Modulation of cholinergic neurotransmission in airways by enkephalin

We compared the effects of methionine enkephalin and leucine enkephalin on contractions of isolated canine tracheal smooth muscle strips induced by field electrical stimulation (ES) and exogenous acetylcholine (approximately 10(-5) M). Methionine and leucine enkephalin (10(-8) to 10(-5) M), when added at the peak of airway contractions induced by ES at 1 Hz, depressed the contractions in a concentration-dependent manner by a maximum of 95 and 99%, respectively. Acetylcholine-induced contractions of similar magnitude were depressed only 4% by methionine enkephalin and 12% by leucine enkephalin. Frequency-response curves (0.5-20 Hz) were also obtained before and after incubation of tracheal strips with 10(-5) M methionine and leucine enkephalin. Enkephalin depressed contractions induced by stimulation at 0.5 and 1 Hz by an average of 98 and 95%, respectively. The inhibitory effect of enkephalin progressively decreased at successively higher stimulus frequencies until at 20 Hz there was no significant difference between airway contractions obtained in the presence and absence of enkephalin. Naloxone (3 X 10(-5) M) antagonized the inhibitory effects of both enkephalins. We conclude that methionine and leucine enkephalins inhibit the release of acetylcholine from the postganglionic parasympathetic neurons that innervate airway smooth muscle.     

Enkephalins are transported by a novel eukaryotic peptide uptake system

We have identified an oligopeptide transporter in the yeast Saccharomyces cerevisiae which mediates the uptake of tetra- and pentapeptides, including the endogenous opioids leucine enkephalin (Tyr-Gly-Gly-Phe-Leu) and methionine enkephalin (Tyr-Gly-Gly-Phe-Met). The transporter is encoded by the gene OPT1. Yeast expressing OPT1 can utilize enkephalins to satisfy amino acid auxotrophic requirements for growth. The transport of radiolabeled leucine enkephalin exhibits saturable kinetics, with a K(m) of 310 microM. Transport activity is optimum at acidic pH and sensitive to reagents which uncouple oxidative phosphorylation, suggesting an energy dependence on the proton gradient. Growth, transport, and chromatographic data indicate that leucine enkephalin is not hydrolyzed in the extracellular medium and as such is translocated intact across the cell membrane. The system is specific for tetra- and pentapeptides and can be inhibited by the opioid receptor antagonists naloxone and naltrexone. To date, this is the first example of a eukaryotic transport system which can use enkephalins as a substrate, opening the possibility that a homologue exists in higher eukaryotes.     

Microdialysis and mass spectrometric monitoring of dopamine and enkephalins in the globus pallidus reveal reciprocal interactions that regulate movement

Pallidal dopamine, GABA and the endogenous opioid peptides enkephalins have independently been shown to be important controllers of sensorimotor processes. Using in vivo microdialysis coupled to liquid chromatography-mass spectrometry and a behavioral assay, we explored the interaction between these three neurotransmitters in the rat globus pallidus. Amphetamine (3 mg/kg i.p.) evoked an increase in dopamine, GABA and methionine/leucine enkephalin. Local perfusion of the dopamine D(1) receptor antagonist SCH 23390 (100 μM) fully prevented amphetamine stimulated enkephalin and GABA release in the globus pallidus and greatly suppressed hyperlocomotion. In contrast, the dopamine D(2) receptor antagonist raclopride (100 μM) had only minimal effects suggesting a greater role for pallidal D(1) over D(2) receptors in the regulation of movement. Under basal conditions, opioid receptor blockade by naloxone perfusion (10 μM) in the globus pallidus stimulated GABA and inhibited dopamine release. Amphetamine-stimulated dopamine release and locomotor activation were attenuated by naloxone perfusion with no effect on GABA. These findings demonstrate a functional relationship between pallidal dopamine, GABA and enkephalin systems in the control of locomotor behavior under basal and stimulated conditions. Moreover, these findings demonstrate the usefulness of liquid chromatography-mass spectrometry as an analytical tool when coupled to in vivo microdialysis.     

