Luminescent gold nanoparticles capped with tiopronin derivatives.

This paper describes the preparation of gold nanoparticles passivated with different tiopronin-like thiol peptide derivatives. The average size of these gold particles falls into the range of 1.7-2.1 nm with narrow dispersity. All the gold nanoparticles exhibit near-IR luminescence in the spectral range of 700-950 nm. The luminescence properties of the gold nanoparticles depend on the structure of the capping ligands; those capped with polar thiol peptides give weaker luminescence in water. Reducing the packing efficiency of the passivating layer by bulky ligands is likely to facilitate the luminescence quenching effects of foreign quenchers and hence weaken the luminescence of the gold nanoparticles.

Nanoparticles comprising a mixed monolayer for specific bindings with biomolecules.

This work presents a strategy of using mixed monolayer protected nanoparticles for specific interactions with target biological molecules. The mixed monolayer is composed of a shielding component and a capture component. The shielding component utilizes ethylene glycol oligomers to prevent nonspecific binding with biomolecules. The capture component is chosen to specifically interact with the target of interest, such as a protein molecule. Such a concept was demonstrated by two synthetic systems. The first one is gold nanoparticles protected by a mixed monolayer of tri(ethylene glycol) thiol (EG(3)-SH) and tiopronin (Tp), which was prepared by a one-step synthesis. Surface chemical composition studies using (1)H NMR spectroscopy revealed that the reactivity of EG(3)-SH is 3 times as high as that of Tp in the nanoparticle formation. Gel electrophoresis analysis identified a critical ratio of (EG(3)-S-)/Tp on the nanoparticle surface above which no nonspecific binding occurred. By further derivatizing Tp into a biotin group, we synthesized Au(-S-EG(3))(n)/Tp-biotin particles that bind specifically to streptavidin with negligible nonspecific binding. The second system is gold nanoparticles protected by a mixed monolayer of EG(3)-SH and glutathione (GSH). By controlling the feeding ratio of EG(3)-SH and GSH, we made Au(-S-EG(3))(n)/GSH particles that bind specifically to gultathione-S-transferase (GST) with negligible nonspecific binding.

Influenza infection causes airway hyperresponsiveness by decreasing enkephalinase.

Ferret tracheal segments were infected with human influenza virus A/Taiwan/86 (H1N1) in vitro. After 4 days, the smooth muscle contractile responses to acetylcholine and to substance P were measured. The response to substance P was markedly accentuated, with a threefold increase in force of contraction at a substance P concentration of 10(-5) M, the highest concentration tested. In contrast, the response to acetylcholine was not affected by viral infection. Histological examination of tissues revealed extensive epithelial desquamation. Activity of enkephalinase (neutral metallo-endopeptidase, EC.3.4.24.11), an enzyme that degrades substance P, was decreased by 50% in infected tissues. Inhibiting enkephalinase activity by pretreating with thiorphan (10(-5) M) increased the response to substance P to the same final level in both infected and control tissues. Inhibiting other substance P-degrading enzymes including kininase II (angiotensin-converting enzyme), serine proteases, and aminopeptidases did not affect the response to substance P. Inhibiting cyclooxygenase and lipoxygenase activity using indomethacin and BW 755c did not affect hyperresponsiveness to substance P. Pretreating tissues with antagonists of alpha-adrenoceptors, beta-adrenoceptors, and H1 histamine receptors (phentolamine 10(-5) M, propranolol 5 X 10(-6) M, and pyrilamine 10(-5) M, respectively) had no effect on substance P-induced contraction. These results demonstrate that infection of ferret airway tissues with influenza virus increases the contractile response of airway smooth muscle to substance P. This effect is caused by decreased enkephalinase activity in infected tissues.

Effect of neutral endopeptidase inhibitors on 3H-substance P binding in rat ileum.

