Effects of bromocriptine on receptor binding of methionine-enkephalin

Bromocriptine inhibited the binding of methionine-enkephalin (ENK) to rat striatal synaptic membranes in a dose-dependent fashion. The bromocriptine IC50 was 16 micrometers. hill coefficients of bromocriptine were 0.7 and 0.3, suggesting that an allosteric effect was involved in bromocriptine inhibition of ENK binding to its receptor. These data suggest that at least a part of the therapeutic antiparkinsonian effect f bromocriptine is its allosteric effects on the ENK receptor, which influences the function of striatal dopamine neurons and/or of striatal cholinergic neurons.     

Evidence for the coexistence and co-release of [met]enkephalin and noradrenaline from sympathetic nerves of the bovine vas deferens

The localization and neurosecretion of methionine-enkephalin was studied in sympathetic nerves of the bovine vas deferens. Immunostaining showed methionine-enkephalin-like immunoreactivity in a network of varicose nerve fibres in the smooth muscle layers of the vas deferens. When vas deferens homogenates were subjected to differential and sucrose density gradient centrifugation, methionineenkephalin was found to parallel the distribution of noradrenaline in the more dense region of the gradient, where “heavy” or large dense-cored vesicles are present. Electron microscopic immunochemistry confirmed this finding and showed methionine-enkephalin-like immunoreactivity in large dense-cored vesicles. The release of methionine-enkephalin upon electrical stimulation was studied in superfusion experiments. The methionine-enkephalin secretion was shown to be Ca²⁺-dependent and was inhibited by adding the adrenergic neuron blocking drug guanethidine to the superfusion medium.

We conclude that in the bovine vas deferens methionine-enkephalin is only present in large dense-cored vesicles of adrenergic neurons and that the peptide is released from these vesicles together with noradrenaline by a Ca²⁺-dependent mechanism.     

Paraganglia of the gallbladder: a report of two cases with an immunohistochemical study

Paraganglionic tissues incidentally observed in the gallbladder are presented. The patients, a 51- and a 55-year-old woman, underwent gallbladder resection for chronic cholecystitis with gallstones. Two and one paraganglionic tissues were observed in the subserosal connective tissue of the two gallbladders, respectively. Immunohistochemically, the chief cells were positive for chromogranin A, and the sustentacular cells were positive for S100 protein. Tyrosine hydroxylase (in two of three), dopamine beta-hydroxylase (in one of three), methionine-enkephalin (in two of three), and leucine-enkephalin (in two of three) were also positive in a small amount of the chief cells. These structures, which slightly resembled adrenal medulla or retroperitoneal paraganglia, might be misunderstood as an infiltration of primary or metastatic carcinoma into the subserosal connective tissue.     

Astrocyte opioid receptors: activation modifies the noradrenaline-evoked increase in 2-[14C]deoxyglucose incorporation into glycogen

Astrocyte-enriched cultures of the newborn rat cortex accumulated 2-[14C]deoxyglucose (2-DG), a proportion portion of which was incorporated into glycogen. Exposure to exogenous noradrenaline resulted in an increase (30%) in the incorporation of 2-[14C]DG into glycogen in these cultures. The extent to which noradrenaline evoked an increase in 2-[14C]DG labelling of glycogen was modified by both morphine and methionine-enkephalin. Whereas morphine augmented the noradrenaline-induced increase in 2-[14C]DG incorporation into glycogen in these cultures, methionine-enkephalin attenuated this response.     

Enkephalins and anaphylactic shock: modulation and prevention of shock in the rat

Wistar rats were sensitized for anaphylactic shock with ovalbumin and treated intraperitoneally with 10 injections of methionine-enkephalin and leucine-enkephalin (4 mg/kg of body weight). Both pentapeptides suppressed the development of systemic anaphylaxis after intravenous injection of a shocking dose of ovalbumin. Methionine-enkephalin completely protected the animals from fatal shock. The level of circulating IgE and precipitating anti-ovalbumin antibodies decreased in rats given 10 injections of enkephalins. Mast cell degranulation was significantly inhibited in these animals as observed at autopsy. A single intraperitoneal injection of 4 mg/kg body weight of methionine-enkephalin given 30 min before the challenge with shock-inducing dose of antigen was also effective, although to a lesser extent, in protecting animals from anaphylactic shock. One injection of leucine-enkephalin did not exhibit significant antishock activity. The results suggest that enkephalins, and methionine-enkephalin in particular, are potent modulators of the complex biochemical processes underlying the anaphylactic shock in the rat.     

