To Endothelin-1-(1-21) in Vitro (Cardiovascular/Vasoconstriction/Vascular Endothelium/Metafloprotease/Endothelin-Converting Enzyme) ELLEN G

Home , Kelatorphan

Proc. Natl. Acad. Sci. USA Vol. 88, pp. 703-707, February 1991 Physiology/Pharmacology Phosphoramidon blocks the pressor activity of porcine big endothelin-1-(1-39) in vivo and conversion of big endothelin-1-(1-39) to endothelin-1-(1-21) in vitro (cardiovascular/vasoconstriction/vascular endothelium/metafloprotease/endothelin-converting enzyme) ELLEN G. MCMAHON*t, MARIA A. PALOMO*, WILLIAM M. MOOREt, JOHN F. MCDONALDt, AND MICHAEL K. STERNt *Searle Research and Development, GD Searle & Co. and tMonsanto Corporate Research, Monsanto Company, 800 North Lindbergh Boulevard, St. Louis, MO 63167 Communicated by Philip Needleman, October 12, 1990 (receivedfor review July 2, 1990)

ABSTRACT In porcine aortic endothelial cells, the 21- Serine-, cysteinyl-, aspartyl-, and metalloproteases have all amino acid peptide endothelin-1 (ET-1) is formed from a 39- been identified as processing enzymes responsible for the amino acid intermediate called "big endothelin-1" (big ET-1) by conversion of precursor proteins to bioactive peptides in a putative ET-converting enzyme (ECE) that cleaves the 39-mer mammalian cells (9). In their original report on the isolation at the bond between Trp-21 and Val-22. Since big ET-1 has only and characterization of ET, Yanagisawa and coworkers (1) 1/100-1/150th the contractile activity of ET-1, inhibition of speculated that ECE is a serine protease with a chymotrypsin- ECE should effectively block the biological effects of ET-1. Big like specificity. Furthermore, we demonstrated that chymo- ET-1 injected intravenously into anesthetized rats produces a trypsin can produce ET-1 from big ET-1, although cleavage at sustained pressor response that presumably is due to conversion Tyr-31 also occurred (10). Ohlstein and coworkers (11) re- of big ET-1 into ET-1 by ECE. We determined the type of ported that two chymotrypsin inhibitors [L-1-tosylamido-2- protease activity responsible for this conversion by evaluating phenylethyl chloromethyl ketone (TPCK) and isatoic anhy- the effectiveness of protease inhibitors in blocking the pressor dride] decreased the amount of ET produced by bovine aortic response to big ET-1 in ganglion-blocked anesthetized rats. The endothelial cells in culture, whereas phenylmethylsulfonyl serine protease inhibitor leupeptin, the cysteinyl protease inhib- fluoride and chymostatin did not block ET production. It is not itor E-64, and the metalloprotease inhibitors captopril and clear from these studies whether ECE is a serine protease or kelatorphan were all ineffective at blocking the pressor response whether other enzymes involved in the processing of prepro- to big ET-1. However, the metalloprotease inhibitors phos- ET are sensitive to inhibition by certain serine protease phoramidon and thiorphan dose-dependently inhibited the pres- et al. (12) that sor response to big ET-1, although phosphoramidon was sub- inhibitors. Recently, Takaoka demonstrated stantially more potent than thiorphan. None of the inhibitors pepsin, an aspartyl protease, cleaves big ET-1 specifically at blocked the pressor response to ET-1 and none had any effect on the bond between Trp-21 and Val-22, although this conversion mean arterial pressure when administered alone. In a rabbit occurred at pH 2.3. Matsumura et al. (13) have further shown lung membrane preparation, ECE activity was identified that that a pepstatin-sensitive enzyme activity in an extract from was blocked by the metalloprotease inhibitors phosphoramidon cultured porcine aortic endothelial cells converts big ET-1 to and 1,10-phenanthroline in a concentration-dependent manner. ET-1. However, the physiological relevance of this conver- This enzyme converted big ET-1 to a species of ET that comi- sion, which occurs only at acidic pH, is not known. More grated on HPLC with ET-1 and produced an ET-like contraction recently, Nichols et al. (14) suggested that ECE is a cysteinyl in isolated rat aortic rings. Our results suggest that the physi- protease, while Ohnaka and coworkers (15) reported that ECE ologically relevant ECE is a metalloprotease. is an EDTA-sensitive neutral metalloprotease. Our strategy to determine the type of protease activity In 1988, Yanagisawa and coworkers (1) reported on the responsible for the conversion ofbig ET-1 to ET-1 in vivo was isolation and characterization of a peptide, endothelin (ET), to evaluate the effectiveness of different classes of protease from the culture supernatant of porcine aortic endothelial inhibitors in blocking the pressor response to big ET-1 injected cells. Now referred to as endothelin-1 (ET-1), this peptide intravenously into ganglion-blocked anesthetized rats. In this contracts vascular smooth muscle in vitro and produces a model, big ET-1 produces a sustained pressor response which sustained pressor response in vivo. In porcine aortic endothe- presumably is due to the conversion of big ET-1 to ET-1 by lial cells, the 21-amino acid peptide ET-1 is formed from a ECE. Our in vivo results indicate that the enzyme responsible 39-amino acid intermediate called "big endothelin-1" (big for the pressor activity of big ET-1 is a metalloprotease ET-1) by a putative ET-converting enzyme (ECE) that hydro- because this activity is blocked by the metalloprotease inhib- lyzes the 39-mer at the bond between Trp-21 and Val-22 to itors phosphoramidon and thiorphan. Furthermore, we have yield