dynorphins and amastatin

The N- and C-terminal regions of dynorphin (Dyn) A (1-17) activate opioid and N-methyl-D-aspartate receptors, respectively. Earlier studies demonstrated that Dyn-converting enzyme cleaved Dyn A (1-17) mainly at the Arg(6)-Arg(7) bond, resulting in the production of N- and C-terminal region peptide fragments, and that this enzyme was not inhibited by a mixture of the three peptidase inhibitors (PIs) amastatin (A), captopril (C), and phosphoramidon (P). The purpose of the present study was to evaluate antinociceptive potential and toxicity with intracerebroventricular administration of Dyn A (1-17) or (1-13) under pretreatment with a mixture of A, C, and P and/or Dyn-converting enzyme inhibitor (p-hydroxymercuribenzoate).. Peptide fragments from Dyn A (1-17) following incubation with membrane preparation under pretreatment with a mixture of the three PIs was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS). Infusion of drugs and peptides into the third ventricle in rats was performed via indwelling cannulae. Induction of antinociception and toxicity by Dyn A (1-17), Dyn A (1-13), Dyn A (1-6), or Dyn A (7-17) were determined by the tail-flick test and induction of barrel rotation, respectively. The effects of the PIs on antinociception and toxicity were evaluated by a dose-response study and a comparison of differences among various combinations of Dyn A (1-17) or Dyn A (1-13) and the three PIs and p-hydroxymercuribenzoate.. MALDI-TOF-MS analysis identified Dyn A (1-6) and Dyn A (1-10) fragments as products following incubation of Dyn A (1-17) with membrane preparation of rat midbrain under pretreatment with a mixture of the three PIs. Pretreatment with a mixture of the three PIs produced an approximately 30-fold augmentation in antinociception induced by low-dose intracerebroventricular administration of Dyn A (1-17) or (1-13) in a μ-, δ- and κ-opioid receptor antagonist-reversible manner, but without signs of toxicity such as barrel rotation in the rat. Dyn A (1-17)-induced antinociception and toxicity was greater than that of Dyn A (1-6), Dyn A (1-13), or Dyn A (7-17) at the same dose. Dyn A (1-17)-induced antinociception and toxicity under pretreatment with various combinations of the three PIs and p-hydroxymercuribenzoate was greater than that with a mixture of the three PIs alone.. These findings suggest that administration of a mixture of the three PIs increases Dyn A (1-17)- or (1-13)-induced antinociception under physiological conditions without toxicity.

Topics: Analgesics; Analgesics, Opioid; Animals; Brain Chemistry; Captopril; Dose-Response Relationship, Drug; Dynorphins; Glycopeptides; Injections, Intraventricular; Male; Pain Measurement; Peptides; Protease Inhibitors; Rats; Rats, Wistar; Receptors, Opioid

Previous in vitro studies showed that the degradation of dynorphin-(1-8) [dyn-(1-8)] by cerebral membrane preparations is almost completely prevented by a mixture of three peptidase inhibitors (PIs), amastatin, captopril and phosphoramidon. In the present investigations, effects of the three PIs on the anti-nociception induced by the intra-third-ventricular (i.t.v.) administration of dyn-(1-8) were examined. The inhibitory effect of dyn-(1-8) on the tail-flick response was increased more than 100-fold by the i.t.v. pretreatment of rats with the three PIs. The inhibition produced by dyn-(1-8) in rats pretreated with any combination of two PIs was significantly smaller than that in rats pretreated with three PIs, indicating that any residual single peptidase could inactivate significant amounts of dyn-(1-8). The antagonistic effectiveness of naloxone, a relatively selective mu-opioid antagonist, indicates that dyn-(1-8)-induced inhibition of tail-flick response in rats pretreated with three PIs is mediated by mu-opioid receptors. Furthermore, mu-receptor-mediated inhibition induced by dyn-(1-8) was significantly greater than that produced by [Met5]-enkephalin in rats pretreated with three PIs. The data obtained in the present investigations together with those obtained in previous studies strongly indicate that dyn-(1-8) not only has well-known kappa-agonist activity but also has high mu-agonist activity.

Topics: Analgesics, Opioid; Analysis of Variance; Angiotensin-Converting Enzyme Inhibitors; Animals; Anti-Bacterial Agents; Captopril; Drug Interactions; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Glycopeptides; Injections, Intraventricular; Male; Naloxone; Narcotic Antagonists; Pain; Pain Measurement; Peptide Fragments; Peptides; Protease Inhibitors; Rats; Rats, Wistar; Receptors, Opioid, mu

