naloxone and ubenimex

Kahweol is a diterpene present in the oil derived from coffee beans. Although several pharmacological activities of kahweol are already well described in the literature, no study was done in order to assess the analgesic activity of this substance. Thus, the aim of this study was to investigate the possible peripheral antinociceptive effect of kahweol. Considering that the opioid peptides have been implicated in peripheral antinociception induced by non-opioidergic compounds, the present study also evaluated the endogenous opioids involvement in this effect.. The rat paw pressure test was used, and hyperalgesia was induced by intraplantar injection of prostaglandin E2 (2μg/paw). All drugs were administered subcutaneously in the hindpaws of male Wistar rats. The expression of β-endorphin was examined by immunohistochemistry in the skin tissue samples of the plantar surface of rat right hindpaws.. Intraplantar injection of kahweol (40 and 80μg) induced significant peripheral antinociception. The antinociceptive effect of kahweol was due to a local peripheral action because the higher dose (80μg/paw) did not produce any effect in the contralateral paw. The opioid receptor antagonist naloxone (50 and 100μg/paw) prevented action of kahweol (80μg/paw) and the aminopeptidases inhibitor bestatin (400μg/paw) potentiated the antinociceptive effect of kahweol (40μg/paw). Furthermore, kahweol treatment increased the intensity of β-endorphin immunoreactivity in the epithelium of rat paws.. The results discussed here provide evidence that kahweol treatment has peripheral antinociceptive effect and suggest that this effect is mediated by the release of endogenous opioids.

Topics: Analgesics; Animals; beta-Endorphin; Coffee; Dinoprostone; Diterpenes; Hyperalgesia; Leucine; Male; Naloxone; Narcotic Antagonists; Opioid Peptides; Pain; Pain Measurement; Peptides; Pressure; Rats; Rats, Wistar; Skin

It is generally believed that NMDA receptor antagonism accounts for most of the anesthetic and analgesic effects of ketamine, however, it interacts at multiple sites in the central nervous system, including NMDA and non-NMDA glutamate receptors, nicotinic and muscarinic cholinergic receptors, and adrenergic and opioid receptors. Interestingly, it was shown that at supraspinal sites, ketamine interacts with the μ-opioid system and causes supraspinal antinociception. In this study, we investigated the involvement of endogenous opioids in ketamine-induced central antinociception. The nociceptive threshold for thermal stimulation was measured in Swiss mice using the tail-flick test. The drugs were administered via the intracerebroventricular route. Our results demonstrated that the opioid receptor antagonist naloxone, the μ-opioid receptor antagonist clocinnamox and the δ-opioid receptor antagonist naltrindole, but not the κ-opioid receptor antagonist nor-binaltorphimine, antagonized ketamine-induced central antinociception in a dose-dependent manner. Additionally, the administration of the aminopeptidase inhibitor bestatin significantly enhanced low-dose ketamine-induced central antinociception. These data provide evidence for the involvement of endogenous opioids and μ- and δ-opioid receptors in ketamine-induced central antinociception. In contrast, κ-opioid receptors not appear to be involved in this effect.

Topics: Aminopeptidases; Analgesics; Animals; Brain; Cinnamates; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hot Temperature; Ketamine; Leucine; Male; Mice; Morphine Derivatives; Naloxone; Naltrexone; Narcotic Antagonists; Nociceptive Pain; Opioid Peptides; Pain Perception; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu

The aim of the present study was to investigate the mechanisms underlying the endogenous control of nociception at a peripheral level during inflammation. Using a pharmacological approach and the rat paw pressure test, we assessed the effect of an intraplantar injection of naloxone, an opioid receptor antagonist, and bestatin, an aminopeptidase inhibitor, on hyperalgesia induced by carrageenan, which mimics an inflammatory process, or prostaglandin E(2) (PGE(2)), which directly sensitizes nociceptors. Naloxone induced a significant and dose-dependent (25, 50 or 100 μg) increase in carrageenan-induced hyperalgesia, but not PGE(2)-induced hyperalgesia. Bestatin (400 μg/paw) significantly counteracted carrageenan-induced hyperalgesia, inducing an increase in the nociceptive threshold compared to control, but it did not modify hyperalgesia induced by PGE(2) injection into the rat paw. Positive β-endorphin immunoreactivity was increased in paw inflammation induced by carrageenan in comparison with the control group. However, PGE(2) did not significantly alter the immunostained area. These results provide evidence for activation of the endogenous opioidergic system during inflammation and indicate that this system regulates hyperalgesia through a negative feedback mechanism, modulating it at a peripheral level.

Topics: Animals; beta-Endorphin; Carrageenan; Dinoprostone; Dose-Response Relationship, Drug; Hyperalgesia; Inflammation; Leucine; Male; Naloxone; Narcotic Antagonists; Opioid Peptides; Pain Threshold; Protease Inhibitors; Rats; Rats, Wistar

