Search

Endothelin-1 1-31 (ET-1 1-31), a novel member of the endothelin family comprising 31 amino acids and derived from the selective hydrolysis of big ET-1 by chymase, directly activates endothelin receptors or converts to ET-1 1-21 by ET converting enzyme (ECE). The cardiovascular effects of central ET-1 1-31 are not identified. The present study was designed to investigate the cardiovascular actions of ET-1 1-31 within the rostral ventrolateral medulla (RVLM) in anesthetized rats. Bilateral injection of ET-1 1-31 (0.5, 1, and 2 pmol for each side) into the rostral ventrolateral medulla produced an initial pressor and/or a long-lasting hypotensive action but did not affect HR. Unilateral microinjection of 2 and 4 pmol of ET-1 1-31 into the rostral ventrolateral medulla only produced a significant (P < 0.05) transient increase in blood pressure by an average of 13 and 12 mm Hg, respectively, whereas unilateral microinjection of 8 pmol of ET-1 1-31 produced a sustained fall in blood pressure (from 92 +/- 6 to 69 +/- 8 mm Hg, P < 0.05). The transient pressor effect of unilaterally injecting ET-1 1-31 (4 pmol) into the rostral ventrolateral medulla was completely abolished by pretreatment with either ETA receptor antagonist BQ123 (83 +/- 2 versus 84 +/- 5 mm Hg, P > 0.05) or ET converting enzyme inhibitor phosphoramidon (99 +/- 5 versus 99 +/- 7 mm Hg, P > 0.05) but not ETB receptor antagonist IRL1038 (89 +/- 6 versus 96 +/- 7 mm Hg, P < 0.05). In addition, prior injection of phosphoramidon also completely abolished the long-lasting hypotension of intra-RVLM ET-1 1-31 (8 pmol) but did not modify the depressor action of intra-RVLM ET-1 1-21 (from 100 +/- 6 to 76 +/- 8 mm Hg, P < 0.05). In conclusion, the current results suggest that the cardiovascular effects of intra-RVLM ET-1 1-31 might be the result of conversion of ET-1 1-31 to ET-1 1-21 through activation of ETA receptors.

Endothelin-1 (ET-1) is generated from big ET-1 by endothelin converting enzyme-1 (ECE-1). This process is inhibited by phosphoramidon through binding to the catalytic domain of ECE-1. There are four isoforms of human ECE-1 (ECE-1a, ECE-1b, ECE-1c and ECE-1d) which possess a conserved catalytic domain. Interestingly, a recent study has shown that in ECE-1b-transfected CHO cells phosphoramidon increases the expression and activity of ECE-1b. It is not known, however, whether phosphoramidon has similar effects on the expression of other ECE-1 isoforms. To address this point, we have established recombinant CHO cell lines that permanently express either human ECE-1a, ECE-1b or ECE-1c. Incubation of CHO/ECE-1a, -1b, and -1c with phosphoramidon (100 microM) for 16 hours markedly elevated the intracellular expression of ECE-1a and ECE-1b, but not ECE-1c protein, as indicated by Western blotting and immunocytochemistry. These increases appear to be due to inhibition of intracellular degradation of the protein because metabolic labeling followed by immunoprecipitation showed ECE-1a and ECE-1b proteins had prolonged half-lives in the phosphoramidon-treated cells. This is further supported by the finding that ECE-1 mRNA levels were unchanged following phosphoramidon treatment. Taken together, our results demonstrate that phosphoramidon differentially affects the expression of three human ECE-1 isoforms.

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.

Bradykinin is a substrate for both neutral endopeptidase 24.11 (NEP) and angiotensin-converting enzyme (ACE). Our previous studies showed that ACE inhibitors can stimulate nitric oxide production in coronary microvessels, which is mediated by local kinins. Whether inhibition of NEP also can affect local vascular NO production has not been established. To determine the role of NEP in the control of NO production, coronary microvessels were isolated from seven mongrel dogs. Two NEP inhibitors, phosphoramidon and thiorphan, and an ACE inhibitor, ramiprilat, were used. Nitrite, the metabolite of NO in aqueous solution, was measured by using the Griess reaction. Phosphoramidon and thiorphan (10(-6) M) increased nitrite production from 80 +/- 6 to 136 +/- 6 and 144 +/- 7 pmol/mg, respectively. Ramiprilat (10(-8) M) increased nitrite production from 78 +/- 6 to 155 +/- 7 pmol/mg wet weight. The effect of these agents on nitrite release was blocked by L-NAME, which inhibits NO synthase, HOE-140, which blocks bradykinin B2-receptor, and dichloroisocoumarin, which blocks kinin-forming enzymes. These results clearly indicate that inhibition of kinin metabolism by using neutral endopeptidase inhibitors increases NO production from coronary microvessels. Thus neutral endopeptidase plays an important role in local kinin-modulated NO production in the coronary microcirculation and NEP inhibitors may be useful clinical tools in treatment of cardiovascular disease.

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.

