Release Of Met-enkephalin Research Articles

Presynaptic opioid κ-receptor and regulation of the release of Met-enkephalin in the rat brainstem

Opioid κ-agonists had much more potent inhibitory effects on the high K +-evoked Met-enkephalin release from rat brain slices than did the μ- or δ-agonists. The opioid κ-antagonist, MR2266 enhanced the evoked release of Met-enkephalin to a greater extent than did μ- or δ-antagonists in vitro and had a potent analgesia in mice in vivo. These findings suggest that the release of Met-enkephalin may be regulated in vitro and in vivo, mainly by presynaptic κ-receptor-mediated mechanisms.

A Met-enkephalin releaser (kyotorphin)-induced release of plasma membrane-bound Ca 2+ from rat brain synaptosomes

The neuropeptide kyotorphin (Tyr-Arg) released 45Ca 2+ from synaptosomal membrane preparations of the rat lower brainstem. This dipeptide also decreased plasma membrane-bound Ca 2+ ([Ca 2+] m), determined using chlorotetracycline, in the slice and synaptosome of the rat lower brainstem. Taken together with other data indicating that kyotorphin induces an increase in synaptosomal intracellular Ca 2+ ([Ca 2+] i) in a Ca 2+ channel-independent manner, it is suggested that kyotorphin may mobilize the [Ca 2+] m and thus increase the [Ca 2+] i in the nerve terminals of the rat lower brainstem.

Chromogranin A synthesis and secretion in chromaffin cells.

A sensitive and selective radioimmunoassay for chromogranin A (Chrg A) has been developed to quantitate content, release, and biosynthesis of this secretory protein in neuroendocrine tissues. An antiserum raised against Chrg A from bovine adrenal medulla was found to detect predominantly only the Mr 70-75 kilodalton Chrg A in its native form, allowing the use of this antiserum as a quantitatively specific probe for Chrg A in cell-free extracts of the adrenal medulla and chromaffin cells. Chrg A comprises about 10% of the total protein of the chromaffin cell. It is released in parallel with Met-enkephalin and catecholamines from the bovine chromaffin cell in primary culture in response to nicotine and nicotinic cholinergic agonists. From 14 to 22% of total Chrg A is released from the cell during a 15-min exposure to a maximally stimulatory dose of nicotine (10-100 microM). Chrg A release on nicotinic stimulation is blocked by D-600 and hexamethonium to the same extent as Met-enkephalin and catecholamine release. The parallel time course and percent release of Chrg A and Met-enkephalin indicate that these secretory polypeptides are contained in, and released from, functionally identical cellular compartments. Chrg A and Met-enkephalin pentapeptide sequences are present in the chromaffin cell at a ratio of about 2:1, although Chrg A is far more abundant on a mass basis. Chrg A and Met-enkephalin biosynthesis appear to be differentially regulated within the chromaffin cell, since chronic treatment of cells with nicotine and forskolin causes an elevation of Met-enkephalin pentapeptide without a concomitant elevation of intracellular levels of Chrg A.

Involvement of the dorsolateral funiculi in the spinal release of Met-enkephalin-like material triggered by heterosegmental noxious mechanical stimuli

The lumbar spinal cord was superfused with artificial CSF at a rate of 0.1 ml/min in halothane anaesthetized rats. Under resting conditions, Met-enkephalin-like material (MELM) found in superfusates corresponded to a spinal release of 4.2 ± 1.4pg Met-enkephalin equivalents per 5 min. During a 30-min period in which pinches were applied to the muzzle, the MELM content in the superfusates increased markedly (by 120.5 ± 32.9%). This effect was totally suppressed following bilateral lesions of the dorsolateral funiculi (DLF), under both chronic and acute conditions. It is concluded that strong mechanical stimuli applied in the trigeminal region can induce the release of MELM within the lumbar spinal cord via mechanisms involving the DLF. This heterosegmental release of Met-enkephalin may participate in the management of pain by methods involving high intensity stimulation.

