phosphoramidon and ubenimex

Intrathecal (i.t.) injection of leucine-enkephalin (Leu-ENK), co-administered with peptidase inhibitors, phosphoramidon (an endopeptidase 24.11 inhibitor), and bestatin (a general aminopeptidase inhibitor), produced behaviors consisting of the biting and/or licking of the hindpaw and the tail along with hindlimb scratching directed toward the flank, which peaked at 10-15 min after an injection. This characteristic behavior was not observed in mice treated with i.t. Leu-ENK alone. We also investigated the effect of the extracellular signal-regulated kinase (ERK) in spinal processing of nociception induced by i.t. co-administration of Leu-ENK with phospharamidon and bestatin. Western blot analysis of phospho-ERK (pERK) showed a significant increase of pERK2 in the lumbar spinal cord in response to i.t. Leu-ENK co-injected with peptidase inhibitors. The MAP kinase-ERK inhibitor, U0126 dose-dependently attenuated the nociceptive behavior and spinal ERK activation to i.t. Leu-ENK co-injected with peptidase inhibitors. Furthermore, the nociceptive behavior and spinal ERK activation evoked by i.t. Leu-ENK in combination with peptidase inhibitors were inhibited by co-administration of the non-selective δ-opioid receptor antagonist, naltrindole, the selective δ2-opioid receptor antagonist, naltriben, the non-competitive N-methyl-D-aspartate (NMDA) antagonist, MK-801 or the non-selective nitric oxide synthase inhibitor, L-NAME, the selective nNOS inhibitor, N(ω)-propyl-L-arginine or the selective iNOS inhibitor, W1400, but not by the selective δ1-receptor antagonist, BNTX (7-benzylidenenaltrexone). These results suggest that spontaneous nociceptive behaviors produced by i.t. co-administration of Leu-ENK with peptidase inhibitors may be induced by an activation of the glutamate-NO-ERK pathway through the δ2-opioid receptor in the dorsal spinal cord.

Topics: Animals; Arginine; Behavior, Animal; Butadienes; Enkephalin, Leucine; Enzyme Activation; Glycopeptides; Injections, Spinal; Leucine; Male; Mice, Inbred Strains; Mitogen-Activated Protein Kinase 1; Naltrexone; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitriles; Nociception; Protease Inhibitors; Receptors, N-Methyl-D-Aspartate; Receptors, Opioid, delta; Spinal Cord

To design a releasable PEGylated TNF-α (rPEG-TNF-α), a cathepsin B-sensitive dipeptide (Val-Cit moiety) was inserted into conventional PEG-modified TNF-α (PEG-TNF-α), facilitating its clinical use for anti-tumor therapy. Comparative pharmacokinetic and pharmacodynamic studies showed that the half-lives of both PEGylated forms of TNF-α were ∼60-fold greater than that of unmodified TNF-α. In addition, the in vitro bioactivity of rPEG-TNF-α was greater than that of PEG-TNF-α with the same degree of PEG modification. Release of TNF-α from rPEG-TNF-α in vitro was dependent on the presence of cathepsin B and was inhibited by a cathepsin B inhibitor. Despite the potent cytotoxicity of unmodified TNF-α against normal cells, its PEGylated forms at higher TNF-α concentrations showed low cytotoxic activity against these cells. In contrast, both forms of PEGylated TNF-α showed potent cytotoxic activity against the B16 and L929 cell lines, with rPEG-TNF-α being 5- and 9-fold more potent, respectively, than PEG-TNF-α. Moreover, rPEG-TNF-α was a more potent in vivo antitumor agent than PEG-TNF-α.

Topics: Animals; Antineoplastic Agents; Cathepsin B; Cell Line, Tumor; Cell Survival; Dipeptides; Dose-Response Relationship, Drug; Drug Compounding; Drug Evaluation, Preclinical; Glycopeptides; Humans; Hydrogen-Ion Concentration; Leucine; Lysosomes; Male; Mice; Mice, Inbred C57BL; Neoplasms, Experimental; Pepstatins; Polyethylene Glycols; Protease Inhibitors; Rats; Time Factors; Tumor Burden; Tumor Necrosis Factor-alpha

Using purified enzyme preparations, we investigated the actions of angiotensin-converting enzyme, aminopeptidase N, and endopeptidase 24.11 on corticotropin-releasing factor (CRF). The effects of inhibition of these enzymes on CRF action in rat anterior pituitary cultures were also determined. Finally, specific inhibitors were used to evaluate ectopeptidase action on the regional brain metabolism of CRF. K(m) values for CRF were 165, 90, and 42 microM for angiotensin-converting enzyme, aminopeptidase N, and endopeptidase 24.11, respectively. A CRF metabolite profile for each enzyme was determined. In pituitary cultures, inhibition of endopeptidase 24.11 and aminopeptidase N potentiated CRF-stimulated release of adrenocorticotropic hormone (ACTH). In rat pituitary and hypothalamus membrane preparations, specific inhibitor experiments indicated that CRF hydrolysis involved members of the neutral endopeptidase and aminopeptidase enzyme families. In cortex membranes, similar peptidase inhibition was without effect. These data support the hypothesis that ectopeptidases play a major role in CRF metabolism and biological function.