We previously described delayed pressor response (DPR) 3 h after endothelin (ET)-1 injection in normotensive rats. In the current study, we examined effects of the ETA receptor antagonist BQ123 (0.01 mumol/kg/min intravenously, i.v.), phosphoramidon (100 mumol/kg i.v.), the neutral endopeptidase inhibitor SQ28603 (112 mumol/kg + 0.04 mumol/kg/min i.v.), the angiotensin-converting enzyme inhibitor enalaprilat (10 mumol/kg i.v.), and the thromboxane receptor antagonist, SQ29548 (0.5 mumol/kg + 0.5 mumol/kg/h i.v.) on DPR. Vehicle and ET-1 (1.0 nmol/kg i.v.) were administered on day 1; vehicle or drug and ET-1 were administered on day 2. BQ123 inhibited DPR 36% (vehicle 44 +/- 5, BQ123 28 +/- 3 mm Hg); phosphoramidon inhibited DPR 56% (vehicle 45 +/- 4, and phosphoramidon 20 +/- 5 mm Hg). DPR was unchanged after SQ28603 (vehicle 39 +/- 2 and SQ28603 44 +/- 2 mm Hg), enalaprilat (vehicle 39 +/- 2 and enalaprilat 38 +/- 7 mm Hg), or SQ29548 (vehicle 46 +/- 6 and SQ29548 43 +/- 3 mm Hg). The results suggest that DPR 3 h after ET-1 injection in rats is mediated in part through ETA receptors. DPR does not appear to involve thromboxane or synthesis of angiotensin II (AII), but may be related to synthesis of ET-1.

We studied the pharmacologic effects of big endothelin-1 (big ET-1), big endothelin-2 (big ET-2), and big endothelin-3 (big ET-3), and characterized the enzyme involved in the conversion of the three peptides in guinea pig upper bronchus (GPUB). ET-1, ET-2 and ET-3 (0.1-300 nM) elicited similar concentration-dependent contractions of GPUB. Big ET-1 and big ET-2, but not big ET-3, also elicited potent concentration-dependent contractions of GPUB. The conversion of big ET-1 to ET-1 in the GPUB is sensitive to phosphoramidon but not to thiorphan or captopril. Phosphoramidon inhibited the conversion of big ET-2 to ET-2, thiorphan had minimal effect, and captopril was without effect. These results suggest that the putative endothelin-converting enzyme (ECE) is involved in the conversion of big ET-1 to ET-1 in GPUB and the conversion of big ET-2 to ET-2 by a nonselective enzymatic process.

We developed a procedure for individual measurement of big endothelin-1 (big ET-1) and the two products of big ET-1 conversion (ET-1 and C-terminal fragment (CTF) of big ET-1 (big ET-1(22-38)), using selective solid-phase extraction and specific radioimmunoassays. These techniques were used to measure the levels of these peptides in extracts of human plasma after brachial artery infusions of big ET-1. Infusion of big ET-1 (50 pmol/min, n = 6) caused a decrease in forearm blood flow from 3.03 to 1.55 ml/dl/min after 60 min (p < 0.05). In agreement, the levels of plasma immunoreactive (IR) big ET-1, ET, and CTF were significantly increased from basal levels in the infused arm (from undetectable to 386 pM, 2.2-7.5 pM, and 0.17-37 pM, respectively; p < 0.05). In the presence of the metalloprotease inhibitor phosphoramidon (30 nmol/min), the increase in IR-ET and the associated vasoconstriction were abolished. However, IR-CTF was still detected, suggesting that either some conversion by phosphoramidon-insensitive endothelin-converting enzyme (ECE) was occurring and/or that CTF was being preserved from further proteolysis by phosphoramidon. These data confirm that exogenous big ET-1 is locally converted to ET-1 and CTF in the human forearm, at least in part by a phosphoramidon-sensitive ECE. Furthermore, because measurable levels of newly synthesized ET-1 are probably rapidly reduced as a result of receptor binding, the assay of IR-CTF may be a more sensitive measure of the overexpression of ET-1 in disease.

The structure-activity relationships of phosphoramidon analogues for inhibition of endothelin-converting enzyme (ECE), neutral endopeptidase 24.11 (NEP), and angiotensin-converting enzyme (ACE) were compared. Phosphoramidon inhibited ECE, NEP, and ACE activities with IC50 values of 3.5, 0.034, and 78 microM, respectively. Removal of the rhamnose moiety of phosphoramidon (dipeptide 3) reduced the potency for ECE (IC50 = 70 microM), whereas the potencies for NEP (0.003 microM) and ACE (0.20 microM) were increased. Addition of 2-(2-naphthyl)ethyl to dipeptide 3 improved the potency for ECE (0.55 microM) but weakened the potency for NEP (0.02 microM), and had no significant change for ACE. Interchange between Leu and Trp abolished the inhibitory activities for ECE and NEP, but the compound remained active for ACE. These results suggest that a hydrophobic group in the P1 position of phosphoramidon analogues increases the potency for ECE significantly, whereas compounds containing a free phosphonic acid are optimal for inhibition of NEP and ACE. Furthermore, an aromatic group in the P'2 position is essential for the inhibition of ECE and NEP, but not ACE.