Enkephalin production by the corpus luteum

In search of Early pregnancy factors, on the base of Kentsin, the sole molecule embryo-specific, we detected the presence of Enkephalin in the Mouse ovary, in Bovine and Human corpus luteum. In the Mouse the Met-Enkephalin release by the ovary seems to be stimulated by the oviduct in presence of the embryo. In vitro Met enkephalin release by Bovine Corpus luteum is about 0.5 to 1. pMole/mg of fresh tissue/24 Hours. This release is not increased in presence of trophoblastic tissue. The content of the fresh tissue is between 0.7 and 1.9 picomoles par gram of Human tissue, and 0.9 picomoles for Bovine tissue. We determine the presence of Leu-Enkephalin and Met-Enkephalin Arg-Gly-Leu. The ratios observed confirm a ProEnkephalin A expression in the Ovary. The roles of these opioid peptides is discussed in term of ovum transport, Granulosa cell physiology and Early pregnancy factors.

歯髄におけるｂｒａｄｙｋｉｎｉｎのｏｐｉｏｉｄ ｐｅｐｔｉｄｅｓ産生・遊離作用

Effect of bradykinin (BK) on the release of met-enkephalin (ME) -like peptides from the rat incisor pulp was examined in in vitro experiments using whole pulp. BK dose-dependently increased the release of ME-like peptides and it was antagonized by Des-Arg9- [Leu8] -BK, a potent BK-antagonist. High K+ did not show any influence on the release. The BK effect was not influenced in Ca++-free medium. These findings suggested that effect of BK on the release of ME-like peptides from the pulp was mediated through B1-receptor and was Ca++-independent. In addition, the BK effect was not affected in presence of ouabain, suggesting that the release may be not active. Kyotorphin, an enkephalin releaser in central nervous system, also increased the content and the release of ME-like peptides in and from the pulp. Interestingly, the kyotorphin effect was attenuated by BK and vice versa. This fact may be explained as an interaction between BK and kyotorphin which may be different in mechanism of action. From the results in the present and the previous studies, the hypothesis that ME-like peptides in the pulp might control pain in a negative feedback mechanism was presented.

Kinetic modeling of hormone release: Application to time-averaged MET-enkephalin release

The time course of hormone concentrations in response to a stimulus can be characterizied with kinetic methods to express rates of hormone release and elimination, total amount of hormone release and expected best times for blood sampling. Routine application of kinetic methods should increase the sensitivity and applicability of hormonal challenge tests. Applications to the TRH test and to prolactin release measurements are reviewed. A new kinetic model for continuously collected samples is applied to measurements of met-enkephalin release in rats. Calculations indicated an initial concentration of 1876 pg/ml and a ventricular volume of distribution of 0.25 ml. After exposure of rats to nitrous oxide, the rate of release of met-enkephalin-like activity into the ventricles rose from 3.4 to 8.3 pg/min.

Invivo and invitro release of endogenous met-enkephalin, substance P and cholecystokinin from rat brain and spinal cord. Effects of peptidase inhibitors

Invivo and invitro release of endogenous met-enkephalin, substance P and cholecystokinin from rat brain and spinal cord. Effects of peptidase inhibitors

Release of Met-enkephalin from rat striatal slices: effect of amphetamine and fipexide

The release of Met-enkephalin immunoreactive material (ME-IR) from rat striatal slices is affected by exposure to amphetamine and fipexide (chloro-4-phenoxy)-2-acetyl-1-(methylene-dixoy 3,4-benzyl-4-pineperazine) a psychostimulant drug with mild dopaminomimetic activity. Both ampethamine and fipexide inhibited in vitro the release of ME-IR. The same effect was observed after in vivo acute administration and is also maintained after chronic treatment. The action of fipexide and amphetamine is still present after reserpine. pretreatment. The results indicate that fipexide may facilitate dopamine neurotransmission by inhibition of an enkephalinergic inhibitory feed-back circuit.

A putative met-enkephalin releaser, kyotorphin enhances intracellular Ca 2+ in the synaptosomes

Kyotorphin (Tyr-Arg) at 1 to 100 μM increased the intracellular [Ca 2+]i, determined with Quin-II in the slice and the entry of 45Ca 2+ entry into synaptosomes of the lower brain stem of the rat. These effects were not antagonized by nifedipine nor verapamil. However, since this dipeptide caused no changes on the membrane potentials of the synaptosomes, measured with Rhodamine 6G, it is suggested that the kyotorphin-induced increase in the [Ca 2+]i may be due not to effects on the voltage dependent Ca 2+ channels and Na +Ca 2+ exchange mechanisms caused by the changes of the membrane potentials, but to the specific receptor (kyotorphin receptor)-mediated mechanisms.

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.