Topics: Adrenocorticotropic Hormone; Algorithms; Amino Acid Sequence; Angiotensin-Converting Enzyme Inhibitors; Animals; Captopril; CD13 Antigens; Cells, Cultured; Cerebral Cortex; Chromatography, High Pressure Liquid; Corticotropin-Releasing Hormone; Glycopeptides; Hypothalamus; In Vitro Techniques; Kinetics; Leucine; Male; Molecular Sequence Data; Neprilysin; Peptidyl-Dipeptidase A; Pituitary Gland; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Thiophenes

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 presence of an extrahypothalamic gonadotropin releasing hormone (GnRH) in human placenta is well known and this decapeptide is presumed to play an important role in the regulation of the function and growth of human placenta. Immunohistochemistry showed that neutral endopeptidase 24.11 (NEP), a candidate of the responsible enzyme of GnRH degradation, is highly expressed on the cell surface of trophoblasts. Hydrolysis of GnRH by human villi was studied by measuring liberated amino acids using high performance liquid chromatography. The GnRH degrading activity was 1.53 times higher after incubation with the membrane fraction of first trimester villi than that after incubation with the membrane fraction of term villi. Phosphoramidon, a potent inhibitor of NEP, reduced the liberated amino acids to about a half, suggesting that NEP is a responsible enzyme for GnRH degradation. Ubenimex, which can inhibit several aminopeptidases, also reduced the liberated amino acids to about 50 per cent. O-phenanthroline, EDTA, and thiorphan could inhibit GnRH degradation but inhibitors of post proline endopeptidase could not. Furthermore, GnRH degrading activity of the membrane fraction was reduced remarkably after the membrane fraction was immunotitrated by anti NEP and anti placental leucine aminopeptidase (P-LAP) IgG. In conclusion, NEP and P-LAP are responsible enzymes for GnRH degradation in human villi.

Topics: Adult; Antibodies, Blocking; Chorionic Villi; Chromatography, High Pressure Liquid; Edetic Acid; Female; Glycopeptides; Gonadotropin-Releasing Hormone; Humans; Intracellular Membranes; Labor, Obstetric; Leucine; Leucyl Aminopeptidase; Neprilysin; Organ Culture Techniques; Phenanthrolines; Pregnancy; Pregnancy Trimester, First; Protease Inhibitors; Thiorphan

Two metalloproteases have recently been linked to the immune response in Lepidoptera. In addition, zinc is highly important in many mammalian immune-related functions. Because of these, we investigated the effect of zinc and two zinc-protease inhibitors on Manduca sexta hemocyte behavior in vitro. Plasmatocytes were significantly more elongated in Grace's medium supplemented with 100 micro m zinc chloride than in the absence of zinc. To test whether zinc-dependent proteases were responsible for the increased length seen in the presence of zinc, we tested two zinc-protease inhibitors, phosphoramidon and bestatin. Each resulted in decreased plasmatocyte length compared to the control, but the distributions of lengths differed with each inhibitor. Each inhibitor also affected plasmatocyte network formation in vitro. This work suggests (1) that at least two different zinc proteases are involved in the cellular defense response of M. sexta, and (2) that zinc should be included in media used for in vitro studies of the immune response.

Topics: Animals; Cell Size; Female; Glycopeptides; Hemocytes; Leucine; Male; Manduca; Microscopy, Phase-Contrast; Protease Inhibitors; Zinc

This study investigated the presence and effects of calcitonin gene-related peptide (CGRP) within the rat and guinea-pig prostate glands. Immunohistochemical studies demonstrated that CGRP immunoreactive nerve fibres are sparsely distributed throughout the prostatic fibromuscular stroma in both species. These CGRP immunopositive nerve fibres shared a similar distribution profile but were not colocalized with tyrosine hydroxylase immunopositive nerve fibres which also innervate the prostatic stroma of these species. Nerve terminals within rat and guinea-pig prostatic tissues were electrically field stimulated (60 V, 0.5 ms, 10 Hz, 20 pulses every 60 s). In guinea-pig preparations, application of human alpha-CGRP, rat adrenomedullin or rat amylin (0.1 nM-1 microM) had no effect on responses to field stimulation. In contrast, both rat and human alpha-CGRP (10 pM-300 nM), rat adrenomedullin (0.3 nM-1 microM) and rat amylin (3 nM-1 microM) concentration-dependently inhibited electrically evoked contractile responses in the rat prostate. The relative order of potency was rat alpha-CGRP=human alpha-CGRP>rat adrenomedullin>rat amylin. The inhibition by rat alpha-CGRP of field stimulation-induced contractions in the rat prostate was competitively antagonized by human CGRP((8-37)) (1, 3 and 10 microM) with a pA(2) of 6.20+/-0.13. Rat alpha-CGRP (10 nM) attenuated contractile responses of the rat prostate to exogenously added noradrenaline (1-100 microM). Inhibitory concentration-response curves to rat alpha-CGRP in rat prostates were unaffected by preincubation in either glibenclamide (10-100 microM), N-nitro-L-arginine methyl ester (L-NAME) (10 microM), bestatin (10 microM), captopril (10 microM) or phosphoramidon (3 microM). Our results indicate that CGRP-induced inhibition of electrically evoked contractions in the rat prostate occurs through activation of postjunctional CGRP(2) receptors which act independently of a K(ATP) channel or nitrergic mechanisms. Degradation of rat alpha-CGRP via peptidases does not appear to occur in the rat prostate.