To elucidate the role of endothelin-1 (ET-1) as an autocrine growth factor for vascular endothelial cells (ECs), we studied the effects of phosphoramidon, an inhibitor of ET-1-converting enzyme, on the production of ET-1-like immunoreactivity (LI), [125I]ET-1 binding activity, ETB receptor mRNA expression, phosphoinositide breakdown, and DNA synthesis in cultured bovine carotid artery ECs. Phosphoramidon dose-dependently decreased ET-1-LI production but reciprocally increased [125I]ET-1 binding capacity. ET-1 and ET-3 equipotently inhibited the binding of [125I]ET-1 only in the presence of phosphoramidon. Northern blot analysis revealed that ETB receptor mRNA expression was more evident in phosphoramidon-treated cells than in nontreated cells. In the presence of phosphoramidon, ET-1 and ET-3 equipotently stimulated inositol 1,4,5-trisphosphate formation and [3H]-thymidine incorporation into cultured ECs. Both phosphoramidon and anti-ET-1 antibody inhibited basal [3H]thymidine incorporation. These data suggest that endogenous ET-1 constitutively secreted by ECs is an autocrine growth factor via ETB receptors.

The role of cardiac endothelin-1 (ET-1) was studied by determining endogenous tissue and coronary ET-1 levels in isolated rat hearts. Hearts were perfused in an upside-down position with a colloid-free buffer and immunoreactive ET-1 was determined in timed collections of coronary effluent (E) and interstitial fluid (transudate, T) produced by the ventricles and appearing on their surface. Basal ET-1 concentrations were 0.2 +/- 0.01 pg/ml (T) and 0.03 +/- 0.002 pg/ml (E), i.e., the T:E concentration ratio was 7. Angiotensin II (0.1 mumol/L) or thrombin (5 U/ml) increased coronary perfusion pressure and ET-1 secretion but had no effect on the T:E ET-1 concentration ratio (5 and 9). In two different protocols of ischemia/reperfusion, T and E concentrations increased up to two- and fivefold, respectively. The T:E ratios were approximately 2, and the highest concentrations in either fluid were < 1 pg/ml. No change in coronary perfusion pressure was observed. In the presence of the ET-1-converting enzyme inhibitor phosphoramidon (1.7 mumol/L), ischemia-induced increases of ET-1 concentrations were attenuated in parallel with a time-dependent rise in coronary perfusion pressure. Therefore, under normoxic conditions and in ischemia/reperfusion, ET-1 is an endogenous vasodilator in the rat heart.

The release of immunoreactive endothelin (ir-ET) by cultured Clara cells isolated from guinea pig lung was investigated. Clara cells were cultured in DMEM-F12 with 10% fetal bovine serum for 4-5 days. The basal release of ir-ET, measured by radioimmunoassay, from cultured guinea pig Clara cells incubated for 2, 6, and 10 h was 59.6 +/- 8.6, 206.0 +/- 25.0, and 291.0 +/- 16.5 pg/ml, respectively. Treatment with phosphoramidon (1 mM) for 45 min decreased the release of ir-ET by 73% and 76% in 6- and 10-h incubation periods, respectively. In addition, treatment with dexamethasone (1 microM) for 24 h significantly attenuated the release of ir-ET by 62% and 71% during 6- and 10-h incubation periods, respectively. Our results show that Clara cells isolated from guinea pig can synthesize significant amounts of ir-ET and that a phosphoramidon-sensitive metalloprotease appears to be responsible for the generation of endothelin. This basal release of ir-ET was significantly attenuated by dexamethasone.

The physiologic significance of endothelin-1 (ET-1) generation in human resistance vessels is unknown. We therefore investigated whether endothelin-converting enzyme (ECE) activity could be demonstrated in human vessels, and the effects of inhibition of the generation or actions of ET-1 on vascular tone in healthy men. Brachial artery infusion of local doses of big ET-1 caused a slow-onset, dose-dependent forearm vasoconstriction that was abolished by co-infusion of the ECE inhibitor phosphoramidon. Phosphoramidon did not affect responses to ET-1. Phosphoramidon caused slow-onset vasodilatation when infused alone, with blood flow increasing by 37% (p = 0.03). Vasoconstriction to ET-1 was completely abolished by co-infusion of the ETA receptor antagonist BQ-123 (p = 0.006), with forearm blood flow tending to increase. Infusion of BQ-123 alone resulted in progressive vasodilatation, with blood flow increasing by 64% (p = 0.007). These results suggest that endogenous generation of ET-1 contributes to the maintenance of vascular tone in states of normal and elevated blood pressure. ECE inhibitors and ETA receptor antagonists may have potential as vasodilators in the treatment of diseases associated with vasoconstriction, such as hypertension and chronic heart failure.

