Phosphoramidon-sensitive Enzyme Research Articles

Potentiation of [Met5]enkephalin-induced antinociception by mixture of three peptidase inhibitors in rat

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. 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, 1-20nmol; PIs, 1-20nmol 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. 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. 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.

Great Increase in Antinociceptive Potency of [Leu5]Enkephalin After Peptidase Inhibition

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: amastatin, captopril, and phosphoramidon. The present in vivo study shows that the inhibitory effect of [Leu5]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 [Leu5]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 [Leu5]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.

The Enhancing Effects of Peptidase Inhibitors on the Antinociceptive Action of [Met5]Enkephalin-Arg6-Phe7 in Rats

Previous in vitro studies have shown that the degradation of [Met5]enkephalin-Arg6-Phe7 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 [Met5]enkephalin-Arg6-Phe7 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 [Met5]enkephalin-Arg6-Phe7 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 [Met5]enkephalin-Arg6-Phe7. 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 [Met5]enkephalin, [Met5]enkephalin-Arg6-Phe7, [Met5]enkephalin-Arg6-Gly7-Leu8, and dynorphin A (1-8), administered intra-third-ventricularly to rats.

Met 5]enkephalin-Arg-Gly-Leu-induced antinociception is greatly increased by peptidase inhibitors

Previous in vitro studies showed that the degradation of [Met 5]enkephalin-Arg-Gly-Leu by cerebral membrane preparations is almost completely prevented by a mixture of three peptidase inhibitors: amastatin, captopril and phosphoramidon. The present investigations showed that the inhibitory effect of [Met 5]enkephalin-Arg-Gly-Leu administered intra-third-ventricularly on the tail-flick response was increased more than 1000-fold by the intra-third-ventricular pretreatment of rats with three peptidase inhibitors. The inhibition produced by the enkephalin octapeptide 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 octapeptide. The present data, together with those obtained from previous studies, clearly show that three types of enzymes, amastatin-, captopril- and phosphoramidon-sensitive enzymes, play important roles in the inactivation of endogenous opioid penta- and octa-peptides administered intra-third-ventricularly to rats.

Breast cancer cell-associated endopeptidase EC 24.11 modulates proliferative response to bombesin.

We have investigated the production, growth and inactivation of gastrin-releasing peptide (GRP)-like peptides in human breast cancer cell lines. Radioimmunoassay detected GRP-like immunoreactivity (GRP-LI) in T47D breast cancer cells but not in the conditioned medium, indicating rapid clearance. No GRP-LI was found in the ZR-75-1 or MDA-MB-436 cells or their conditioned medium. High-performance liquid chromatography (HPLC) analysis of the GRP-LI in the T47D cells revealed a major peak, which co-eluted with GRP18–27, and a minor more hydrophilic peak. In vitro stimulation of T47D cell growth by bombesin (BN) was enhanced to 138% of control levels (bombesin alone) by the addition of the selective endopeptidase EC 3.4.24.11 inhibitor phosphoramidon (0.1 ng ml−;1). Fluorogenic analysis using whole cells confirmed low levels of this phosphoramidon-sensitive enzyme on the T47D cells. This enzyme, previously unreported in human breast cancer cells, significantly modulates both T47D growth and its response to BN-induced growth. © 1999 Cancer Research Campaign

A Rapid and Selective Endothelin-Converting Enzyme Assay: Characterization of a Phosphoramidon-Sensitive Enzyme from Guinea Pig Lung Membrane

Endothelin-1 (ET-1) is the most potent vasoconstrictor hormone known to date. Biosynthesis of ET-1 from its precursor big endothelin-1 (BET-1) is mediated by endothelin-converting enzyme (ECE), a phosphoramidon-sensitive metalloprotease. We have established a simple, rapid, and selective assay for the evaluation of ECE activity. This assay is based on the quantitative determination of [ 125I]ET-1 released from (3-[ 125I] idotyrosyl 13)BET-1 by binding to the membrane-bound endothelin (ET) receptor. Using this assay we have discovered that guinea pig lung membrane (GPLGM) contains a phosphoramidon-sensitive ECE. Treatment of GPLGM with 0.06% lubrol increased ECE activity and ET binding of the membrane preparation. Lubrol-treated GPLGM (L-GPLGM) contains a high density of ET binding sites ( B max = 2000 fmol/mg protein) and shows no proteolytic activity for degredation of ET-1. At protein concentrations suitable for measurement of ECE activity (0.2 mg/ml), L-GPLGM contains a high concentration of ET receptors and shows a rapid rate of ET-1 binding to the membrane preparation (binding equilibrium in <5 min). Thus, we have utilized L-GPLGM preparation for both ECE activity and measurement of ET-1 binding in a single-step assay for the determination of enzyme activity. ECE activity of L-GPLGM was fully inhibited by phosphoramidon, 1,10-phenanthroline, and EDTA. Class-specific inhibitors of serine, cysteine, and aspartic proteases showed no significant effect on ECE activity in L-GPLGM. These results show that GPLGM contains exclusively a phosphoramidon-sensitive ECE.

