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Purpose: 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)., Methods: 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., Results: 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., Conclusion: 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.

Purpose: Previous in vitro studies have shown that degradation of opioid peptides during incubation with cerebral membrane preparations is almost completely prevented by a mixture of three peptidase inhibitors (PIs), namely, amastatin, captopril, and phosphoramidon. In the present in vivo study, we evaluate the effects of intrathecal administration of these PIs on antinociception by [Met(5)]enkephalin (ME) or PIs themselves., Methods: Drugs were administered into the thoracolumbar level of the spinal cord in the intrathecal space in rat. Induction of antinociception was measured by the tail immersion assay, with 55 °C as the nociceptive stimulus. Effects of PIs on antinociception were evaluated by dose-response study (ME, 1-20 nmol; PIs, 1-20 nmol each), by comparison of differences among two combinations of PIs (amastatin and captopril; captopril and phosphoramidon; amastatin and phosphoramidon) and three PIs (amastatin, captopril, and phosphoramidon), and by using opioid receptor selective antagonists., Results: Intrathecal administration of ME with these three PIs or PIs alone significantly and dose dependently increased antinociception in a μ- and δ-opioid receptor antagonist-reversible manner; moreover, the degree of antinociception with a combination of any two of these was less than that with all three, indicating that any residual single peptidase could inactivate significant amounts of ME., Conclusion: The present data, together with those of earlier studies, clearly demonstrate that amastatin-, captopril-, and phosphoramidon-sensitive enzymes play an important role in inactivation of opioid peptides at the spinal level.

Objective: Previous in vitro studies have shown that the degradation of [Leu5]enkephalin during incubation with cerebral membrane preparations is almost completely prevented by a mixture of three peptidase inhibitors such as amastatin, captopril, and phosphoramidon. The present in vivo study was performed to examine the antinociceptive effect of [Leu5]enkephalin administered intrathecally under pretreatment with these three peptidase inhibitors., Methods: A tail-flick test was used to determine the nociceptive threshold after administration of [Leu5]enkephalin. The time-course profiles of the latency to flick the tail and the area under the time response curve were evaluated for the antinociceptive action of the drug., Results: The antinociceptive effect of [Leu5]enkephalin administered intrathecally on the tail-flick test was increased more than 100-fold under i.t. pretreatment with three peptidase inhibitors. The antinociceptive effect produced by [Leu5]enkephalin in rats pretreated with any single peptidase inhibitor increased antinociception compared to that with saline. The antinociceptive potency of [Leu5]enkephalin under pretreatment with any combination of two peptidase inhibitors was smaller than that in rats pretreated with three peptidase inhibitors together. These results indicate that any residual single peptidase inactivates significant amounts of [Leu5]enkephalin., Conclusion: The present data, together with those of earlier studies, clearly demonstrate that amastatin-, captopril-, and phosphoramidon-sensitive enzymes play an important role in the inactivation of [Leu5]enkephalin administered intrathecally in rat.

Novel glycopeptides containing amino acids such as valine and alanine were designed, synthesized and tested for inhibition of the wild type C-SA HIV-1 protease enzyme. The incorporation of dipeptide sequences Val-Ala/Ala-Val to the sugar B-amino acid at two side chain positions resulted in a series of nine novel compounds. Compounds 3a, 3b, 3c, 3d, 4a, 4b and 5 displayed significant activities against the HIV protease enzyme. The glycopeptides are orders of magnitude less toxic to human MT-4 cells than lopinavir. Computational results were in good agreement with the experimental HIV-PR activity. The sugar hydroxyl group at the C(3) position interacts with the enzymatic Asp25/Asp25' residues. The docked position of the inhibitor is preserved during MD simulations and at least five hydrogen bond forms between the inhibitor and the enzymatic pocket. The results provide a platform for the progress of more effective carbohydrate supported inhibitors of HIV-1 and other aspartic proteases., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Intranasal administration is emerging as a reliable and non-invasive method to bypass the blood-brain barrier and deliver drugs to the brain. This approach has been primarily used to explore therapeutic avenues for neurological diseases. However, intranasal administration could also be used to create animal models of brain disease. Beta-amyloid peptide (Aβ) accumulation is a key feature of Alzheimer's disease (AD), and the most common models of AD are transgenic mice expressing mutant human genes linked to familial AD. An alternative model of amyloidosis utilizes intracerebroventricular infusion of thiorphan or phosphoramidon to block the activity of key Aβ degrading enzymes (NEP, NEP2) resulting in accumulation of Aβ. Here, we demonstrate that intranasal administration of phosphoramidon produces significantly elevated cerebral Aβ levels in wild-type mice. Furthermore, intranasal phosphoramidon administration in double knockout mice lacking NEP and NEP2 also showed increased levels of Aβ(40). These data show that intranasal delivery of drugs can be used to model AD and suggest that other phosphoramidon-sensitive peptidases are degrading Aβ in NEP/NEP2-deficient mice.

Previous in vitro studies have shown that the degradation of [Leu(5)]enkephalin during incubation with cerebral membrane preparations is almost completely prevented by a mixture of three peptidase inhibitors: amastatin, captopril, and phosphoramidon. The present in vivo study shows that the inhibitory effect of [Leu(5)]enkephalin administered intra-third-ventricularly on the tail-flick response was increased more than 500-fold by the intra-third-ventricular pretreatment with the three peptidase inhibitors. The antinociceptive effect produced by the [Leu(5)]enkephalin in rats pretreated with any combination of two peptidase inhibitors was significantly smaller than that in rats pretreated with the three peptidase inhibitors, indicating that any residual single peptidase could inactivate significant amounts of the [Leu(5)]enkephalin. The present data, together with those obtained from previous studies, clearly demonstrate that amastatin-, captopril-, and phosphoramidon-sensitive enzymes play important roles in the inactivation of short endogenous opioid peptides, such as penta-, hepta-, and octa-peptides, administered intra-third-ventricularly to rats.

Previous in vitro studies have shown that the degradation of [Met(5)]enkephalin-Arg(6)-Phe(7) during incubation with cerebral membrane preparations is largely prevented by a mixture of three peptidase inhibitors: amastatin, captopril, and phosphoramidon. The present in vivo study shows that the inhibitory effect of [Met(5)]enkephalin-Arg(6)-Phe(7) administered intra-third-ventricularly on the tail-flick response was increased more than 1000-fold by the intra-third-ventricular pretreatment with three peptidase inhibitors. The antinociceptive effect produced by the [Met(5)]enkephalin-Arg(6)-Phe(7) in rats pretreated with any combination of two peptidase inhibitors was significantly smaller than that in rats pretreated with three peptidase inhibitors, indicating that any residual single peptidase could inactivate significant amounts of the [Met(5)]enkephalin-Arg(6)-Phe(7). The present data, together with those obtained from previous studies, clearly show that amastatin-, captopril-, and phosphoramidon-sensitive enzymes play important roles in the inactivation of endogenous opioid peptides, such as [Met(5)]enkephalin, [Met(5)]enkephalin-Arg(6)-Phe(7), [Met(5)]enkephalin-Arg(6)-Gly(7)-Leu(8), and dynorphin A (1-8), administered intra-third-ventricularly to rats.