Experiments were designed to investigate the reactivity of vascular smooth muscle to endothelins (ETs) in veins taken from human placentas immediately after delivery. The placental veins were cut into rings and suspended between two stirrups in conventional organ chambers (filled with aerated, modified Krebs-Ringer bicarbonate solution) for isometric recording of tension. ET-1 and ET-3 caused concentration-dependent contractions of the isolated human placental veins. The responses induced by ET-1 were greater than those evoked by ET-3 and were not significantly affected by BQ-123, a selective inhibitor of ETA receptors. Contractions to big ET-1 were obtained in rings both with and without endothelium; they were inhibited by phosphoramidon, an inhibitor of endothelin-converting enzyme. These findings indicate that the conversion of the precursor of ET-1 can occur in human placental veins. The receptors mediating the contraction of human placental veins to endothelins do not belong to the ETA subtype; the response to the peptides is probably mediated in part by an uncharacterized ET-receptor subtype and in part by ETB receptors. The output of big ET-1 in the vascular wall or from surrounding tissues in the placenta could be involved in the regulation of venous tone in this organ.

Endothelin (ET) is generated from prepro-ET initially by dibasic pair proteolysis, followed by a specific proteolytic cleavage between Trp21 and Val22. Currently, intense research is focused on the investigation of a metalloprotease that is inhibited by phosphoramidon (PHOS) only, but not by other protease inhibitors. In this report, we show that ET binding was increased significantly in cultured Swiss 3T3 fibroblasts with PHOS pretreatment. The effect of PHOS was dose- and time-dependent. Other protease inhibitors, such as thiorphan, pepstatin-A, E-64, phenylmethyl sulfonyl fluoride, and aprotinin, failed to exert a similar effect. Control experiments indicated that the effect of PHOS was not due to inhibition of 125I-ET-1 degradation. Binding studies using whole cells or membranes prepared from cell show that ET binding sites increased from 23,000 to 133,000 sites/cell in control versus PHOS-treated cells. The effect of PHOS treatment on the ET receptor may be due to the inhibition of a protease responsible for ET-receptor processing. This effect is likely to complicate interpretation of results from studies using PHOS to block the putative ET-converting enzyme.

Endothelin-1 (ET-1) is a powerful renal vasoconstrictor peptide that could be implicated in acute renal failure. The aim of this study was to test the effects of the endothelin-converting enzyme (ECE) inhibitor phosphoramidon on pressor responses to ET-1 and its precursor, big ET-1, in isolated perfused rat kidneys and in pithed rats. In Tyrode-perfused rat kidneys, both big ET-1 (0.2-0.4 nmol) and ET-1 (0.01-0.03 nmol) evoked dose-dependent constrictions. Phosphoramidon (10 microM) selectively inhibited the pressor responses to big ET-1 without altering those to ET-1, norepinephrine, angiotensin I (AT-I), or angiotensin II (AT-II). The metalloprotease inhibitor thiorphan, but not the angiotensin-converting enzyme (ACE) inhibitor perindoprilate, also selectively inhibited the renal constrictions caused by big ET-1 but not those induced by ET-1. In vivo, both big ET-1 and ET-1 (0.5-2 nmol/kg) evoked pressor responses that were augmented by indomethacin (15 mg/kg) and L-NNA (1 mg/kg/min). Phosphoramidon selectively inhibited the pressor responses to big ET-1 (ID50: 78 micrograms/kg/min) without affecting those to ET-1, AT-I, or AT-II. These data illustrate that the pressor responses to big ET-1 in the rat, both in vivo and in vitro, are due to its conversion into ET-1 by a phosphoramidon-sensitive ECE. In the rat, phosphoramidon selectively inhibits ECE but not ACE both in vitro and in vivo.

Although many agents have been shown to stimulate release of endothelin (ET) in vitro, the factors regulating and affecting production and release of ET in intact animals remain obscure. Experiments were conducted to determine the effect of surgical preparation and infusion of plasma and/or saline on circulating immunoreactive (ir)-ET concentrations and to characterize the mechanism of endotoxin-induced increases in plasma ir-ET. Male Sprague-Dawley rats (210-325 g) anesthetized with Inactin were placed on a surgical heating table for a period of about 2 h before blood was collected via puncture of the abdominal aorta. The plasma concentration of ir-ET was 8.1 +/- 0.9 pg/ml in rats that did not undergo surgical preparation or receive any further treatment. Standard surgical preparation, as for renal clearance experiments (catheters in the trachea, femoral artery, femoral vein, and bladder), resulted in a dramatic rise in ir-ET to 17.7 +/- 3.0 pg/ml (p < 0.05). Infusion of plasma from donor rats (5 ml/kg over 20 min) resulted in an additional significant increase to 32.6 +/- 3.5 pg/ml (p < 0.05). In contrast, infusion of saline (0.9% NaCl) in a similar manner produced no further increase in ir-ET levels (23.2 +/- 3.2 pg/ml). Infusion of endotoxin in anesthetized, surgically prepared rats significantly increased ir-ET. This increase was not blocked by a platelet-activating factor antagonist (Esai 6123), despite blockade of endotoxin-induced hypotension. Phosphoramidon, which has been shown to block in vivo conversion of exogenous big ET to ET, was unable to prevent endotoxin-induced increases in ir-ET.(ABSTRACT TRUNCATED AT 250 WORDS)