Methionine-enkephalin as immunomodulator therapy in human immunodeficiency virus infections: Clinical and immunological effects

Enkephalins have been shown to enhance T cell-mediated immune responses and natural killer-cell activityin vitro. We have studied the effects of infusions of methionine-enkephalin on immune functions and clinical courses in seven patients with various stages of infection with human immunodeficiency virus (HIV). All patients were clinically stable at the time of entry into the study. Each received 10 µg/kg of methionine-enkephalin in an intravenous infusion three times weekly for up to 12 weeks. Evaluation of cellular immunity (T-cell subsets,in vitro interleukin-2 production and interleukin-2 receptor expression, T-cell responses to mitogens and antigens, and delayed-hypersensitivity skin tests) as well as clinical and toxicity monitoring was performed prior to treatment, at 2-week intervals during treatment, and after the cessation of treatment. Increases in interleukin-2 receptor expression were seen on lymphocytes collected on one occasion from each of two patients 30 min postinfusion. Studies done 24 hr after infusions revealed increases in interleukin-2 production in one patient, but when pre- and posttreatment values were compared there were no significant changes in numbers of circulating T cells of any phenotype or in T-cell responses to mitogens or antigens. None of the patients with Kaposi's sarcoma had regression of tumor; one patient dropped out of the study at week 5 because of deteriorating clinical status and progression of tumor. There were no adverse reactions or evidence of toxicity. We conclude that methionine-enkephalin appears to enhance temporarily selected immune responses in patients with HIV infection, however, in the schedule used in this study it was not clinically efficacious.     

Serum acetylcholinesterase possesses trypsin-like and carboxypeptidase B-like activity

Acetylcholinesterase (AChE, EC 3.1.1.7) purified from the electric organ of eel possesses a protease activity resembling that of a neuropeptide processing enzyme. To examine whether any mammalian AChEs possess a similar protease activity, the enzyme was purified, 110,000-fold from foetal bovine serum. Purified serum AChE cleaved 2 synthetic peptide substrates in a manner resembling the combined actions of trypsin-like and carboxypeptidase B-like enzymes. A synthetic fragment of preproenkephalin A (residues 97-107) containing a complete methionine-enkephalin sequence was cleaved by serum AChE to yield free methionine-enkephalin. The carboxypeptidase action of AChE was weakly stimulated by the presence of 100 microM CoCl2 suggesting the requirement of a metal ion for complete activity. The results support the hypothesis that in many tissues AChE may act as a neuropeptide processing enzyme.     

Effect of intrathecal capsaicin analogues on the immunofluorescence of peptides and serotonin in the dorsal horn in rats

The intrathecal administration of capsaicin, a homovanillylamide derivative, has been demonstrated to cause analgesia in response to thermal stimuli. This analgesia has been correlated with a profound depletion of spinal substance P, a putative primary afferent transmitter. We studied the effects of capsaicin, a series of capsaicin analogues, piperine and kainic acid on the immunohistochemical staining of substance P, cholecystokinin, somatostatin, methionine-enkephalin and serotonin. Capsaicin and an analogue 1-nonenoyl-vanillylamide significantly elevated the tail flick latency and when the spinal cords of the rats were analyzed immunohistochemically, a profound depletion of substance P and cholecystokinin was observed. The spinal somatostatin-immunoreactivity of these rats was slightly reduced. Piperine also depleted substance P and reduced somatostatin staining but did not alter the staining intensity or density of cholecystokinin, methionine-enkephalin or serotonin. Kainate-depleted methionine-enkephalin but did not alter any other neuropeptides studied or serotonin. These results may indicate a link between capsaicin-induced analgesia and the concomitant depletion of cholecystokinin and substance P.     