Topics: Adrenomedullin; Amyloid; Angiotensin-Converting Enzyme Inhibitors; Animals; Anti-Ulcer Agents; Calcitonin Gene-Related Peptide; Captopril; Dose-Response Relationship, Drug; Electrophysiology; Enzyme Inhibitors; Glyburide; Glycopeptides; Guinea Pigs; Humans; Hypoglycemic Agents; Immunohistochemistry; Islet Amyloid Polypeptide; Leucine; Male; NG-Nitroarginine Methyl Ester; Norepinephrine; Peptides; Prostate; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Time Factors; Tyrosine 3-Monooxygenase; Vasoconstrictor Agents; Vasodilator Agents

Since kinins kallidin (KD) and bradykinin (BK) appear to have cardioprotective effects ranging from improved hemodynamics to antiproliferative effects, inhibition of kinin-degrading enzymes should potentiate such effects. Indeed, it is believed that this mechanism is partly responsible for the beneficial effects of angiotensin-converting enzyme (ACE) inhibitors. In the heart, enzymes other than ACE may contribute to local degradation of kinins. The purpose of this study was to investigate which enzymes are responsible for the degradation of KD and BK in human heart tissue.. Cardiac membranes were prepared from the left ventricles of normal (n=5) and failing (n=10) hearts. The patients had end-stage congestive heart failure as the result of coronary heart disease or idiopathic dilated cardiomyopathy. Heart tissue was incubated with KD or BK in the presence or absence of enzyme inhibitors. We found no difference in the enzymes responsible for kinin metabolism or their activities between normal and failing hearts. Thus KD was mostly converted into BK by the aminopeptidase M-like activity. When BK was used as substrate, it was converted into an inactive metabolite BK-(1-7) mostly (80% to 90%) by the neutral endopeptidase (NEP) activity, with ACE unexpectedly playing only a minor role. The low enzymatic activity of ACE in the cardiac membranes, compared with that of NEP, was not due to chronic ACE inhibitor therapy, because the cardiac ACE activities of patients, whether receiving ACE inhibitors or not, and of normal subjects were all equal.. The present in vitro study shows that in human cardiac membranes, the most critical step in kinin metabolism, that is, inactivation of BK, appears to be mediated mostly by NEP. This observation suggests a role for NEP in the local control of BK concentration in heart tissue. Thus inhibition of cardiac NEP activity could be cardioprotective by elevating the local concentration of BK in the heart.

Topics: Angiotensin-Converting Enzyme Inhibitors; Anti-Bacterial Agents; Bradykinin; Captopril; CD13 Antigens; Dipeptides; Female; Glycopeptides; Heart Failure; Humans; Kallidin; Leucine; Male; Membrane Proteins; Middle Aged; Myocardium; Neprilysin; Peptides; Peptidyl-Dipeptidase A; Protease Inhibitors; Substrate Specificity

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

Neural membrane fractions, prepared from brain-subesophageal ganglion complexes of the adult lepidopteran Lymantria dispar, contain at least two peptidases capable of metabolizing locust adipokinetic hormone-I in vitro. The initial fragments, pGlu1-Leu2-Asn3 and Phe4-Thr5-Pro6-Asn7-Trp8-Gly9-Thr10, result from the action of an endopeptidase with properties similar to those reported for neutral metalloendopeptidase in Schistocerca gregaria and mammalian endopeptidase 24.11. The heptapeptide is further degraded by an aminopeptidase that exhibits kinetic properties similar to those described for aminopeptidase 3.4.11.2. These enzymes appear to be responsible for the first two steps in AKH catabolism in L. dispar.

