

Monoclonal antibodies to enkephalins were established by immunization of mice with met-enkephalin, leu-enkephalin or both. Twenty-three clones with a high titer were classified into 6 types according to the binding properties to enkephalins and their derivatives. Antibody LM 239 showed binding characteristics similar to opiate receptor. It has a very high affinity to enkephalins and their derivatives which have a potent opioid activity, but a low affinity to enkephalin derivatives which devoid of opioid activity. The binding of 3H-met-enkephalin to the antibody was inhibited by naloxone and morphine, although the ID50 values were considerably higher than the Ka values of the alkaloids to opiate receptor.     

Effects of enkephalins on perfusion pressure in isolated hindlimb preparations

A series of studies were conducted to determine the effects of leucine-(leu-) enkephalin and methionine-(met-) enkephalin on perfusion pressure. These experiments utilized isolated perfused femoral arterial preparations in pentobarbital-anesthetized cats. The enkephalins were administered intraarterially into the femoral artery and changes in perfusion pressure recorded. Leu-enkephalin in doses of 1 μg to 320 μg produced significant dose-dependent decreases in perfusion pressure (4.0 ± 1.3% with 1 μg to 19.1 ± 2.1% with 320 μg). Similar declines in perfusion pressure (5.2 ± 2.4% with 1 μg to 21.7 ± 4.1% with 320 μg) were observed following the administration of met-enkephalin. Pretreatment with naloxone (3 mg/kg) antagonized the effects of both enkephalins. Diphenhydramine (2 mg/kg) effectively antagonized the leu-enkephalin elicited decline in perfusion pressure but blocked the effects of met-enkephalin only at lower agonist doses. Propranolol treatment (4 mg/kg) did not alter the pressure responses to either enkephalin. The results of the study show that intraarterially administered enkephalins exert a vasodilatory effect on vasculature in skeletal muscle which may be direct, indirect or both. The differential antagonism of the effects of the two enkephalins suggest that the two opioids act through different receptors or multiple receptors.     

Immunocytochemical localization of enkephalin in the lateral septum of the guinea-pig brain

By the use of antisera to met-enkephalin and leu-enkephalin, enkephalin-containing structures were visualized in the lateral septum of the guinea-pig brain. The present results do not reveal immunoreactive perikarya in this area. The immunostaining is exclusively located in numerous nerve fibers and endings mostly encompassing neuronal perikarya, which accounts for the fact that at the light-microscopic level cellular somata appear to be immunostained. The immunoreactive terminals and fibers contain granules approximately 110 nm in diameter and synaptic vesicles. The origin and the functional role of these numerous enkephalin terminals remain to be established.     

Isolation of human met-enkephalin and two groups of putative precursors (2K-pro-met-enkephalin) from an adrenal medullary tumour

Human met-enkephalin from an adrenal medullary tumour has been purified and characterised. Two groups of putative human met-enkephalin precursors (MW ～ 2000) were also isolated and were only opiate active after trypsinisation and yielded met-enkephalin on further treatment with carboxypeptidase B. Amino acid analyses of these peptides show they are not related to β-LPH nor any other reported putative enkephalin precursors. Their amino acid compositions suggest they are structurally closely related and may represent two groups of heterologous peptides.     

Release of immunoreactive met-enkephalin by intraventricular beta-endorphin in anesthetized rats

The release of immunoreactive met-enkephalin and leu-enkephalin from the spinal cord by intraventricular injection of different doses of beta-endorphin was studied using the intrathecal perfusion technique. The intraventricular beta-endorphin elicited the release of immunoreactive met-enkephalin from the spinal cord in a dose dependent manner. Immunoreactive leu-enkephalin in the spinal perfusate was not increased after intraventricular beta-endorphin injection. Both immunoreactive met-enkephalin and leu-enkephalin in the spinal cord were not changed by low doses (2-6 micrograms) of beta-endorphin but were increased markedly by 60-70% after high doses of beta-endorphin (32-64 micrograms). It is likely that the biosynthesis of enkephalins was also increased after intraventricular beta-endorphin injection. Intraventricular naloxone, 30 micrograms did not induce any release of immunoreactive met-enkephalin from the spinal cord and did not block the release of immunoreactive met-enkephalin induced by intraventricular beta-endorphin, 15 micrograms.     

Immunohistochemical localization of opioid peptides in the brain of the leech Theromyzon tessulatum

Summary By use of antisera directed against met-enkephalin, leu-enkephalin, dynorphin or -neoendorphin, immunoreactive structures were visualized in the central nervous system and proboscis of the leech Theromyzon tessulatum. Their distribution in the various compartments of the supra- and subesophageal ganglia was mapped. No correspondence could be established between the neurons containing met- or leu-enkephalin-like substances and the different types of neurosecretory cells classically described in Hirudinea. Successive localization of leu- and met-enkephalin on the same section revealed that these two peptides occur in different neurons. Only one cell located in compartment 6 of the supraesophageal ganglion was both dynorphin- and leu-enkephalin-positive. The other dynorphinimmunoreactive cells were not stained with the anti-leuenkephalin serum. The -neoendorphin-immunopositive cells were leu-enkephalin immunonegative and vice versa.     