From the membrane fraction of cultured human renal adenocarcinoma (ACHN) cells, two endothelin-2-converting enzymes (ECE-2A and ECE-2B) were solubilized with detergent Lubrol PX and separated by hydrophobic butyl fast-performance liquid chromatography. The pH range of the converting activity of ECE-2B for big endothelin-1 (big ET-1), big ET-2, or big ET-3, was very narrow, and the optimal pH for each substrate was significantly different; the pH optimum for big ET-1 was 6.8 and that for big ET-2 or big ET-3 was 6.4. The ET-converting activity was abolished by phosphoramidon, 1,10-phenanthroline and EDTA but was not inhibited by thiorphan, E-64, leupeptin, PCMS, p-APMSF, or pepstatin A. The conversion efficiency for big ET-2 or big ET-3 by ECE-2B was approximately one-eighth of that for big ET-1. The molecular weight of ECE-2B was estimated to be 400 kDa by gel filtration. Because these characteristics of ECE-2B are very similar to those of ET-1-converting enzyme (ECE-1) in endothelial cells, these results raise the possibility that ECE-2B is identical to ECE-1 and that a single ECE physiologically converts all big ETs to the corresponding ETs in ET-producing cells.

The pharmacology of human big endothelin-1 (big ET-1) and big ET-3 was compared in five pharmacologic models: perfused rat and guinea pig lungs, perfused rabbit kidney, and in the rat and the guinea pig in vivo (blood pressure monitoring). In these models, big ET-1 consistently induced concentration- or dose-dependent pharmacologic effects sensitive to phosphoramidon (vasopressor or prostanoid-releasing effects). In contrast, big ET-3, dissolved in either phosphate-buffered saline (pH 7.4) or 0.1% acetic acid, was inactive in all the models used in this study. In addition, the activity of big ET-3 was also assessed in the prostatic portion of the rat vas deferens. In this model, although big ET-1 induced a phosphoramidon-sensitive increase of the twitch response of the tissue to electrical stimulation, big ET-3, dissolved either in phosphate-buffered saline or acetic acid, remained inactive. Our results, presented in the above-mentioned models, illustrate the capacity of the phosphoramidon-sensitive endothelin-converting enzyme (ECE) to discriminate between human big ET-1 and big ET-3.

The activity of human big endothelin-1 (bET-1) or endothelin-1 (ET-1) was investigated in the isolated perfused kidney of the rabbit. In some experiments the effluent superfused a rabbit jugular vein (RbJV) or rat colon (RC), and bolus doses of bET-1 or ET-1 were administered either directly over the tissue (OT) or through the kidney (TK). When injected OT, the contractile responses of the RbJV to ET-1 were > or = 100-200 times more than those to bET-1. However, at least 10 times the ET-1 dose given OT was needed TK to produce an equivalent contraction of the RbJV, showing that ET-1 was being inactivated or removed by the kidney. Injections of bET-1 TK produced increases in perfusion pressure of a magnitude 1/25th of those of ET-1. In some experiments administration of bET-1 TK was associated with the release into the perfusate of an ET-1-like factor that contracted the RbJV, but not the RC (used to detect any angiotensin II release), at doses that had little effect when administered OT, suggesting that bET-1 was being activated by the kidney. Phosphoramidon (10 microM) infused TK blocked (92 +/- 1% inhibition) the renal responses to bET-1 and reduced the overflow of ET-1-like material onto the tissues without affecting ET-1-induced renal vasoconstriction. Incubation of bET-1 with rabbit renal cortical microsomes (100 micrograms protein) resulted in the generation of ET-1-like activity, as assessed by bioassay, which was inhibited by phosphoramidon (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular localization of membrane-bound endothelin-converting enzyme (ECE) was examined in rat lung by sucrose-gradient ultracentrifugation coupled with organelle marker studies. Lung microsomal fraction was prepared and fractionated by ultracentrifugation through a linear sucrose gradient. Sedimentation profiles of marker enzymes for plasma membrane, Golgi, lysosome, and mitochondria showed that these organelles were measurably separated from each other. A major portion of phosphoramidon-sensitive ECE activity was distributed with a single peak at the approximately 1.05-1.2 M sucrose region, where it appeared to be cosedimented with membrane vesicles that contained the two different marker enzymes for Golgi apparatus. These microsomal vesicles also seemed to contain the majority of endogenous immunoreactive ET-1 found in the lung. These findings suggest that a majority of the ECE activity in rat lung may be responsible for the intracellular conversion of big ET-1 during its transit through the secretory pathways.