C-type natriuretic peptide (CNP), a local regulator of vascular tone and cell proliferation, is eliminated from the circulation via NPR-C receptors and neutral endopeptidase enzyme (NEP, EC. 3.4.24.11). The increased contractility of coronary arteries in different cardiovascular diseases made us study the possible enhancement of vasodilator capacity of exogenously added CNP with concomitant NEP inhibition on porcine coronary arteries in vitro. CNP (0.006-1.4 microM) concentration dependently relaxed the U46619 (0.07-0.4 microM) precontracted preparations in an almost equally effective manner in the presence and absence of functional endothelium with maximum effects of about 40%. The combined NEP/endothelin-converting enzyme inhibitor (NEP/ECE inhibitor), phosphoramidon (10 microM) or the specific inhibitor of the NEP, thiorphan (10 microM) resulted in an enhanced magnitude of CNP-induced relaxation without significant change in the EC50 both on endothelium intact and endothelium deprived preparations. The inhibition of endothelin receptors by PD 142893 (10 microM) enhanced the relaxing effect of CNP in the presence but not in the absence of functional endothelium indicating a functional antagonism between CNP and endothelin. Our results suggest that inhibition of CNP degradation may endue this endogenous peptide with therapeutic potency in cardiovascular diseases.

Objective: Several metallopeptidases have been implicated in both term and preterm parturition. We hypothesize that endotoxin-induced preterm delivery can be prevented by the administration of a metallopeptidase inhibitor., Study Design: We used an animal model of endotoxin-induced preterm delivery in timed pregnancy C57Bl/6 mice. Test animals received lipopolysaccharide followed by phosphoramidon, either every 1.5 or every 3 hours. Control mice received lipopolysaccharide followed by buffer injections at the same intervals. The primary outcome was a preterm delivery rate., Results: The rate of preterm delivery for the control animals was 88.0% compared with the treatment groups of 45.5% for the mice that received phosphoramidon every 3 hours and 30.8% for the group that received it every 1.5 hours (P<.01)., Conclusion: The administration of a metallopeptidase inhibitor resulted in a decreased rate of preterm delivery in this animal model.

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.

The aim of this study was to investigate whether the blocking of endothelin-converting enzyme (ECE) activity offers a new approach to inhibiting the development of cerebral vasospasm after subarachnoid hemorrhage (SAH) by preventing transformation of big endothelin-1 (big ET-1) to vasoactive endothelin-1 (ET-1). The effect of potential ECE inhibitors was determined in vitro by measurement of isometric contractions, induced by big ET-1, in isolated rat basilar arteries. Intact and de-endothelialized endothelium (E+ and E-, respectively) segments were examined after preincubation with the putative ECE inhibitors: phosphoramidon (10(-4) M), and [22D-Val]big ET-1 [16-38] (10(-5) M and 10(-6) M). Additionally, the effect of [D-Val22]big ET-1 [16-38] was investigated in rabbits after intracisternal application in order to inhibit the contraction of the basilar artery induced by (2x10(-6) M) big ET-1. Application of 10(-4)-M phosphoramidon resulted in a statistically significant decrease in big ET-1-induced contraction in E+ and E- segments; 10(-5)-M and 10(-6)-M [22D-Val]big ET-1 [16-38] in E- segments produced no statistically significant effect. The application of 10(-6)-M [22D-Val]big ET-1 [16-38] in E+ segments caused increased contractions, as shown by the shift to the left of the concentration-effect curve (CEC). In the rabbit group pretreated with [D-Val22]big ET-1 [16-38] (2x10(-5) M) (n=8), the angiographically measured diameter of the basilar artery increased from 0.63+/-0.12 mm to 0.66+/-0.12 mm. In the control group (n=8), this diameter decreased from 0.71+/-0.13 mm to 0.57+/-0.15 mm. This corresponded to an increase in vessel diameter of 5.24+/-9.89% in the treatment group and a decrease of 19.54+/-15.81% in the control group (P=0.002). The present study indicates the existence of functional ECE activity in rat basilar artery, which differs in the endothelium and the smooth muscle layer. These results demonstrate that [D-Val22]big ET-1 [16-38] has a potent ECE-inhibitory effect, preventing cerebral vasospasm in rabbit basilar artery by inhibiting the transformation of big ET-1 to vasoactive ET-1 after intracisternal application in vivo, whereas no inhibitory effect was detectable in rat basilar artery in vitro. Therefore, further studies of the biochemical nature of cerebrovascular ECE activity are required.

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.

Chagas' disease is an important cause of cardiomyopathy. Endothelin-1, a vasoactive peptide has been implicated in the pathogenesis of chagasic cardiomyopathy. C57BL/6 x 129sv and CD1 mice were thus, infected with trypomastigotes of Trypanosoma cruzi (Brazil strain) and these infected mice were compared with infected mice treated with phosphoramidon. This compound inhibits endothelin-converting enzyme and neutral endopeptidases and does not affect the growth of the parasite in culture. Phosphoramidon was given in a dose of 10mg/kg for the initial 15 days post-infection None of the C57Bl/6 x 129sv mice died as a result of infection. However, there was marked myocardial inflammation and fibrosis in infected, untreated mice. The hearts of the infected, phosphoramidon-treated mice showed significantly less pathology. Cardiac magnetic resonance imaging of infected mice revealed right ventricular dilation that was less severe in those treated with phosphoramidon. Phosphoramidon-treated CD1 mice survived the acute infection. Transthoracic echocardiography demonstrated left ventricular dilation and reduced percent fractional shortening and relative wall thickness. These alterations were also attenuated as a result of phosphoramidon treatment. These data suggest that endothelin-1 contributes to the pathogenesis of chagasic cardiomyopathy and interventions that inhibit the synthesis of endothelin-1 and/or neutral endopeptidase might have a protective effect on myocardial structure and function in murine Chagas' disease.

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.

Background: The balance between proteolysis and protease inhibition in the formation and breakdown processes of the extracellular matrix plays a major role in tumor cell invasion. An understanding of this relationship gave rise to the therapeutic concept of lowering tumor cell invasion by inhibiting protease activity. Phosphoramidon is an unspecific proteinase inhibitor. This experimental study investigated the effect of intraperitoneal phosphoramidon administration on tumor growth in a laparoscopic rat model., Methods: In the first phase of the study, we investigated the influence of phosphoramidon on tumor cell invasion in a collagen matrix gel chamber in vitro. In a second experiment, a suspension of colon carcinoma cells (CC531) was introduced into the peritoneal cavity of male WAG rats. Prior to laparoscopy (at 6 mmHg for 20 min), the animals were randomized to two groups. At the start of laparoscopy, the test substance was applied intraperitoneally (group 1: controls, 1 ml 0.9% NaCl; group 2: 250 mg phosphoramidon in 1 ml 0.9% NaCl). Three weeks after the injection of tumor cells, the animals were autopsied and the tumor mass determined., Results: In comparison with the control group (tumor weight 7.42 +/- 1.01 g), intraperitoneal tumor growth in the experimental group was significantly (p < 0.001) reduced by the application of phosphoramidon (tumor weight, 3.22 +/- 1.06 g). Phosphoramidon also significantly (p < 0.05) reduced tumor cell invasion through the matrix gel., Conclusion: The proteinase inhibitor phosphoramidon reduced tumor cell invasion in vitro and tumor cell growth in vivo in this laparoscopic rat model.