Topics: Amino Acid Sequence; Aminopeptidases; Animals; Anti-Bacterial Agents; Edetic Acid; Endopeptidases; Ganglia, Invertebrate; Glycopeptides; In Vitro Techniques; Insect Hormones; Lepidoptera; Leucine; Molecular Sequence Data; Peptides; Protease Inhibitors; Thiorphan

The hydrolysis of arginine vasopressin (AVP) by human placental subcellular fractions and pregnancy sera was studied in the presence of selective inhibitors and the antibody against pregnancy serum oxytocinase (P-LAP) (EC 3.4.11.3) by measuring liberated amino acids by high-performance liquid chromatography (HPLC). AVP degradation by placental subcellular fractions and pregnancy sera was inhibited by bestatin. The IC50 values of bestatin on AVP degradation by placental subcellular fractions and pregnancy sera were similar to that of this inhibitor on the P-LAP measured by L-Leu-p-nitroamnilide as a substrate (LAP activity), which we reported previously. Our immunotitration study clearly showed that the initiating and responsible protease in AVP degradation in human placenta and pregnancy serum is P-LAP. Since N-benzylcarbonyl-valyl-prolinal (Z-Val-prolinal), a selective inhibitor of post-proline endopeptidase, and phosphoramidon, a putative endopeptidase-24.11 inhibitor, could not significantly influence the degradation of AVP by placental microsomal fractions. Neither enzyme seems to be actively involved in AVP degradation.

Topics: Amino Acid Sequence; Arginine Vasopressin; Cell Fractionation; Cystinyl Aminopeptidase; Cytoplasm; Dipeptides; Edetic Acid; Female; Glycopeptides; Humans; Hydrolysis; Leucine; Lysosomes; Mercaptoethanol; Microsomes; Molecular Sequence Data; Placenta; Pregnancy; Protease Inhibitors

Met-enkephalin concentration-dependently and transiently inhibited the ileal twitch contraction and this inhibition gradually recovered with time. Bacitracin, phosphoramidon, thiorphan and captopril did not influence the twitch inhibition of met-enkephalin, but bestatin increased the twitch inhibitory potency of met-enkephalin and terminated it in a manner which almost paralleled that of untreated tissue. Transient inhibition of twitch contraction after tetanic stimulation (post-tetanic twitch inhibition) was obtained. Bestatin increased the potency of met-enkephalin and this was terminated within 2 min. Phosphoramidon tended to increase the potency and delayed the termination of post-tetanic twitch inhibition. Bacitracin, thiorphan and captopril did not influence either the potency or the termination of post-tetanic twitch inhibition. Morphine-induced twitch inhibition was not influenced by bacitracin, bestatin or phosphoramidon. These results suggest that bestatin-sensitive aminopeptidase and phosphoramidon-sensitive enkephalinase take part in post-tetanic twitch inhibition, acting in a different mode of action, and have an important role in the termination of the pharmacological action of endogenous opioids (post-tetanic twitch inhibition) in MPLM. This different mode of response of bestatin and phosphoramidon upon post-tetanic twitch inhibition may underlie that aminopeptidase is a more soluble enzyme and enkephalinase is membrane-bound in myenteric plexus-longitudinal muscle (MPLM).

Topics: Aminopeptidases; Animals; Bacitracin; Captopril; Dose-Response Relationship, Drug; Electric Stimulation; Endorphins; Enkephalin, Methionine; Glycopeptides; Guinea Pigs; Ileum; Leucine; Male; Morphine; Muscle Contraction; Muscle, Smooth; Protease Inhibitors; Thiorphan

Endothelin, a potent vasoactive peptide originally isolated from cultured porcine endothelial cells, (1) elicits hemodynamic and glycogenolytic actions in perfused rat liver; (2) evokes phosphoinositide signaling in hepatic cells; and (3) stimulates synthesis of mediators in Kupffer cells and glucose production in hepatocytes. Recently, we characterized receptor(s) for endothelin on hepatocytes (C. R. Gandhi, R. H. Behal, S. A. K. Harvey, T. Nouchi, and M. S. Olson, Biochem. J. 287, 897-904, 1992). Both hepatocytes and Kupffer cells rapidly internalize [125I]endothelin-1 ([125I]ET-1). In the present study we exposed primary cultures of hepatocytes or Kupffer cells to [125I]ET-1 and analyzed the radiolabeled metabolites which appeared in the cell medium. Six metabolites were separated by high-performance liquid chromatography (HPLC) from hepatocyte medium; these peaks had approximate elution times of 5 (free iodide), 22, 35, 37, 38, and 41 min, respectively, whereas the elution time for [125I]ET-1 was 43 min. The kinetics of formation of the metabolites, and experiments using excess unlabeled ET-1, both showed that internalization of the native peptide by hepatocytes is required for the metabolism of [125I]ET-1 into metabolite, and for the subsequent deiodination of metabolite. The formation of metabolites does not require internalization of the native peptide. In Kupffer cells, the cell medium contained only metabolite and metabolite. Internalization of the native peptide was required for the formation of metabolite but not for metabolite. Three classes of [125I]ET-1 metabolites from hepatic cells also were separated by sequential precipitation with trichloroacetic acid (TCA) and with silver nitrate. This procedure facilitated multiple rapid assays of [125I]ET-1 metabolism. Phosphoramidon, an inhibitor of neutral metalloendopeptidases, did not affect significantly the binding or the metabolism of [125I]ET-1 by hepatocytes or Kupffer cells. The aminopeptidase inhibitor bacitracin strongly attenuated [125I]ET-1 metabolism by hepatocytes, with a concomitant increase in the intracellular content of [125I]ET-1. These data suggest that enzymes capable of endothelin degradation are present both on the surface and in the intracellular compartment of hepatic cells, and that endothelin is not metabolized by neutral endopeptidase 24.11 in the liver.