Role of endogenous opioids in soman (pinacolyl methylphosphonofluoridate)-induced antinociception

The effect of soman poisoning on the levels of methionine enkephalin and beta-endorphin in mice and rats were determined. Soman poisoning produced no significant effect on methionine enkephalin levels in the striatum of rats or mice or beta-endorphin levels in the pituitary gland of mice. In rats beta-endorphin levels were significantly reduced 24 hr post soman poisoning, but returned to control levels by 48 hr. In vitro, the hydrolysis of leucine enkephalin by aminopeptidase was virtually complete by 30 min and found to be the major route of degradation. The release of TYR-GLY-GLY in the presence or absence of puromycin (10 microM) was found to be low (less than or equal to 2.0%). A minor effect on TYR release in the presence of GLY-GLY-PHE-MET (50 microM) was insignificant. Preincubation of mouse striatum homogenates with soman (1 or 10 microM) did not inhibit the hydrolysis of leucine enkephalin. These results suggest that the long term antinociception following soman exposure is not due to either altered concentration of endogenous opioid-like substances or inhibition of the enzymes responsible for their degradation.     

Distribution of a Methionine Enkephalin-Like Substance in Gonads of Drosophila melanogaster

Summary

By means of the peroxidase-antiperoxidase (PAP) technique, methionine enkephalin immunoreactivity was demonstrated in ovaries and testes of Drosophila melanogaster. In all ovaries the germarium is immunopositive. Immunopositivity appears in the follicle cells from stage 6 and 7 and in the ooplasm from stage 7. Increasing immunoreaction was visualised in nurse cells of all successive stages. From stages 11 and 12 on, immunoreactivity is absent. Apart from the mature spermatozoa, all cells of the spermatogenic line are immunopositive. No immunoreactivity is found with antisera against leucine enkephalin and β-endorphin. Possible functions of the methionine enkephalin-like material are discussed.     

Enkephalins increase dopamine levels in the CNS of a marine mollusc.

Intracardiac administration of methionine enkephalin and leucine enkephalin increased dopamine but not serotonin levels in the CNS of the marine mollusc $\text{Mytilus}$$\text{edulis}$. Naloxone blocked the effects of the enkephalins. These responses displayed a time dependent desensitization to methionine enkephalin. The study suggests the presence of an opiate receptor mechanism in this invertebrate species.     

甲硫氨酸脑啡肽对猴免疫缺陷病毒早期感染引起CEM x1 74细胞凋亡的可能作用(英文)

探讨短期甲硫氨酸脑啡肽作用对SIV感染CEMx174细胞凋亡的可能作用 .用MTS法测定甲硫氨酸脑啡肽对感染CEMx174细胞存活率的影响 ,流式细胞仪分析SIV诱导细胞凋亡及其甲硫氨酸脑啡肽的作用 ,并测定了cAMP含量、PKA活性和组蛋白磷酸化的水平 .SIV能够显著减少CEMx174细胞数 ,甲硫氨酸脑啡肽可以提高感染细胞的存活率 .AnnexinⅤ结合实验显示 ,1μmol L甲硫氨酸脑啡肽可以增加存活细胞的比率 ,减少凋亡细胞数 .甲硫氨酸脑啡肽降低正常细胞和感染细胞cAMP的含量和PKA活性 ,但是在感染组较为明显 .在感染的情况下 ,磷酸化的组蛋白增加 ,甲硫氨酸脑啡肽可以减少其含量 .甲硫氨酸脑啡肽的作用可以被纳酪酮所拮抗 .研究结果提示 ,甲硫氨酸脑啡肽对SIV感染引起早期细胞凋亡的作用涉及cAMP PKA信号传递过程     

ATP-stimulated degradation of endogenous proteins in cell-free extracts of BHK 21/C13 fibroblasts. A key role for the proteinase, macropain, in the ubiquitin-dependent degradation of short-lived proteins.

Baby hamster kidney (BHK) 21/C13 cell proteins, labeled with [35S]methionine, [14C]leucine or [3H]leucine in intact cells, were degraded in soluble, cell-free extracts by an ATP-stimulated process. The stimulatory effect of ATP appeared to require ATP hydrolysis and was mediated to a large extent by ubiquitin. Although the cell extracts contained endogenous ubiquitin, supplementation with exogenous ubiquitin increased ATP-dependent proteolysis by up to 2-fold. Furthermore, antibodies against the E1 ubiquitin conjugating enzyme specifically inhibited both conjugation of [125I]ubiquitin to endogenous proteins and ATP/ubiquitin-dependent proteolysis. Addition of purified E1 to antibody-treated extracts restored conjugation and proteolysis. Proteins containing the amino acid analogues canavanine and azatryptophan were also degraded in vitro by an ATP/ubiquitin-dependent process but at a rate up to 2-fold faster than normal proteins. These results indicate that soluble, cell-free extracts of BHK cells can selectively degrade proteins whose rates of degradation are increased in intact cells. Treatment of cell-free extracts with antibodies against the high molecular weight proteinase, macropain, also greatly inhibited the ATP/ubiquitin-dependent degradation of endogenous proteins. Proteolysis was specifically restored when purified macropain L was added to the antibody-treated extracts. Treatment of cell extracts with both anti-macropain and anti-E1 antibodies reduced ATP/ubiquitin-dependent proteolysis to the same extent as treatment with either antibody alone. Furthermore, proteolysis could be restored to the double antibody treated extracts only after addition of both purified E1 and macropain. These results provide strong evidence for an important role for macropain in the ATP/ubiquitin-dependent degradation of endogenous proteins in BHK cell extracts.     

