尾壳核内注射脑啡肽和bestatin对大鼠操作式条件...

Female Wistar rats were trained in a Skinner-box, 30 trials per day in a dark room to establish operant defence conditioning. Training started with a light (15 s), then combined with footshock for further 8 s. When the rats learned to press the key to avoid footshock within 15 s, conditioned response was considered established. After the rats reached a conditioning rate (CR) above 80% for 5 days, cannulae were implanted into caudate-putamen. Two to three days later, Met-enkephalin (MEK) or bestatin (an aminopeptidase inhibitor) was injected bilaterally into caudate-putamen. 30 min, 2 h, 24 h and 48 h after injection, conditioning tests were conducted, with each session consisting of 30 trials. Control experiments were done when 0.9% NaCl (NS) was injected. After injection of NS, CR maintained above 80% in all 4 test sessions. MEK (60 ng/rat) or bestatin (10 micrograms/rat) significantly lowered the CR during the 30 min and 2 h test session. In the latter case, the latency (L) was also prolonged. However both CR and L returned to the control level in the 24 h and 48 h test sessions. Naloxone (2 mg/kg, i.p.) blocked the conditioning-depression effect of bestatin. No significant alteration was seen in locomotor activity after MEK or bestatin injection. The results suggest that enkephalin in caudate-putamen may be involved in the regulation of retrieval of conditioning. Bestatin mimics the effect of MEK on conditioning reflex probably by increasing production of endogenous enkephalin.     

The involvement of opioid peptides in stress-induced analgesia in the slug Arion ater

Application of tail-pinch stress to the terrestrial slug, Arion ater, produced a significant increase in the response time when tested on the hot-plate for foot-lifting response. The analgesia was completely reversed by injections of the opiate antagonists, naltrexone and ICI 174864, in a dose-dependent manner. Analgesia could also be elicited by the injection into the foot of beta-endorphin and the enkephalin analogues, DAGO and DADLE. No effect was seen with dynorphin A (1-8) or dynorphin A (1-17). The stress-induced analgesia disappeared after 30 minutes but could be maintained for 100 min following the injection of a mixture of bestatin and the enkephalinase inhibitor, N-carboxymethyl-L-phenylalanyl-L-leucine. This work suggests that in the slug, a physical stressor produces an analgesia which may be due to the release of endogenous opiates.     

Enkephalin degrading enzymes are present in the electric organ of Torpedo californica

Two proteolytic activities that degrade [Leu5]enkephalin were found in Torpedo californica electric organ. One is a soluble aminopeptidase that degrades enkephalin at the Tyr1-Gly2 peptide bond, and the second is an endopeptidase that degrades enkephalin at the Gly3-Phe4 peptide bond. The aminopeptidase is inhibited by low concentrations of puromycin and bestatin. More than 60% of the endopeptidase is associated with the particulate fraction and is almost completely inhibited by low concentrations of captopril (SQ 14225) or SQ 20881 (potent inhibitors of angiotensin converting enzyme). Thiorphan and phosphoramidon (potent enkephalinase inhibitors) are much less effective. The pattern of cleavage and inhibition of the particulate endopeptidase thus resembles that of angiotensin converting enzyme.     

Degradation of kyotorphin by a purified membrane-bound-aminopeptidase from monkey brain: potentiation of kyotorphin-induced analgesia by a highly effective inhibitor, bestatin

Kyotorphin (Tyr-Arg) was rapidly degraded in rat brain homogenates and the Vmax and Km were 29.4 nmol/mg protein/min and 16.6 microM, respectively. This degradation was effectively inhibited by bestatin (IC 50; 0.08 microM) and p-chloromercuribenzoate (IC 50; 0.70 microM). Kyotorphin was also degraded by a membrane-bound aminopeptidase from monkey brains. The Vmax and Km of kyotorphin degradation by the aminopeptidase were 20.0 nmol/mg protein/min and 29.2 microM, respectively. The degradation of kyotorphin was also inhibited effectively by bestatin (KI; 0.4 microM). Co-administration with bestatin 50 micrograms (i.cist.) potentiated the analgesic effects of kyotorphin (i.cist.) by 4.8 times, and these effects were abolished by pretreatment with naloxone 0.5 mg/kg s.c. These results suggest that potentiation of analgesia by bestatin may be due to the protection against the degradation of kyotorphin and released enkephalin by a membrane-bound aminopeptidase.     