Topics: Animals; Bacitracin; Cells, Cultured; Chromatography, High Pressure Liquid; Endothelins; Glycopeptides; Iodine Radioisotopes; Kidney; Kinetics; Kupffer Cells; Leucine; Liver; Lung; Male; Rats; Rats, Sprague-Dawley

1. Met-enkephalin is degraded by peptidases present in the hemolymph fluid and hemocyte membrane suspension of Mytilus edulis. Degradation of Met-enkephalin is rapid in the fluid and slower in the membrane. 2. Aminopeptidase activity is bestatin sensitive in hemocyte membrane and highest in the fluid of the hemolymph, which appears to have a component which is insensitive to inhibitor. 3. ACE activity is found only in the fluid of the hemolymph. 4. Carboxypeptidase and NEP (CD10: "enkephalinase") are membrane bound and the former appears to predominate. Phosphoramidon inhibits not only NEP, as expected, but the invertebrate carboxypeptidase as well.

Topics: Amino Acid Sequence; Aminopeptidases; Animals; Bivalvia; Carboxypeptidases; Cell Membrane; Chromatography, High Pressure Liquid; Enkephalin, Methionine; Glycopeptides; Hemocytes; Hemolymph; Leucine; Membrane Proteins; Molecular Sequence Data; Neprilysin; Peptidyl-Dipeptidase A; Substrate Specificity

The hydrolysis of oxytocin by human placental subcellular fractions was studied in the presence of selective inhibitors by measuring liberated amino acids by high performance liquid chromatography (HPLC). Oxytocin degradation by microsomal and lysosomal fractions was inhibited by bestatin, amastatin and puromycin. The IC50 values of these inhibitors on oxytocin degradation by both fractions were similar to those of these inhibitors on the human placental aminopeptidase M measured by L-Leu-p-nitroanilide as a substrate (LAP activity), which we reported previously. However, purified aminopeptidase M from human placental microsomal fractions could not liberate any amino acid from oxytocin. Since phosphoramidon (1 mumol/l), a putative metalloendopeptidase inhibitor, and N-benzylcarbonyl-valyl-prolinal (Z-Val-prolinal) (14 mumol/l), a selective inhibitor of post-proline endopeptidase, could not significantly influence the degradation of oxytocin by either subcellular fractions, neither enzyme seems to be actively involved in oxytocin degradation. These results strongly suggested the existence of oxytocinase(s) other than the above three enzymes in microsomal and/or lysosomal fractions of human placenta.

Topics: Amino Acids; Aminopeptidases; Anti-Bacterial Agents; CD13 Antigens; Chromatography, High Pressure Liquid; Cystinyl Aminopeptidase; Female; Glycopeptides; Humans; Leucine; Leucyl Aminopeptidase; Lysosomes; Microsomes; Oligopeptides; Oxytocin; Peptides; Placenta; Pregnancy; Prolyl Oligopeptidases; Puromycin; Serine Endopeptidases; Subcellular Fractions

We studied the effect of substance P (SP) on the electric properties of cultured canine tracheal epithelium and its possible modulation by neutral endopeptidase (NEP) by Ussing's short-circuited technique in vitro. Addition of SP (5 x 10(-6) M) to the mucosal side increased short-circuit current (SCC) from 5.1 +/- 0.9 to 10.3 +/- 2.2 microA/cm2 (mean +/- SE; p less than 0.01), which was accompanied by increases in transepithelial potential difference and conductance. The effect of the mucosal SP on SCC was dose-dependent, with the maximal increase from the baseline value being 5.8 +/- 1.0 microA/cm2 observed at 5 x 10(-5) M. The NEP inhibitor phosphoramidon (10(-5) M) did not affect these responses. On the other hand, SCC was not altered by the addition of SP to the submucosal side. However, it was increased dose-dependently in the presence of phosphoramidon (10(-5) M) but not in the presence of captopril, bestatin or leupeptin. This stimulatory effect of submucosal SP was abolished by furosemide, diphenylamine-2-carboxylate and Cl-free medium, but not by amiloride. These results suggest that SP may selectively stimulate Cl secretion across the airway epithelium and that this effect may be modulated by submucosal NEP.