To determine whether neutral endopeptidase regulates the binding of substance P to the receptors, and if so, what the mechanism is, we determined the effect of neutral endopeptidase inhibitors, thiorphan and phosphoramidon, on specific binding of 3H-substance P to homogenates of rat ileum. Specific binding was of high affinity and was saturable (dissociation constant, KD = 2.4 +/- 0.17 nM and number of maximal binding sites, Bmax = 101.1 +/- 5.5 fmol/mg protein), and the receptor subtype was substance P-P type. Neutral endopeptidase inhibitors increased the specific binding to up to 160% of control (P less than 0.005). Neutral endopeptidase inhibitors prevented the degradation of 3H-substance P during the binding assay and increased the amount of 3H-substance P remaining in the assay system to up to 4.5-fold of control (P less than 0.005), but did not significantly change the KD or Bmax values of specific binding. Protease inhibitors of kininase II, serine proteinases, or thiol proteinases did not significantly change either specific binding or the amount of 3H-substance P remaining in the assay system. We conclude that neutral endopeptidase regulates the binding of substance P to the receptors and that it does so by decreasing the amount of substance P available to the receptors, without significantly changing the affinity or the number of receptors.

Labelling and exploration of the active site of enkephalinase (EC 3.4.24.11) in kidney membranes with [3H]thiorphan as ligand.

[3H]Thiorphan, a potent inhibitor of enkephalinase (EC 3.4.24.11), was used to label the enzyme in membranes from rat kidney cortex an to explore its specificity at the active site. [3H]Thiorphan binding occurred reversibly, with low non-specific binding and to a single class of sites. The dissociation constant, determined by either kinetics or saturation studies was approximately 0.4 nM. The ratio of the maximal velocity of enkephalinase with enkephalins as substrates to the maximal binding of [3H]thiorphan was consistent with the catalytic constant of the enzyme. Enkephalinase inhibitors competed with [3H]thiorphan and had inhibitory constants in agreement with the corresponding values derived from measurement of the enzyme catalytic activity, whereas inhibitors of other metallopeptidases were ineffective. The inhibitory potencies of a series of systematically varied oligopeptides regarding [3H]thiorphan binding and enkephalinase activity were also highly correlated. Structure-activity relationships among competitors indicated that the main subsites of enkephalinase are: (1) the hydrophobic pocket in P'1, the requirements of which are best satisfied by aromatic amino acid side chain residues (2) the P'2 subsite, the requirements of which are best satisfied by amino acids with a short, uncharged side chain and a free terminal carboxyl group. This novel binding assay should facilitate the exploration of the active site of enkephalinase and the development of new inhibitors.

Neutral endopeptidase and angiotensin converting enzyme inhibitors potentiate kinin-induced contraction of ferret trachea.

Neutral endopeptidase (NEP) (enkephalinase, EC 3.4.24.11) and angiotensin converting enzyme (ACE) are two peptidases that can cleave the C-terminal dipeptide bradykinin(8-9) from bradykinin. To determine whether these peptidases play roles in modulating kinin-induced contractions in the airways, we studied the effects of captopril, an ACE inhibitor, and of leucine-thiorphan and phosphoramidon, two NEP inhibitors, on the contractile responses to bradykinin and lysyl-bradykinin in isolated segments of ferret trachea. Bradykinin and lysyl-bradykinin-induced contractions in a concentration-dependent fashion (P less than .001), with a threshold of 10(-7) M and 5 x 10(-7) M, respectively. In contrast, the bradykinin(8-9) and the N-terminal heptapeptide bradykinin(1-7), the major fragments of hydrolysis of bradykinin by NEP and ACE, had a very weak or no effect on tracheal contraction in concentrations as great as 10(-5) M. Captopril, leucine-thiorphan and phosphoramidon (each inhibitor at 10(-5) M, 15 min) shifted the concentration-response curves to lower concentrations by approximately 1 to 1.5 log U (P less than .05). Both NEP inhibitors and the ACE inhibitor potentiated the response to bradykinin in a concentration-dependent fashion (P = .0001), and the combination of phosphoramidon and captopril resulted in an additive potentiation of bradykinin-induced contraction (P less than .02). [D-Pro2-D-Trp7,9]-substance P, a substance P antagonist, did not modify the potentiation of bradykinin-induced contraction by NEP inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Substance P-induced bronchoconstriction in the guinea pig. Enhancement by inhibitors of neutral metalloendopeptidase and angiotensin-converting enzyme.