FMRF-NH2-like mammalian peptide precipitates opiate-withdrawal syndrome in the rat

Yang et al. (14) have isolated from mammalian brain an octapeptide FLFQPQRF-NH2 (F-8-F-NH2) with certain antiopiate properties. Third ventricular injection of 2 micrograms of this peptide together with the aminopeptidase inhibitor bestatin precipitated an opiate-withdrawal syndrome in morphine-dependent but not in nondependent rats. Third ventricular injection in nondependent rats of 15 micrograms of the peptide together with bestatin induced a morphine-withdrawal-like behavioral syndrome. This syndrome was not produced by injection of bestatin or saline vehicle alone and was preventable by injection of 3.5 mg/kg morphine sulphate SC.     

Role of aminopeptidases in enkephalin catabolism: comparative study of the regional distribution of aminopeptidases and enkephalinase A in the rat brain

In order to elucidate the role of aminopeptidases in enkephalin catabolism in rat brain, the local distribution of two types of cerebral cellular membrane aminopeptidases (puromycin-sensitive and puromycin-insensitive ones) and of the enkephalin system marker, enkephalinase A, was studied. It was found that the distribution patterns of the former enzymes differ essentially from that of enkephalinase A. Study of coupling between the enzymatic activities in different regions of rat brain revealed a strong correlation between the activities of puromycin-insensitive aminopeptidase and enkephalinase A in midbrain (including hypothalamus). It was supposed that in midbrain the role of aminopeptidase M in intrasynaptic inactivation of enkephalins is much more conspicuous than in other regions of rat brain. The puromycin-sensitive aminopeptidase activity does not seem to play a role in enkephalin catabolism.     

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, Arg°-Met-enkephalin was the most potent inhibitor for the aminoenkephalinases, with an IC₅₀ of about 0.6 μM, it was more effective than bestatin (IC₅₀=0.8–1.0 μM). This inhibition was partly due to substrate competition. Arg°-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-β-naphthylamide by the membrane aminoenkephalinase. Since Arg°-Met-enkephalin did not inhibit leucine aminopeptidase, it was a more selective inhibitor than bestatin of Met-enkephalin breakdown by aminopeptidases. Arg°-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.     

Metabolism and absorption enhancement of methionine enkephalin in human nasal epithelium

The objective of this study was to investigate absorption enhancing approaches for systemic delivery of methionine enkephalin via the nose. Absorption promotion of methionine enkephalin in the presence of protease inhibitors (bestatin, puromycin) and absorption enhancers (glycocholate, dimethyl-beta-cyclodextrin) were investigated in human nasal epithelium. Co-administration of the peptide with protease inhibitors and absorption enhancers resulted in a remarkable increase in Met-Enk permeation (4- to 94-fold). The increase was proportional to transepithelial resistance reduction and permeation of paracellular marker dye. Perturbation of the epithelial tight junctions seen in vitro may not occur in vivo due to mucus protection and mucociliary clearance.     

Influence of peptidase inhibitors on the apparent agonist potency of delta selective opioid peptides in vitro.

Several peptides of diverse structure, reported to possess high affinity and selectivity for the delta opioid receptor, were studied using the mouse isolated vas deferens preparation to determine the effect of peptidase inhibition on their apparent potency. The peptides evaluated included [Leu5] enkephalin, the cyclic enkephalin analogs [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Pen2,p-F-Phe4,D-Pen5]enkephalin (F-DPDPE), the linear enkephalin analogs [D-Ala2,D-Leu5]enkephalin (DADLE) and [D-Ser2(O-tBu), Leu5,Thr6]enkephalin (DSTBULET), and the naturally occurring amphibian peptides Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (dermenkephalin), Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 (deltorphin I) and Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 (deltorphin II). Concentration-response curves were determined for each peptide in the absence and presence of a combination of the peptidase-inhibiting agents bacitracin, bestatin, and captopril. A wide range of potencies was observed, both in the control state and in the presence of peptidase inhibition. The synthetic enkephalin analogs demonstrated small increases in potency with peptidase inhibition (no increase in the case of DPDPE), whereas the naturally occurring peptides were markedly increased in potency, up to as much as 123-fold for dermenkephalin. In the presence of peptidase inhibition, deltorphin II was the most potent peptide tested (IC50 = 1.13 x 10(-10) molar), and as such is the most potent delta opioid agonist reported to date. Stability to metabolism must be considered in the design and evaluation of in vitro experiments using peptides of this type.     