Distribution of methionine-enkephalin in the minipig brainstem

We have studied the distribution of immunoreactive cell bodies and axons are containing methionine-enkephalin in the minipig brainstem. Immunoreactive axons were widely distributed, whereas the distribution of perikarya was less widespread. A high or moderate density of axons containing methionine-enkephalin were found from rostral to caudal levels in the substantia nigra, nucleus interpeduncularis, nucleus reticularis tegmenti pontis, nucleus dorsalis raphae, nucleus centralis raphae, nuclei dorsalis and ventralis tegmenti of Gudden, locus ceruleus, nucleus sensorius principalis nervi trigemini, nucleus cuneatus externalis, nucleus tractus solitarius, nuclei vestibularis inferior and medialis, nucleus ambiguus, nucleus olivaris inferior and in the nucleus tractus spinalis nervi trigemini. Immunoreactive perikarya were observed in the nuclei centralis and dorsalis raphae, nucleus motorius nervi trigemini, nucleus centralis superior, nucleus nervi facialis, nuclei parabrachialis medialis and lateralis, nucleus ventralis raphae, nucleus reticularis lateralis and in the formatio reticularis. We have also described the presence of perikarya containing methionine-enkephalin in the nuclei nervi abducens, ruber, nervi oculomotorius and nervi trochlearis. These results suggest that in the minipig the pentapeptide may be involved in many physiological functions (for example, proprioceptive and nociceptive information; motor, respiratory and cardiovascular mechanisms).     

Neuroimmunomodulation with enkephalins: enhancement of human natural killer (NK) cell activity in vitro

To further define the effects of enkephalins on immune function, the effect of methionine-enkephalin and leucine-enkephalin on natural killer cell (NK) activity in isolated human peripheral blood lymphocytes was investigated. Incubation of lymphocytes with either enkephalin resulted in significant increases in natural killer cell activity. At effector:target cell ratios of 11:1 methionine-enkephalin significantly (P less than 0.05) enhanced NK activity at dilutions of 10(-6), 10(-8), 10(-10), and 10(-14) mg/ml, while leucine-enkephalin significantly (P less than 0.05) enhanced NK activity at dilutions of 10(-4), 10(-6), 10(-8), 10(-10), and 10(-14) mg/ml. Cells from individuals with low NK activity showed greater percentage increases in NK activity following enkephalin than did cells from individuals with high NK activity.     

Electrophysiological properties of catecholaminergic neurons in the norepinephrine-deficient mouse

To determine how norepinephrine affects the basic physiological properties of catecholaminergic neurons, brain slices containing the substantia nigra pars compacta and locus coeruleus were studied with cell-attached and whole-cell recordings in control and dopamine beta-hydroxylase knockout (Dbh -/-) mice that lack norepinephrine. In the cell-attached configuration, the spontaneous firing rate and pattern of locus coeruleus neurons recorded from Dbh -/- mice were the same as the firing rate and pattern recorded from heterozygous littermates (Dbh +/-). During whole-cell recordings, synaptic stimulation produced an alpha-2 receptor-mediated outward current in the locus coeruleus of control mice that was absent in Dbh -/- mice. Normal alpha-2 mediated outward currents were restored in Dbh -/- slices after pre-incubation with norepinephrine. Locus coeruleus neurons also displayed similar changes in holding current in response to bath application of norepinephrine, UK 14304, and methionine-enkephalin. Dopamine neurons recorded in the substantia nigra pars compacta similarly showed no differences between slices harvested from Dbh -/- and control mice. These results indicate that endogenous norepinephrine is not necessary for the expression of catecholaminergic neuron firing properties or responses to direct agonists, but is necessary for auto-inhibition mediated by indirect alpha-2 receptor stimulation.     

