phosphoramidon and Hypertension

Topics: Animals; Aspartic Acid Endopeptidases; Benzazepines; Benzofurans; Drug Design; Endothelin-Converting Enzymes; Glycopeptides; Heart Failure; Humans; Hypertension; Metalloendopeptidases; Mice; Mice, Knockout; Myocardial Infarction; Organophosphonates; Phenylalanine; Tetracyclines; Tetrazoles

Angiotensin-converting enzyme (kininase II [ACE]) inhibitors are capable of potentiating bradykinin (BK) effects by enhancing the actions of bradykinin on B(2) receptors independent of blocking its inactivation. To investigate further the importance of ACE kininase activity on BK-induced vasodilation, we investigated the effect of inhibiting ACE, as well as other kininases, on both BK metabolism and vasodilator effect in preparations that exhibit increased ACE activity. Mesenteric arterial beds obtained from 1-kidney, 1-clip hypertensive rats presented augmented ACE and angiotensin I converting activities compared with normotensive rats. The isolated and perfused mesenteric beds were exposed to BK for 15 minutes in the absence or in the presence of kininase inhibitors; then, the perfusate was collected for analysis of the products of BK metabolism by high-performance liquid chromatography. BK was metabolized to the fragments BK(1-8), BK(1-7), and BK(1-5), and the recovery of intact BK was reduced by 47% in the hypertensive group. Recovery of BK was increased in both groups in the presence of a kininase I inhibitor and in the hypertensive group by neutral endopeptidase 24.11 inhibitor; however, ACE inhibition did not affect BK metabolism in both groups. In contrast, only the ACE inhibitor potentiated the vasodilator effect of BK in a mesenteric bed preconstricted with phenylephrine; the increase in BK effect, nevertheless, was not greater in arteries from hypertensive rats that presented an increased ACE activity when compared with those in the normotensive group. These data demonstrated that ACE inhibitor-induced potentiation of BK vasodilator effects is not related to their actions on BK degradation.

Topics: 3-Mercaptopropionic Acid; Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Bradykinin; Drug Synergism; Enzyme Inhibitors; Glycopeptides; Hypertension; In Vitro Techniques; Lysine Carboxypeptidase; Male; Mesenteric Arteries; Metalloendopeptidases; Neprilysin; Peptide Fragments; Peptidyl-Dipeptidase A; Protease Inhibitors; Rats; Rats, Wistar; Vasodilation; Vasodilator Agents

The purpose of this study was to determine whether tissue neutral endopeptidase (NEP) 24.11 activity, a membrane-bound metalloenzyme widely distributed in the peripheral circulation that cleaves and inactivates vasodilator peptides, is increased in spontaneously hypertensive hamsters relative to genetically/age-matched normotensive hamsters. Mean arterial pressure and heart rate were 163 +/- 11 mm Hg and 312 +/- 7 beats/min in spontaneously hypertensive hamsters and 99 +/- 3 mm Hg and 302 +/- 10 beats/min in normotensive hamsters, respectively (mean +/- SEM). NEP 24.11 activity is significantly increased in the kidney, cheek pouch, and spinotrapezius muscle, and significantly decreased in the heart and aorta of spontaneously hypertensive hamsters relative to controls (P < .05). Lung and brain NEP 24.11 activity is similar in both groups. Renal NEP 24.11 activity increases and to a similar extent in spontaneously hypertensive and normotensive hamsters as chloride anion concentration in the assay buffer is increased. Substituting citrate for chloride anion significantly attenuates renal NEP 24.11 activity. Taken together, these data indicate that NEP 24.11 activity in spontaneously hypertensive hamsters is increased in two organs that contribute appreciably to peripheral vascular resistance, skeletal muscle, and kidney. We suggest that the spontaneously hypertensive hamster is a suitable model to study the role of skeletal muscle and renal NEP 24.11 in regulating vasomotor tone in essential hypertension.

Topics: Animals; Anions; Chlorides; Cricetinae; Glycopeptides; Hypertension; Kidney; Neprilysin; Osmolar Concentration; Protease Inhibitors; Thiorphan

Experiments on isolated strips of rat v. portae revealed that endothelin participates in the development of myogenic reactions of vascular smooth muscles. Endothelin is known to be released by endotheliocytes and to stimulate the contraction of vascular smooth muscle cells. Rats with inherited arterial hypertension showed significant decrease of the stimulating effect of endothelin-1 upon the contraction of vascular smooth muscles. At the same time, the effect of phosphoramidon, an inhibitor of endothelin-converting enzyme, upon the length-tension curve of the strips of v. portae is more marked in hypertensive vs. control rats. The data obtained testify to the fact that the increase of vascular tone in the hypertensive rats is most likely to be conditioned by the involvement of endothelin.