Xylazine is an alpha(2)-adrenoceptor agonist extensively used in veterinary and animal experimentation. Evidence exists that alpha(2)-adrenoceptor agonists can activate opioid receptors via endogenous opioid release. Considering this idea and the multiple alpha(2) subtypes currently known (alpha(2A), alpha(2B), alpha(2C) and alpha(2D)), the aim of this study was to investigate which alpha(2) receptor subtype mediates xylazine-induced peripheral antinociception and possible opioid receptor and endogenous opioid involvement. The rat pressure test was used; the hyperalgesia was induced by intraplantar injection of prostaglandin E(2) (2 microg). Xylazine was administered locally (25, 50 and 100 microg) into the right hind paw of Wistar rat alone and after either alpha(2)-adrenoceptor antagonist yohimbine (5, 10 and 20 microg/paw), the alpha(2) antagonists to alpha(2A), alpha(2B), alpha(2C) and alpha(2D) subtypes (BRL 44 480, imiloxan, rauwolscine and RX 821002; 20 microg/paw, respectively) the opioid receptor antagonist naloxone (12.5, 25 and 50 microg) and the enkephalinase inhibitor bestatin (400 microg/paw). Intraplantar injection of xylazine (50 and 100 microg) induced peripheral antinociception; however, a dose of 25 microg/paw did not significantly reduce the hyperalgesic effect. Yohimbine, rauwolscine and naloxone prevented action of xylazine 100 microg/paw. BRL 44 480, imiloxan and RX 821002 were ineffective in blocking xylazine antinociception. Bestatin (400 microg/paw) potentiated the antinociceptive effect of xylazine 25 microg/paw. The present results provide evidence that the peripheral antinociceptive effect of xylazine probably results from activation of alpha(2C)-adrenoceptors and also by the release of endogenous opioids that act on their receptors.

Topics: Adrenergic alpha-2 Receptor Agonists; Adrenergic alpha-2 Receptor Antagonists; Analgesics; Animals; Dinoprostone; Dose-Response Relationship, Drug; Hyperalgesia; Idazoxan; Imidazoles; Leucine; Male; Naloxone; Opioid Peptides; Pain; Pain Measurement; Rats; Rats, Wistar; Xylazine; Yohimbine

This study was performed to compare the effects of naloxone (NLX) and post-tetanic stimulation on isolated guinea-pig ileum followed by prolonged exposure to morphine and bestatin. Morphine or bestatin alone did not induce any responses. In the presence of 1 microM morphine, the challenge with 1 microM naloxone caused quick contraction and post-tetanic contraction. A longer duration of these NLX-induced contraction and post-tetanic contractions was observed at the 6th stimulation compared to those after the 2nd stimulation. By contrast, the addition of bestatin, an aminopeptidase inhibitor, did not induce any NLX-induced contraction, although the same results for post-tetanic contraction as those of morphine were observed. These different effects of morphine and bestatin on NLX-induced contraction and post-tetanic contraction in the ileum may be due to different mechanisms of action in the opioid system. That in turn may suggest the possibility that bestatin has a physical dependence liability.

Topics: Animals; Drug Combinations; Electric Stimulation; Guinea Pigs; Ileum; In Vitro Techniques; Leucine; Male; Morphine; Muscle Contraction; Muscle, Smooth; Naloxone; Narcotic Antagonists; Random Allocation; Tetany

Topics: Animals; Evoked Potentials; Female; In Vitro Techniques; Leucine; Membrane Potentials; Naloxone; Narcotic Antagonists; Neurons; Nociceptin; Opioid Peptides; Oxytocin; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Supraoptic Nucleus; Thiorphan; Vasopressins

L-Arginine produces central antinociception by acting as a precursor of kyotorphin (L-tyrosyl-L-arginine), a [Met5]enkephalin releaser. This study investigated the antinociceptive activity of L-ornithine, a metabolite of L-arginine. L-Ornithine given s.c. at 300-1000 mg/kg suppressed carrageenin-induced hyperalgesia in rats in a naloxone-reversible manner. L-Ornithine and L-arginine, when given i.c.v. at 10-100 mu g/mouse, elicited antinociception even in intact mice, the effects being abolished by naloxone or naltrindole, and potentiated by bestatin, an inhibitor of aminopeptidase and kyotorphinase. The antinociception induced by i.c.v. L-ornithine was also inhibited by i.c.v. L-leucyl-L-arginine, a kyotorphin receptor antagonist, but was resistant to intracisternal anti-kyotorphin serum. L-Tyrosyl-L-ornithine, a synthetic dipeptide, (1-10 mu g/mouse, i.c.v.), exerted kyotorphin-like antinociception in mice. These findings suggest that L-ornithine produces L-arginine-like antinociception via kyotorphin receptors. However, this effect does not appear to be mediated by kyotorphin itself, but most likely by L-tyrosyl-L-ornithine, a putative dipeptide.

Topics: Analgesia; Animals; Arginine; Carrageenan; Citrulline; Dipeptides; Dose-Response Relationship, Drug; Excipients; Hyperalgesia; Injections, Intraventricular; Leucine; Male; Mice; Naloxone; Naltrexone; Narcotic Antagonists; Ornithine; Pain Measurement; Protease Inhibitors; Rats

We examined the effects on urine outflow rate after microinjections of thiorphan (a carboxypeptidase inhibitor) and bestatin (an aminopeptidase inhibitor) into the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei of anesthetized hydrated rats to determine the possible role of neuropeptides in the regulation of urine production. After individual microinjection of the peptidase inhibitors into the nuclei, only thiorphan at 100 nmol administered into the PVN significantly decreased the urine outflow rate. Two consecutive microinjections of the peptidase inhibitors at 100 nmol each into the nuclei induced potent antidiuresis. These effects after microinjections of the peptidase inhibitors into the PVN and SON were diminished by pretreatment with [Sar1,Ile8]angiotensin (ANG) II (an ANG II receptor antagonist) and naloxone (an opioid receptor antagonist) in the PVN and with [Sar1,Ile8]ANG II in the SON, respectively. A vasopressin (AVP) receptor antagonist, d(CH2)5-D-Tyr(Et)VAVP (i.v.), completely blocked the antidiuresis by microinjections of the peptidase inhibitors into both the nuclei. Urinary osmotic pressure was significantly increased by consecutive microinjections of the peptidase inhibitors into the PVN and SON. These results suggest that endogenously-released ANG II and opioid peptides in the PVN and ANG II in the SON regulate urine production mediated through increased AVP release.