Effects of Phosphoramidon and Pepstatin a on the Secretion of Endothelin-I and Big Endothelin-I by Human Umbilical Vein Endothelial Cells: Measurement by Two-Site Enzyme-Linked Immunosorbent Assays

1. Two-site enzyme-linked immunosorbent assays have been developed for the rapid, sensitive and non-isotopic measurement of endothelin-1 and big endothelin-1. The sensitivities of detection were 0.5 and 0.3 fmol/well, with ED50 values of 13 and 12 fmol/well for the endothelin-1 and big endothelin-1 assays, respectively. Each assay is highly selective for its corresponding antigen. The ET-1 assay showed no detectable cross-reactivity with ET-1-(1-20), indicating that the assay only recognizes the 21-amino acid biologically active peptide. 2. The two assays were used to measure the effects of two classes of protease inhibitor on the basal release of enothelin-1 and big endothelin-1 from cultured first-passage human umbilical vein endothelial cells. 3. The secretion of both peptides was time-dependent over 12 h. The metalloprotease inhibitor phosphoramidon (1 x 10(-4) mol/l) significantly reduced the amount of endothelin-1 secreted into the medium (P < 0.05), with a concomitant increase in the secreted levels of big endothelin-1 (P < 0.01). The aspartyl protease inhibitor, pepstatin A, also caused a significant decrease in the secretion of endothelin (P < 0.05). However, unlike phosphoramidon, there was no increase in the levels of big ET-1 compared with the controls. At these concentrations, neither inhibitor affected the viability of the cells as indicated by Trypan Blue exclusion. 4. The two assays permit the direct measurement of endothelin-1 and its precursor, and will be of use in the elucidation of the putative human endothelin-converting enzyme(s).(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphoramidon potentiates the contractile response to endothelin-3, but not endothelin-1 in isolated airway tissue.

1. Phosphoramidon (10 microM) markedly increased the contractile response to endothelin-3 in human and rabbit bronchus in vitro. In human tissue the contractile response to 0.3 microM endothelin-3 was significantly increased from 54 +/- 12% to 137 +/- 34% (of the response to 1 nM acetylcholine) in the presence of phosphoramidon. Similarly, in rabbit isolated bronchus, the endothelin-3-induced response was increased from 34 +/- 5% to 61 +/- 7%. 2. In addition, the potency (as measured by EC30 values) of this peptide in human and rabbit airways was significantly augmented in the presence of the enzyme inhibitor. The geometric mean EC30 value was decreased from 53 nM (95% CI:15, 190) to 8 nM (95% CI:3, 23) in human bronchus and from 150 nM (95% CI:89, 250) to 23 nM (95% CI:11, 50) in rabbit tissue. 3. Neither the potency nor the response (at 0.3 microM) to endothelin-3 in canine bronchial rings was altered after incubation of the tissue in phosphoramidon. 4. A previous study carried out in human airways has implied that the difference in potency between endothelin-1 and endothelin-3 may be attributed to a heterogeneous endothelin receptor population. The results of our study, while also demonstrating this difference in potency, have shown that this marked difference, as well as that obvious in rabbit airway tissue can be abolished in the presence of phosphoramidon. 5. Phosphoramidon produced no change in the cumulative concentration-response curve for endothelin-1 in airway tissue from the three species studied. 6. These results suggest that a phosphoramidon-sensitive enzyme (probably neutral endopeptidase) found in lung, may be responsible for local degradation of endothelin-3, but not endothelin-l in human and rabbit isolated bronchus.

The effects of phosphoramidon on the regional haemodynamic responses to human proendothelin [1–38] in conscious rats

1. Cardiovascular responses to human proendothelin [1-38], in the absence and presence of phosphoramidon, were studied in conscious Long Evans rats, chronically instrumented for the continuous recording of heart rate, systemic arterial blood pressure and renal, mesenteric and hindquarters blood flows. 2. A dose of 0.1 nmol kg-1 human proendothelin [1-38] caused a slight pressor effect (maximum 5 +/- 2 mmHg), but a clear bradycardia (maximum -29 +/- 7 beats min-1). Renal haemodynamics were unchanged but there was mesenteric vasoconstriction and a vasodilation followed by a vasoconstriction in the hindquarters. 3. A dose of 1.0 nmol kg-1 human proendothelin [1-38] caused a gradual hypertension (maximum 42 +/- 4 mmHg at 10 min) and a profound bradycardia (-149 +/- 10 beats min-1 at 30 min). There were gradual but marked, renal and hindquarters vasoconstrictions, whereas there was a substantial mesenteric vasoconstriction that was relatively rapid in onset. 4. In 2 animals, administration of human proendothelin [1-38] at a dose of 10 nmol kg-1 caused an initial hypotension followed by a rapidly-developing pressor effect; there were renal and mesenteric vasoconstrictions and vasodilatation followed by vasoconstriction in the hindquarters. These changes were very similar to those seen following injection of endothelin-1 (0.1 nmol kg-1). 5. Phosphoramidon (2 mumol kg-1) had no cardiovascular effects itself and it did not affect significantly the pressor or mesenteric vasoconstrictor effects of human proendothelin [1-38], but it reduced the bradycardia and renal and hindquarters vasoconstrictor responses. A higher dose of phosphoramidon (lOnmolkg-') caused significant attenuation of all the responses to human proendothelin [1-38], but a substantial mesenteric vasoconstrictor response still occurred under these conditions. 6 The results are consistent with the involvement of phosphoramidon-sensitive enzyme systems in the conversion of human proendothelin [1-38] to endothelin-1 in vivo. In addition, considering the different patterns of responses to human proendothelin [1-38] in the effector tissues studied (heart, and renal, mesenteric and hindquarters vascular beds), and the differential degrees of inhibition of them by phosphoramidon, it is likely that the effects of human proendothelin [1-38] were due to its local (rather than systemic) conversion to endothelin-1 by processes with differing degrees of susceptibility to phosphoramidon.