Several studies have shown that phosphoramidon (PHOS) reduces the release of endothelin-1 (ET-1) from cultured endothelial cells. Moreover, the main endothelin-converting enzyme (ECE) activity in these cells is a membrane-bound metallopeptidase that is also inhibited by PHOS. We have investigated further the role of the PHOS-sensitive ECE in the conversion of big ET-1 to ET-1. ET-1 was measured using a radioimmunoassay specific for the C-terminal ET[16-21] sequence. The effect of PHOS on the production of ET-1 from endogenous precursors was determined using cultured bovine aortic endothelial cells (BAECs) and the human endothelial cell line (EA.hy 926). The concentrations of ET-1 accumulating in the medium over 24 h from BAECs were lowered by PHOS (-27% 10 microM, -76% 100 microM). In contrast, with EA.hy 926 cells, the same concentrations of PHOS increased by five- to sixfold the amount of ET-1 present in the medium after 24-h incubation. In other experiments, incubation of big ET-1 (1 microM) with intact BAECs or EA.hy 926 cells resulted in the generation of ET-1, and with both cell types this was inhibited by PHOS (IC50: BAECs = 6.4 microM; EA.hy 926 = 0.26 microM). These results are consistent with both cell types having a PHOS-sensitive ECE that is readily accessible to exogenous big ET-1 and is therefore probably located on the plasma membrane. Furthermore, another intracellular ECE may play a part in the endogenous intracellular formation of ET-1 in EA.hy 926 cells.

Like endothelin-1 (ET-1), its immediate human precursor big ET-1 (1-100 nM) increased the rate of spontaneous phasic contractions and caused graded tonic contractions of isolated rat uterus strips. The tonic contraction to big ET-1 (10 nM) was markedly blocked by phosphoramidon (100 microM), which did not modify the response to an equipotent concentration of ET-1 (3 nM). Responses to big-ET-1 (30 nM) were abolished in calcium-free medium, but those to ET-1 (10 nM) were only reduced by this condition. The EC50 of big ET-1 for inducing tonic contraction was only sevenfold greater than that of ET-1, and both peptides produced a maximal response similar to that evoked by KCl 80 mM. ET-3 was much less potent. The selective ETA receptor antagonist BQ-123 (40-600 nM) caused graded rightward shifts of the ET-1 curve without affecting the maximal response, yielding a Schild plot with a slope not different from unity and a pA2 value of 7.76. BQ-123 (100 nM) did not affect contractions induced by oxytocin (5 nM), acetylcholine (3 microM), or bradykinin (0.3 nM), but inhibited responses to both big ET-1 and ET-1. Therefore, the rat uterus contains a phosphoramidon-sensitive, calcium-dependent endothelin-converting enzyme that readily converts big ET-1 into ET-1, which then contracts the myometrium via activation of ETA receptors.

We studied whether endothelin (ET) isopeptides have any effects on adenylate cyclase activity in cultured bovine endothelial cells (ECs). Both ET-1 and ET-3 dose-dependently inhibited cAMP formation stimulated by forskolin and isoproterenol, although the inhibitory effect of ET-1 was less potent than that of ET-3. In contrast, ET-1 and ET-3 almost equipotently inhibited forskolin-stimulated cAMP formation in bovine EC pretreated with phosphoramidon, a putative ET-1 converting-enzyme inhibitor. These data suggest that endothelial ETB receptors are functionally coupled to adenylate cyclase, possibly via Gi protein.

To facilitate studies of the intracellular processing of endothelin-1 (ET-1) in endothelial cells we developed an enrichment procedure for the isolation of transport or secretory vesicles containing ET-1. Cultured bovine aortic endothelial cells (BAECs) were disrupted using a tight-fitting Dounce homogenizer, and subcellular fractions were isolated by sucrose-density-gradient ultracentrifugation. ET-1 immunoreactivity (ET-IR) in the fractions was measured with a specific ET[16-21] radio-immunoassay (RIA). The major peak of ET-IR was consistently localized at the 1.0/1.2 M sucrose interface (10.96 fmol/10(6) cells) in each preparation of subcellular fractions. The mean level of ET-IR at this interface, expressed as a percentage of the total, was 47.9 +/- 3.5% (n = 6). This finding provides strong evidence for the existence of a vesicle that transports ET-1 within BAECs, which may be an important site for endogenous ET-1 processing. A number of studies have reported that the final processing step in ET-1 biosynthesis by the hypothetical endothelin-converting enzyme (ECE) is inhibited by phosphoramidon (PHOS). Therefore, levels of PHOS-sensitive ECE activity in each fraction were assessed in parallel with ET-IR levels. The ECE activity associated with the peak of ET-IR (2.67 pmol ET-1/h/10(6) cells) was insensitive to 100 microM PHOS. However, the peak of ECE-like activity (10.4 pmol ET-1/h/10(6) cells) localized in the 0.8 M sucrose band was inhibited by 79% in the presence of 100 microM PHOS.(ABSTRACT TRUNCATED AT 250 WORDS)

The endothelin-converting enzyme (ECE) activity present in endothelial cells and rat and human brains was characterized using a selective and rapid bioassay for endothelin-1 (ET-1) or endothelin-3 (ET-3) together with a sensitive enzyme-linked immunosorbent assay. We found that ECE activity was predominantly in the membrane fraction of endothelial cells from which it could be extracted by treatment with detergent. In rat brain tissue, the ECE activity was in the membrane fraction and was not solubilized by detergent treatment. Further dissection of the brain revealed that there was a strong localization of ECE activity in the hypothalamus, midbrain, and medulla oblongata in agreement with earlier observations of ET-like immunoreactivity and binding sites. Experiments with human brain tissue also showed the presence of ECE activity. In conclusion, our studies confirmed the presence of ECE activity within endothelial cells, and showed ECE to be localized in brain tissue in sites consistent with the selective distribution of the ET-1 synthetic pathway. In all tissues studied, the ECE activity was significantly inhibited by phosphoramidon or ethylenediaminetetra-acetate.