Beta-Endorphin-(1-27) inhibits the spinal beta-endorphin-induced release of Met-enkephalin.

The effect of intraventricular beta-endorphin-(1-27) on the spinal release of Met-enkephalin induced by intraventricular beta-endorphin was studied using the intrathecal superfusion technique in urethane anesthetized rats. Intraventricular injection of beta-endorphin at a dose of 15 micrograms released Met-enkephalin from the spinal cord. This release of Met-enkephalin induced by beta-endorphin was significantly reduced by beta-endorphin-(1-27), 60 micrograms, injected intraventricularly. Injection of beta-endorphin (1-27) itself did not cause any release of Met-enkephalin. The finding is in line with the previous report that beta-endorphin (1-27) inhibited the analgesia induced by beta-endorphin.

Low doses of naloxone produce analgesia in the mouse brain by blocking presynaptic autoinhibition of enkephalin release.

The involvement of presynaptic autoinhibition of Met-enkephalin release in naloxone-induced analgesia was studied. In both acetic acid writhing and tail-flick tests in mice, naloxone produced biphasic effects, analgesia at very low doses (1 microgram/kg s.c. or 1 ng intracisternal) and hyperalgesia at higher doses (100 micrograms/kg s.c. or 100 ng intracisternal). Morphine at 10(-6) to 10(-5)M depressed the high K+-evoked release of Met-enkephalin from slices of the rat brainstem by 12.5-55.9% of control, while naloxone at 10(-6)M significantly enhanced the release by 80.6%. These findings strongly suggest that in the mouse brain a very low dose of naloxone produces analgesia by blocking autoinhibition of enkephalin release.

Lack of inhibitory effect of aprotinin and bacitracin on the spinal release of Met-enkephalin induced by intraventricular beta-endorphin

We have previously reported that administration of beta-endorphin intraventricularly in the rat increases the release of immunoreactive Met-enkephalin from the spinal cord. To further eliminate the possibility that the increase in Met-enkephalin might arise from the degradation of beta-endorphin injected, the effect of peptidase inhibitors, aprotinin and bacitracin, on the spinal fluid content of Met-enkephalin released by intraventricular beta-endorphin was studied using an intrathecal perfusion technique in urethane anesthetized rats. Inhibition of peptidases by intraventricular aprotinin and bacitracin did not decrease nor enhance the increased content of Met-enkephalin in the spinal perfusate produced by intraventricular beta-endorphin. The result indicates that the Met-enkephalin arises from neuronal release in the spinal cord rather than from degradation of the beta-endorphin injected intraventricularly.

Local and remote effects of intra-caudate administration of GABA-related drugs on met-enkephalin release in the basal ganglion

The possible influence of GABAergic systems on the activity of enkephalinergic neurones within the basal ganglia was examined by measuring the release of Met-enkephalin in the caudate nuclei and pallida of halothane-anesthesized cats treated by intra-caudate applications of GABA-related drugs. Depending on the concentration used, GABA exerted local stimulatory (at 10 μM of the amino acid) or inhibitory (at 0.5 mM) action on Met-enkephalin release in the cat caudate nucleus. Only the inhibition was reproduced by the GABA agonists muscimol (1 μM) and (−)-baclofen (10 μM) and by diazepam (10 μM). Conversely, the intra-caudate application of the GABA antagonist bicuculline enhanced markedly the local release of the pentapeptide. Complementary studies using slices of the rat straitum (caudate nucleus + putamen) revealed that a low concentration of GABA (10 μM) tended to increase the K +-evoked efflux of Met-enkephalin, whereas a high concentration of the amino acid exerted a strong inhibitory effect on the peptide release. Such in vivo and in vitro findings suggest that the GABA-induced inhibition of Met-enkephalin release took place via the stimulation of specific GABA A and GABA B receptors within the caudate nucleus, whereas the GABA-induced increase of the peptide release might involve some intracellular regulatory processes in striatal neurones containing both GABA and enkephalins. In addition to altering the local release of Met-enkephalin, intra-caudate applications of GABA-related drugs affected the peptide release in the ipsilateral globus pallidus and contralateral basal ganglia. The observed changes suggest that GABA A, but not GABA B, receptors participated in some tonic inhibitory influence of striatal GABAergic neurones on the striato-pallidal enkephalinergic system. Furthermore, the present results confirmed previous studies (Bourgoin et al. 2) showing that GABAergic neurones can contribute to some bilateral modulation of enkephalinergic neurones within the basal ganglia.