Potentiation by thiorphan and bestatin of the naloxone-insensitive analgesic effects of neurotensin and neuromedin n

Neurotensin induced significant antinociceptive activity as measured in a variety of nociceptive tests 10 and 30 min following intracerebroventricular (i.c.v.) injection in mice. The lowest effective peptide doses were 25 ng in the writhing test, 25–50 ng in the tail-flick test, 50–100 ng in the hot-plate test and 2000 ng in the tail electrical stimulation test. The neurotensin related hexapeptide neuromedin N also displayed antinociceptive properties but only in the writhing and tail-flick tests. Furthermore, as compared to neurotensin, the neuromedin effects required higher doses. ED₅₀'s for neurotensin and neuromedin in the writhing test were 70 ng and 1070 ng, respectively. Separate or combined injections of the endopeptidase 24.11 (enkephalinase) inhibitor thiorphan (l0μg) and the aminopeptidase inhibitor bestatin (50μg) did not affect tail-flick latencies. In contrast, i.c.v. injection of thiorphan together with an ineffective dose of neurotensin (25 ng) resulted in a significant antinociceptive effect. Bestatin did not modify tail-flick latencies in neurotensin-treated mice whether in the absence or presence of thiorphan. On the contrary, each of these peptidase inhibitors promoted antinociceptive effects of subthreshold doses of neuromedin (lμg) in the tail-flick test. Maximal antinociception was obtained by combining both inhibitors, thus conferring antinociceptive effects to neuromedin doses that were as low as 10 ng. Naloxone (0.5–2 mg/kg, s.c.) did not significantly reduced the antinociceptive effects of combinations of neurotensin and thiorphan and of neuromedin, thiorphan and bestatin. The data show that both neurotensin and neuromedin elicit analgesia in mice through an opiate independent mechanism. Furthermore, like enkephalin, neuromedin is readily degraded by brain endopeptidase 24.11 and bestatin sensitive aminopeptidase(s), whereas the resistance of neurotensin to aminopeptidase attack confers to this peptide a broader spectrum and longer duration of action than its congener neuromedin.     

Formation of [Leu5]Enkephalin from Dynorphin A(1–8) by Rat Central Nervous Tissue In Vitro

[3H]Dynorphin A(1-8) is readily metabolised by rat lumbosacral spinal cord tissue in vitro, affording a variety of products including a significant amount (20% recovered activity) of [3H][Leu5]enkephalin. In the presence of the peptidase inhibitors bestatin, captopril, thiorphan, and leucyl-leucine, [3H][Leu5]enkephalin was the major metabolic product, accounting for 60% of recovered activity. Production of [3H][Leu5]enkephalin was seen across all gross brain regions. The enzyme responsible for the cleavage has an optimal substrate length of 8-13 amino acids and is inhibited by N-[1-(RS)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, a site-directed inhibitor of the metalloendopeptidase EC 3.4.24.15. However the enzymic breakdown also has properties in common with involvement of endo-oligopeptidase A. Possible consequences of the formation of [Leu5]-enkephalin from the smaller dynorphins are discussed.     

Novel carboxypeptidase for processing of animal nerve tissue enkephalins

New carboxypeptidase which catalyzes the C-terminal arginine splitting from hexapeptide (an enkephalin precursor) Tyr-Gly-Gly-Phe-Met-Arg is revealed in the cat nerve tissue. The enzyme is activated by cobalt ions, the pH action optimum being 7.6. A relatively high activity of carboxypeptidase in the hypothalamus, midbrain and medulla oblongata permits supposing that it is the key enzyme of the enkephalin processing in areas of the limbic brain system.     