We tested the effects of the neutral metalloendopeptidase (NEP) inhibitor, thiorphan (0.17, 0.5, and 1.7 mg i.v), and the angiotensin-converting enzyme (ACE) inhibitor, captopril (0.5, 1.7, and 5.0 mg i.v.), on the bronchoconstrictor response to rapid intravenous infusions of substance P (0.1 to 30 nmol/kg) in anesthetized, mechanically ventilated guinea pigs. The decreases in pulmonary conductance and dynamic compliance caused by substance P were greater in animals treated with either thiorphan or captopril than in control animals. Thiorphan (0.5 mg) had no effect on airway responsiveness to intravenously administered methacholine, whereas captopril (1.7 mg) caused a small increase in methacholine responsiveness. Both drugs significantly increased the recovery of immunoreactive substance P in arterial plasma after exogenous administration of the peptide. We conclude that degradation of substance P by both NEP and ACE is important for determining the magnitude of the bronchoconstriction caused by intravenous administration of this neuropeptide. These data suggest that conditions associated with diminished peptidase activity could result in enhanced responses to stimuli which cause the release of endogenous substance P.

Release of a dynorphin A (1-8) degrading enzyme from the spinal cord by intraventricular morphine in the rat.

Intraventricular injection of morphine sulfate, 40 micrograms, released an enzyme from the spinal cord into the perfusate which degraded dynorphin A (1-8) and, to a lesser extent, dynorphin A (1-13) in urethane anesthetized rats. The enzyme did not degrade dynorphin A (1-17), Met-enkephalin, Leu-enkephalin, substance P and neurotensin. This dynorphin A (1-8) degrading enzyme was inhibited by aprotinin, thiorphan, and, to a lesser extent, by bacitracin but was not inhibited by bestatin. A kinetic study of the interaction between dynorphin A (1-8) and aprotinin with the enzyme indicated that it is competitive in nature. The pharmacological significance of the findings is still unknown.

Inhibition of enkephalin degradation attenuated stress-induced motor suppression (conditioned suppression of motility).

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.

Influence of enkephalinase inhibitors on gastric emptying in mice depends on the nature of the meal.

The effects of two enkephalinase inhibitors (thiorphan and acétorphan) and DALAMIDE on gastric emptying of fat or non-fat meals were evaluated in mice. When administered intraperitonally at low doses (0.1 and 0.2 mg/kg) 30 min prior to a fatty (milk) meal, both thiorphan and acetorphan increased significantly (P less than 0.01) gastric emptying; these effects were maximal for 0.2 and 0.1 mg/kg respectively and decreased progressively to be not significant for doses higher than 5 mg/kg for thiorphan and 0.5 mg/kg for acetorphan. Similarly DALAMIDE given IP increased significantly (P less than 0.05) gastric emptying at doses of 0.5 and 1 mg/kg while a slowing of gastric emptying was obtained for 10 times higher doses. The effects of thiorphan (0.2 mg/kg) and DALAMIDE (0.5 mg/kg) were blocked by previous administration of naloxone (0.3 mg/kg) and methyl-naloxone (0.5 mg/kg) while only naloxone (0.3 mg/kg) blocked the slowing effect of high dose of DALAMIDE. Administered prior to a non-fat meal, thiorphan (1 mg/kg) stimulated gastric emptying and inhibited it at higher dosage (10 mg/kg). Neither acetorphan nor DALAMIDE at similar dosages affected the gastric emptying of a non-fat meal and the effects of thiorphan (1 and 0.1 mg/kg) were not blocked by naloxone (0.3 mg/kg). It is concluded that enkephalinase inhibitors (thiorphan and acetorphan) administered systemically stimulate the gastric emptying of a fat meal by increasing enkephalin levels in peripheral tissues, while thiorphan exhibits non-opiate effects on gastric emptying of a non-fat meal.