Topics: Aminopeptidases; Angiotensin II; Animals; Carboxypeptidases; Diuresis; Hemodynamics; Hypothalamus; Leucine; Male; Naloxone; Protease Inhibitors; Rats; Rats, Wistar; Receptors, Angiotensin; Receptors, Opioid; Thiorphan

The role of endogenous opioids in modulating pain transmission in amphibians was examined by two methods known to activate endogenous opioids in mammals. Analgesia was assessed using the acetic acid test in the Northern grass frog, Rana pipiens. One or 2 h of immobilization produced a significant analgesia lasting for at least 90 min. Systemic, but not spinal, administration of naloxone before immobilization prevented the analgesic effects seen in saline-pretreated controls. Spinal administration of the enkephalinase inhibitor, thiorphan, but not bestatin (both at 100 nmol/frog), produced significant analgesia. The analgesic effect of thiorphan was blocked by coadministration of intraspinal naloxone. These data are the first to suggest a role for endogenous opioid modulation of noxious stimuli in lower vertebrates by examination of stress-induced analgesia and the action of agents that inhibit enkephalin degradation.

Topics: Acetates; Acetic Acid; Analgesia; Animals; Immobilization; Injections, Spinal; Leucine; Naloxone; Neprilysin; Pain Measurement; Protease Inhibitors; Rana pipiens; Stress, Psychological; Thiorphan

We have investigated the effect of bestatin and thiorphan on growth of murine transplantable tumors, survival time, activity of natural killer (NK) cells and macrophages in mice. The injections of thiorphan at the does of 0.5 or 5 micrograms per mouse retarded tumor growth and prolonged survival period in B16 melanoma bearing animals. Pretreatment with naloxone (an unspecific antagonist of opioid receptors) blocked the tumor growth inhibition induced by the treatment with bestatin and thiorphan what could suggest a contribution of endogenous enkephalin in this antitumor effect. The percentage of mice bearing B16 melanoma tumor in the group treated with thiorphan at doses of 0.5, 5 or 50 micrograms per mouse was lower in comparison with control animals in a dose-dependent manner. The treatment with thiorphan at concentrations of 0.4-1.6 mg/ml inhibited growth of cultured in vitro B16 melanoma cell in comparison with control culture. Thiorphan added to the medium at concentration of 0.5 microgram/ml or administered 4 times at the dose of 0.5 microgram/mouse augmented NK lymphocyte activity.

Topics: Animals; Antineoplastic Agents; Drug Synergism; Female; Killer Cells, Natural; Leucine; Macrophages; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Naloxone; Protease Inhibitors; Thiorphan

Two analogues of the aminopeptidase inhibitor bestatin, Z 4212 (N-[(2S, 3R)-3-Amino-2-hydroxy-4-(4-methylsulphonyl-phenyl)-1-oxobutyl]-1- aminocyclopentanecarboxylic) and Z 1796 ((2S)-N-[(2S,3R)-3-Amino-2-hydroxy-4-(4-methylsulphonyl-phenyl)-1- oxobutyl]-L-leucine) were found to behave as hypoalgesics when intracerebroventricularly (i.c.v.) administered to mice in the hot-plate test. At high doses, Z 4212 was also found to reduce the pain threshold after intraventricular (i.v.) administration. Hypoalgesia induced by bestatin analogues was prevented by prior treatment with the opiate receptor blocker naloxone. Thiorphan, a potent inhibitor of NEP, was found to enhance the hypoalgesic effect of low doses of either Z 4212 or Z 1796. These results indicate that both the major opioid-degrading peptidases, i.e. aminopeptidases and neutral endopeptidase (NEP), are individually implicated in the hypoalgesia induced by peptidase inhibitors. In vitro studies showed that these new bestatin analogues readily inhibit aminopeptidases in membranes from mouse c. striatum whereas more than 1000 times the concentration was required for NEP to be blocked. Ex vivo experiments showed that, at variance with bestatin, the hypoalgesic action of Z 4212 or Z 1796 appeared to implicate central aminopeptidases but not NEP, so partially sparing the metabolism of other NEP substrates that might produce additional alterations (substance P and ANP). On the basis of the antitumour and immunomodulatory actions of bestatin, these new analogues might be potentially useful as mixed antitumour and hypoalgesic agents in malignancy.