The enzymatic activity of endothelin-converting enzymes (ECE) was altered to determine the potential effect of endothelins (ET) on cerebral blood flow measured by laser Doppler flowmetry (CBF(LDF)) and the resultant neural damage of rats, made hypoxic via breathing 12% O(2) for 35 min. Intrastriatal administration of phosphoramidon (PRN, 5 microM), a dual inhibitor of ECE and neutral endopeptidase (NEP), significantly increased infarct volume to hypoxia with a significant attenuation of CBF(LDF). However, intrastriatal thiorphan (TRN, 5 microM), an inhibitor of NEP, had no effect on the CBF(LDF) responses or infarct volume induced by the hypoxic challenge. These findings showed that inhibition of ECE by PRN interfered with the vasodilator activity of ET to the hypoxic response that increased neural damage, thus suggesting that PRN-sensitive ECE is functionally active in the modulation of cerebral blood flow in rats undergoing hypoxia.

The effects of the metalloproteinase inhibitors thiorphan and R-94138 on the matrilysin-catalyzed hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2) [MOCAc-PLGL(Dpa)AR] were examined. The inhibitor constants (K(i)) of thiorphan and R-94138 for matrilysin at pH 7.5, 25 degrees C were determined to be 11.2 and 7.65 microM, respectively. From the temperature dependence of the K(i) values at pH 7.5, the standard enthalpy change (Delta H degrees ') values for the binding of matrilysin with thiorphan and R-94138 were determined to be -(18.2 +/- 0.9) and (1.65 +/- 1.07) kJ x mol(-1), respectively. The binding of matrilysin to thiorphan is exothermic and the free energy change in the complex formation depends mainly on the change in enthalpy, while the binding to R-94138 is endothermic and typically entropy-driven. Hydrophobic interactions are suggested to contribute significantly to the binding of matrilysin to R-94138 as well as to the substrate. The pH dependence of the K(i) value suggests that at least two ionizing groups with pK(a) values of 4.5 and 9.1--9.3 are involved in the binding. The matrilysin activity is regulated by ionizing groups with pK(a) values of 4.3 and 9.6. Both inhibition and hydrolysis are suggested to be controlled by the same residues in matrilysin, most likely Glu 198 and Tyr 219, respectively.

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.

In the skin, keratinocytes synthesize and secrete endothelin-(ET-1), a potent vasoconstrictor peptide which acts also as a growth factor for most skin cells. The aim of the study was to test the effects of UVA1 and the associations UVA1/B on the expression of ET-1 in normal human keratinocytes and to determine whether exogenously added prostaglandin E2 (PGE2) regulated ET-1 expression. As ET-1 is susceptible to degradation, we also evaluated whether ET-1 secretion was modulated by peptidase inhibitors. Our results showed that UVA1 (365 nm) did not modify the levels of preproET-1 mRNA and protein. Moreover, the associations UVA1+UVB or UVB+UVA1 down-regulated the overexpression of secreted ET-1 induced by UVB alone. PGE2 at 10(-5) M reduced the expression of ET-1 at the mRNA and protein levels but did not exert any significant modification at lower concentrations from 10(-10) to 10(6) M. Phosphoramidon, an endothelin converting enzyme (ECE) inhibitor, drastically decreased the amount of ET-1 accumulating in the culture medium in basal conditions or after UVB irradiation. Conversely, thiorphan, a specific inhibitor of neutral endopeptidase (NEP), rather increased the levels of ET-1 secretion mainly after UVB irradiation. Taken together, the results showed that normal human keratinocytes secrete and partly degrade ET-1 through ECE and NEP pathways and pointed out a differential regulation of ET-1 by UVB and UVA1 radiations without any noticeable role for PGE2.

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.

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.

Three putative metalloprotease inhibitors were synthesized and tested for their ability to inhibit the catalytic activity of botulinum neurotoxin B light chain (BoNT/B LC). The compounds were designed to emulate the naturally occurring metalloprotease inhibitor phosphoramidon, which has been reported to be a weak antagonist of BoNT/B action. All three analogs contained the dipeptide Phe-Glu in place of Leu-Trp of phosphoramidon and possessed a phenyl, ethyl or methyl group in place of the rhamnose sugar of the parent compound. The inhibitors were evaluated in a cell-free assay based on the detection of a fluorescent product following cleavage of a 50-mer synaptobrevin peptide ([Pya(88)] S 39-88) by BoNT/B LC. This peptide corresponds to the hydrophilic core of synaptobrevin-2 and contains a fluorescent analog L-pyrenylalanine (Pya) in place of Tyr(88). Cleavage of [Pya(88)] S 39-88 by BoNT/B LC gives rise to fragments of 38 and 12 amino acid residues. Quantification of BoNT/B-mediated substrate cleavage was achieved by separating the 12-mer fragment (FETSAAKLKRK-Pya) that contains the C-terminal fluorophore and measuring fluorescence at 377 nm. The results indicate that the phenyl-substituted synthetic compound ICD 2821 was slightly more active than phosphoramidon, but analogs with methyl or ethyl substitutions were relatively inactive. These findings suggest that phosphonate monoesters may be useful for providing insights into the structural requirement of BoNT/B protease inhibitors.

We compared the enzymatic inactivation of major circulating forms of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Both ANP and BNP induced a significant increase in cyclic GMP (cGMP) formation in cultured epithelial cell line derived from porcine kidney, LLC-PK1. The cGMP formation stimulated by ANP in LLC-PK1 cells was significantly decreased by pre-treatment of the peptide with rat renal brush-border membranes, and the inactivation of ANP was inhibited by neutral endopeptidase inhibitors, phosphoramidon and S-thiorphan. BNP exhibited greater resistance to enzymatic inactivation than did ANP. In addition, phosphoramidon potentiated the natriuresis with a low dose (7.5 pmol min(-1) kg(-1)) of ANP but not of BNP in rats. These results suggest that enzymatic degradation of natriuretic peptides is highly dependent on peptide structure, and that the affinity of BNP to neutral endopeptidase is less than that of ANP.

Increasing evidence suggests that angiotensin-converting enzyme (ACE) inhibitors can increase vascular nitric oxide (NO) production. Recent studies have found that combined inhibition of ACE and neutral endopeptidase (NEP) may have a greater beneficial effect in the treatment of heart failure than inhibition of ACE alone. Amlodipine, a calcium channel antagonist, has also been reported to have a favorable effect in the treatment of patients with cardiac dysfunction. The purpose of this study was to determine whether and the extent to which all of these agents used in the treatment of heart failure stimulate vascular NO production. Heart failure was induced by rapid ventricular pacing in conscious dogs. Coronary microvessels were isolated from normal and failing dog hearts. Nitrite, the stable metabolite of NO, was measured by the Griess reaction. ACE and NEP inhibitors and amlodipine significantly increased nitrite production from coronary microvessels in both normal and failing dog hearts. However, nitrite release was reduced after heart failure. For instance, the highest concentration of enalaprilat, thiorphan, and amlodipine increased nitrite release from 85 +/- 4 to 156 +/- 9, 82 +/- 7 to 139 +/- 8, and 74 +/- 4 to 134 +/-10 pmol/mg (all *p <.01 versus control), respectively, in normal dog hearts. Nitrite release in response to the highest concentration of these two inhibitors and amlodipine was reduced by 41% and 31% and 32% (all #p <.01 versus normal), respectively, in microvessels after heart failure. The increase in nitrite induced by either ACE or NEP inhibitors or amlodipine was entirely abolished by Nw-nitro-L-arginine methyl ester, HOE 140 (a B2-kinin receptor antagonist), and dichloroisocoumarin (a serine protease inhibitor) in both groups. Our results indicate that: 1) there is an impaired endothelial NO production after pacing-induced heart failure; 2) both ACE and NEP are largely responsible for the metabolism of kinins and modulate canine coronary NO production in normal and failing heart; and 3) amlodipine releases NO even after heart failure and this may be partly responsible for the favorable effect of amlodipine in the treatment of heart failure. Thus, the restoration of reduced coronary vascular NO production may contribute to the beneficial effects of these agents in the treatment of heart failure.