Involvement of caspase-3 pathway in anti-apoptotic action of methionine enkephalin on CEM x 174 cells in prolonged infection with simian immunodeficiency virus in vitro

The roles of methionine enkephalin, as an immunomodulator, on immunodeficiency virus-induced apoptosis of lymphocytes during prolonged infection are still unclear. In the present study, we evaluated the effects of methionine enkephalin on the viability, the profile of cell cycle and apoptosis, as well as the expression of apoptosis-related genes in CEM x 174 cells infected with simian immunodeficiency virus for 72 h. Our data demonstrated that methionine enkephalin maintains the viability of cells during the period of prolonged infection. Following co-incubation with the virus, CEM x 174 cells were arrested at S phase, with increased mortality as a result of apoptosis. Methionine enkephalin could abolish virus-induced over-expression of caspase-3. Taken together all findings, we conclude that methionine enkephalin may maintain the viability of SIV-infected cells via suppressing the expression of caspase-3, which may have clinical implications in opioid peptide therapy for AIDS.     

Double-enkephalins—Synthesis,activity on guinea-pig ileum,and analgesic effect

We have synthesized enkephalin analogues in which C-terminal methionine or leucine residues are replaced by a second active fragment of the enkephalin analogue. Synthesis of two compounds is described: in one, two fragments of a D-Ala²-enkephalin analogue are connected by a -NH-NH-bridge, and in the other, three methylene groups are incorporated between the amino groups. The first compound is a very potent inhibitor of electrically induced contractions of guinea-pig ileum and produces a strong analgesia when administered intraperitoneally in mice. The second compound is less active on the ileum and fails to produce analgesia after systemic injection. The double-enkephalins may interact with μ-receptors.     

Fast-atom-bombardment mass spectra of enkephalins.

The positive- and negative-ion mass spectra of [methionine]enkephalin and [leucine]enkephalin have been obtained by using a fast-atom-bombardment source described previously by Barber, Bordoli, Sedgwick & Tyler [(1981) J. Chem. Soc. Chem. Commun., in the press]. This technique has allowed the spectra to be obtained without conversion of the enkephalins into volatile derivatives. The fast-atom-bombardment spectra show good pseudo-molecular-ion sensitivity and fragmentation that can be interpreted on the basis of the known molecular structure.     

The destruction of allogeneic tumour cells by antibody and adherent cells from peritoneal cavities of mice.

Antisera to a DBA2 lymphoma (SL2) were raised in C57 black mice. The sera contained cell-dependent antibodies which lysed SL2 cells in conjunction with a monolayer of adherent peritoneal cells from unimmunised mice. The strongest lytic reaction was observed when the three components of the system, monolayer, target, and antiserum, were incubated together. The free antibody was not cytophilic for macrophages. It combined specifically with the target cell but precoated SL2 cells were not lysed effectively, probably because cells in the monolayer also accelerated the inactivation of antibody on the surface of the target cell.     

Voltage-regulated calcium channels involved in the regulation of enkephalin synthesis are blocked by phorbol ester treatment