Pharmacological analysis of the mechanism of action for colonic contraction induced by neurotensin, substance P and methionine-enkephalin

Infusion of neurotensin, substance P and methionine-enkephalin induces colonic contraction in the cat. The present study was performed to investigate the effect of various pharmacological blocking agents on colonic contraction evoked by these peptides infused by the i.a. or i.v. route. The contractions caused by infusions of neurotensin were blocked by tetrodotoxin (1 micrograms kg-1 i.a.), hexamethionium (10 mg kg-1 i.v.), atropine (0.1 mg kg-1 i.v.) or somatostatin (100 pmol min-1 i.a.), but not by haloperidol, methysergide, mepyramine, cimetidine or naloxone. The contractile effect of substance P on the colon was abolished by the substance P receptor antagonist (D-Arg1, D-Pro2, D-Trp7,9, Leu11)-substance P (70 nmol min-1 i.a.). No other blockers used, such as tetrodotoxin, hexamethonium, atropine, mepyramine, cimetidine, methysergide, naloxone or somatostatin inhibited the response to substance P. Methionine-enkephalin produced a colonic contraction that was completely blocked by naloxone (1 mg kg-1 i.a.). Both atropine (0.1 mg kg-1 i.v.) and somatostatin (100 pmol min-1 i.a.) reduced the contractile response. However, tetrodotoxin, hexamethonium, mepyramine, cimetidine and methysergide did not affect the response to methionine-enkephalin. All adrenergic blockers tested, that is, guanethidine, propranolol and phentolamine, increased the contractile responses to the peptides. The results indicate that the colonic contraction induced by neurotensin is mediated via nervous cholinergic pathways. Substance P induces colonic contraction, probably by a direct effect on smooth muscle substance P receptors. Methionine-enkephalin induces colonic contraction which could be blocked by naloxone. However, a cholinergic or peptidergic link may also be involved in the response to methionine-enkephalin.     

Synaptic association between enkephalin-containing axon terminals and proopiomelanocortin-containing neurons in the arcuate nucleus of rat hypothalamus

By employing an electron microscopic dual immunolabeling technique, a synaptic association between neurons containing immunoreactive adrenocorticotropin (ACTH) and axonal terminals containing immunoreactive methionine-enkephalin octapeptide (Enk-8) was found in the arcuate nucleus of the rat hypothalamus. The axonal terminals contained many small clear vesicles and some large cored vesicles. At the synaptic portions, membrane specialization was asymmetric.     

Phencyclidine decreases methionine-enkephalin content in rat striatum

The acute administration of phencyclidine induced a decrease in methionine-enkephalin levels in rat striatum, as measured by specific radioimmunoassay. The maximum decrease (about 40% with respect to controls) was obtained at a dose of 20 mg/kg, 30 min after intraperitoneal injection. These results suggest a possible involvement of enkephalins in the central effects of phencyclidine.     

Co-existence of unrelated peptides in oxytocin and vasopressin terminals of rat neurohypophyses: Immunoreactive methionine5-enkephalin-, leucine5- enkephalin- and cholecystokinin-like substances

In neurohypophyses of normal rats and of vasopressin-deficient Brattleboro rats, applying immunocytochemical methods, we have found (1) in oxytocin terminals co-existing methionine-enkephalin-, cholecystokinin- and possibly leucine-enkephalin-like substances and (2) in vasopressin terminals, co-existing leucine-enkephalin-like material. The correspondence between the enkephalin immunoreactivity patterns and the oxytocin or vasopressin immunoreactivity pattern in serial 0.5 μ thick sections was so close that occurrence of enkephalin in separate nerves appears unlikely. At a subcellular level the enkephalin- and the cholecystokinin-like immunoreactivities were localized mainly in the neurosecretory granules that also contain oxytocin or vasopressin; there was no evidence for separate enkephalingranule populations. The intensity and frequency of leucine-enkephalin immunostaining in vasopressin terminals was much enhanced by treatment of the deplasticized sections with trypsin prior to incubation with antibodies. This suggests incorporation of the leucine-enkephalin sequence into longer peptide chains, presumably dynorphin and/or α-neo-endorphin. In oxytocin endings tryptic cleavage enhanced cholecystokinin-like immunoreactivity, while methionine-enkephalin immunostaining was as intense without enzyme treatment.The opioid peptide forms of vasopressin neurons appear to be elongated chains containing exclusively the leucine-enkephalin sequence, whereas in oxytocin neurons methionine-enkephalin as free pentapeptide seems to prevail. Association of enkephalins with vasopressin and oxytocin was also indicated when methionine- and leucine-enkephalin contents of neurointermediate lobes of homozygous Brattleboro rats were compared with peptide stores in heterozygous controls in a high pressure liquid chromatography system and highly specific leucine- and methionine-enkephalin radioimmunoassays. We assume that the opioid peptides in the oxytocin system and in the vasopressin system are derived from different opioid precursor molecules.     