The intra-third-ventricular (i.t.v.) administration of [Met5]-enkephalin (enk) to rats pretreated i.t.v. with three peptidase inhibitors (PIs), amastatin, captopril and phosphoramidon, inhibited the tail-flick response. The enk-induced inhibition was augmented by increasing the doses of the three PIs, with the maximum inhibition being attained at the doses of 10 nmol each. The enk-induced inhibition in rats pretreated with any combination of two PIs, however, were markedly smaller than that in rats pretreated with all three PIs, indicating that three kinds of enzymes all played important roles in the inactivation of enk. The inhibitory effect of enk on the tail-flick response in rats pretreated with the three PIs at doses of 10 nmol each was approximately tenfold higher than that of morphine. The relative anti-nociceptive potencies of enk and morphine were similar to the relative inhibitory potencies obtained previously with the isolated guinea pig ileum pretreated with the three PIs, indicating that the hydrolysis of the i.t.v. administered enk was largely prevented by the three PIs. However, the magnitude of the enk-induced inhibition in rats pretreated s.c. with the three PIs indicated that the hydrolysis of enk injected i.t.v. was not largely prevented by the s.c. administration of three PIs at doses up to 10 micromol each/kg.

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.

Coronary dysfunctions identified in the presence of chronic heart failure are an important pathophysiologic abnormality that influences the prognosis of the disease. Because the endothelin pathway plays a significant role in the increased peripheral vascular tone associated with heart failure, we hypothesized that the endothelin pathway may be involved in the abnormal coronary vasomotion associated with this pathologic condition. Experiments were carried out in failing hearts (UM-X7.1 cardiomyopathic hamsters, aged 225-250 days) and normal hearts (Syrian LVG hamsters, also aged 225-250 days). Isolated hearts were perfused at constant flow and exposed to the blocker of the generation of endothelin-1 (ET-1), phosphoramidon (10 microM infusion), as well as to the selective ET(A)-receptor antagonist BQ 123 (10 microM infusion) and to a selective ET(B)-receptor antagonist BQ 788 (1 microM infusion). Coronary and cardiac effects of exogenous ET-1 (0.01-100 pmol) were also studied. Phosphoramidon, BQ 788, and BQ 123 did not altered coronary perfusion pressure either in normal or in failing hearts, whereas cardiac contractility was significantly impaired in the presence of phosphoramidon and BQ 123. Coronary sensitivity to exogenous ET-1 did not demonstrate a significant difference between normal and failing hearts [median effective concentration (EC50), 7 pmol in failing hearts vs. 12 pmol in normal hearts; p = NS]. In the presence of exogenous ET-1, cardiac contractility was significantly increased in both groups. In normal hearts, the exogenous ET-1-induced increase in coronary perfusion pressure was completely antagonized by BQ 123, whereas combined administration of BQ 788 and BQ 123 was necessary to induce complete inhibition in failing hearts. The positive inotropic effect elicited by exogenous ET-1 (EC50) was completely abolished in the presence of BQ 123, whereas BQ 788 had no significant effect. Results indicate that the endothelin pathway does not play a significant role in the altered coronary vasomotion observed in this model of chronic heart failure. On the contrary, the endothelin pathway appears to participate in the maintenance of myocardial contractility. According to these observations, administration of an inhibitor of ET-1 synthesis, as well as the use of an ET(A)-receptor antagonist, may be contraindicated in the presence of poor left ventricular function because the endothelin pathway contributes significantly to the maintenance of cardiac contractility.

We have previously found that human chymase cleaves big endothelins at the Tyr31-Gly32 bond and produces 31-amino acid long endothelins-(1-31), without any further degradation products. In this study, we investigated the effect of synthetic endothelin-1-(1-31) on the intracellular free Ca2+ concentration ([Ca2+]i) in cultured human coronary artery smooth muscle cells. Endothelin-1-(1-31) increased [Ca2+]i in a concentration-dependent manner (10(-14) to 10(-10) M). This endothelin-1-(1-31)-induced [Ca2+]i increase was not affected by phosphoramidon (N-(alpha-Rhamnopyranosyloxyhydroxyphosphinyl)-L-Leucyl-L-Tryptoph an), an inhibitor of endothelin-converting enzyme. It was, however, inhibited by 10(-10) M BQ123 (Cyclo-(-D-Trp-D-Asp(ONa)-Pro-D-Val-Leu-)), an endothelin ET(A) receptor antagonist, but not by 10(-10) M BQ788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-yMeLeu-D-Trp(COOM e)-D-Nle-ONa), an endothelin ET(B) receptor antagonist. These results suggest that endothelin-1-(1-31) by itself exhibits vasoactive properties probably through endothelin ET(A) receptors. Since human chymase has been reported to play a role in atherosclerosis, endothelin-1-(1-31) may be one of the candidate substances for its cause.

The contents of [Met5]-enkephalin-Arg6-Phe7 (met-enk-RF) and its six hydrolysis products: Y, YG, YGG, YGGF, YGGFM, and YGGFMR were estimated after incubating met-enk-RF with either a guinea-pig ileal or striatal membrane fraction for various times at 37 degrees C. After 45 min incubation with either ileal or striatal membranes, met-enk-RF was completely hydrolyzed, yielding Y as the major product. Incubation with either membrane preparation for 60 min in the presence of the aminopeptidase inhibitor amastatin hydrolyzed 90 or 92% of met-enk-RF, respectively, with YGG being the major product. If the dipeptidyl carboxypeptidase I inhibitor captopril is also included in the incubation, met-enk-RF hydrolysis decreases by about half for both membranes, with YGG remaining the major product. Inclusion of three peptidase inhibitors, amastatin, captopril, and phosphoramidon (inhibition of endopeptidase-24.11) further reduced met-enk-hydrolysis, with 87% or more remaining intact. This shows that met-enk-RF was mainly 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, estimations of [Leu5]-enkephalin (leu-enk), alpha- and beta-neoendorphins (alpha- and beta-neoends), and dynorphin B (dyn B) contents after incubating the individual peptides with striatal membrane for 60 min in the presence of the three peptidase inhibitors showed that 98, 32, 5, and 23%, respectively, remained intact. Our previous studies together with the data obtained here show that one group of endogenous opioid peptides: met-enk, leu-enk, met-enk-RF, met-enk-RGL, and dyn A-(1-8) are largely or almost exclusively hydrolyzed by the three enzymes, amastatin-sensitive aminopeptidase, captopril-sensitive dipeptidyl carboxypeptidase I, and phosphoramidon-sensitive endopeptidase-24.11, and indicate that an unidentified fourth enzyme(s) is involved in the hydrolysis of another group of peptides: alpha-neoend, beta-neoend, and dyn B.