Topics: Amiloride; Animals; Anti-Bacterial Agents; Biological Transport, Active; Captopril; Cells, Cultured; Complement C1; Dogs; Dose-Response Relationship, Drug; Electric Conductivity; Female; Furosemide; Glycopeptides; In Vitro Techniques; Ion Channel Gating; Leucine; Leupeptins; Male; Membrane Potentials; Neprilysin; Substance P; Time Factors; Trachea

Topics: Animals; Capsaicin; Captopril; Glycopeptides; Guinea Pigs; Ileum; Leucine; Muscle Contraction; Muscle, Smooth; Receptors, Neurokinin-2; Receptors, Neurotransmitter; Receptors, Tachykinin; Serotonin; Substance P

Topics: Animals; Cerebral Cortex; Dose-Response Relationship, Drug; Electroencephalography; Electroshock; Enkephalins; Glycopeptides; Leucine; Male; Mice; Mice, Inbred Strains; Neprilysin; Nitrous Oxide; Rats; Rats, Inbred Strains; Sleep Deprivation; Sleep, REM; Substance Withdrawal Syndrome; Thiorphan

To study the effect of atrial natriuretic factor (ANF) on airway ciliary motility, we measured ciliary beat frequency by a photoelectric method in response to ANF in cultured tracheal epithelial cells from rabbits. Addition of ANF but not [Tyr8]ANF-(5-27) decreased ciliary beat frequency in a dose-dependent fashion; the maximal decrease from the baseline value was 24.1 +/- 1.5% (+/- SE, P less than 0.001), and a half-maximal inhibitory concentration (IC50) was 3 x 10(-12) M. Inhibition of neutral endopeptidase activity by phosphoramidon (10(-6) M) or thiorphan (10(-6) M) potentiated the effect of ANF so that the dose-response curve for ANF was shifted to lower concentrations by approximately 0.5 log units (P less than 0.05, in each case). The inhibition of ciliary motility induced by ANF was not affected by the blockade of arachidonic acid metabolism with indomethacin, piroxicam, or nordihydroguaiaretic acid, but it was blocked by methylene blue, a soluble guanylate cyclase inhibitor, and was potentiated by M & B 22948, a guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase inhibitor. The intracellular cGMP levels were increased by ANF, an effect that was further potentiated by phosphoramidon or thiorphan. These results suggest that ANF inhibits ciliary motility presumably through a guanylate cyclase-dependent regulatory pathway and that neutral endopeptidase may play a role in modulating the ANF effect on airway mucociliary transport function.

Topics: Animals; Atrial Natriuretic Factor; Captopril; Cells, Cultured; Cilia; Enzyme Inhibitors; Epithelium; Glycopeptides; Indomethacin; Kinetics; Leucine; Leupeptins; Male; Masoprocol; Methylene Blue; Movement; Piroxicam; Purinones; Rabbits; Thiorphan; Trachea

Modulation of baroreceptor reflex (BRR) by endogenous substance P (SP) in the brain was investigated in rats anesthetized with pentobarbital sodium. Intracerebroventricular administration of the undecapeptide (15 or 30 nmol) and its antagonist, (D-Pro2, D-Trp7,9)-SP (30 or 60 nmol) or SP antiserum (1:20), respectively, promoted a significant increase and decrease in the sensitivity of BRR response. Prolonging the endogenous activity of SP with the aminopeptidase blocker, bestatin (200 nmol) or with the endopeptidase-24.11 inhibitor, phosphoramidon (200 nmol) significantly augmented the same reflex. Combining the undecapeptide with either peptidase blocker, moreover, promoted additional potentiation of the BRR response. On the other hand, simultaneous administration of bestatin and (D-Pro2, D-Trp7,9)-SP produced a reduction of the augmented effect of bestatin on the sensitivity of BRR response. Bilateral microinjection of SP (600 pmol) or an antiserum against SP (1:20) into the nucleus tractus solitarius (NTS) elicited respectively an enhancement of and reduction in the BRR response. These data suggest that neurons that contain SP may participate in central cardiovascular control by tonically enhancing the sensitivity of the BRR response, possibly via an action on the NTS.

Topics: Animals; Brain; Drug Interactions; Glycopeptides; Immune Sera; Injections, Intraventricular; Leucine; Male; Microinjections; Pressoreceptors; Rats; Rats, Inbred Strains; Substance P; Time Factors

The effects of peptidase inhibitors were examined upon behavioural responses including scratch, bite and lick produced by intrathecal (IT) injection of substance P (SP) and neurokinin A (NK A) in mice. Phosphoramidon (0.002-2.0 nmol), an endopeptidase-24.11 inhibitor, simultaneously injected with SP or NK A, remarkably enhanced and prolonged SP- or NK A-induced behavioural response in a dose-dependent manner. The behavioural response to SP was significantly increased by 2.0 nmol of bestatin, an aminopeptidase inhibitor, but not by 1.0 nmol. Captopril, an angiotensin-converting enzyme inhibitor, was without effect on both tachykinin-induced responses. When phosphoramidon was injected together with bestatin and captopril which have no significant effect alone, SP- or NK A-induced behavioral response was significantly increased. These data suggest that endopeptidase-24.11 may be an important enzyme responsible for terminating of SP- or NK A-induced behavioral response at the spinal cord level.