Topics: Aminopeptidases; Analgesics; Animals; Cycloleucine; Drug Interactions; Enkephalin, Methionine; Injections, Intraventricular; Leucine; Male; Mice; Naloxone; Neprilysin; Pain Measurement; Protease Inhibitors; Thiorphan

The possible existence of a feedback control by endogenous opioids of the spinal release of met-enkephalin-like material was assessed in vivo, in halothane-anesthetized rats whose intrathecal space was continuously perfused with an artificial cerebrospinal fluid supplemented with various opioid-related drugs. Both the intrathecal perfusion of the mu agonist D-Ala2-D-MePhe4-Gly-ol5-enkephalin (DAGO) (10 microM) and the delta agonist Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET) (10 microM) produced a significant inhibition of the spinal outflow of met-enkephalin-like material. The effect of DAGO, but not that of DTLET, could be prevented by naloxone (10 microM), and, conversely, the effect of DLTET, but not that of DAGO, was no longer observed in the presence of naltrindole (10 microM). Therefore naloxone and naltrindole acted as potent and selective mu and delta antagonists, respectively, when perfused at 10 microM in the intrathecal space of halothane-anesthetized rats. As expected from the lack of a tonic opioid control of spinal enkephalinergic neurones, neither naloxone nor naltrindole alone affected the spontaneous outflow of met-enkephalin-like material. However, naltrindole, but not naloxone, markedly increased the spinal overflow of met-enkephalin-like material due to intrathecal administration of either porcine calcitonin (10 microM) or the peptidase inhibitors thiorphan (10 microM) plus bestatin (20 microM). These data suggest that delta, but not mu, receptors are involved in a phasic opioid inhibitory control of the release of met-enkephalin-like material in the rat spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcitonin; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Methionine; Enkephalins; Feedback; Indoles; Injections, Spinal; Leucine; Male; Morphinans; Naloxone; Naltrexone; Oligopeptides; Radioimmunoassay; Rats; Rats, Inbred Strains; Spinal Cord; Thiorphan

In anesthetized, artificially ventilated cats, pretreated with the muscarinic antagonist scopolamine, the effect of naloxone on the efficacy of nicotinic transmission in the superior cervical ganglion was used as a test of endogenous opioid release by the preganglionic axons. The cervical sympathetic trunk (CST) was split into two bundles. The compound action potential (CAP), evoked by supramaximal low-frequency stimulation (0.25 Hz) of one CST bundle, was recorded from a postganglionic nerve of the superior cervical ganglion. Partial block with hexamethonium was used to reduce the 'safety factor' of nicotinic transmission. A conditioning train (5 Hz, 40 s, supramaximal) to the other CST bundle (heterosynaptic conditioning) inhibited the CAP. At the peak of the inhibition the CAP was attenuated by 51 +/- 4%. Recovery was 90% complete in 201 +/- 28 s. The inhibition was antagonized in a dose-dependent manner by i.v. naloxone with an apparent IC50 of 60 +/- 12 micrograms/kg. The maximum effect obtained with naloxone was an 80% decrease in the magnitude of the inhibition. Magnitude and duration of the heterosynaptic inhibition were related to frequency, duration and intensity of the conditioning train. Naloxone-sensitive inhibition was observed with frequencies of the conditioning train as low as 0.5 Hz. A train (5 Hz, 40 s) to the postganglionic nerves produced a naloxone-insensitive depression of the orthodromic test CAP which was of smaller amplitude and duration than when the conditioning train was applied to the preganglionic axons.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Action Potentials; Animals; Axons; Captopril; Cats; Electric Stimulation; Female; Ganglia, Sympathetic; Hexamethonium; Hexamethonium Compounds; Leucine; Male; Naloxone; Protease Inhibitors; Receptors, Nicotinic; Synapses; Synaptic Transmission; Thiorphan

In the present study we report the effects of inhibitors of enkephalin-degrading peptidases on spontaneous locomotion in mice and the involvement of delta opioid receptors in these effects. Animals received intracerebroventricularly (i.c.v.) or intravenously (i.v.) enkephalinase inhibitors (thiorphan and acetorphan), aminopeptidase inhibitors (bestatin and carbaphethiol) or mixed peptidase inhibitors (kelatorphan). The i.c.v. co-administration of bestatin and thiorphan (50 micrograms + 50 micrograms) induced an increase in both the horizontal and vertical components of locomotion. A similar pattern was observed after the i.c.v. administration of kelatorphan (8.5-50 micrograms) or the i.v. co-administration of acetorphan and carbaphethiol (5 mg/kg + 10 mg/kg). The opiate antagonist naltrexone (1 mg/kg, s.c.) failed to reverse the excitolocomotor effects of kelatorphan or of bestatin and thiorphan and antagonized only partially the effects of acetorphan and carbaphethiol. Naloxone (2 mg/kg-10 mg/kg, s.c.) partially reversed the increase in locomotion elicited by bestatin and thiorphan. The pretreatment with the delta opioid antagonists ICI 154,129 (20 micrograms, i.c.v.) or ICI 174,864 (2-4 micrograms, i.c.v.) strongly decreased the effects of all the peptidase inhibitors we tested. These results suggest that endogenous enkephalins may control via delta opioid receptors the horizontal and vertical components of locomotor activity in mice.