The effects of i.v. injections of two endogenous tachykinins, substance P (SP) and neuropeptide gamma and the highly selective tachykinin agonists [Sar9,Met(O2)11]-SP, [Lys5,MeLeu9, Nle10]-NKA(4-10) and senktide, on total lung resistance (RL), dynamic lung compliance (Cdyn) and systemic blood pressure, were compared in the anaesthetized rabbit. Senktide, the NK-3 receptor selective agonist, had no effect on RL, Cdyn or blood pressure. The other four agonists caused dose-dependent increases in RL and Cdyn, with [Sar9,Met(O2)11]-SP being the most potent agonist in producing changes in the absence of phosphoramidon. This suggested that NK-1 receptors play an important role in these responses. [Sar9, Met(O2)11]-SP, SP and neuropeptide gamma also decreased blood pressure. Phosphoramidon (1 mg/kg) potentiated the changes in RL and Cdyn evoked by [Sar9,Met(O2)11]-SP and SP, with very marked enhancement of responses to neuropeptide gamma. Responses to [Lys5, MeLeu9,Nle10]-NKA(4-10) were unaffected, suggesting that this NK-2 selective agonist may not be catabolized by neutral endopeptidase (NEP). In the presence of phosphoramidon, the non-peptide tachykinin NK-1 receptor selective antagonist CP 96345 (80 nmol/kg) reduced all responses to [Sar9,Met(O2)11]-SP and SP, whereas the NK-2 selective antagonist SR 48968 (40 nmol/kg) inhibited the bronchomotor but not the vasodepressor responses to neuropeptide gamma and [Lys5,MeLeu9, Nle10]-NKA(4-10). The fall in blood pressure induced by neuropeptide gamma was diminished by CP 96345, whereas bronchoconstriction was unaffected, indicating possible differences in NK-1 receptors in the vasculature and airways. Electrical stimulation of the distal ends of vagus nerves caused increases in RL which were abolished by atropine (1 mg/kg)., (Copyright 1998 Academic Press Limited)

The human megakaryoblastic cell lines HEL, MEG-01, and DAMI express preproendothelin-1 mRNA. This investigation was designed to find out whether they could also express endothelin-converting enzyme (ECE) and release mature endothelin (ET). RT-PCR applied to RNA isolated from the cell lines amplified fragments of the expected size. The amplified cDNA of MEG-01 was submitted to restriction enzymes, which generated the expected subfragments. Membrane ECE activity was phosphoramidon-sensitive, in contrast to the cytosolic activity capable of producing ET-1 from big ET-1. The three cell lines produced ir-ET in a time-dependent manner. These results show that human megakaryoblastic cell lines express functional, phosphoramidon-sensitive and insensitive ECE activity and produce mature ET.

Endothelin-1 (ET-1) may be involved in the upregulation of cerebroarterial resistance under pathologic conditions, most notably in the development of vasospasm after subarachnoid hemorrhage. Therefore, blocking the contractile action of ET-1 by receptor antagonists may prove to be a new and worthwhile approach. However, decreasing synthesis and release of ET-1 by blocking the endothelin-converting enzyme (ECE) activity may also prove worthwhile. In this study we have therefore investigated the effect of several putative ECE inhibitors in isolated rat basilar artery by measuring isometric contraction after application of big ET-1, the precursor peptide which is not vasoactive in itself. In the presence of phosphoramidon (10(-4) M in segments with an intact endothelium or 5 x 10(-4) M in de-endothelialized segments), there was only a small shift to the right of the concentration-effect curve for big ET-1. Similarly, 10(-3) M thiorphan (a selective inhibitor of the neutral endopeptidase) did not affect big ET-1-induced contraction, both alone and in combination with phosphoramidon (10(-3) M). When the big ET-1 analogue [22Phe]big ET-1[19-37] was applied, an increase in resting tension occurred irrespective of whether or not the endothelium was present. Furthermore, in the presence of 10(-5) M [22Phe]big ET-1[19-37], contraction induced by big ET-1 was not affected in de-endothelialized segments but rather was enhanced in endothelium-intact segments. These results suggest the presence of functional ECE activity in the rat basilar artery wall. However, such activity could not be markedly inhibited with different putative enzyme inhibitors. Therefore, the chemical nature of the cerebroarterial ECE activity must be further elucidated before rational development of efficient ECE inhibitors for treatment of cerebral vasospasm becomes possible.

Part of the contractile response of adenosine in the asthmatic airways may be due to the activation of peptidergic pathways with subsequent local release of spasmogenic neuropeptides. At present, little is known about the potential role of lung peptidases in modulating adenosine-induced airway dysfunction in humans in vivo. We have, therefore, investigated the change in bronchial reactivity to adenosine 5'-monophosphate (AMP), after treatment with inhaled phosphoramidon, a potent neutral endopeptidase (NEP) inhibitor, in a double-blind, placebo-controlled, randomized study of 12 asthmatic subjects. Subjects attended on six separate occasions, during which concentration response studies with inhaled AMP and methacholine were carried out, initially in the absence of treatment and then after nebulized phosphoramidon sodium salt (10[-5] M) or matched placebo 5 min prior to a bronchoprovocation test with AMP or methacholine. Agonist responsiveness was expressed as the provocative concentration of AMP or methacholine producing a 20% fall in FEV1 from baseline (PC[20,AMP] or PC[20,meth], respectively). When compared to placebo, phosphoramidon failed to potentiate the airway response to AMP. The geometric mean (range) PC20 AMP value of 23.4 (4.4-190.6) mg x mL(-1) after placebo was not significantly different from that of 20.7 (45-100.9) mg x mL(-1) obtained after phosphoramidon. The lack of change in bronchial reactivity to adenosine 5'-monophosphate after phosphoramidon indicates that endogenous airway neutral endopeptidase may not be of physiological importance in modulating the contractile response of adenosine in the airways. Thus, the present data do not support the view that activation of peptidergic pathways with subsequent local release of spasmogenic neuropeptides is important in the airway response to adenosine

Inhibition of peptidases within the lungs not only potentiates the effects of neuropeptides released from C-fibers but also the effects of bradykinin and capsaicin both of which stimulate C-fibers. To determine if peptidase inhibition potentiates C-fiber activation, we challenged pulmonary C-fibers in rats with capsaicin or bradykinin before and after inhibition of neutral endopeptidase (NEP) or angiotensin converting enzyme (ACE). Inhibition of NEP by phosphoramidon (10 mg/kg, i.v.) potentiated the effect of capsaicin (0.5-1 micrograms, i.v.) on C-fiber activity but did not change the response to bradykinin (1-2 micrograms, i.v.). Inhibition of ACE by captopril (5 mg/kg, i.v.) potentiated C-fiber activation by either bradykinin or capsaicin. Aerosol administration of either phosphoramidon (1 x 10(-5) M, 2 min) or captopril (4.6 x 10(-3) M, 2 min) potentiated C-fiber activation by capsaicin aerosol (1.6 x 10(-4) M, 1 min) but not by bradykinin aerosol (9.4 x 10(-5) M, 1 min). Therefore, inhibition of NEP or ACE may potentiate airway obstructive mechanisms initiated by C-fiber stimulation.