Topics: Aggression; Animals; Behavior, Animal; Captopril; Dose-Response Relationship, Drug; Glycopeptides; Injections, Spinal; Leucine; Male; Mice; Mice, Inbred Strains; Neurokinin A; Substance P; Tachykinins

Substance P is a neuropeptide released in vivo from the substantia nigra, the principal substance P nerve terminal region in the rat brain. Its inactivation was investigated in a purified nigral synaptic membrane preparation. The membrane-bound enzyme shares many features with the endopeptidase 24-11 (EC 3.4.24.11): 1) hydrolysis of peptide bonds Gln6-Phe7, Phe7-Phe8 and Gly9-Leu10, 2) sensitivity to the inhibition by phosphoramidon and 3) relative affinity for substance P. Bestatine and captopril inhibit only the hydrolysis of the metabolites. These results suggest that substance P is inactivated in substantia nigra by endopeptidase 24-11 and that a bestatin-sensitive aminopeptidase and angiotensin converting enzyme may play a role in subsequent degradation of the substance P metabolites.

Topics: Animals; Captopril; Enzyme Activation; Glycopeptides; Hydrolysis; Leucine; Neprilysin; Peptidyl-Dipeptidase A; Rats; Rats, Inbred Strains; Substance P; Substantia Nigra; Substrate Specificity; Synaptic Membranes

We investigated the activity of bombesin (BN), neuromedin-C (NM-C) and neuromedin-B (NM-B) on serotonin (5-HT) release and reuptake in rat hypothalamus (HYP) in vitro. BN and NM-C but not NM-B (all 1 microM) decreased K+ evoked 3H-5-HT release from superfused HYP slices by 25%. Bacitracin (BCN, 2 micrograms/ml), a nonspecific peptidase inhibitor, reversed the inhibitory effect of BN on K+ evoked 3H-5-HT release. Phosphoramidon (PAN, 10 microM) an endopeptidase 24.11 inhibitor, abolished the inhibitory effect of BN, but not NM-C, on K+ evoked 3H-5-HT release. The peptidyl dipeptidase A inhibitor enalaprilat (ENP, 10 microM), enhanced both BN and NM-C inhibition of 3H-5-HT release. Bestatin (BST, 10 microM) had no effect on BN or NM-C inhibitory activity on 3H-5-HT release. Neither BN, NM-C nor NM-B affected reuptake of 3H-5-HT into HYP synaptosomes alone or in combination with any of the peptidase inhibitors, nor did these peptides alter the ability of fluoxetine to inhibit 3H-5-HT uptake. These data suggest: a) that BN-like peptides may alter neurotransmission in the HYP by acting presynaptically on the 5-HT release mechanism; b) a similarity in the structural requirements for the BN induced inhibition of 5-HT release and BN evoked thermoregulatory disturbances; and c) that peptidases may selectively augment or reduce pharmacologic activity of BN-like peptides upon CNS administration.

Topics: Amino Acid Sequence; Aminopeptidases; Angiotensin-Converting Enzyme Inhibitors; Animals; Bacitracin; Bombesin; Drug Interactions; Enalapril; Enalaprilat; Glycopeptides; Hypothalamus; Leucine; Male; Molecular Sequence Data; Neurokinin B; Peptide Fragments; Protease Inhibitors; Rats; Rats, Inbred Strains; Serotonin

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

Serum succinyl (Ala)3-p-nitroanilide hydrolyzing elastase-like activity which elevates in patients with obstructive jaundice, is due to the joint action of two enzymes: first, succinyl (Ala)3-p-nitroanilide is cleaved to succinyl (Ala)2 and Ala-p-nitroanilide by metalloendopeptidase, and then Ala-p-nitroanilide is cleaved to Ala and p-nitroaniline by aminopeptidase. We adopt a new assay method for serum endopeptidase activity using HPLC.

Topics: Adult; Aminopeptidases; Anti-Bacterial Agents; Cholestasis; Chromatography, High Pressure Liquid; Diabetes Mellitus; Endopeptidases; Female; Glycopeptides; Humans; Immunodiffusion; Kidney; Leucine; Male; Metalloendopeptidases; Neoplasms; Oligopeptides; Peptides; Protease Inhibitors

Intracarotid (IC) injection of bestatin produced a dose-dependent biphasic change in blood pressure (BP) of the rat, consisting of an initial short-lasting fall followed by a long-lasting increase. This effect was regularly depressed or abolished by IV injection of naloxone. IC injection of Leu-enkephalin also produced a biphasic BP response, with the same characteristics as that produced by IC injection of bestatin. This effect was also easily blocked by IV injection of naloxone. IC injection of bestatin significantly potentiated the BP response to IC injection of Leu-enkephalin. This potentiated response was blocked by naloxone. IC injection of both bestatin and phosphoramidon, whether separately or in combination, significantly depressed the hypertensive response to physostigmine. This depressive action of bestatin and phosphoramidon on physostigmine hypertension can be significantly antagonized or even reversed by IV injection of naloxone. IC injection of both bestatin and phosphoramidon did not affect the BP response to either acetylcholine or catecholamines. It is concluded that bestatin and phosphoramidon, injected into the carotid artery, inhibit the activity of aminopeptidase and "enkephalinase", thus producing an accumulation of enkephalins in the central nervous system. These enkephalins activate opioidergic receptors in the brain, but concomitantly produce a depression of the cholinergic-adrenergic interaction in the central nervous system, which is known to be a prerequisite for the hypertensive response to physostigmine in the rat.