Topics: Aminopeptidases; Animals; CD13 Antigens; Dipeptides; Drug Synergism; Enkephalin, Leucine; Enkephalins; Injections, Intravenous; Injections, Intraventricular; Leucine; Locomotion; Male; Mice; Naloxone; Neprilysin; Phenylcarbamates; Prodrugs; Receptors, Opioid; Receptors, Opioid, delta; Sulfhydryl Compounds; Thiorphan

This study utilized inhibitors of the enzymatic degradation of enkephalins to investigate the possibility that this class of opioid peptides contributes to the stress-induced antinociception seen in inflamed peripheral tissues of rats with Freund's complete adjuvant-initiated unilateral hind paw inflammation. Following a 1-min cold water swim stress, rats previously injected in both hind paws with vehicle showed a transient elevation of paw pressure threshold, which was much greater in inflamed than in noninflamed paws and returned to control levels within 15 min. This preferential antinociception was significantly pronounced and prolonged in rats previously injected bilaterally with a cocktail of the enkephalinase inhibitors thiorphan (0.2 mg intraplantar) and bestatin (0.2 mg intraplantar). The enhancement of stress-induced antinociception by thiorphan/bestatin was dose-dependently antagonized by tertiary naloxone (0.125-2 mg kg-1 s.c.). Evidence for a peripheral site of action of enkephalin-like peptides in this model was provided by the antagonism of the actions of thiorphan/bestatin by quaternary naltrexone (10-20 mg kg-1 s.c.). Systemic administration of the orally active enkephalinase inhibitor SCH 34826 (5-40 mg kg-1 i.p.) was also able to dose-dependently potentiate the preferential stress-induced antinociception in a naloxone (1 mg kg-1 s.c.) reversible manner.

Topics: Analgesia; Animals; Dioxolanes; Dipeptides; Enkephalins; Inflammation; Leucine; Male; Naloxone; Naltrexone; Neprilysin; Rats; Rats, Inbred Strains; Receptors, Opioid; Stress, Physiological; Swimming; Thiorphan

Acetorphan, a parenterally active enkephalinase inhibitor, induced dose-dependently a naloxone-reversible analgesia on the hot-plate jump test in DBA/2J (DBA2) mice but was devoid of effects in C57BL/6J (C57) mice. By contrast, acetorphan increased locomotion in both strains; however, the DBA2 strain was much more sensitive than C57 mice to the locomotor stimulant effect. The increased locomotion was antagonized by naloxone in both strains. These data suggest that endogenous enkephalins modulate nociception and locomotion in the two inbred strains differently.

Topics: Analgesics; Animals; Cerebral Ventricles; Enkephalins; Injections, Intraventricular; Leucine; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Motor Activity; Naloxone; Pain; Protease Inhibitors; Reference Values; Species Specificity; Thiorphan

Mice exhibit a marked suppression of motility when they are placed in the same cage in which they had previously received an electric shock. This suppression of motility is believed to be stress-induced and is a conditioned response. A decrease in the Met-enkephalin levels and a decrease in the dopamine (DA) turnover in the striatum of these "conditioned suppression" groups have been exhibited. The present study investigates whether inhibition of enkephalin degradation induced by an enkephalinase A and/or an aminopeptidase inhibitor attenuates a conditioned suppression of motility. The effects of thiorphan and bestatin, both alone and in combination, were investigated. Thiorphan alone (25, 50 and 100 micrograms i.c.v.) significantly attenuated the conditioned suppression of motility in a dose-dependent manner, but not bestatin (25, 50 and 100 micrograms i.c.v.) alone. The combination of these drugs (25 and 50 micrograms, each, i.c.v.) also significantly reduced the conditioned suppression of motility in a dose-dependent manner. The attenuation of conditioned suppression of motility induced by thiorphan and bestatin was antagonized by naloxone (5 mg/kg s.c.) and pimozide (100 micrograms/kg i.p.). In addition, the combination of thiorphan and bestatin reversed the decreases of Met-enkephalin levels and the decreases of DA turnover in the striatum in conditioned suppression group. These results suggest that attenuation of the conditioned suppression of motility induced by thiorphan and bestatin may be directly proportional to the increases of endogenous opioid peptide contents, and that the effect of these drugs may be related to the striatal DAergic system.

Topics: Amino Acids, Sulfur; Aminopeptidases; Animals; Corpus Striatum; Dopamine; Enkephalin, Methionine; Enkephalins; Leucine; Male; Metalloendopeptidases; Mice; Motor Activity; Naloxone; Neprilysin; Pimozide; Stress, Physiological; Thiorphan; Tiopronin

The effects of various peptidase inhibitors were examined upon the K+-evoked overflow of substance-like immunoreactive material (SPLI) from slices of rat substantia nigra in order to assess the possible involvement of "enkephalinase," angiotensin-converting enzyme (ACE) and calpain in the enzymatic inactivation of endogenous substance P in brain tissues. The calpain inhibitor leupeptin and the enkephalinase inhibitors thiorphan and phosphoramidon increased markedly SPLI overflow, whereas the two ACE inhibitors, captopril and enalaprilat (up to 10 microM in the superfusing medium), were inactive. Surprisingly kelatorphan, which inhibits not only enkephalinase but also aminopeptidase and dipeptidylaminopeptidase activities, was less potent than thiorphan or phosphoramidon to enhance SPLI overflow. However, in the presence of ICI-154129 or naloxone to block opiate receptors, kelatorphan was as potent as thiorphan, therefore suggesting some negative influence of endogenous opioids on SPLI release with kelatorphan but not thiorphan. In agreement with this interpretation, the direct stimulation of delta opiate receptors by deltakephalin was found to significantly reduce SPLI overflow. Furthermore, an increased outflow of [Met]enkephalin-like material was observed from substantia nigra slices superfused with kelatorphan but not thiorphan. These results indicate that endogenous substance P released within the substantia nigra is very probably inactivated by enkephalinase and calpain, but not ACE. They also demonstrate that endogenous opioids can exert a negative control upon substance P release in this brain region.