Injection of substance P (SP) in a rat hindpaw induced extravasation of 125I-labelled albumin in both hindpaws and salivation. Intravenous injection of SP dose-dependently increased vascular permeability. This latter effect was increased in rat paws by captopril, an inhibitor of angiotensin-converting enzyme (ACE), administered locally in combination with diprotin A, an inhibitor of an dipeptidyl(amino)peptidase IV (DAP IV) or phosphoramidon, an inhibitor of neutral endopeptidase (NEP). The increase in permeability induced by SP was inhibited by RP 67580, a NK-1-receptor antagonist. Intravenous injection of capsaicin induced labelled albumin extravasation in rat paws. This effect was increased by combination of captopril with diprotin A or phosphoramidon, but not by captopril associated with amastatin, an inhibitor of aminopeptidase M (AmM). It was suppressed by RP 67580. Injection of collagenase in rat paws triggered a swelling and a local plasma exudation. These responses were reduced by RP 67580 but not by RP 68651, its inactive enantiomer. They were increased by combination of captopril with diprotin A or phosphoramidon in normal rats. The potentiating effects of captopril and diprotin A were suppressed by RP 67580 in normal rats but did not develop in kininogen-deficient rats. The oedema induced by collagenase was also increased by lisinopril, another ACE inhibitor, administered locally in combination with apstatin, an inhibitor of aminopeptidase P (AmP). In rats pretreated by methysergide, collagenase-induced oedema was reduced and can be increased by captopril, by lisinopril, administered alone or by lisinopril associated with apstatin. It is concluded that SP is mainly inactivated in rat paws by ACE, DAP IV and NEP. In collagenase-induced oedema, a low amount of SP would be released from afferent nerve terminals by bradykinin formed in low amounts. Bradykinin is inactivated in rat paws by ACE and AmP. In collagenase-oedema, the pro-inflammatory effects of bradykinin are concealed by the effects of the other mediators.

1. The role of the metalloendopeptidase EC 3.4.24.15 (EP 24.15) in peptide metabolism in vivo is unknown, in part reflecting the lack of a stable enzyme inhibitor. The most commonly used inhibitor, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP-AAY-pAB, Ki = 16 nM), although selective in vitro, is rapidly degraded in the circulation to cFP-Ala-Ala, an angiotensin converting enzyme (ACE) inhibitor. This metabolite is thought to be generated by neutral endopeptidase (NEP; EC 3.4.24.11), as the Ala-Tyr bond of cFP-AAY-pAB is cleaved by NEP in vitro. In the present study, we have examined the role of NEP in the metabolism of cFP-AAY-pAB in vivo, and have tested a series of inhibitor analogues, substituted at the second alanine, for both potency and stability relative to the parent compound. 2. Analogues were screened for inhibition of fluorescent substrate cleavage by recombinant rat testes EP 24.15. D-Ala or Asp substitution abolished inhibitory activity, while Val-, Ser- and Leu-substituted analogues retained activity, albeit at a reduced potency. A relative potency order of Ala (1) > Val (0.3) > Ser (0.16) > Leu (0.06) was observed. Resistance to cleavage by NEP was assessed by incubation of the analogues with rabbit kidney membranes. The parent compound was readily degraded, but the analogues were twice (Ser) and greater than 10 fold (Leu and Val) more resistant to cleavage. 3. Metabolism of cFP-AAY-pAB and the Val-substituted analogue was also examined in conscious rabbits. A bolus injection of cFP-AAY-pAB (5 mg kg-1, i.v.) significantly reduced the blood pressure response to angiotensin I, indicating ACE inhibition. Pretreatment with NEP inhibitors, SCH 39370 or phosphoramidon, slowed the loss of cFP-AAY-pAB from the plasma, but did not prevent inhibition of ACE. Injection of 1 mg kg-1 inhibitor resulted in plasma concentrations at 10 s of 23.5 microM (cFP-AAY-pAB) and 18.0 microM (cFP-AVY-pAB), which fell 100 fold over 5 min. Co-injection of 125I-labelled inhibitor revealed that 80-85% of the radioactivity had disappeared from the circulation within 5 min, and h.p.l.c. analysis demonstrated that only 25-30% of the radiolabel remained as intact inhibitor at this time. Both analogues were cleared from the circulation at the same rate, and both inhibitors blunted the pressor response to angiotensin I, indicative of ACE inhibition. 4. These results suggest that both NEP and other clearance/degradation mechanisms severely limit the usefulness of peptide-based inhibitors such as cFP-AAY-pAB. To examine further EP 24.15 function in vivo, more stable inhibitors, preferably non-peptide, must be developed, for which these peptide-based inhibitors may serve as useful molecular templates.

Endothelin converting enzyme was partially purified by detergent extraction and ion exchange chromatography from porcine aortic endothelial cells. This kinetically homogeneous preparation catalyzes the hydrolysis of porcine big endothelin-1 to endothelin-1 with a pH optimum of 7. Human big endothelins-1, -2, and -3 are also hydrolyzed, but at progressively lower rates. Fragments of big porcine endothelin-1 comprising residues 16-39 and 16-29 are good substrates, but additional C-terminal truncations are devoid of substrate activity. Endothelin converting enzyme is characteristically inhibited by phosphoramidon and other metalloproteinase inhibitors including EDTA, o-phenanthroline, and diethylpyrocarbonate, but not by inhibitors of other classes of proteases or thiorphan. The inhibition by phosphoramidon is competitive with big porcine endothelin-1 suggestive of a common binding site for substrate and inhibitor. A number of novel analogs of phosphoramidon were synthesized by modifying various regions of the molecule and tested for inhibitory activity. The most potent of these, a methylphosphonic acid, has an IC50 of 0.05 microM.

Endothelin-converting enzyme is a phosphoramidon-sensitive membrane metallopeptidase that catalyses the final step in biosynthesis of the potent vasoactive endothelin peptides. Immunomagnetic separation technology and immunohistochemistry have been used to demonstrate the co-localisation of endothelin-converting enzyme with the established ectoenzyme, aminopeptidase N, on the surface of endothelial cells. Unlike aminopeptidase N, however, endothelin-converting enzyme is seen to associate in clusters on the plasma membrane which can be distinguished from caveolae both biochemically and immunologically. Pre-treatment of endothelial cells with the metallopeptidase inhibitors phosphoramidon or thiorphan in the range 0.01-100 microM produced a dose-dependent increase in the levels of endothelin-converting enzyme protein and its accumulation in an intracellular compartment. No corresponding change in the levels of endothelin-converting enzyme-1 mRNA was detected under these conditions, nor in the levels of the closely related metalloenzyme, endopeptidase-24.11. The phosphoramidon and thiorphan-dependent increase is not due to direct inhibition of endothelin-converting enzyme not endopeptidase-24.11 but, rather, to an inhibition of the selective turnover of endothelin-converting enzyme protein.

To determine whether lysylbradykinin (LBK, kallidin) causes bronchoconstriction in animals and if peptidase inhibitors modulate the response, we studied the effects of LBK administered by aerosol in rats and assessed whether pretreatment with aerosolized solutions of enalaprilat, an inhibitor of angiotensin converting enzyme (ACE), or phosphoramidon, an inhibitor of endopeptidase 24.11 (EP 24.11, neutral endopeptidase), altered the response. Accordingly, LBK-induced bronchoconstriction was measured in anesthetized, mechanically ventilated, specific pathogen-free, Sprague-Dawley rats by body plethysmography and followed by continuous determination of lung resistance (RL) and maximal expiratory flow (MEF). Incremental doses of aerosolized LBK were administered by nebulization to obtain a concentration that caused a 5-15% increase in RL, which was designated the BC10 dose. We found that pretreatment with aerosolized enalaprilat (1 mM) 3 min prior to a BC10 dose of LBK significantly increased RL as compared to the BC10 dose alone (129 +/- 4.1% vs. 105 +/- 2.4%, P < 0.002, n = 4) and significantly decreased MEF (83 +/- 1.5% vs. 97 +/- 1.4%, P < 0.008, n = 4). Following pretreatment with aerosolized phosphoramidon (1 mM), significant increases in RL (113 +/- 1.4% vs. 106 +/- 1.6%, P < 0.019, n = 7) and decreases in MEF (92 +/- 0.9% vs. 95 +/- 0.9%, P < 0.035, n = 7) were observed (paired Student's t-test). The above findings demonstrate the effects of LBK on airway caliber for the first time in an animal model, and suggest that ACE and EP 24.11 contribute to degradation of the peptide in the airway.