Amelioration of naloxone-precipitated opioid withdrawal symptoms by peripheral administration of the enkephalinase inhibitor acetorphan.

The effects of 60 min pretreatment with the enkephalinase inhibitor acetorphan were assessed on naloxone-precipitated (2.5 mg/kg IP) abstinence in chronically morphinized rats. In addition, the antinociceptive activity of the compound was investigated in mice. Intraperitoneal injection (50 mg/kg) in rats attenuated some aspects of the opioid withdrawal syndrome such as burrowing, wet dog shakes, squeal on touch hostility, tachypnoea, ptosis and rough hair, whereas jumping and escape behaviour were significantly increased in acetorphan-treated animals. No effect was observed on withdrawal hypothermia or acute weight loss. Similarly, chronic dosing with acetorphan after withdrawal produced no significant effect on body weight. Acetorphan (50 mg/kg IP) failed to produce any antinociceptive activity in the mouse tail immersion test, but potentiated the antinociceptive effect of D-Ala2-D-Leu5-enkephalin. These results are discussed in terms of acetorphan crossing the blood-brain barrier before being hydrolysed to thiorphan, thus yielding opioid withdrawal relieving effects.

Role of endogenous enkephalins in locomotion evidenced by acetorphan, an "enkephalinase" inhibitor.

To investigate whether endogenous enkephalins modulate locomotion we studied the effect of the systemic administration of acetorphan, a parenterally active "enkephalinase" inhibitor. Locomotor activity in mice and rats was considered as an index to the activity of mesolimbic dopaminergic neurons. Acetorphan injected i.v. induced an increase in locomotion, mice and rats presenting a similar behavioral response. Naloxone, at low doses, blocked the enhanced motor response. The increased locomotion was antagonized by a pretreatment with haloperidol or potentiated by GBR 12783, a potent and specific inhibitor of dopamine (DA) uptake. The neurotoxic lesion of the mesolimbic DA system with 6-hydroxydopamine abolished the effect of acetorphan. These data suggest that the locomotor hyperactivity induced by the enkephalinase inhibitor results from the protection of local endogenous enkephalins and may be mesolimbic DA-dependent.

Enkephalinase inhibitor potentiates mammalian tachykinin-induced contraction in ferret trachea.

To determine the roles of endogenous enkephalinase (EC.3.4.24.11) in regulating tachykinin-induced contraction of airway smooth muscle, the authors studied the effects of the enkephalinase inhibitor leucine-thiorphan on the contractile responses to substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) in isolated ferret tracheal smooth muscle segments. Leucine-thiorphan shifted, in concentration-dependent fashions, the dose-response curves to all tachykinins to lower concentrations. Leucine-thiorphan changed the rank order of tachykinin potency from NKA greater than SP greater than NKB to NKA = NKB greater than SP. Removal of the epithelium slightly enhanced the contractile responses to SP and NKA but not to NKB. Atropine shifted the dose-response curves of all tachykinins to higher concentrations. Each tachykinin increased the contractile response to electrical field stimulation (5 Hz, 20 sec of duration, 20 V) in a dose-dependent fashion. This effect was not altered by hexamethonium, indomethacin, BW755C or naloxone but was potentiated by leucine-thiorphan and inhibited by the tachykinin receptor antagonist (D-Pro2, D-Trp7,9)-SP and by atropine. Because tachykinins did not affect contractile responses to acetylcholine significantly, their effects were probably on presynaptic postganglionic nerves. Captopril, bestatin and leupeptin did not alter contractile responses, suggesting that angiotensin converting enzyme, aminopeptidases and serine proteinases did not modulate tachykinin-induced effects. Enkephalinase immunofluorescence was found in the smooth muscle and epithelium and confirmed the authors' finding of enkephalinase-like activity in the muscle. The results suggest that tracheal enkephalinase is an important modulator of tachykinin-induced effects.

