phosphoramidon and norbinaltorphimine

Sialorphin is an exocrine and endocrine signaling mediator, which has been identified by a genomic approach. It is synthesized predominantly in the submandibular gland and prostate of adult rats in response to androgen steroids and is released locally and systemically in response to stress. We now demonstrate that the cell surface molecule to which sialorphin binds in vivo in the rat kidney is the membrane-anchored neutral endopeptidase (neprilysin; NEP, EC 3.4.24.11). NEP plays an important role in nervous and peripheral tissues, as it turns off several peptide-signaling events at the cell surface. We show that sialorphin prevents spinal and renal NEP from breaking down its two physiologically relevant substrates, substance P and Met-enkephalin in vitro. Sialorphin inhibited the breakdown of substance P with an IC50 of 0.4-1 microM and behaved as a competitive inhibitor. In vivo, i.v. sialorphin elicited potent antinociceptive responses in two behavioral rat models of injury-induced acute and tonic pain, the pin-pain test and formalin test. The analgesia induced by 100-200 mcicrog/kg doses of sialorphin required the activation of mu- and delta-opioid receptors, consistent with the involvement of endogenous opioid receptors in enkephalinergic transmission. We conclude that sialorphin protects endogenous enkephalins released after nociceptive stimuli by inhibiting NEP in vivo. Sialorphin is a natural systemically active regulator of NEP activity. Furthermore, our study provides evidence that it is a physiological modulator of pain perception after injury and might be the progenitor of a new class of therapeutic molecules.

Topics: Amino Acid Sequence; Analgesics; Animals; Enkephalin, Methionine; Formaldehyde; Glycopeptides; Kidney; Leucine; Male; Membrane Proteins; Molecular Sequence Data; Naltrexone; Neprilysin; Pain; Pain Measurement; Prostate; Protease Inhibitors; Protein Precursors; Rats; Rats, Wistar; Receptors, Opioid, delta; Receptors, Opioid, mu; Salivary Proteins and Peptides; Spinal Cord; Submandibular Gland; Substance P; Thiorphan; Wounds and Injuries

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

The antinociceptive effect of intrathecally (i.t.) administered protease inhibitors was tested against capsaicin (800 ng) injected into the dorsal surface of a hindpaw. Both p-hydroxymercuribenzoate (2-8 nmol), a cysteine protease inhibitor, and phosphoramidon (1-4 nmol), an endopeptidase 24.11 inhibitor in the presence of bestatin (0.25 nmol) an aminopeptidase inhibitor, administered i.t. 60 min prior to the injection of capsaicin produced a dose-dependent reduction of the capsaicin-induced paw licking and biting response. p-Hydroxymercuribenzoate (4 nmol)-induced antinociception was significantly antagonized by nor-binaltorphimine, a selective kappa-opioid receptor antagonist, but not by naltrindole, a selective delta-opioid receptor antagonist. On the other hand, phosphoramidon (4 nmol) /bestatin-induced antinociception was significantly antagonized by naltrindole, but not by nor-binaltorphimine. The results indicate that the antinociceptive effect of p-hydroxymercuribenzoate may be due to the inhibition of a cysteine protease degrading endogenous dynorphins whereas phosphoramidon in the presence of bestatin blocks the degradation of enkephalins.

Topics: Animals; Capsaicin; Dose-Response Relationship, Drug; Drug Combinations; Glycopeptides; Hindlimb; Hydroxymercuribenzoates; Injections, Spinal; Leucine; Male; Mice; Mice, Inbred Strains; Naltrexone; Narcotic Antagonists; Pain; Protease Inhibitors; Receptors, Opioid; Time Factors