Topics: Animals; Captopril; Dipeptides; In Vitro Techniques; Leucine; Male; Metalloendopeptidases; Naloxone; Neprilysin; Oligopeptides; Peptidyl-Dipeptidase A; Potassium; Protease Inhibitors; Rats; Rats, Inbred Strains; Substance P; Substantia Nigra; Thiorphan; Tiopronin

BAM 18 is a derivative of the opioid precursor proenkephalin A. Although it exists in rat and guinea-pig brain in relatively high concentrations, its physiological function is presently unknown. In the present study we have determined the opioid receptor selectivity of this peptide using radioligand binding and peripheral tissue bioassay. When selective binding conditions were used, BAM 18 bound to the mu opioid receptor with an affinity three times that of the kappa opioid receptor and over 10 times that of the delta opioid receptors (Ki = 0.29, 0.75, and 3.2 nM respectively). BAM 18 also displayed mixed receptor selectivity in in vitro bioassay. Ke values for naloxone antagonism of BAM 18 agonist activity in the electrically stimulated guinea-pig ileum and the mouse vas deferens were 4.3 and 9.9 nM, respectively. These data indicate that BAM 18 binds to all three opioid receptor subtypes with a selectivity profile of mu greater than kappa greater than delta.

Topics: Animals; Biological Assay; Dose-Response Relationship, Drug; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalin, Methionine; Enkephalins; Guinea Pigs; In Vitro Techniques; Leucine; Male; Naloxone; Protein Precursors; Radioligand Assay; Receptors, Opioid; Structure-Activity Relationship

Cholecystokinin octapeptide (CCK 8) produced significant antinociception in the tail immersion test in the rat, paw pressure test in the rat and acetylcholine-induced writhing test in the mouse after subcutaneous (s.c.) administration. In the hot plate test, CCK 8 (s.c.) showed antinociceptive activity if the latency to lick was used as the end point but if the latency to jump was recorded the antinociceptive effects were not evident. Cholecystokinin tetrapeptide (CCK 4) was shown to be antinociceptive in the writhing but not in the hot plate test after subcutaneous administration and appeared to be less potent than CCK 8 when tested under the same conditions. Antinociception induced by CCK 8 in the hot plate test (lick) could also be demonstrated after direct intracerebroventricular (i.c.v.) injection and this observation was also made with the CCK-related peptide FMRF amide. Antinociception induced by CCK 8 (which did not appear to be associated with reduced locomotor activity) was evident 5 min after intraventricular injection and was maximal at 10 min, the effect lasting over a 30-45 min period. The antinociceptive effect of CCK 8 was antagonised by naloxone and was potentiated by simultaneous administration of the peptidase inhibitors bestatin, thiorphan and captopril.

Topics: Animals; Captopril; FMRFamide; Gastrins; Leucine; Male; Mice; Naloxone; Neuropeptides; Pain; Protease Inhibitors; Rats; Rats, Inbred Strains; Reaction Time; Sincalide; Tetragastrin; Thiorphan; Tiopronin

Intracarotid (IC) injection of bestatin produced a dose-dependent biphasic change in blood pressure (BP) of the rat, consisting of an initial short-lasting fall followed by a long-lasting increase. This effect was regularly depressed or abolished by IV injection of naloxone. IC injection of Leu-enkephalin also produced a biphasic BP response, with the same characteristics as that produced by IC injection of bestatin. This effect was also easily blocked by IV injection of naloxone. IC injection of bestatin significantly potentiated the BP response to IC injection of Leu-enkephalin. This potentiated response was blocked by naloxone. IC injection of both bestatin and phosphoramidon, whether separately or in combination, significantly depressed the hypertensive response to physostigmine. This depressive action of bestatin and phosphoramidon on physostigmine hypertension can be significantly antagonized or even reversed by IV injection of naloxone. IC injection of both bestatin and phosphoramidon did not affect the BP response to either acetylcholine or catecholamines. It is concluded that bestatin and phosphoramidon, injected into the carotid artery, inhibit the activity of aminopeptidase and "enkephalinase", thus producing an accumulation of enkephalins in the central nervous system. These enkephalins activate opioidergic receptors in the brain, but concomitantly produce a depression of the cholinergic-adrenergic interaction in the central nervous system, which is known to be a prerequisite for the hypertensive response to physostigmine in the rat.

Topics: Animals; Antibiotics, Antineoplastic; Blood Pressure; Carotid Arteries; Catecholamines; Enkephalin, Leucine; Female; Glycopeptides; Injections, Intra-Arterial; Leucine; Male; Naloxone; Physostigmine; Rats; Rats, Inbred Strains

The enkephalins, found in high levels in the superficial dorsal horn where they are associated with the terminals of afferent nociceptive fibres, are rapidly degraded by at least 3 peptidases. The use of inhibitors of these peptidases allows effects mediated by endogenous enkephalins to be observed. We report here the inhibitory effects on spinal nociceptive transmission of bestatin, a non-specific aminopeptidase inhibitor, thiorphan, an inhibitor of enkephalinase and kelatorphan, a mixed inhibitor of aminopeptidases, particularly aminopeptidase M, enkephalinase and dipeptidylaminopeptidase. The agents were applied intrathecally, directly onto the spinal cord, in halothane-anaesthetized intact rats. Bestatin (n = 23 neurones) produced weak inhibitions of C-fibre-evoked activity (maximum 17% inhibition) whereas thiorphan (n = 20) produced maximal 25% inhibitions. Kelatorphan (n = 32) produced maximal 46% inhibitions which were naloxone reversible. All 3 agents caused dose-dependent effects which were relatively selective for C-fibre evoked responses, sparing A beta-fibre inputs. The results are discussed in relation to roles of the enkephalins in sensory modulation and the potential of these compounds as novel therapeutic agents.