Expression of the human proenkephalin gene in mouse pituitary cells: accurate and efficient mRNA production and proteolytic processing.

A recombinant plasmid containing the human proenkephalin gene ligated to pBR322 was introduced into a mouse pituitary cell line (AtT-20D16v) that normally expresses pro-opiomelanocortin but not proenkephalin. The plasmid was introduced by co-transformation with the G418-selectable plasmid, pRSVneo. Stable transformants were isolated and analyzed for the presence of the human proenkephalin gene. AtT-20 transformants which had one or more copies of the human proenkephalin gene integrated stably into the mouse chromosomal DNA expressed a 1.45 kb mRNA identical in size to human proenkephalin mRNA. Primer extension analysis indicated that the human proenkephalin gene was accurately and efficiently transcribed from its own promoter. AtT-20 transformants that expressed the 1.45 kb human proenkephalin mRNA also expressed proenkephalin protein and cleaved the protein to form free Met-enkephalin. This is of particular interest because these cells do not cleave all of the available pairs of basic amino acids in the endogenous protein, pro-opiomelanocortin, the precursor to ACTH, beta-endorphin and melanocyte stimulating hormones. The release of both ACTH and Met-enkephalin from these cells is stimulated by corticotropin releasing factor, a natural secretagogue for ACTH, indicating that the two classes of peptide share a related secretory pathway.

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.

Release of immunoreactive met-enkephalin from the spinal cord by intraventricular β-endorphin but not morphine in anesthetized rats

Effect of β-endorphin and morphine injected intraventricularly on the release of immunoreactive Met-enkephalin, Leu-enkephalin and dynorphin 1–13 from the spinal cord was studied in anesthetized rats. Intraventricular β-endorphin, 16 μg, caused a marked spinal release of immunoreactive Met-enkephalin and to a much lesser extent, of immunoreactive Leu-enkephalin while intraventricular morphine, 40 μg, did not cause any significant release of immunoreactive enkephalins. The release of immunoreactive Met-enkephalin was not blocked by the pretreatment with 5 mg/kg naloxone, i.p. Immunoreactive dynorphin 1–13 was not released by either β-endorphin or morphine. High performance liquid chromatographic analysis indicated that immunoreactive Met-enkephalin released by β-endorphin had a retention time identical to [ 3H]Met-enkephalin. These findings in conjunction with previous pharmacological studies suggest different modes of pharmacological action for β-endorphin and morphine.

Carboxypeptidase activity in synaptic vesicles isolated from striatum and cortex of calf brain

A carboxypeptidase activity has been found in synaptic vesicles (secretory granules) isolated from the cortex and striatum of calf brain which removes amino acids from the carboxy terminus of enkephalin-containing (EC) peptides. The formed enkephalin molecules are not further degraded by this enzyme activity. The preparations were found to be free of cytoplasmic and lysosomal constituents as determined by marker enzyme activities. The vesicle preparations of both cortex and striatum showed differences in the degradation velocities of the various EC peptides depending on size and charge of the amino acid present at the carboxy terminus. The pH optimum of the release of Met-enkephalin from Met-enkephalin-Arg 6 has been shown to be between pH 5 and 6. The enzyme activity is inhibited by thiol-blocking agents such as p-hydroxymercuribenzoate and copper ions, but only slightly by metal-chelating agents.

Chronic ethanol induces changes in opiate receptor function and in met-enkephalin release

Ethanol induces supersensitivity of striatal delta-opiate receptor sites labelled by 3H-Etorphine. This effect may be ascribed to the diminished enkephalin release detected in striatal slices after chronic ethanol consumption. On the other hand, K d values for 3H-Met-enkephalin and 3H-DHM (mu-opiate receptors) specific binding are enhanced. The different sensitivity of the two classes of opiate receptors to ethanol may be due to specific effects on enkephalinergic transmission. It has been hypothesized that the decrease of 3H-Met-enkephalin and 3H-DHM affinity for their receptors takes place because endogenous substances from ethanol metabolism (for example salsolinol) behave as μ opioid agonists. This hypothesis is confirmed by “in vitro” studies demonstrating that salsolinol displaces 3H-Met-enkephalin and 3H-DHM but not 3H-DADLE binding. On the contrary, it seems that delta-receptors become supersensitive because of the decreased endogenous peptide release.