Topics: Acetylcholine; Animals; Aspartic Acid Endopeptidases; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Hypertension; In Vitro Techniques; Metalloendopeptidases; Muscle Contraction; Muscle, Smooth, Vascular; Portal Vein; Protease Inhibitors; Rats; Rats, Inbred SHR

We reported previously that the endothelin converting enzyme (ECE) inhibitor phosphoramidon lowers mean arterial pressure (MAP) when infused in conscious, spontaneously hypertensive rats (SHRs). In this study we determined the dose-response relationship for this action in SHRs and in a high-renin hypertensive model, the renal artery-ligated rat. We also determined whether the ETA receptor antagonist BQ-123 (cyclo [D-Trp-D-Asp-Pro-D-Val-Leu]) might lower MAP in hypertensive rats. Phosphoramidon lowered MAP by 9 +/- 4, 31 +/- 4, and 40 +/- 4 mm Hg after 5 h when infused in SHRs at 10, 20, and 40 mg/kg/h. This lowering of MAP was associated with dose-related inhibition of the pressor response to a bolus intravenous injection of big ET (1-39) at 1 nmol/kg. BQ-123 also lowered MAP in SHRs (by 25 +/- 3 mm Hg), but only at a very high dose (50 mg/kg/h for 5 h). At this dose, BQ-123 blocked the pressor response to a bolus intravenous injection of ET-1 (1 nmol/kg), but the blockade was incomplete. Phosphoramidon infused in conscious, renal hypertensive rats lowered MAP by 31 +/- 9, 46 +/- 8, and 54 +/- 1 mm Hg after 5 h at 10, 20, and 40 mg/kg/h, respectively. This lowering of MAP was associated with blockade of the pressor response to big ET (1-39). BQ-123 did not lower MAP in renal hypertensive rats when infused at 30 mg/kg/h for 5 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Angiotensin I; Animals; Aspartic Acid Endopeptidases; Blood Pressure; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Hypertension; Hypertension, Renal; Male; Metalloendopeptidases; Molecular Sequence Data; Neprilysin; Peptides, Cyclic; Protein Precursors; Rats; Rats, Inbred SHR; Rats, Sprague-Dawley

Aspergillomarasmine-A and -B (AM-A and -B), which were isolated from the cultured broth of an unidentified fungus N877, showed apparent inhibition against endothelin-converting enzyme (ECE) from bovine endothelial cells as measured by the formation of endothelin-1 (ET-1) converted from big endothelin-1 (bET-1), with IC50 values of 3.4 and 2.5 microM for AM-A and -B, respectively. EDTA also inhibited ECE (IC50 = 1.1 microM), but the inhibitions by AM-A, AM-B and EDTA were each abolished by the addition of 10 microM Zn2+ to the reaction mixture. In mice, AM-A and -B dose-dependently (10-50 mg/kg, i.v.) caused significant prolongation of the latency to sudden death induced by i.v. bET-1 (25 nmol/kg), but not that by ET-1 (5 nmol/kg), accompanied by a decrease in plasma immunoreactive ET-1 formation, while EDTA (24 mg/kg) failed to do so. In mice, the LD50 value of AM-A was calculated to be 159.8 mg/kg, i.v., which was much larger than that of EDTA (28.5 mg/kg, i.v.), indicating the low toxicity of AM-A. AM-A (30 mg/kg, i.v.) also suppressed bET-1-induced hemoconcentration and hypertension in mice and rats, respectively. These findings suggest that although ECE inhibition by AM-A was mainly attributable to its chelating activity, it showed apparent in vivo activities due to ECE inhibition with low toxicity.

Topics: Animals; Aorta; Aspartic Acid; Aspartic Acid Endopeptidases; Cattle; Edetic Acid; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Glycopeptides; Hypertension; In Vitro Techniques; Lethal Dose 50; Male; Metalloendopeptidases; Mice; Mice, Inbred ICR; Rats; Rats, Sprague-Dawley

Phosphoramidon inhibits both endothelin converting enzyme (ECE) and neutral metalloendopeptidase (NEP). The contribution of ECE and NEP inhibition to the antihypertensive effects of phosphoramidon was investigated in SHR and DOCA-salt hypertensive rats. SCH 32615, an active acid of the potent and selective NEP inhibitor prodrug SCH 34826 was used as a reference compound. Intravenous infusion of SCH 32615 (1.0 mg/kg/min x 2 hr) or phosphoramidon (0.3 and 1.0 mg/kg/min x 2 hr) did not reduce blood pressure (BP) in conscious SHR. The combination of SCH 32615 (100 mg/kg + 1.0 mg/kg/min) and phosphoramidon (0.3 mg/kg/min) also did not alter BP in SHR. In comparison, the BP of conscious DOCA-salt rats was significantly reduced by phosphoramidon (0.01, 0.1 and 1.0 mg/kg/min x 2 hr) (-28 +/- 6, -51 +/- 5 and -85 +/- 6 mmHg, respectively). SCH 32615 (100 mg/kg, i.v.) over 5 min followed by a sustained infusion of 1.0 mg/kg/min for 2 hr also reduced BP by 49 +/- 7 mmHg (P < .05) in DOCA-salt rats. However, phosphoramidon (0.1 mg/kg/min x 2 hr) failed to cause a further reduction in BP in DOCA-salt rats concurrently receiving SCH 32615. In contrast, a higher dose of phosphoramidon (0.3 mg/kg/min) in combination with SCH 32615 caused a greater reduction in BP in DOCA-salt rats than SCH 32615 alone. In anesthetized normotensive rats, phosphoramidon (0.01-1.0 mg/kg/min x 30 min) dose-dependently inhibited the BP responses to big endothelin-1 (BET-1) without blocking the pressor responses to ET-1. SCH 32615 failed to attenuate the pressor responses to either BET-1 or ET-1. The results indicate that SCH 32615 lacks in vivo ECE inhibitory activity. It is concluded that the antihypertensive action of SCH 32615 and low doses of phosphoramidon can be attributed to the inhibition of NEP which may presumably cause an accumulation of ANF. In comparison, at higher doses phosphoramidon causes a further reduction of BP in DOCA-salt hypertensive rats by inhibition of endothelin bioconversion.