The amounts of dynorphin-(1-8) [dyn-(1-8)] and its seven hydrolysis products, Y, YG, YGG, YGGF, YGGFL, YGGFLR and YGGFLRR, were estimated after incubating dyn-(1-8) with a membrane fraction from either guinea-pig ileum or striatum for various times at 37 degrees C. The major hydrolysis products during the initial 5-min incubation were YGGFLR and Y, which indicates that dipeptidyl carboxypeptidase and aminopeptidase activities were mainly involved in the hydrolysis. After 60 min of incubation, dyn-(1-8) was completely hydrolyzed in both membrane preparations. When the ileal and the striatal preparations were incubated for 60 min in the presence of both captopril, a dipeptidyl carboxypeptidase inhibitor, and amastatin, an aminopeptidase inhibitor, 63.8 and 49.3% of dyn-(1-8), respectively, were hydrolyzed. The YGG fragment was the major hydrolysis product in both preparations. When the ileal and the striatal membrane fractions were incubated with dyn-(1-8) in the presence of three peptidase inhibitors, captopril, amastatin and phosphoramidon (an inhibitor of endopeptidase-24.11), approximately 95% of the opioid octapeptide remained intact in both cases. This shows that dyn-(1-8) was almost exclusively hydrolyzed by three enzymes, amastatin-sensitive aminopeptidase, captopril-sensitive dipeptidyl carboxypeptidase I and phosphoramidon-sensitive endopeptidase-24.11, in both ileal and striatal membranes. Additionally, the Ke (equilibrium dissociation constant) values of selective antagonists against dyn-(1-8) and its initial main hydrolysis product YGGFLR in two isolated preparations pretreated with the three peptidase inhibitors indicate that the latter acts on mu receptors in guinea pig ileum but delta receptors in mouse vas deferens and the former acts on kappa receptors in both preparations. It is indicated, therefore, that in the absence of peptidase inhibitors endogenously released dyn-(1-8) acts either through dyn-(1-8) itself on kappa receptors or through YGGFLR on mu or delta receptors depending on both the three peptidase activities and the three receptor type densities at the target synaptic membrane.

Topics: Animals; Anti-Bacterial Agents; Biotransformation; Captopril; Chromatography, High Pressure Liquid; Dynorphins; Electrochemistry; Glycopeptides; Guinea Pigs; Hydrolysis; Hypothalamic Hormones; In Vitro Techniques; Kinetics; Male; Membranes; Mice; Mice, Inbred ICR; Peptide Fragments; Peptides; Protease Inhibitors; Receptors, Opioid, delta; Receptors, Opioid, kappa

Proteolytic processing of prodynorphin-derived peptides in rat brain was studied with the help of high performance size exclusion chromatography (SEC) connected to electrospray ionization mass spectrometry. Extracts from rat striatum were incubated with individual synthetic dynorphin peptides. Dynorphin A was the most resistant to proteolytic cleavage, converting slowly to Leu-enkephalin (0.3 pmol/min), whereas dynorphin B was processed to this pentapeptide at a 10(4)-fold higher rate. Minor cleavage was also observed between Arg6-Arg7. Alphaneoendorphin was also rapidly metabolized to Leu-enkephalin (6 nmol/min) and, to a lesser extent, to Leu-enkephalinArg6. This new strategy for studying peptidases can easily be adapted to identification of components present in body fluids.

Topics: Animals; Anti-Bacterial Agents; Chromatography, Gel; Corpus Striatum; Dynorphins; Endorphins; Enkephalin, Leucine; Enkephalins; Male; Mass Spectrometry; Peptide Hydrolases; Peptides; Protein Precursors; Rats; Sensitivity and Specificity; Tissue Extracts

Inactivation of dynorphin-(1-8) in three in vitro isolated preparations, guinea-pig ileum, mouse vas deferens and rabbit vas deferens, was estimated by employing the relatively specific inhibitors of enkephalin-hydrolyzing enzymes. All three enzyme inhibitors, amastatin, captopril and phosphoramidon, significantly enhanced the inhibitory potency of dynorphin-(1-8) in the three isolated preparations. The magnitude of the enhancement of the dynorphin potency by captopril was significantly higher than that by either amastatin or phosphoramidon in guinea-pig ileum; that by amastatin was significantly higher than that by either captopril or phosphoramidon in rabbit vas deferens; and that by amastatin was similar to that by captopril, but significantly higher than that by phosphoramidon in mouse vas deferens. The Ke values of three antagonists, naloxone, Mr 2266 and ICI 154129, against dynorphin-(1-8) in the presence of the three peptidase inhibitors indicated that dynorphin-(1-8) acted on kappa receptors in guinea-pig ileum and on both kappa and delta receptors in mouse vas deferens. Since amastatin, captopril and phosphoramidon produced the naloxone-reversible inhibition of contractions of guinea-pig ileum in the presence of dynorphin-(1-8), all three dynorphin-inactivating enzymes were indicated to be located very close to kappa receptors.

Topics: Aminopeptidases; Animals; Anti-Bacterial Agents; Captopril; Dynorphins; Glycopeptides; Guinea Pigs; Hypothalamic Hormones; In Vitro Techniques; Male; Mice; Mice, Inbred ICR; Muscle Contraction; Muscle, Smooth; Neprilysin; Oligopeptides; Peptide Fragments; Peptides; Peptidyl-Dipeptidase A; Protease Inhibitors; Rabbits

In human cerebrospinal fluid, aminopeptidase, dipeptidyl aminopeptidase, dipeptidyl carboxypeptidase, and carboxypeptidase which were capable of hydrolyzing enkephalins were detected. Among these enzymes, two distinct aminopeptidase, designated C-AP1 and C-AP2, were partially purified. These enzymes were not purified thoroughly, but the characteristics of C-AP2 were similar to those of an aminopeptidase purified from monkey brain. But the inhibitory activity of amastatin on C-AP2 was stronger, and that of substance P was negligible. On the other hand, characteristics of C-Ap1 were extremely differ from those of C-AP2 or an aminopeptidase purified from monkey brain. C-AP1 had an optimum pH more in the acidic range (the highest at pH 6.0) and was not inhibited by any of the protease inhibitor tested including bestatin and amastatin.

Topics: Aminopeptidases; Angiotensins; Anti-Bacterial Agents; Chromatography; Dynorphins; Enkephalin, Leucine; Humans; Oligopeptides; Peptide Fragments; Peptides; Protease Inhibitors; Secretin; Substance P