The purpose of present study was to determine whether peptidase activity affects the release of substance P (SP) and calcitonin gene-related peptide (CGRP) from spinal cord slices. When slices were exposed to various inhibitors of endopeptidase 24.11, the resting and capsaicin-stimulated release of SP were less than 0.04% and 0.20% total content per minute, respectively. Resting CGRP release was approximately 0.10% and stimulated release was 0.40%. The combination of 20 microM bacitracin, 100 microM phenylalanylalanine (Phe-Ala), and 50 microM p-chloromercuriphenylsulfonic acid (PCMS) significantly increased both resting and stimulated release of SP and CGRP at least two- or threefold. Doubling the concentration of PCMS and Phe-Ala did not further improve peptide release. These results demonstrate that recovery of peptides released from spinal cord slices is dependent in part on activity of multiple peptidases in the tissues.

Diaspirin cross-linked hemoglobin (DCLHb) is a promising hemoglobin-based, oxygen-carrying resuscitative solution. DCLHb (400 mg/kg, iv) produces significant cardiovascular effects, along with an increase in plasma endothelin-1 (ET-1) level, when administered to conscious or anesthetized rats. Present studies were performed to determine whether the cardiovascular effects of DCLHb are due to an increase in the conversion of proendothelin-1 (1-38) (proET-1) to ET-1 by endothelin-converting enzyme (ECE). The regional circulatory and systemic hemodynamic effects of proET-1 (20 micrograms/kg, iv) and DCLHb (400 mg/kg, iv) were determined by using a radioactive microsphere technique in control rats and rats pretreated with phosphoramidon (ECE inhibitor). Administration of proET-1 produced an immediate increase in mean arterial pressure (MAP)(52%) and total peripheral resistance (TPR) (55%); stroke volume (SV) and cardiac output were not affected in the initial phase but were decreased subsequently. Heart rate (HR) was not affected after administration of proET-1. A significant increase in blood flow to the heart (39%), brain (46%), kidneys (74%), portal system (40%), and gastrointestinal tract (GIT) (42%) was also observed after administration of proET-1. Vascular resistance was found to be significantly increased in the mesentery and pancreas (168%) and in the musculoskeletal system (147%) and decreased in the kidneys (-11%) after administration of proET-1. Phosphoramidon (4 mg/kg, iv) pretreatment attenuated the increase in MAP and TPR induced by proET-1. Phosphoramidon pretreatment significantly attenuated the proET-1-induced increase in blood flow to the heart, brain, kidneys, portal system, and GIT. The increase in vascular resistance induced by proET-1 in the mesentery and pancreas and in the musculoskeletal system was also attenuated by phosphoramidon. DCLHb increased MAP (63%) and TPR (54%) without affecting HR. DCLHb increased blood flow to the heart (95%), GIT (45%), portal system (43%), and skin (79%) and increased vascular resistance in the musculoskeletal system (58%). In phosphoramidon-treated rats, DCLHb increased MAP (99%), HR (25%), cardiac output (37%), and TPR (60%). DCLHb increased blood flow to the heart (104%), brain (66%), kidneys (49%), GIT (59%), portal system (63%), and skin (100%) when administered to phosphoramidon-treated rats. Phosphoramidon did not attenuate any of the DCLHb-induced cardiovascular effects. It is concluded that proET-1 increases blood flow to various organs and that phosphoramidon, an ECE inhibitor, could block the proET-1-induced increases in regional blood flow.(ABSTRACT TRUNCATED AT 400 WORDS)

Diabetic complications are mostly vascular and involve alteration in blood vessel reactivity and permeability. The contribution of the latter dysfunction to the development of target organ damage has not been thoroughly examined. In this study, we verify the acute effect of three peptidase inhibitors (phosphoramidon: N-(alpha-rhamnopyranosylhydroxyphosphinyl)-Leu-Trp, thiorphan: 3-mercapto-2-benzyl-propanoylglycine, and SQ 28,603: N-(2-mercaptomethyl)-1-oxo-3-phenylpropyl]- < or = b-alanine; each at a dose of 2 mg/kg), as well as captopril ([2S]-1-[3-mercapto-2-methyl-propionyl]-L-proline; 10 mg/kg), and an aminopeptidase inhibitor (amastatin: ([(2S,3R)-3-amino-2-hydroxy-5-methylhexanoyl]-Val-Val-Asp; 2 mg/kg) on capillary extravasation abnormalities in the streptozotocin-induced diabetic rat using the Evans blue method. Untreated diabetic rats exhibited a significant enhancement of Evans blue extravasation in the duodenum, upper bronchus, pancreas and skin (175 +/- 19, 94 +/- 4, 95 +/- 9 and 47 +/- 10 micrograms/g dry tissue respectively compared to 67 +/- 9, 44 +/- 5, 47 +/- 4, and 6 +/- 2 micrograms/g dry tissue). The three endopeptidase inhibitors normalized capillary permeability in all tissues. Also, treatment with captopril was associated with complete correction of capillary dysfunction in the skin and partially in the duodenum, upper bronchus, and pancreas. These findings indicate for the first time that the use of neutral endopeptidase inhibitors may be beneficial in preventing capillary abnormalities in this diabetic rat model.

1. The vasoconstrictor peptide, endothelin-1 (ET-1) and a biologically inactive C-terminal fragment (CTF) are generated from an intermediate big ET-1 by a putative ET converting enzyme, sensitive to phosphoramidon. We have developed a procedure using selective solid-phase extraction and specific radioimmunoassays to measure the levels of immunoreactive (IR) big ET-1 and the products of conversion (ET-1 and CTF) in human plasma. These techniques have been used to determine the levels of the three peptides in venous plasma following local infusions of ET-1 and big ET-1, both alone and together with phosphoramidon. 2. Infusion of ET-1 into the brachial artery (5 pmol min-1) significantly increased (P < 0.05) IR ET levels from a basal level of 2.3 pM to 55.2 pM in plasma from the infused arm after 60 min of infusion. This corresponded with a marked decrease in forearm blood flow from a basal level of 2.6 ml dl-1 min-1 to 1.7 ml dl-1 min-1. The levels of IR big ET-1 and CTF were unchanged. Co-infusion of phosphoramidon (30 nmol min-1) with ET-1 had no significant effect on the plasma IR levels of ET, big ET-1, CTF, or blood flow. 3. Big ET-1 (50 pmol min-1) significantly increased (P < 0.05) venous concentrations of all three IR peptides after 60 min compared to basal (ET: from 2.2 to 7.7 pM, big ET-1; from 0 to 386.0 pM, CTF: from 0.2 to 37.0 pM). Forearm blood flow decreased significantly (P<0.05) from a basal level of 3.0 ml dl-1 min-1 to 1.6 ml dl-1 min-1.4. When phosphoramidon was co-infused with big ET-1, both the rise in IR ET and associated vasoconstriction were abolished. However, IR CTF was still detected, suggesting that either some conversion by phosphoramidon-insensitive enzyme(s) was occurring, and/or that CTF was being protected from further degradation by phosphoramidon.5. These data show that in the human forearm the activity of a phosphoramidon-sensitive ET converting enzyme is at least in part responsible for the vasoconstrictor properties of exogenous big ET-1. Furthermore, because measurable levels of newly synthesized ET-1 are likely to be rapidly reduced in the blood/plasma through receptor binding, assay of IR big ET-1 and CTF may be a more sensitive measure of ET-1 generation in disease.