Endothelin-Converting Enzyme and Its in vitro and in vivo Inhibition

We investigated endothelin (ET)-converting enzyme and its localization in the vasculature. The membrane and cytosol fractions of cultured endothelial cells of bovine carotid artery contain phosphoramidon-sensitive ET-converting enzymes, and their molecular weights are about 100 and 540 kDa, respectively. The specific conversion of big ET-1 by these enzymes proceeds at pH 7.0 +/- 0.5, and it is inhibited by EDTA, o-phenanthroline, and phosphoramidon. Big ET-3 is converted by the membrane enzyme at a rate about one-tenth that of big ET-1, but it is not converted by the cytosol enzyme. Big ET-1 (but not ET-1)-induced hypertension in rats was remarkably suppressed by pretreatment with phosphoramidon, and big ET-1 (but not ET-1)-induced contraction of isolated coronary arteries, either with or without the endothelium, was substantially suppressed by phosphoramidon. These results suggest an essential role of phosphoramidon-sensitive enzyme(s) in the vascular conversion of big ET-1, and the existence of such enzymes also in nonendothelial cells. We found three converting enzymes operating at different optimal pH values in noncultured vascular smooth muscle cells; two pepstatin-sensitive, cytosolic acid proteinases and a phosphoramidon-sensitive neutral enzyme(s) in the membrane and cytosol. All of these findings strongly suggest the importance of phosphoramidon-sensitive neutral enzymes in the vascular conversion of big ET-1.

Functional evidence for the presence of a phosphoramidon-sensitive enzyme in rat brain that converts big endothelin-1 to endothelin-1

Endothelin-1 (ET-1) is produced from its precursor, big endothelin-1 (BigET-1), by a putative endothelin-converting enzyme (ECE), but it is not known whether the nzyme is present in the brain. This study was conducted to examine the central hemodynamic effects of BigET-1 and to indirectly determine the presence of an ECE in rat brain. Cardiovascular efffects of centrally administered BigET-1 and ET-1 were examined in anesthetized, ventilated rats. BigET-1 (100 pmol) or ET-1 (10 pmol) applied to the IV ventricle produced similar prolonged decreases in mean arterial pressure (MAP) and renal blood flow (RBF). Thus, peak decreases with BigET-1 were (mean ± S.E.): MAP=−35 ± 4%; RBF=−27 ± 5%, while those with ET-1 were: MAP=−36 ± 5%; RBF=−29 ± 9%. Pretreatment with phosphoramidon, a metalloprotease inhibitor (90 nmol), abolished the hemodynamic responses elicited by BigET-1 (MAP=−9 ± 2%; RBF=−3 ±2%) but not those produced by ET-1. These data indicate that: i) conversion of BigET-1 to ET-1 in the brain is essential for the expression of hemodynamic actions and ii) a metalloprotease capable of converting BigET-1 to ET-1 is present in rat brain.

Neuropeptide-degrading endopeptidase activity of locust (Schistocerca gregaria) synaptic membranes.

Locust adipokinetic hormone (AKH, pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2) was used as the substrate to measure neuropeptide-degrading endopeptidase activity in neutral membranes from ganglia of the locust Schistocerca gregaria. Initial hydrolysis of AKH at neural pH by peptidases of washed neural membranes generated pGlu-Leu-Asn and Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2 as primary metabolites, demonstrating that degradation was initiated by cleavage of the Asn-Phe bond. Amastatin protected the C-terminal fragment from further metabolism by aminopeptidase activity without inhibiting AKH degradation. The same fragments were generated on incubation of AKH with purified pig kidney endopeptidase 24.11, and enzyme known to cleave peptide bonds that involve the amino group of hydrophobic amino acids. Phosphoramidon (10 microM), a selective inhibitor of mammalian endopeptidase 24.11, partially inhibited the endopeptidase activity of locust neural membranes. This phosphoramidon-sensitive activity was shown to enriched in a synaptic membrane preparation with around 80% of the activity being inhibited by 10 microM-phosphoramidon (IC50 = 0.2 microM). The synaptic endopeptidase was also inhibited by 1 mM-EDTA, 1 mM-1,10-phenanthroline and 1 microM-thiorphan, and the activity was maximal between pH 7.3 and 8.0. Localization of the phosphoramidon-sensitive enzyme in synaptic membranes is consistent with a physiological role for this endopeptidase in the metabolism of insect peptides at the synapse.