Topics: Action Potentials; Animals; Dipeptides; Enkephalins; Leucine; Male; Naloxone; Nerve Fibers; Neural Inhibition; Neurons, Afferent; Pain; Protease Inhibitors; Rats; Rats, Inbred Strains; Spinal Cord; Thiorphan; Tiopronin

In plexus containing preparations of the longitudinal muscle of the guinea-pig ileum, an inhibitory action of tetracyclines on twitch-responses to electrical field stimulation was found. Tetracycline, chlortetracycline, minocycline and doxycycline, but not oxytetracycline (0.02 to 1.6 mmol/l) caused a concentration-dependent presynaptic inhibition of acetylcholine release. The inhibitory effect of the tetracyclines was also obtained after ganglion block by hexamethonium (30 mumol/l). The inhibitory effect of the tetracyclines was not antagonized by piperoxan (2 mumol/l) or yohimbine (1 mumol/l) and was partly reduced by the presence of naloxone (1 to 50 nmol/l). After exposing the preparation the peptidase inhibitors, i.e., to the combination of bestatin (10 mumol/l), captopril (10 mumol/l) and thiorphan (0.3 mumol/l), the inhibitory effect of tetracyclines was significantly increased. From these results it would appear that twitch-inhibition caused by tetracycline, chlortetracycline, minocycline and doxycycline is mainly mediated via the release of endogenous opioids from the myenteric plexus.

Topics: Animals; Captopril; Drug Interactions; Electric Stimulation; Guinea Pigs; In Vitro Techniques; Leucine; Muscle Contraction; Muscle, Smooth; Myenteric Plexus; Naloxone; Protease Inhibitors; Tetracyclines; Thiorphan; Tiopronin

We investigated the effects of thiorphan, a specific inhibitor of enkephalinase A and bestatin, a specific inhibitor of aminopeptidase, on the stereotyped behaviors induced by phencyclidine (PCP) in cannulated rats. The PCP-induced turning was significantly potentiated when thiorphan (50 micrograms) and bestatin (50 micrograms) were injected simultaneously into the rat lateral ventricle. The increase of PCP-induced turning was completely antagonized by the pretreatment with naloxone. Thiorphan (50 micrograms) or bestatin (50 micrograms) alone failed to potentiate PCP-induced turning. Thiorphan and/or bestatin did not affect significantly the PCP-induced head weaving and back-pedalling except that thiorphan (50 micrograms) potentiated the PCP-induced head weaving 15-18 min after the PCP injection. The combination of thiorphan and bestatin alone did not induce any behavioral change. These results suggest that thiorphan and bestatin produce an increase of endogenous enkephalins and that as a result, PCP-induced turning may be enhanced.

Topics: Amino Acids, Sulfur; Animals; Drug Synergism; Leucine; Male; Naloxone; Phencyclidine; Rats; Rats, Inbred Strains; Stereotyped Behavior; Thiorphan; Time Factors; Tiopronin

To confirm that endogenous opioid-peptidergic systems and monoaminergic systems participate in the regulation of pain, the effects of a narcotic antagonist naloxone, inhibitors of enkephalin-degrading enzymes and monoaminergic blockers on persistent pain induced by formalin were investigated. Rats were given formalin into the plantar region of the left hind-paw and the duration of licking responses was measured at periods of 0-10 min (period I), 10-30 min (period II), 30-60 min (period III) and 60-120 min (period IV). Naloxone was administered systemically through mini-osmotic pumps and there was an enhanced licking response at period III and a tendency toward enhancement at period IV. The duration of the licking response at period III was increased when naloxone was given into the fourth ventricle in a dose of 30 nmol/rat, but not when it was injected into the lumbo-sacral subarachnoid space in doses of 30 and 300 nmol/rat. Thiorphan and bestatin injected simultaneously into the fourth ventricle (50 micrograms/rat) depressed the licking response at period III. Intrathecal injection of phentolamine significantly enhanced the licking response at periods I-III and produced a similar, but weaker effect at period IV. Intrathecal injection of propranolol and methysergide did not affect the response. These results suggest that opioid-peptidergic systems in the brain stem and noradrenergic systems in the spinal cord (via alpha-adrenoceptors) participate in the regulation of pain.

Topics: Animals; Endorphins; Formaldehyde; Injections, Spinal; Leucine; Male; Medulla Oblongata; Naloxone; Norepinephrine; Pain; Rats; Rats, Inbred Strains; Spinal Cord; Thiorphan; Time Factors; Tiopronin

The endogenous opioid peptide enkephalin (EK) is known to be degraded mainly by two enzymes, the dipeptidyl carboxypeptidase 'enkephalinase' and aminopeptidase. Microinjection of the enkephalinase inhibitor thiorphan or the aminopeptidase inhibitor bestatin into the nucleus accumbens of the rabbit produced a dose-dependent analgesic effect. This analgesic effect was totally reversed by the narcotic antagonist naloxone or by antibodies against [Met5]enkephalin (MEK) administered to the same site. Antibodies against [Leu5]enkephalin were not effective. Moreover, microinjection of thiorphan or bestatin into the nucleus accumbens resulted in a marked potentiation of the aftereffect of electroacupuncture (EA) produced analgesia, as well as the analgesia induced by a small dose of morphine. It is concluded that the analgesic effect elicited by EA and morphine is mediated, at least in part, by MEK-like immunoreactive substance(s) in the nucleus accumbens.

