dynorphins and ubenimex

Inflammatory pain can be controlled by endogenous opioid peptides. Here we blocked the degradation of opioids in peripheral injured tissue to locally augment this physiological system. In rats with hindpaw inflammation, inhibitors of aminopeptidase N (APN; bestatin) or neutral endopeptidase (NEP; thiorphan), and a dual inhibitor, NH(2)-CH-Ph-P(O)(OH)CH(2)-CH-CH(2)Ph(p-Ph)-CONH-CH-CH(3)-COOH (P8B), were applied to injured paws. Combined bestatin (1.25-5 mg)/thiorphan (0.2-0.8 mg) or P8B (0.0625-1 mg) alone elevated mechanical nociceptive thresholds to 307 and 227% of vehicle-treated controls, respectively. This analgesia was abolished by antibodies to methionine-enkephalin, leucine-enkephalin, and dynorphin A 1-17, by peripherally restricted and by selective μ-, δ-, and κ-opioid receptor antagonists. Flow cytometry and photospectrometry revealed expression and metabolic activity of APN and NEP on macrophages, granulocytes, and sciatic nerves from inflamed tissue. Radioimmunoassays showed that inhibition of leukocytic APN and NEP by bestatin (5-500 μM)/thiorphan (1-100 μM) combinations or by P8B (1-100 μM) prevented the degradation of enkephalins. Blockade of neuronal peptidases by bestatin (0.5-10 mM)/thiorphan (0.1-5 mM) or by P8B (0.1-10 mM) additionally hindered dynorphin A 1-17 catabolism. Thus, leukocytes and peripheral nerves are important sources of APN and NEP in inflamed tissue, and their blockade promotes peripheral opioid analgesia.

Topics: Alanine; Amino Acid Sequence; Animals; Antibodies; CD13 Antigens; Dose-Response Relationship, Drug; Dynorphins; Enkephalin, Leucine; Enkephalin, Methionine; Enzyme Inhibitors; Flow Cytometry; Hindlimb; Inflammation; Leucine; Leukocytes; Male; Narcotic Antagonists; Neprilysin; Neurons; Opioid Peptides; Pain; Pain Threshold; Phosphinic Acids; Rats; Rats, Wistar; Receptors, Opioid; Thiorphan

Previous studies have demonstrated neuroprotective effects of the opioid peptide dynorphin (dyn) 1-13 in focal cerebral ischemia. The passage of dyn 1-13 across the blood-brain barrier (BBB) was studied by a modification of the Oldendorf technique in the normal rat and cat, as well as in a feline model of experimentally induced focal cerebral ischemia. In the rat, dyn 1-13 penetration of the BBB could not be detected by this technique, even in the presence of peptidase inhibitors. In contrast, dyn 1-13 did cross the BBB into the normal cat hippocampus, cortex and cerebellum. The passage of dyn 1-13 across the BBB was greater in cats with experimentally induced focal cerebral ischemia. Some of the tritium-labeled material which crossed the BBB was confirmed by high performance liquid chromatography to be dyn 1-13. These studies support the hypothesis that the therapeutic effects observed after the peripheral administration of dyn 1-13 to cats with focal cerebral ischemia can be produced by a central mechanism of action.

Topics: Analgesics, Opioid; Animals; Anti-Bacterial Agents; Antiviral Agents; Aprotinin; Bacitracin; Blood-Brain Barrier; Cats; Chromatography, High Pressure Liquid; Dynorphins; Leucine; Male; Neuroprotective Agents; Peptide Fragments; Radionuclide Imaging; Rats; Rats, Sprague-Dawley; Sucrose; Tyrosine

Conversion of the octapeptide dynorphin (Dyn) A-(1-8) to Leu5-enkephalin (LE) by endopeptidase EC 3.4.24.15 (EP-24.15) in vivo was examined using the technique of ventriculocisternal perfusion. Peptides were administered intracerebroventricularly in the presence or absence of the EP-24.15 inhibitor N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFPAAF-pAB) via cannulae placed into the lateral ventricle of urethane-anesthetized rats. The concentration of Dyn-like peptides and LE within the CSF was monitored by radioimmunoassay in samples of CSF taken from a second cannula placed in the cisterna magna. In the absence of inhibitor, less than 5% of the Dyn A-(1-8) administered was recovered in CSF. Immunoreactive LE, which is normally not found in CSF, increased rapidly in content following Dyn A-(1-8) infusion, an observation suggesting that the larger peptide is converted to LE. When the inhibitor cFPAAF-pAB was coadministered with Dyn A-(1-8), the concentration of immunoreactive Dyn A-(1-8) after 5 min was 40 times higher than that found in the absence of inhibitor. The angiotensin converting enzyme inhibitor captopril reduced the degradation of Dyn A-(1-8) to a much lesser degree. The inhibitor of EP-24.15 also afforded some protection of other Dyn-like peptides. No EP-24.15 activity was found in rat CSF, whereas high activity was found in the choroid plexus. Taken together, these data clearly indicate that an ectoenzyme form of EP-24.15 rapidly converts intracerebroventricularly administered Dyn-like peptides to LE.