Topics: Animals; Antihypertensive Agents; Aspartic Acid Endopeptidases; Blood Pressure; Consciousness; Desoxycorticosterone; Dipeptides; Drug Therapy, Combination; Endothelin-Converting Enzymes; Glycopeptides; Hypertension; Male; Metalloendopeptidases; Neprilysin; Rats; Rats, Inbred SHR

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Edema; Glycopeptides; Humans; Hypertension; Natriuresis; Neprilysin; Substrate Specificity; Tissue Distribution

Neutral endopeptidase 3.4.24.11 (NEP) has been identified as the major atrial natriuretic factor (ANF) degrading enzyme in rat kidney, therefore, suggesting a possible role for this enzyme in blood volume and pressure regulation. Various experimentally induced and genetically hypertensive rat models have been used to test NEP inhibitors. The presence of different isoforms of NEP in the various hypertensive rat models would have relevance when searching for novel NEP inhibitors. Therefore, we compared the properties of NEP in kidney cortex homogenates in order to test for possible differences in the following hypertensive rat models and their appropriate controls: spontaneously hypertensive rats (SHR), Wistar Kyoto strain (WKY), DOCA-salt hypertensive rats, and Sprague Dawley control rats (SD). No relevant differences were found when comparing the following parameters: (1) specific activity (mean: 204 U/mg protein), (2) Michaelis constant (mean: 280 microM), (3) IC50 of thiorphan (mean: 6.5 nM) and phosphoramidon (mean: 54 nM), (4) pH profiles (optimum at pH 8.0), (5) heat inactivation profiles (half-life 20 min at 65 degrees C), (6) immunotitration of kidney cortex homogenates, (7) molecular weight as determined by gel filtration (92,000 Dalton) and (8) affinity chromatography with concanavalin A. Without evidence for the presence of different NEP isoforms, it is unlikely that divergent findings in DOCA-salt rats and SHR using a given NEP inhibitor are due to isoforms of NEP.

Topics: Animals; Body Weight; Desoxycorticosterone; DNA; Glycopeptides; Hot Temperature; Hydrogen-Ion Concentration; Hypertension; Kidney Cortex; Neprilysin; Organ Size; Protein Denaturation; Proteins; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Sprague-Dawley; Salts; Thiorphan

In porcine aortic endothelial cells, the 21-amino acid peptide endothelin-1 (ET-1) is formed from a 39-amino acid intermediate big endothelin (big ET) by a putative endothelin-converting enzyme (ECE) that cleaves the 39-mer at the Trp21-Val22 bond. Because big ET has less than 1% of the contractile activity of ET-1, inhibition of ECE should effectively block the biological effects of big ET. Big ET injected intravenously into anesthetized rats produces a sustained pressor response that presumably is due to conversion of big ET to ET-1 by ECE. We determined the type of protease activity responsible for this conversion by evaluating the effectiveness of protease inhibitors in blocking the pressor response to big ET in ganglion-blocked anesthetized rats. The serine protease inhibitor leupeptin, the cysteinyl protease inhibitor E-64, and the metalloprotease inhibitors captopril and kelatorphan were ineffective at blocking the pressor response to big ET. However, the metalloprotease inhibitors phosphoramidon and thiorphan both dose-dependently inhibited the pressor response to big ET, although phosphoramidon was substantially more potent than thiorphan. None of the inhibitors blocked the pressor response to ET-1 and none had any effect on blood pressure when administered alone as an i.v. bolus to the ganglion-blocked anesthetized rat. However, phosphoramidon infused intravenously at 20 mg/kg/h for 4 h lowered the mean arterial pressure (MAP) in conscious spontaneously hypertensive rats (SHRs) whereas kelatorphan at the same dose did not. Our results suggest that ECE is a novel metalloprotease and that ECE inhibitors could have therapeutic potential for the treatment of hypertension.

Topics: Animals; Aspartic Acid Endopeptidases; Blood Pressure; Dose-Response Relationship, Drug; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Glycopeptides; Hypertension; Male; Metalloendopeptidases; Protease Inhibitors; Protein Precursors; Rats; Rats, Inbred SHR; Rats, Inbred Strains; Rats, Inbred WKY