Topics: Amino Acids, Sulfur; Analgesia; Animals; Dose-Response Relationship, Drug; Enkephalin, Methionine; Immune Sera; Leucine; Male; Microinjections; Morphine; Naloxone; Nucleus Accumbens; Rabbits; Septal Nuclei; Thiorphan; Time Factors; Tiopronin; Transcutaneous Electric Nerve Stimulation

The hypothermic effect of neurotensin (0.1 microgram) injected i.c.v. in mice is potentiated by the enkephalinase inhibitor thiorphan (10 micrograms, i.c.v.). This potentiation is not reversed by systemic naloxone. The hypothermic effect of neurotensin is not modified by the amino-peptidase inhibitor bestatin (50 micrograms, i.c.v.) nor by the angiotensin-converting enzyme inhibitor captopril (50 micrograms, i.c.v.). These data indicate the involvement of enkephalinase in the inactivation of neurotensin, at least when it is injected i.c.v.

Topics: Amino Acids, Sulfur; Animals; Body Temperature; Captopril; Drug Synergism; Endopeptidases; Injections, Intraventricular; Leucine; Male; Mice; Naloxone; Neprilysin; Neurotensin; Thiorphan; Tiopronin

Kyotorphin(KTP) and its analog D-kyotorphin(D-KTP) which is resistant to enzyme degradation produced dose-dependent analgesic effects in the tail-pinch test when applied locally to the periaqueductal gray (PAG), nucleus reticularis paragigantocellularis(NRPG) and lumbosacral subarachnoid space(LSS) near the spinal dorsal horn in rts. ED50 values of both dipeptides at the PAG, NRPG and LSS were: 59.0, 105 and 52.6 micrograms/rat for KTP, and 6.2, 8.8 and 10.6 micrograms/rat for D-KTP, respectively. Pretreatment with naloxone(1 mg/kg s.c.) significantly inhibited the analgesic effects of KTP and D-KTP at the PAG and LSS but not at the NRPG. Simultaneous administration of bestatin (2.5 or 50 micrograms) with KTP enhanced the analgesic effect of KTP in the PAG and LSS but not in the NRPG. These findings suggest that the analgesic actions of KTP and D-KTP result from two different mechanisms, 1)enkephgalin-releasing mechanism in the PAG and spinal dorsal horn, and 2)a mechanism without involvement of enkephalin-releasing actions in the NRPG.

Topics: Analgesics; Animals; Central Nervous System; Endorphins; Enkephalins; Leucine; Male; Naloxone; Periaqueductal Gray; Rats; Rats, Inbred Strains; Reticular Formation; Spinal Cord

The analgesic activity of some opioid peptides which display a relative selectivity for either the mu-receptor subtype, [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAGO) or the delta-receptor subtype. [D-Ala2, D-Leu5]enkephalin (DADLE), [D-Ser2, Leu5]enkephalyl-Thr (DSLET) and [D-Thr2, Leu5]enkephalyl-Thr (DTLET) is highly correlated with their affinity for central or peripheral mu- but not delta-receptors. Moreover their analgesic effects as well as those elicited by degrading enzyme inhibitors (bestatin + thiorphan) of endogenous enkephalins were easily antagonized by naloxone with similar pA2 values but not by the delta-antagonist ICI 154,129. Therefore the analgesia produced by opioid peptides including endogenous enkephalins is likely connected to mu-receptor stimulation. Finally, there was no obvious potentiation by delta-agonists of the analgesia resulting from either administration of the mu-agonist morphine or endogenous enkephalins. This suggested that in the hot plate test, there is no modulation of the effect resulting from mu-receptor stimulation by a delta-receptor interaction. Likewise, enkephalinergic activity such as that due to thiorphan + bestatin does not appear to be regulated through mu- or delta-receptor stimulation.

Topics: Analgesics; Animals; Enkephalin, Leucine; Enkephalins; Guinea Pigs; In Vitro Techniques; Leucine; Male; Mice; Morphine; Muscle Contraction; Muscle, Smooth; Nalorphine; Naloxone; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Thiorphan; Tiopronin

In the mouse 'behavioral despair' test the immobility time was shortened by [D-Ala2,Met5]enkephalin at doses which did not modify locomotor activity. Similarly, the inhibitors of the enzymes degrading enkephalins, thiorphan and/or bestatin were effective. Their effect was antagonized by naloxone. We conclude that endogenous enkephalins are implicated in the 'behavioral despair' test.

Topics: Aminopeptidases; Animals; Behavior, Animal; Enkephalin, Methionine; Enkephalins; Leucine; Male; Mice; Motor Activity; Naloxone; Protease Inhibitors; Thiorphan; Tiopronin

Thiorphan, an 'enkephalinase' inhibitor, and bestatin, an aminopeptidase inhibitor, decreased the turnover of noradrenaline and increased that of dopamine and serotonin in mouse brain regions. All effects were prevented by naloxone, indicating that when protected from the action of inactivating neuropeptidases endogenous enkephalins might affect monoaminergic neurons in the same way as exogenous opiates.

Topics: alpha-Methyltyrosine; Amino Acids, Sulfur; Aminopeptidases; Animals; Biogenic Amines; Brain; Enkephalins; Leucine; Male; Methyltyrosines; Mice; Naloxone; Protease Inhibitors; Thiorphan; Tiopronin