Naloxone-reversible antidiarrheal effects of enkephalinase inhibitors.

Thiorphan and acetorphan, two potent inhibitors of enkephalinase (EC 3.4.24.11 membrane-metalloendopeptidase) significantly reduced the castor oil-induced diarrhea in rats when administered intravenously (or orally, for acetorphan) but not when administered intracerebroventricularly. These effects were more marked during the 90 min period following the castor oil challenge but were still significant up to 4-8 h after the latter. Acetorphan was about 6 times more potent than thiorphan. The antidiarrheal activity of both compounds was completely prevented in rats receiving naloxone subcutaneously but not intracerebroventricularly (in the case of thiorphan). In contrast to loperamide, a peripherally acting opiate receptor agonist, the enkephalinase inhibitors did not significantly reduce gastrointestinal transit as measured in the charcoal meal test. The antidiarrheal activity of enkephalinase inhibitors therefore seems attributable to protection of endogenous opioids, presumably outside the brain, and to reduction of intestinal secretion rather than transit.

Enkephalinase is involved in the degradation of endogenous substance P released from slices of rat substantia nigra.

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.

Enantiomers of thiorphan and acetorphan: correlation between enkephalinase inhibition, protection of endogenous enkephalins and behavioral effects.

The relationships between various properties of inhibitors of enkephalinase (membrane metalloendopeptidase, EC 3.4.24.11) i.e., enzyme inhibition, protection of endogenous enkephalins, antinociceptive activity and stimulation of locomotor activity was investigated by comparing the relative potencies of the two enantiomers of Thiorphan and acetorphan, its parenterally active prodrug. In vitro (R)- and (S)-Thiorphan were almost equipotent in inhibiting enkephalinase activity (Ki, 1.7 and 2.2 nM, respectively) or thermolysin activity (Ki, 13 and 6 microM, respectively) whereas the (R)-isomer was 44-fold less potent than the (S)-isomer on ACE activity (Ki 4800 and 110 nM, respectively). When tested on slices of rat globus pallidus in the presence of bestatin, to block the aminopeptidase pathway of enkephalin degradation, both Thiorphan enantiomers ensured a complete protection of endogenous (Met5)enkephalin released by depolarization and a suppression of the increase in the extracellular levels of Tyr-Gly-Gly, a characteristic enkephalin metabolite. These two effects occurred at EC50 values of the two enantiomers (10 nM in both cases), consistent with the idea that they were due to enkephalinase inhibition. After i.v. administration of the acetorphan enantiomers to mice, the enkephalinase activity of a rapidly prepared striatal membrane fraction was reduced in a dose-dependent manner with similar "ex vivo" ED50 values (1.0 and 0.3 mg/kg for the (R)- and (S)-isomer, respectively). In contrast the ACE activity of the same preparation was reduced in a significant manner only by (S)-acetorphan (ED50 value of 11 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Enkephalinase inhibitor potentiates substance P- and electrically induced contraction in ferret trachea.

To determine the role of endogenous enkephalinase (EC 3.4.24.11) in regulating peptide-induced contraction of airway smooth muscle, we studied the effect of the enkephalinase inhibitor, leucine-thiorphan (Leu-thiorphan), on responses of isolated ferret tracheal smooth muscle segments to substance P (SP) and to electrical field stimulation (EFS). Leu-thiorphan shifted the dose-response curve to SP to lower concentrations. Atropine or the SP antagonist [D-Pro2,D-Trp7,9]SP significantly inhibited SP-induced contractions in the presence of Leu-thiorphan. Leu-thiorphan increased the contractile responses to EFS dose dependently, an effect that was significantly inhibited by the SP antagonist [D-Pro2,D-Trp7,9]SP. SP, in a concentration that did not cause contraction, increased the contractile responses to EFS. This effect was augmented by Leu-thiorphan dose dependently and was not inhibited by hexamethonium or by phentolamine but was inhibited by atropine. Because contractile responses to acetylcholine were not significantly affected by SP or by Leu-thiorphan, the potentiating effects of SP were probably on presynaptic-postganglionic cholinergic neurotransmission. Captopril, bestatin, or leupeptin did not augment contractions, suggesting that enkephalinase was responsible for the effects. These results suggest that endogenous tachykinins modulate smooth muscle contraction and endogenous enkephalinase modulates contractions produced by endogenous or exogenous tachykinins and tachykinin-induced facilitation of cholinergic neurotransmission.