In the present study, we explored whether or not a neutral endopeptidase inhibitor provokes cough in awake guinea-pigs. Inhalation of phosphoramidon at a concentration of 10(-6) M did not cause cough, but increasing the concentration to 10(-5) M caused cough with a latency of about 10 to 12 min. Inhalation of enalapril, an angiotensin-converting enzyme inhibitor, did not cause cough, even at high concentrations of 10(-5) M and 10(-4) M. Pretreatment with phosphoramidon (10(-5) M) significantly increased the number of coughs caused by substance P and capsaicin. Capsaicin-induced coughs were more easily produced in bronchitic guinea-pigs than in normal guinea-pigs. However, there was no significant difference in the number of phosphoramidon-induced coughs between normal and bronchitic guinea-pigs. Phosphoramidon-induced coughs were significantly depressed by codeine (20 mg/kg, p.o.) and CP96345 (2 mg/kg, i.v.). The present results provide new evidence for the proposed idea that neutral endopeptidase may regulate the occurrence of cough.

Background: Bradykinin is a potent vasoactive peptide which has been proposed as an important inflammatory mediator in asthma since it provokes potent bronchoconstriction in asthmatic subjects. Little is known at present about the potential role of lung peptidases in modulating bradykinin-induced airway dysfunction in vivo in man. The change in bronchial reactivity to bradykinin was therefore investigated after treatment with inhaled phosphoramidon, a potent neutral endopeptidase (NEP) inhibitor, in a double blind, placebo controlled, randomised study of 10 asthmatic subjects., Methods: Subjects attended on six separate occasions at the same time of day during which concentration-response studies with inhaled bradykinin and histamine were carried out, without treatment and after each test drug. Subjects received nebulised phosphoramidon sodium salt (10(-5) M, 3 ml) or matched placebo for 5-7 minutes using an Inspiron Mini-neb nebuliser 5 minutes before the bronchoprovocation test with bradykinin or histamine. Agonists were administered in increasing concentrations as an aerosol generated from a starting volume of 3 ml in a nebuliser driven by compressed air at 8 1/min. Changes in airway calibre were measured as forced expiratory volume in one second (FEV1) and responsiveness as the provocative concentration causing a 20% fall in FEV1 (PC20)., Results: Phosphoramidon administration caused a transient fall in FEV1 from baseline, FEV1 values decreasing 6.3% and 5.3% on the bradykinin and histamine study days, respectively. When compared with placebo, phosphoramidon elicited a small enhancement of the airways response to bradykinin, the geometric mean PC20 value (range) decreasing from 0.281 (0.015-5.575) to 0.136 (0.006-2.061) mg/ml. In contrast, NEP blockade failed to alter the airways response to a subsequent inhalation with histamine, the geometric mean (range) PC20 histamine value of 1.65 (0.17-10.52) mg/ml after placebo being no different from that of 1.58 (0.09-15.21) mg/ml obtained after phosphoramidon., Conclusions: The small increase in bronchial reactivity to bradykinin after phosphoramidon exposure suggests that endogenous airway NEP may play a modulatory role in the airways response to inflammatory peptides in human asthma.

Escherichia coli endotoxin (0.1 to 1000 micrograms/ml) stimulated the release of neutrophil chemotactic activity (P < 0.001) and induced bronchial epithelial cell (BEC) cytotoxicity assessed by lactate dehydrogenase release (P < 0.001). Endotoxin (100 micrograms/ml) inhibited BEC accumulation (P < 0.001). In the present study, we investigated the role of proteolytic activity of BECs per se in response to endotoxin. Several structurally and functionally different antiproteases, alpha 1 protease inhibitor, soybean trypsin inhibitor, two chloromethyl ketone derivatives (N-tosyl-L-lysine chloromethyl ketone and methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone), and L-658,758, a neutrophil elastase inhibitor, attenuated the release of neutrophil chemotactic activity and lactate dehydrogenase (P < 0.01). alpha 1-Protease inhibitor and N-tosyl-L-lysine chloromethyl ketone attenuated the inhibition of BEC accumulation by endotoxin (P < 0.001). The proteolytic enzyme activity measured by synthetic substrates revealed that endotoxin significantly augmented the serine proteolytic activity in the cell layers. Culture supernatant fluids and cell lysates of BECs in the presence of endotoxin solubilized 14C-labeled casein. These data suggest that responses of BECs to endotoxin may involve activation of cellular proteolytic activity.

Substance P (SP) evokes fluid secretion and plasma extravasation when applied to the nasal mucosa of rats. SP and another tachykinin, neurokinin A (NKA), are degraded in vitro by neutral endopeptidase (NEP) and angiotensin-1-converting enzyme (ACE). In this study, NKA or SP were applied locally to the nasal mucosa of rats. Subsequent fluid secretion was measured by a filter paper technique. Plasma exudation was derived as the recovery of intravenous (i.v.) administered 125I-albumin from the fluid-containing filter papers. In order to inhibit enzymatic degradation of the tachykinins by NEP and ACE, the rats were treated with i.v. administered phosphoramidon or captopril respectively or their combination. SP evoked fluid secretion that was augmented by phosphoramidon and further enhanced by adding captopril. NKA evoked nasal fluid secretion less effectively than SP and the effect was unaffected by peptidase inhibition. SP, but not NKA, evoked increased plasma exudation but only after pre-treatment with phosphoramidon.

Endothelin-1 (ET-1) levels are elevated in human breast tumours compared with normal and benign tissues, and in the presence of insulin-like growth factor 1 (IGF-I) ET-1 is a potent mitogen for human breast fibroblasts. In this study we have examined the ability of intact human breast cancer cell lines to process exogenously added big ET-1 (1-38) to the active mature ET-1 peptide by using a specific radioimmunometric assay. In both hormome-dependent (MCF-7, T47-D) and hormone-independent (MDA-MB-231) breast cancer cell lines the putative endothelin-converting enzyme (ECE) exhibited apparent Michaelis-Menten kinetics when converting added big ET-1 to ET-1. Both basal ET-1 production and exogenously added big ET-1 to ET-1 conversion were greatly reduced in all three cell lines in response to the metalloproteinase inhibitor phosphoramidon but were insensitive to other classes of protease inhibitors. Inhibition was also observed when cells were incubated in the presence of the divalent cation chelators 1,10-phenanthroline and EDTA. In MCF-7 cells the optimal pH for the ECE activity using a saponin cell permeabilisation procedure was found to residue within a narrow range of 6.2-7.26. Our results indicate that human breast cancer cells contain a neutral phosphoramidon-sensitive metalloproteinase which can process big ET-1 to ET-1. In the breast this conversion could contribute substantially to the local extracellular levels of this proposed paracrine breast fibroblast mitogen.