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.

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.

Since the discovery of endothelin-1 (ET-1), its involvement in cerebral vasospasm after subarachnoid hemorrhage (SAH) has been suspected. We performed various experiments, first to demonstrate the presence of ET in both patients and dogs with SAH, and second to examine the effects of ET synthesis inhibition in experimental vasospasm. Here we report that ET was present in both plasma and cerebrospinal fluid (CSF) in SAH, but did not correlate with vasospasm. However, ET was locally expressed in the vascular endothelium in vasospasm. Several therapeutic approaches causing the inhibition of ET synthesis were effective in preventing the development of vasospasm. Such approaches utilized drugs that inhibited RNA and DNA synthesis. Among them, actinomycin D treatment was most effective. We also utilized phosphoramidon, a recently found conversion inhibitor of big ET to ET. However, this product failed to ameliorate the development of vasospasm. Therefore, although we cannot yet conclude that ET is the main cause of cerebral vasospasm, it may, at least, act as one of the modifying factors in cerebral vasospasm.

When big endothelin-1 (big ET-1, 1-39) was incubated with the membrane fraction obtained from cultured endothelial cells (ECs) at pH 7.0 for 6 h, the immunoreactive (ir) ET in the reaction mixture was markedly increased. Phosphoramidon, a metalloproteinase inhibitor, as well as metal chelators specifically suppressed the above increase. Using reverse-phase high-performance liquid chromatography, ir-ET was confirmed to be ET-1[1-21]. In addition, we noted that the alterations in ET-1 correlated with those in the C-terminal fragment (CTF, 22-39) of big ET-1. When cultured ECs were incubated with phosphoramidon, time-dependent secretion of ET-1 and CTF from the cells was markedly suppressed. In contrast, the secretion of big ET-1 was increased by phosphoramidon. Thiorphan, a specific inhibitor of neutral endopeptidase 24.11, was without effect on the secretion of ET-related peptides. Moreover, phosphoramidon potently inhibited the hypertensive effect of big ET-1 without affecting the ET-1-induced hypertension in anesthetized rats. From these findings, it seems reasonable to consider that phosphoramidon-sensitive and membrane-bound metalloproteinase, which is not a neutral endopeptidase 24.11, is the most plausible candidate for big ET-1-converting enzyme in vivo.

Both the soluble and membrane fractions prepared from cultured bovine endothelial cells (ECs) possessed the converting activities to metabolize big endothelin-1 (big ET-1) to endothelin-1 (ET-1) at neutral pH. Metal chelators inhibited the activities of both fractions, whereas phosphoramidon, a metalloprotease inhibitor, strongly inhibited only the activity of the membrane fraction. Phosphoramidon reduced the secretion of ET-1 and concomitantly enhanced the release of big ET-1 from cultured ECs. The incubations of big ET-1, big ET-2, and big ET-3 with cultured ECs resulted in their conversions to mature ETs. Phosphoramidon also abolished these conversions. These results indicate that vascular endothelium can convert not only endogenous big ET-1 but also exogenous big ET isopeptides to their mature ETs through a phosphoramidon-sensitive neutral metalloprotease.

Human big endothelin (big ET) and endothelin-1 (ET-1) induce similar increases in left ventricular systolic pressure in the anesthetized rabbit. Unlike ET-1, human big ET does not induce an initial transient hypotension. Human big ET (3 nmol/kg) inhibits ADP-induced platelet aggregation ex vivo by 60% whereas ET-1 at 1 nmol/kg inhibits platelet aggregation by more than 80%. The C-terminal fragment, big ET[22-38] (3 nmol/kg), has no antiaggregatory properties. Inhibition of ex vivo platelet aggregation by human big ET and ET-1 was not seen in rabbits pretreated with indomethacin (5 mg/kg). Human big ET (10(-7) M) or ET-1 (2.5 x 10(-9)-10(-8) M) induced the release of prostacyclin (PGI2) from rabbit, guinea pig, and rat lungs. Phosphoramidon (50 microM, infused 45 min prior to and during administration of peptides) inhibited the prostanoid-releasing properties of human big ET without affecting the release induced by ET-1. Intravascular administration of human big ET (1 nmol/kg) significantly increased the circulating levels of immunoreactive ET-1 (ir-ET-1) for 30 min whereas administration of ET-1 at the same concentration increased the plasma level of ir-ET-1 for 5 min only. Our results suggest that human big ET is converted to ET-1 in the rabbit in vivo. We further suggest that to induce the release of prostanoids in perfused lungs, human big ET needs to be converted to ET-1 by a phosphoramidon-sensitive endothelin-converting enzyme (ECE).

