dynorphins and phosphoramidon

Brain region-specific expression of proteolytic enzymes can control the biological activity of endogenous neuropeptides and has recently been targeted for the development of novel drugs, for neuropathic pain, cancer, and Parkinson's disease. Rapid and sensitive analytical methods to profile modulators of enzymatic activity are important for finding effective inhibitors with high therapeutic value. Combination of in situ enzyme histochemistry with MALDI imaging mass spectrometry allowed developing a highly sensitive method for analysis of brain-area specific neuropeptide conversion of synthetic and endogenous neuropeptides, and for selection of peptidase inhibitors that differentially target conversion enzymes at specific anatomical sites. Conversion and degradation products of Dynorphin B as model neuropeptide and effects of peptidase inhibitors applied to native brain tissue sections were analyzed at different brain locations. Synthetic dynorphin B (2pmol) was found to be converted to the N-terminal fragments on brain sections whereas fewer C-terminal fragments were detected. N-ethylmaleimide (NEM), a non-selective inhibitor of cysteine peptidases, almost completely blocked the conversion of dynorphin B to dynorphin B(1-6; Leu-Enk-Arg), (1-9), (2-13), and (7-13). Proteinase inhibitor cocktail, and also incubation with acetic acid displayed similar results. Bioconversion of synthetic dynorphin B was region-specific producing dynorphin B(1-7) in the cortex and dynorphin B (2-13) in the striatum. Enzyme inhibitors showed region- and enzyme-specific inhibition of dynorphin bioconversion. Both phosphoramidon (inhibitor of the known dynorphin converting enzyme neprilysin) and opiorphin (inhibitor of neprilysin and aminopeptidase N) blocked cortical bioconversion to dynorphin B(1-7), wheras only opiorphin blocked striatal bioconversion to dynorphin B(2-13). This method may impact the development of novel therapies with aim to strengthen the effects of endogenous neuropeptides under pathological conditions such as chronic pain. Combining histochemistry and MALDI imaging MS is a powerful and sensitive tool for the study of inhibition of enzyme activity directly in native tissue sections.

Topics: Animals; Brain; Cysteine Endopeptidases; Dynorphins; Endorphins; Glycopeptides; Humans; Neuropeptides; Oligopeptides; Parkinson Disease; Protease Inhibitors; Rats; Salivary Proteins and Peptides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

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

The antinociceptive effects of intracerebroventricularly (i.c.v.) administered dynorphin A, an endogenous agonist for kappa-opioid receptors, in combination with various protease inhibitors were examined using the mouse formalin test in order to clarify the nature of the proteases involved in the degradation of dynorphin A in the mouse brain. When administered i.c.v. 15 min before the injection of 2% formalin solution into the dorsal surface of a hindpaw, 1-4 nmol dynorphin A produced a dose-dependent reduction of the nociceptive behavioral response consisting of licking and biting of the injected paw during both the first (0-5 min) and second (10-30 min) phases. When co-administered with p-hydroxymercuribenzoate (PHMB), a cysteine protease inhibitor, dynorphin A at the subthreshold dose of 0.5 nmol significantly produced an antinociceptive effect during the second phase. This effect was significantly antagonized by nor-binaltorphimine, a selective kappa-opioid receptor antagonist, but not by naltrindole, a selective delta-opioid receptor antagonist. At the same dose of 0.5 nmol, dynorphin A in combination with phosphoramidon, an endopeptidase 24.11 inhibitor, produced a significant antinociceptive effect during both phases. The antinociceptive effect was significantly antagonized by naltrindole, but not by nor-binaltorphimine. Phenylmethanesulfonyl fluoride (PMSF), a serine protease inhibitor, bestatin, a general aminopeptidase inhibitor, and captopril, an angiotensin-converting enzyme inhibitor, were all inactive. The degradation of dynorphin A by mouse brain extracts in vitro was significantly inhibited only by the cysteine protease inhibitors PHMB and N-ethylmaleimide, but not by PMSF, phosphoramidon, bestatin or captopril. The present results indicate that cysteine proteases as well as endopeptidase 24.11 are involved in two steps in the degradation of dynorphin A in the mouse brain, and that phosphoramidon inhibits the degradation of intermediary delta-opioid receptor active fragments enkephalins which are formed from dynorphin A.

Topics: Animals; Brain; Cell Extracts; Drug Interactions; Dynorphins; Glycopeptides; Hydroxymercuribenzoates; Injections, Intraventricular; Mice; Naltrexone; Narcotic Antagonists; Nociceptors; Pain; Pain Measurement; Protease Inhibitors; Rats

Earlier studies indicate that nitrous oxide antinociception is mediated by opioid receptors, and we have hypothesized that nitrous oxide stimulates a neuronal release of an endogenous opioid peptide (EOP) that stimulates opioid receptors. To further test this hypothesis, male NIH Swiss mice were pretreated intracerebroventricularly with rabbit antisera to opioid peptides or with various inhibitors of peptidases involved in the degradation of EOPs. Mice were subsequently exposed to three different concentrations of nitrous oxide in oxygen, and their antinociceptive responsiveness was measured using the acetic acid abdominal constriction test. Nitrous oxide antinociception was significantly attenuated by 24-h pretreatment with antisera to various fragments of dynorphin (DYN) but not by antisera against methionine-enkephalin (ME) or beta-endorphin (beta-EP). In other experiments, nitrous oxide antinociception was significantly enhanced by 30-min pretreatment with phosphoramidon, an inhibitor of endopeptidase 24.11, which has been implicated in DYN degradation, but not bestatin or captopril, which inhibit aminopeptidase and angiotensin-converting enzyme, respectively. The latter enzymes have been implicated in degradation of certain EOPs albeit not DYN. These findings support the hypothesis that nitrous oxide antinociception in the mouse abdominal constriction test is mediated by endogenous DYN acting in the central nervous system.

Topics: Analgesics; Animals; Dose-Response Relationship, Drug; Dynorphins; Glycopeptides; Immune Sera; Male; Mice; Nitrous Oxide; Opioid Peptides; Pain Measurement; Protease Inhibitors; Rabbits

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

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

The effect of peptidase inhibitors on the degradation of [3H]-bradykinin by rat hypothalamic slices was studied using HPLC to separate and identify the products. The degradation appears to be mainly mediated by an enzyme which cleaves the peptide at the Phe5-Ser6 bond and is inhibited by 1,10-phenanthroline, dynorphin(1-13) and carboxyphenylethyl-Ala-Ala-Phe-p-aminobenzoate. This suggest the involvement of a membrane bound variant of the soluble metalloendopeptidase (EC3.4.24.15) isolated from rat brain which degrades neurotensin, angiotensin and other neuropeptides as well as bradykinin.

Topics: 4-Aminobenzoic Acid; Animals; Bradykinin; Captopril; Chromatography, High Pressure Liquid; Dynorphins; Endopeptidases; Glycopeptides; Hypothalamus; Leucine; Male; Metalloendopeptidases; Neurotensin; Oligopeptides; para-Aminobenzoates; Peptide Fragments; Phenanthrolines; Rats; Rats, Inbred Strains