Relationship between the inhibitory potencies of thiorphan and retrothiorphan enantiomers on thermolysin and neutral endopeptidase 24.11 and their interactions with the thermolysin active site by computer modelling.

The inhibitory potency of separate enantiomers of thiorphan and retrothiorphan has shown that several particularities of the active site of thermolysin are also present in the neutral endopeptidase 24.11, "enkephalinase", such as its ability: i) to recognize a retroamide bond as well as a standard amide bond, ii) to interact similarly with residues in P1' position of either R or S configuration in the thiorphan series but contrastingly to discriminate between the R and S isomers in the retrothiorphan series. These four inhibitors were modellized in the thermolysin active site and their spatial arrangement compared with that of a thiol inhibitor co-crystallized with thermolysin. In all cases, the essential interactions involved in the stabilization of the bound inhibitor were conserved. However, the bound (R) retrothiorphan displayed unfavorable intramolecular contacts, accounting for its lower inhibitory potency for the two metallopeptidases.

Phelorphan, an inhibitor of enzymes involved in the biodegradation of enkephalins, affected the withdrawal symptoms in chronic morphine-dependent rats.

Intracerebroventricular administration of phelorphan (158 nmol/2 microliters), a blocker of dipeptidylaminopeptidase (enkephalinase B) and other enzymes involved in the enkephalin biodegradation, inhibited in chronic morphine-dependent rats, the occurrence of some of the naloxone-precipitated withdrawal symptoms. This effect of phelorphan was compared with an equimolar dose of the dipeptidyl-carboxypeptidase inhibitor (enkephalinase A), thiorphan. The results indicate that both drugs decrease some of the naloxone-precipitated withdrawal symptoms (writhing, digging, head hiding, chewing, diarrhoea and Straub tail), while others were potentiated (penile licking) or unaltered (wet dog shakes, grooming and rearing). In addition, phelorphan compared with the controls or thiorphan, pretreated animals, increased the frequency of paw tremor, head shakes, scratching, erection and ejaculation, but other symptoms were decreased (stretching) or unaltered (teeth chattering). The results are discussed in light of the differences in permeability and specificity of the two enkephalinase inhibitors. Furthermore, these data support the hypothesis that the use of enkephalinase inhibitors might be a promising way for the attenuation of the severity of the withdrawal syndrome.

Analgesic effect of actinonin, a new potent inhibitor of multiple enkephalin degrading enzymes.

Actinonin, previously isolated as an antibiotic and shown to be an inhibitor of aminopeptidase M (EC 3.4.11.2), has now been shown to inhibit three enkephalin-degrading enzymes from guinea-pig striatum. The values of IC50 were 0.39 microM for striatal membrane aminopeptidase ("enkephalin-aminopeptidase") and 5.6 microM striatal membrane neutral endopeptidase ("enkephalinase A"). Furthermore, soluble dipeptidylaminopeptidase in a rat whole brain homogenate was also inhibited by actinonin with the IC50 value of 1.1 microM. Actinonin administered intracisternally (i.cist., 50 micrograms) or intraperitoneally (i.p., 100 mg/kg), potentiated the analgesic action of met-enkephalin (50 micrograms i.cist.). analgesia by a tail-flick test. The potentiating activity of actinonin i.p. to met-enkephalin analgesia was almost the same potency as that of thiorphan, whereas the inhibitory activity of actinonin against enkephalinase A was 1/1000 that of thiorphan. Actinonin alone, administered either i.cist. or i.p., showed an analgesic action as estimated by the tail-flick test.