Treatment of bovine chromaffin cells with 40 mM KCl stimulates a 3-fold increase in total methionine enkephalin immunoreactivity (medium plus cells) and a 4-fold increase in proenkephalin mRNA (mRNAenk). These effects of KCl, which are dependent on extracellular calcium, can be blocked by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), although release of methionine enkephalin appears less affected. Using fura-2-loaded chromaffin cells and a dual-excitation wavelength spectrofluorometer, we have examined whether the actions of KCl and TPA on methionine enkephalin synthesis and release can be explained by changes in intracellular free calcium ([Ca2+]i). KCl produced a rapid 600 nM increase in [Ca2+]i from resting levels of approximately 170 nM. Subsequently, [Ca2+]i declined to a new steady-state plateau which was approximately 275 nM higher than the original resting levels. The postdepolarization plateau of [Ca2+]i was reduced by TPA, (-)-(R)-202,791 (a dihydropyridine calcium channel antagonist), and LaCl3 (a nonselective calcium channel blocker). TPA also inhibited potentiation of the KCl-stimulated plateau of [Ca2+]i due to (+)-(S)-202,791, a calcium channel agonist. In contrast, TPA had no effect on resting [Ca2+]i and only slightly inhibited the initial rapid KCl-stimulated increase in [Ca2+]i. The inhibitory effects were maintained for 24 h in the continuous presence of TPA. We conclude 1) that TPA inhibits enkephalin synthesis by inactivating dihydropyridine-sensitive voltage-dependent calcium channels, 2) that these channels alone maintain elevated [Ca2+]i following KCl depolarization, and 3) that sustained elevation in [Ca2+]i is necessary in order to increase enkephalin synthesis in KCl-treated chromaffin cells.     

Structural and immunochemical studies of the lipopolysaccharides of Salmonella strains with both antigen O4 and antigen O9.

Two Salmonella hybrid strains, SL5313 (Salmonella typhimurium with a D.rfb+ gene cluster) and SL5396 (S. enteritidis with a B.rfb+ gene cluster), each expressing both O-antigen 4 (of serogroup B) and O-antigen 9 (of serogroup D) were studied by immunofluorescence using a mixture of O4-specific mouse monoclonal and O9-specific rabbit polyclonal antibodies. Bound antibodies, detected by anti-mouse antibody labelled with fluorescein isothiocyanate and anti-rabbit antibody labelled with tetramethylrhodamine isothiocyanate showed that more than 98% of the bacteria expressed both the O4 and O9 epitopes. Phenol-water-extracted lipopolysaccharide from batch-grown cultures subjected to sugar and methylation analyses by gas-liquid chromatography and mass spectrometry were shown to contain abequose (of the O4 epitope) and tyvelose (of the O9 epitope) in ratios of 1:1.5 and 1:2.5 for SL5313 and SL5396, respectively. Isolated polysaccharide chains, obtained by weak-acid hydrolysis of the lipopolysaccharides, were found to contain both O4 and O9 specificities in the same molecule, since polysaccharide bound to O4 antibody attached to a solid-phase-adsorbed O9-specific antibody and vice versa. This demonstrates that in strains SL5313 and SL5396 O chains containing both O4 repeating units (from S. typhimurium) and O9 units (from S. enteritidis) are present.     

Human large granular lymphocytes express high affinity receptors for murine monoclonal antibodies of the IgG3 subclass

We have evaluated the ability of murine monoclonal antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) by human lymphoid cells. Purified large granular lymphocytes (LGL) and interleukin 2-dependent cloned LGL lines having a CD2+/CD16+/CD3- phenotype were tested as effector cells in an ADCC assay by using a family of IgG isotype switch variant anti-Thy-1.1 antibodies against 51Cr-labeled Thy-1.1+ murine SL2 thymoma target cells, a system in which human cells have no spontaneous cytotoxicity. Cytotoxicity was greatest when using the IgG3, followed in rank order by the IgG2a and IgG2b. No cytotoxicity was observed with the IgG1 antibody. Because the antigen-binding regions of the antibodies are identical, the differences in cytotoxicity directly reflect the relative affinity of LGL Fc receptors for each antibody isotype.     

Influence of antibody isotype on passive serotherapy of lymphoma

We assessed the in vivo anti-tumor effectiveness of monoclonal antibodies of different isotypes. Starting with a hybridoma cell secreting an IgG3 anti-Thy-1.1 antibody, we isolated three variant hybridoma cell lines secreting anti-Thy-1.1 antibody of the IgG1, IgG2a, and IgG2b isotypes. Each antibody displayed identical antigen binding properties, but differed in their ability to mediate in vitro lysis of Thy-1.1+ AKR/J SL2 lymphoma cells. In assays of complement dependent cytotoxicity, the relative activity of each antibody isotype was IgG2a = IgG2b greater than IgG3 greater than IgG1. In assays of antibody-dependent cell-mediated cytotoxicity when using non-immune spleen cells as effectors, the relative activities were IgG2a greater than or equal to IgG2b greater than IgG1 greater than IgG3. Infusion of equivalent amounts of each antibody (1.5 mg) in AKR/Cum (Thy-1.2+) mice inoculated subcutaneously with 3 X 10(5) AKR/J SL2 lymphoma cells resulted in significant inhibition of tumor growth only in mice treated with IgG2a antibody. However, the antibodies were cleared at different rates, with the IgG2a antibody having the slowest clearance. When antibody doses were adjusted to achieve equivalent serum levels 24 hr after infusion, all of the antibody isotypes exhibited at least some anti-tumor activity, although IgG2a antibody was again the most effective. These studies demonstrate that the difference in anti-tumor activity between antibodies of different isotypes may result from differences both in their serum clearance rate and their ability to interact with host effector mechanisms.     