Adrenal growth and ontogeny of adrenal substance P, enkephalins and catecholamines in the ovine fetus

Biologically active peptides have been identified in the adrenal glands of several adult mammalian species. Some of these peptides appear to modulate the nicotine-induced catecholamine release from the adrenal medulla. The present study was carried out to investigate the presence and ontogeny of the peptides substance P, met-enkephalin and leu-enkephalin in the ovine fetal adrenal gland from 70 to 140 days gestation (term = 145-150 days). Concurrently, the growth of the fetal adrenal as well as the gestational changes in catecholamine content were determined. The maternal adrenal glands were also studied for comparison. The ovine fetal adrenal gland increased in weight with advancing gestation at a single exponential rate. Total adrenal substance P content correlated with gestational age, while met-enkephalin, leu-enkephalin and total catecholamine contents correlated with adrenal weight. The adrenal content (normalized as per unit protein) of substance P was highest in the young fetuses at 70 days gestation, decreased progressively towards term and, in the adult levels were significantly lower than those measured in the fetuses. The contents of met-enkephalin and leu-enkephalin were low in the young fetuses at 70 days gestation, but reached high levels at 130 to 140 days gestation. Maternal adrenal contents of the two enkephalins were significantly lower than those measured in the near-term fetal adrenal. Total catecholamine content in the fetal adrenal medulla increased as the fetus matured. Norpinephrine was the primary catecholamine present in the medulla of fetuses at 70 and 80 days gestation, while epinephrine was the major one in the adult.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leu-enkephalin-like material in nerves and enterochromaffin cells in the gut

Summary The distribution and cellular localization of leu-enkephalin in the gut and pancreas was studied by immunohistochemistry using two different antisera, one specifically directed against leu-enkephalin and the other cross reacting with met-enkephalin. The results were identical with both antisera. In all species examined, enkephalin-immunoreactive material was found in nerves of the smooth muscle, particularly numerous in the myenteric plexus. Here, immunoreactive nerve cell bodies were observed occasionally. In addition, enkephalin-immunoreactive material was demonstrated in gut endocrine cells of chicken, mouse, rat, pig and monkey but not of guinea pig, cat and man. Enkephalin cells were detected also in the exocrine parenchyma of the porcine pancreas. They were rare in the gut of mouse, rat and monkey but numerous in the antrum and duodenum of pig where they were identified as 5-hydroxytryptamine-storing enterochromaffin cells. The enkephalin-containing cells of the porcine antrum and duodenum were defined ultrastructurally by the consecutive semithin/ultrathin section technique. The ultrastructural features were typical of enterochromaffin cells, the most characteristic ones being the irregular shape and high electron density of the cytoplasmic granules. The immunoreactive material was confined to the cytoplasmic granules.     

Leu-enkephalin-like material in nerves and enterochromaffin cells in the gut. An immunohistochemical study.

The distribution and cellular localization of leu-enkephalin in the gut and pancreas was studied by immunohistochemistry using two different antisera, one specifically directed against leu-enkephalin and the other cross reacting with met-enkephalin. The results were identical with both antisera. In all species examined, enkephalin-immunoreactive material was found in nerves of the smooth muscle, particularly numerous in the myenteric plexus. Here, immunoreactive nerve cell bodies were observed occasionally. In addition, enkephalin-immunoreactive material was demonstrated in gut endocrine cells of chicken, mouse, rat, pig and monkey but not of guinea pig, cat and man. Enkephalin cells were detected also in the exocrine parenchyma of the porcine pancreas. They were rare in the gut of mouse, rat and monkey but numerous in the antrum and duodenum of pig where they were identified as 5-hydroxytryptamine-storing enterochromaffin cells. The enkephalin-containing cells of the porcine antrum and duodenum were defined ultrastructurally by the consecutive semithin/ultrathin section technique. The ultrastructural features were typical of enterochromaffin cells, the most characteristic ones being the irregular shape and high electron density of the cytoplasmic granules. The immunoreactive material was confined to the cytoplasmic granules.     

The structure and expression of the preproenkephalin gene.

Enkephalins are pentapeptides with opioid activity which are found in a wide variety of tissues. Studies of enkephalin-containing peptides from the adrenal gland have established that the mature pentapeptides are derived by proteolytic processing of a precursor protein. We have shown that human adrenal medullary tumours contain mRNA which can be translated in vitro to yield a single major enkephalin precursor. The sequence of cloned cDNA shows that the preproenkephalin mRNA encodes four copies of met-enkephalin, two copies of met-enkephalin extended sequences and one copy of leu-enkephalin; each copy is flanked by paired basic amino acids which are presumably recognised by the processing protease. We have used the cloned human cDNA as a hybridisation probe to detect the corresponding mRNAs in rat adrenal gland and, in smaller amounts, in rat brain. We have been unable to detect in brain any other cross-hybridising mRNAs which might encode other putative precursor proteins.