Topics: Animals; Brain; Captopril; Cerebral Ventricles; Choroid Plexus; Dynorphins; Enkephalin, Leucine; Kinetics; Leucine; Male; Metalloendopeptidases; Neprilysin; Oligopeptides; Peptide Fragments; Peptidyl-Dipeptidase A; Perfusion; Rats; Rats, Inbred Strains

The binding of radiolabelled ligands with high affinity for kappa-opioid binding sites has been studied in homogenates of lumbo-sacral spinal cord from the rat. The unselective ligands [3H]bremazocine and [3H]diprenorphine labelled a large number of sites which could not be fully resolved in terms of mu-, delta- and kappa-types by displacement assays. In particular binding at the kappa-site appeared anomalous in that sites which could be identified as high affinity kappa-type represented only 40% of total kappa-binding, defined using the unselective [3H]ligands. This was confirmed by the low levels of binding seen with the kappa-agonists [3H]dynorphin A(1-9) and [3H]U-69593. In guinea-pig cord, under conditions in which binding to mu- and delta-sites was suppressed, [3H]dynorphin A(1-9) and [3H]U-69593 labelled only 60% of the kappa population, defined by the [3H]unselective ligands. The reasons for the observed discrepancies are discussed.

Topics: Animals; Benzeneacetamides; Benzomorphans; Captopril; Diprenorphine; Dynorphins; Guinea Pigs; In Vitro Techniques; Leucine; Ligands; Male; Peptide Fragments; Pyrrolidines; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, kappa; Spinal Cord

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

Eighteen endogenous opioid peptides, all containing the sequence of either Met5- or Leu5-enkephalin, were tested for their ability to modify nicotine-induced secretion from bovine adrenal chromaffin cells. ATP released from suspensions of freshly isolated cells was measured with the luciferin-luciferase bioluminescence method as an index of secretion. None of the peptides affected 5 microM nicotine-induced ATP release at 10 nM. Three peptides inhibited secretion at 5 microM: dynorphin1-13, dynorphin1-9, and rimorphin inhibited by 65%, 37%, and 29% respectively. Use of peptidase inhibitors (bestatin, thiorphan, bacitracin, or 1,10-phenanthroline) did not result in any of the other peptides showing potent actions on the nicotinic response, although bestatin and thiorphan did enhance the inhibitory actions of dynorphin1-13 and dynorphin1-9 by 20-30%. Nicotine-induced secretion of endogenous catecholamines from bovine chromaffin cells cultured for 3 days was also studied to assess any selective actions of the peptides on adrenaline or noradrenaline cell types. Dynorphin1-13 was 1,000-fold more potent than Leu5-enkephalin at inhibiting endogenous catecholamine secretion. Dynorphin1-13 was slightly more potent at inhibiting noradrenaline release than adrenaline release whereas Leu5-enkephalin showed the opposite selectivity. The structure-activity relationships of opioid peptide actions on the chromaffin cell nicotinic response are discussed in relation to the properties of the adrenal opioid binding sites.

Topics: Adenosine Triphosphate; Adrenal Medulla; Amino Acid Sequence; Animals; Bacitracin; Cattle; Dynorphins; Endorphins; Enkephalin, Leucine; Enkephalin, Methionine; Epinephrine; Guinea Pigs; Leucine; Nicotine; Norepinephrine; Peptide Fragments; Phenanthrolines; Rabbits; Receptors, Opioid; Thiorphan; Tiopronin

[3H]-Dynorphin A (1-8) was degraded in brain homogenates at 25 degrees and even at 0 degree C. The peptidase inhibitors bestatin and captopril almost completely protected[3H]-dynorphin A (1-8) from degradation at 0 degree C but had only little effect on binding at this temperature. At 25 degrees C, the binding of [3H]-dynorphin A (1-8) was markedly improved by addition of bestatin, captopril and L-leucyl-L-arginine, which afforded some, but not complete protection from degradation. The results of saturation binding assays at 25 degrees C in the presence of the peptidase inhibitors were variable. However, it was found from saturation binding assays at 0 degree C that the maximum binding capacity for [3H]-dynorphin A (1-8) at the kappa-site is similar to that of [3H]-(-)-bremazocine and [3H]-dynorphin A (1-9).

Topics: Animals; Brain; Captopril; Dynorphins; Guinea Pigs; Leucine; Peptide Fragments; Receptors, Opioid; Receptors, Opioid, kappa