Exposure of phosphoramidon-treated (0.1 mM solution by aerosol for 15 min) guinea pigs to an aerosol of endothelin-1 (ET-1) (10 micrograms/ml) for 30 min induced after 18-24 h an enhanced bronchopulmonary response (BR) to rechallenge with ET-1 (3 micrograms/ml) administered by aerosol. Compared with saline-exposed animals, aerosol exposure of guinea pigs to ET-1 (10 micrograms/ml) for 30 min did not alter the dose-related BR to acetylcholine (ACh) administered by aerosol 18-24 h following challenge with the peptide. In phosphoramidon-treated guinea pigs, ET-1 (5 micrograms/ml) exposure for 30 min evoked a slight but non-significant enhancement of the ACh-induced BR. Administered by aerosol in phosphoramidon-treated guinea pigs, ET-1 did not induce eosinophil accumulation in the lung, as demonstrated by examination of histological preparations and the assessment of the cell composition of bronchoalveolar lavages. The present data indicate that in spite of treatment of guinea pigs with phosphoramidon, ET-1 administration does not lead to the development of bronchial hyperresponsiveness.

In pentobarbital-anesthetized rats endothelin-1 (ET-1), endothelin-2 (ET-2), endothelin-3 (ET-3), and mouse ET-2 (mET-2), in contrast to human big ET-1 (h-big ET), administered as i.v. bolus injections (0.25 nmol/kg i.v.) produced rapidly appearing and short-lasting blood pressure decreases. This effect was markedly inhibited (80-100%) after an 8-min i.v. infusion (0.1 nmol/kg/min over 10 min) of any of the ET studied, but not by h-big ET, the precursor of ET-1. Similarly, in pithed rats given a 10 min i.v. infusion of an equipressor dose (0.1 nmol/kg/min) of ET-1 or h-big ET, the hypotensive effects of ET-1 were entirely blocked only in the group of animals pretreated with ET-1. In pithed rats, ET-1 (0.25 nmol/kg i.v.) and h-big ET (0.5 nmol/kg i.v.) produced equivalent maximal pressor responses and the same pattern of increase in systemic, hindquarter, and renal vascular resistance. However, ET-1 was three times more potent than h-big ET as a vasoconstrictor of the mesenteric bed. Also the pressor response to h-big ET, but not ET-1 (0.25 nmol/kg i.v.), was markedly inhibited by the metalloprotease inhibitor phosphoramidon (5 mg/kg i.v.). These results indicate that the hypotensive effects of ET isopeptides have a common mechanism because they elicit cross tachyphylaxis, although h-big ET did not inhibit the decrease in blood pressure produced by ET-1. A possible explanation for this finding is that h-big ET has intrinsic pressor activity but does not have affinity for receptors mediating the vasodepressor effects of ET isoforms. Alternatively, h-big ET is converted into ET-1 too slowly to yield biophase concentrations of ET-1 necessary for lowering blood pressure and developing tachyphylaxis to ET-isoform-induced hypotension. Finally, if the pressor effects of h-big ET are mediated by ET-1 formation, phosphoramidon can be considered as an inhibitor of the endothelin-converting enzyme.

Several processes participate in the clearance of atrial natriuretic peptide (ANP) from the circulation, one of which is enzymatic degradation. Endoprotease EC 3.4.24.11 (NEP 24.11), present within the kidney in high concentration, has been shown in vitro to degrade ANP. Phosphoramidon and thiorphan, two potent NEP 24.11 inhibitors, have been shown to prevent the enzymatic degradation of ANP. The purpose of the present study was to determine if phosphoramidon or thiorphan would alter the in vivo time course of the pharmacologic effects of ANP. The magnitude and duration of the ANP-induced increase in urine output and sodium and cyclic GMP excretion were examined with and without either thiorphan or phosphoramidon. Six separate groups of anesthetized rats received either a low, medium, or high infusion rate of thiorphan or phosphoramidon. Renal responses to ANP were potentiated and prolonged during the low phosphoramidon infusion (3 Ki) and the medium thiorphan infusion (150 Ki). At high inhibitor infusion rates in the anesthetized rat, ANP elicited a marked depressor response. In the conscious spontaneously hypertensive rat (SHR), a 15-min intravenous (i.v.) infusion of ANP (1 microgram/kg/min) lowered mean arterial pressure (MAP 23 +/- 6 mm Hg), with an approximately 35-min duration of action. A simultaneous i.v. infusion of phosphoramidon (high dose) produced both a potentiation (33 +/- 3 mm Hg) and a prolongation (greater than 65 min to return to baseline) of the depressor response. These data lend support to the hypothesis that enzymatic breakdown of ANP may play an important role in regulating the actions of atrial natriuretic peptide.