Endogenous methionine enkephalin may play an anticonvulsant role in the seizure-susceptible El mouse

After the intracisternal injection of three protease inhibitors which prevent the degradation of methionine enkephalin (amastatin, Des-Pro²-bradykinin, and phosphoramidon) and a mixture of these protease inhibitors, we investigated the effect on convulsive seizures in the seizure-susceptible El mouse. We also measured the cerebral methionine enkephalin content by high-performance liquid chromatography coupled with radioimmunoassay. Protease inhibitors significantly decreased both the incidence of seizures and the seizure score in El mice in a dose-dependent manner. This anticonvulsant effect was reversed by naloxone (2 mg/kg, sc). The cerebral methionine enkephalin content increased significantly after the administration of protease inhibitors in comparison with saline injection. These findings suggest that it was not protease inhibitors but instead increase of endogenous methionine enkephalin that reduced the incidence of seizures and the seizure score in El mice. Together with our previous data, the present findings support our hypothesis that a deficit in anticonvulsant endogenous methionine enkephalin is involved in the pathogenesis of seizures in the El mouse.     

Region-dependent G-protein activation by mu-, delta 1- and delta 2-opioid receptor agonists in the brain: comparison between the midbrain and forebrain

The ability of selective mu- ([D-Ala2, NHPhe4, Gly-ol]enkephalin: DAMGO), delta1- ([D-Pen2, Pen5]enkephalin: DPDPE) and delta2- ([D-Ala2]deltorphin II: DELT II) opioid receptor agonists to activate G-proteins in the midbrain and forebrain of mice and rats was examined by monitoring the binding of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS). The levels of [35S]GTPgammaS binding stimulated by DAMGO in the mouse and rat midbrain were significantly greater than those by DPDPE or DELT II. However, relatively lower levels of stimulation of [35S]GTPgammaS binding by all of the agonists than would have been predicted from the receptor densities were observed in either the limbic forebrain or striatum of mice and rats. The effects of DAMGO, DPDPE and DELT II in all three regions were completely reversed by selective mu-, delta1- and delta2-antagonists, respectively. The results indicate that the levels of mu-, delta1- and delta2-opioid receptor agonist-induced G-protein activation in the midbrain are in good agreement with the previously determined distribution densities of each receptor type. Furthermore, the discrepancies observed in the forebrain might reflect differential catalytic efficiencies of receptor-G-protein coupling.     

The effect of naloxone on the activation, inducible by chronic alcoholic intoxication, of the membrane-bound form of enkephalin convertase in the midbrain and hypothalamus of rats]

The activity of membrane-bound and soluble enkephalin convertase was determined with dansyl-Phe-Leu-Arg as substrate in midbrain, including hypothalamus, of Wistar rats, who were given ethanol (20% solution i.g., 9-15 g/kg per day during 4 days) or naloxone (2 mg/kg i.p. twice a day during 4 days) or their combination. It was shown that activation of membrane-bound enzyme, observed after alcohol treatment of rats, didn't develop by combined ethanol-naloxone administration. It's supposed that alcohol-stimulating effect on this enzyme realizes throughout the hyperstimulation of opioid receptors by enkephalins and, possible, by other opioid-active compounds.     

Organic anion transporters involved in the excretion of bestatin in the kidney

Bestatin, a dipeptide, a low molecular weight aminopeptidase inhibitor, has been demonstrated to be an immunomodulator with an antitumor activity. However, the transporter-mediated renal excretion of bestatin is not fully understood. The purpose of this study was to elucidate the transporter-mediated renal excretion mechanism for bestatin. The plasma concentration of bestatin was increased markedly and both the accumulative renal excretion and renal clearance of bestatin were decreased significantly after intravenous administration of bestatin in combination with probenecid. p-Aminohippuric acid (PAH), a substrate of organic anion transporter (OAT) 1, benzylpenicillin (PCG), a substrate of OAT3 and JBP485, a substrate of OAT1 and OAT3, reduced the uptake of bestatin in rat kidney slices and in hOAT1- or hOAT3-HEK 293 cells. The accumulation of bestatin in hOAT1-HEK and hOAT3-HEK 293 cells was significantly greater than that in vector-HEK, and the K(m) and V(max) were 0.679 ± 0.007 mM and 0.807 ± 0.006 nmol/mg protein/30s for OAT1, 0.632 ± 0.014 mM and 1.303 ± 0.015 nmol/mg protein/30s for OAT3 respectively. PAH and JBP485 inhibited significantly the uptake of bestatin in hOAT1-HEK with the K(i) values of 92 ± 9 μM and 197 ± 21 μM; and PCG, JBP485 inhibited significantly the uptake of bestatin in hOAT3-HEK 293 cells with the K(i) values of 88 ± 12 μM and 160 ± 16 μM. Our results are novel in demonstrating for the first time that OAT1 and OAT3 are involved in the renal excretion of bestatin.     