Electron microscopic study of the optic tentacle collar cells of the snail Helix aspersa and immunolocalization of methionine enkephalin-like material in their granular content

An ultrastructural and immunocytological study was carried out on the collar cells of the optic tentacle of Helix aspersa. These cells are supposed to be the source of a reproduction controlling hormone. The immunocytological study was performed using an anti-methionine enkephalin antibody obtained from rabbits in our laboratory. The collar cells are characterized by an enlarged rough endoplasmic reticulum, numerous mitochondria and Golgi bodies surrounded by secretory vesicles, suggesting an intense synthesizing activity. Their principal feature consists of numerous various-sized granules where methionine enkephalin immunoreactivity is localized. No classical neurosecretory granules are observed while synapse-like structures are often encountered. The cells should not be regarded as neurosecretory cells but rather as glandular cells which could ensure different functions, one in relation to reproduction, and another in relation to perception processes, particularly as they contain methionine enkephalin-like material.     

Electron microscopic study of the optic tentacle collar cells of the snail Helix aspersa and immunolocalization of methionine enkephalin-like material in their granular content

Summary— An ultrastructural and immunocytological study was carried out on the collar cells of the optic tentacle of Helix aspersa. These cells are supposed to be the source of a reproduction controlling hormone. The immunocytological study was performed using an anti-methionine enkephalin antibody obtained from rabbits in our laboratory. The collar cells are characterized by an enlarged rough endoplasmic reticulum, numerous mitochondria and Golgi bodies surrounded by secretory vesicles, suggesting an intense synthesizing activity. Their principal feature consists of numerous various-sized granules where methionine enkephalin immunoreactivity is localized. No classical neurosecretory granules are observed while synapse-like structures are often encountered. The cells should not be regarded as neurosecretory cells but rather as glandular cells which could ensure different functions, one in relation to reproduction, and another in relation to perception processes, particularly as they contain methionine enkephalin-like material.     

Tyr-MIF-1 and Met-enkephalin share a saturable blood-brain barrier transport system

Previous studies have shown that methionine enkephalin and Tyr-MIF-1 are transported from the brain to the blood by a saturable, stereospecific, carrier-mediated process. It was not established by these studies whether Tyr-MIF-1 and methionine enkephalin were transported by the same system or by separate, but overlapping systems. This issue was investigated in anesthetized mice receiving injections containing both 131I-methionine enkephalin and 125I-Tyr-MIF-1 into the lateral ventricle of the brain. Mice were decapitated and the brain to blood transport rate was derived from the residual counts in the brain. It was found that in individual mice, the transport rate for Tyr-MIF-1 correlated highly with the transport rate for methionine enkephalin but not with the transport of iodide. This shows that the transport of Tyr-MIF-1 is closely coupled to the transport of methionine enkephalin but dissociable from the brain to blood transport of iodide. Furthermore, the inability of varying doses of Tyr-MIF-1 or of methionine enkephalin to preferentially self-inhibit is radiolabeled form in comparison with the other peptide shows that, functionally, only a single system exists. Aluminum, a noncompetitive inhibitor of Tyr-MIF-1 transport, was also without preferential inhibition. Thus, under the conditions of these studies, only a single system could be functionally demonstrated for the transport of both Tyr-MIF-1 and methionine enkephalin.     

Evidence for morphine and morphine-like alkaloid responses resistant to naloxone blockade in the rat vas deferens.

The application of morphine or surrogates to the isolated rat vas deferens maintained at 37° C in Tyrode solution, produced an increase in the electrically induced muscular twitch. In contrast, leucine enkephalin or D-alanine₂methionine enkephalinamide produced a dose-dependent inhibition of the muscular twitch. The effect of morphine and derivatives was not antagonized by naloxone, but the depression caused by the opiate pentapeptides or β-Endorphin was readily antagonized and reversed by naloxone. Tolerance developed to the $\text{in vitro}$ effect of morphine; vasa deferentia obtained from tolerant-dependent rats were about six times less sensitive to the effect of morphine and about five times less sensitive to the depression caused by leucine enkephalin as compared to their respective paired, placebo implanted control rats.