Adrenal release of catecholamines and Met-enkephalin before and after stress as measured by a novel in vivo dialysis method in the rat

A dialysis tubing was implanted in the adrenal gland of rats. Adrenomedullary secretion products were dialysed into Ringer solution in the awake animal. Catecholamines (CAs) and methionine-enkephalin (Met-Enk) were measurable under resting conditions. Epinephrine, norepinephrine and Met-Enk-like immunoreactive material increased following application of an immobilization stress for 5 min. The results demonstrate that adrenomedullary CAs and peptides are coreleased. They also demonstrate that Met-Enk-like immunoreactive material is released under stress.     

Human spinal neurons: innervation by both substance P and enkephalin

The distribution of the peptides substance P and methionine-enkephalin in relation to neurons in the human thoracic and lumbar spinal cord has been studied with the unlabeled peroxidase anti-peroxidase method employing antibodies directed against substance P and methionine-enkephalin. The peptides have been localized in varicosities and terminal-like processes in close apposition to the soma and proximal dendrites of neurons in the marginal zone, the intermediolateral nucleus, intermediomedial nucleus, the reticular nucleus of lamina V and the ventral horn; some cells in all these regions are innervated by both peptides.The findings provide morphological evidence of these peptides in the human spinal cord. The demonstration of innervation by both peptides of single neurons in laminae I and V provides some morphological basis to models that postulate the interaction of substance P and enkephalin in pain modulation. The innervation of other neuron types indicates that these neuropeptides may be involved in other spinal functions as well.     

Effects of morphine and opioid peptides on sensitivity to acetylcholine of dialysed snail neurons

The effects of intracellular and extracellular applications of morphine (in concentrations from 10(-3) to 10(-5) M), leucine-enkephalin and methionine-enkephalin (10(-6) to 10(-8) M) were studied in unidentified acetylcholine-sensitive dialysed neurons of a snail under voltage clamp. Morphine produced inward membrane currents, while enkephalins did not. Both morphine and enkephalins altered the effect of acetylcholine on postsynaptic acetylcholine receptors; intracellular application of these substances being much more effective than extracellular application. This suggested that opioid peptides take part in the regulation of cholinergic synaptic transmission.     

Further studies of the changes in alkaline secretion, transepithelial potential difference and net fluid transport induced by the heat-stable enterotoxin of Escherichia coli (STa) in the rat jejunum in vivo

A pH-stat technique was used to study the mechanisms underlying the intestinal alkalinization evoked by the heat-stable enterotoxin of Escherichia coli (STa) in the rat denervated jejunum in vivo. In addition, concomitant changes of transepithelial potential difference and fluid transport were also investigated. To test the possible involvement of the enteric nervous system in the STa-stimulated alkaline secretion and potential difference, the mesenteric nerves were electrically stimulated or neuropeptide Y or methionine-enkephalin was infused intravenously. None of these interventions inhibited to any large extent the STa-stimulated alkaline secretion, whereas a greater suppression was noted on the concomitantly increased potential difference. Furthermore, neuropeptide Y but not methionine-enkephalin significantly inhibited STa-induced jejunal fluid secretion although neuropeptide Y was without effect on basal fluid transport. It is concluded that the enteric nervous reflex(es) which are of significant importance in explaining STa-evoked fluid secretion plays a minor role in controlling alkaline secretion. Furthermore, alkaline secretion may not contribute to the increase in potential difference caused by STa Amiloride (10(-4) or 10(-3) M) had no effect on the STa-stimulated alkaline secretion, implying that some cellular mechanism other than an inhibition of Na+/H+ exchanger explains the observed response. Similarly, acetazolamide had no effect on the STa-stimulated alkaline secretion or potential difference, suggesting that the secreted alkaline is of extracellular origin rather than from the cellular metabolism in the enterocytes.