Responses of neurons of the frontal area of the cortex of the rabbit to electric stimulation of certain limbic-mesencephalic structures after administration of substance P

In rabbits tested on behavioural reactions by electrical stimulation of certain limbic-midbrain structures, intravenous injection of substance P (30 mcg/kg) led after 10 min of silent period to a decrease of spontaneous neuronal activity in the frontal cortex. Convergence of excitations arising from the lateral hypothalamus, the dorsal hippocampus and the midbrain reticular formation was also found to decrease after the substance P injection. Analysis of neuronal responses allowed to establish that substance P markedly changed the ascending excitations of the lateral hypothalamus and was less effective for the influences from the midbrain reticular formation.     

EARLY RESPONSE OF RAT BRAIN TRYPTOPHAN HYDROXYLASE ACTIVITY TO CYCLOHEXIMIDE, PUROMYCIN AND CORTICOSTERONE

Abstract— The activity of tryptophan hydroxylase was measured in whole homogenates of midbrain and forebrain areas of the rat brain. A significant elevation of tryptophan hydroxylase in midbrain and forebrain was found within 1 h after injection of corticosterone hemisuccinate Na salt (10mg/kg) into normal rats. A further elevation of tryptophan hydroxylase at 4 h after injection occurred only in the midbrain region. A rapid alteration of tryptophan hydroxylase was also observed following intracistemal injection of a protein synthesis inhibitor, cydoheximide. A significant depression of 50% of normal levels occurred both in midbrain and forebrain regions within 1 h. However. 4 h after injection only the midbrain tryptophan hydroxylase level was depressed, and this depression was 16% of normal levels. This temporal and spatial pattern following cydoheximide injection was not the result of changes in the ability of cydoheximide to inhibit in vivo protein synthesis since [³H]valine incorporation into protein was shown to be equally depressed at both 1 and 5 h in both the midbrain and forebrain. Puromycin blocked [³H]valine incorporation into proteins in the midbrain and forebrain. but only caused a depression of 16% of tryptophan hydroxylase in the midbrain at 4 h. The aminonucleoside derivative of puromycin has no effect on protein synthesis or on tryptophan hydroxylase. Cydoheximide had no effect on tryptophan hydroxylase in vitro. The data suggest that cydoheximide and corticosterone produce an early (1 h) effect on tryptophan hydroxylase unrelated to de novo protein synthesis in regions known to contain perikaryon (midbrain) and axon terminals (forebrain) of 5-HT-containing neurons. The later (4h) effects of these two compounds and puromycin on tryptophan hydroxylase in the perikaryon (midbrain) region of 5-HT-containing neurons probably result from alteration in de novo protein synthesis. The half time of tryptophan hydroxylase in midbrain region is calculated to be 12 h.     

Purification and characterization of enkephalinase B from rat brain membrane

Enkephalinase B from rat brain membrane which hydrolyzes enkephalin at the Gly-Gly bond was purified about 9400-fold to apparent electrophoretic homogeneity. The enzyme, which has a molecular weight of 82,000, consists of a single polypeptide chain. The enzyme has a pH optimum of 6.0-6.5 and is stable in the neutral pH region. The Km values of Met-enkephalin and Leu-enkephalin for this enzyme were 5.3 X 10(-5) M and 5.0 X 10(-5) M, respectively. The enzyme was inactivated by metal chelators, EDTA and o-phenanthroline and restored by the addition of divalent metal ions, Zn2+, Mn2+ or Fe2+, but was not inhibited by bestatin, amastatin, phosphoramidon or captopril. The enzyme hydrolyzed Met-enkephalin and Leu-enkephalin effectively. Although the enzyme belongs to the dipeptidyl aminopeptidase class, enkephalin-related peptides such as Leu-enkephalin-Arg, dynorphin (1-13) or alpha-endorphin and other biologically active peptides examined were hardly, or not at all, hydrolyzed. It was assumed that enkephalinase B functions mainly in enkephalin degradation in vivo.