Topics: Animals; Antibiotics, Antineoplastic; Blood Pressure; Carotid Arteries; Catecholamines; Enkephalin, Leucine; Female; Glycopeptides; Injections, Intra-Arterial; Leucine; Male; Naloxone; Physostigmine; Rats; Rats, Inbred Strains

The enkephalin-hydrolyzing peptidases in mouse vas deferens were studied and compared with those in guinea pig ileum which had been characterized in the previous study. The present results showed that three distinct peptidases, bestatin-sensitive aminopeptidase, captopril-sensitive peptidyl dipeptidase A, and thiorphan-sensitive endopeptidase-24.11, played a critical role in the inactivation of enkephalin in mouse vas deferens, being consistent with the previous results obtained with guinea pig ileum. However, the data in both previous and present studies showed that the activity of the bestatin-sensitive aminopeptidase relative to that of either the captopril-sensitive peptidyl dipeptidase A or the thiorphan-sensitive endopeptidase-24.11 in guinea pig ileum was higher than that in mouse vas deferens, while the activity of either peptidyl dipeptidase A or endopeptidase-24.11 relative to that of aminopeptidase in mouse vas deferens was higher than that in guinea-pig ileum. In contrast to these three enzymes, both L-tyrosyl-L-tyrosine-sensitive dipeptidyl aminopeptidase and D-phenylalanine-sensitive carboxypeptidase were suggested not to be involved significantly in the inactivation of enkephalin in mouse vas deferens as well as guinea pig ileum.

Topics: Amino Acids, Sulfur; Aminopeptidases; Animals; Captopril; Dipeptides; Dose-Response Relationship, Drug; Endopeptidases; Enkephalin, Methionine; Glycopeptides; Guinea Pigs; Ileum; In Vitro Techniques; Leucine; Male; Mice; Mice, Inbred Strains; Muscle Contraction; Neprilysin; Peptidyl-Dipeptidase A; Phenylalanine; Thiorphan; Tiopronin; Vas Deferens

Utilizing both thin-layer chromatography and high pressure liquid chromatography, it was determined that a vascular plasma membrane preparation contains a carboxypeptidase capable of converting kinins (B2 agonists) to des(Arg)kinins (B1 agonists) by hydrolysis of C-terminal Arg. The plasma membrane carboxypeptidase also converted Leu5-enkephalin-Arg6 to Leu5-enkephalin. Carboxypeptidase activity was significantly higher in cultured endothelial (1.47 +/- 0.4 units/mg) than in cultured smooth muscle cells (0.16 +/- 0.4 units/mg). Both the vascular and endothelial activities had neutral pH optima and were activated 4- to 5-fold by 0.1 mM CoCl2. The carboxypeptidase N inhibitor MERGETPA (D-L-mercaptoethanol-3-guanidino-ethylthiopropanoic acid) inhibited the plasma membrane bound carboxypeptidase with an I50 of 0.3 microM. Conversion was also inhibited by o-phenanthroline and EDTA, whereas inhibitors of aminopeptidases (bestatin, puromycin), endopeptidases (phosphoramidon), "enkephalinase" (ZINCOV) or enkephalin convertase (PCMS) were without effect. The affinity of the endothelial plasma membrane carboxypeptidase for bradykinin (Km = 56.8 +/- 4.7 microM) was higher than that for Leu5-enkephalin-Arg6 (Km = 92.7 +/- 10.1 microM), whereas the maximal rates of conversion (calculated per mg of endothelial plasma membrane protein) were similar (17.1 and 21.3 nmoles/min/mg respectively). These results demonstrate that a carboxypeptidase is present on the cell surface of vascular endothelium which can convert kinins and enkephalins in the micro-environments of vascular cell surface receptors.

Topics: 3-Mercaptopropionic Acid; Animals; Carboxypeptidases; Cell Membrane; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Edetic Acid; Endothelium; Enkephalins; Glycopeptides; Hydrogen-Ion Concentration; Kinetics; Kinins; Leucine; Lysine Carboxypeptidase; Microscopy, Phase-Contrast; Muscle, Smooth, Vascular; Phenanthrolines; Puromycin; Swine

Topics: Aminopeptidases; Animals; Brain; Corpus Striatum; Endopeptidases; Glycopeptides; Hydroxamic Acids; Leucine; Mice; Neprilysin; Organophosphorus Compounds; Peptides; Protease Inhibitors; Puromycin