ET-1-(1-21) and the C-terminal fragment, big ET-1-(22- partially purified an ECE from rabbit lung membranes. This 39) (2). Since big ET-1 has only 1/100-1/150th the contractile enzyme converts big ET-1 to a molecular species that comi- activity ofET-1 (3), inhibition ofECE should effectively block grates with ET-1 on HPLC and produces an ET-like contrac- the biological effects of ET-1. This could be an important tion in isolated vascular smooth muscle. This conversion is therapeutic strategy for the treatment of hypertension, acute also blocked by the metalloprotease inhibitors phosphorami- renal failure, myocardial infarction, and coronary and cerebral don and 1,10-phenanthroline. Our results suggest that the vasospasm-diseases in which an overproduction ofET might physiologically relevant ECE is a metalloprotease. play an important pathophysiological role (4-8). Abbreviations: ET, endothelin; ECE, ET-converting enzyme; MAP, The publication costs of this article were defrayed in part by page charge mean arterial pressure; NEP, neutral endopeptidase EC 3.4.24.11 payment. This article must therefore be hereby marked "advertisement" (enkephalinase). in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 703 Downloaded by guest on October 1, 2021 704 Physiology/Pharmacology: McMahon et al. Proc. Natl. Acad. Sci. USA 88 (1991) MATERIALS AND METHODS delivery system. A 5-,um Vydac C18 column was used to In Vivo Experiments. Male Sprague-Dawley rats (200-250 separate big ET-1 from ET-1. Elution was performed by using g) were anesthetized with Inactin (100 mg/kg of body weight a binary gradient consisting of 0.02% CF3COOH in water i.p.), and catheters (PE-50) were inserted in a femoral artery (solvent A) and 0.02% CF3COOH in acetonitrile (solvent B). and vein for measurement of mean arterial pressure (MAP) The elution profile incorporated a linear gradient from 0 to and administration of drugs, respectively. Autonomic neu- 35% solvent B in 15 min followed by isocratic elution at 35% rotransmission was blocked by treatment with mecamyl- solvent B for 15 min and a linear gradient from 35% to 63% amine (3 mg/kg i.v.) and atropine (400 pg/kg i.v.). The rats solvent B over 15 min at a flow rate of 0.5 ml/min. Eluates were allowed to recover for 45 min after implantation of the were monitored by absorbance at 215 nm. With this system, catheters. Subsequently, either big ET-1 or ET-1 at 1 nmol/ big ET-1 and ET-1 were completely resolved with retention kg with or without protease inhibitor, or protease inhibitor times of 34.3 and 36.8 min, respectively. alone, was administered i.v. Various amounts of protease HPLC assays to monitor the conversion of big ET-1 to inhibitors were administered as an i.v. bolus 10-15 sec prior ET-1 used the soluble enzyme fraction prepared as described to the administration of big ET-1 or ET-1. above but with further purification by ammonium sulfate Porcine big ET-1-(1-39) and both human and porcine fractionation. Thus, in a typical example, a 10-ml aliquot of ET-1-(1-21) were obtained from Peptides International (Lou- the soluble enzyme fraction was brought to 33% saturation in isville, KY) and were serially diluted from 100 ,uM stock ammonium sulfate, equilibrated for 30 min, and centrifuged solutions (in distilled water) with 0.05% bovine serum albu- at 11,000 x g for 20 min. The supernatant was brought to 65% min in 0.9%o saline. Leupeptin, phosphoramidon, and E-64 saturation and then centrifuged at 11,000 x g for 20 min. The were obtained from Peptides International, and captopril was resulting pellet was dissolved in 4.0 ml of50mM Tris'HC1 (pH a gift from Squibb. Racemic thiorphan {(±)-N-[1-oxo-2- 7.5) and was dialyzed against the same buffer for 12 hr. This (mercaptomethyl)-3-phenyl-propyl] glycine} was synthesized enzyme preparation had a total protein concentration of 2.4 by a modification of previously described procedures (16, mg/ml and contained ECE activity as monitored by the rat 17). Kelatorphan {(R,S)-[3-(N-hydroxy)carboxamido-2- aortic ring assay. benzylpropanoyl]-L-alanine} was synthesized by the method The conversion of big ET-1 to ET-1 was demonstrated by of Fournie-Zaluski et al. (18). using the following reaction conditions. In a final volume of In Vitro Experiments. Preparation of a soluble enzyme 1.0 ml, the assay mixture contained 3.75 ,tM big ET-1-(1-39), fraction from rabbit lung. Frozen rabbit lungs were thawed 5 uM thiorphan, 50 mM Tris HC1 (pH 7.5), and 480 ,.g of and homogenized at 40C in 4 vol of 0.25 M sucrose/5 mM soluble rabbit lung membrane protein. Identical reactions Tris HCI, pH 7.5 by treatment with an Omni-mixer homog- were run in the presence of 100 ,uM phosphoramidon. The enizer at maximum speed (twice for 45 sec), followed by reaction mixture was placed in an Ultra Wisp automatic treatment with a Polytron mixer (four times for 30 sec). The sampler maintained at 37°C. At 1-hr intervals, 100-,lI aliquots homogenate was filtered twice through two layers of cheese- were removed and immediately assayed by HPLC. cloth and centrifuged at 5000 x g for 20 min. The pellet was washed with 0.5 vol of the homogenization buffer, and the RESULTS combined supernatants were centrifuged at 15,000 x g for 20 In Vivo Experiments. Injection of big ET-1 at 1 nmol/kg min. The supernatant obtained was then centrifuged at produced a large sustained pressor response in the ganglion- 100,000 x g for 60 min. The pellet was washed with 50 mM blocked anesthetized rat (Fig. 1, top tracing). The increase in Tris HCl (pH 7.5) and centrifuged at 100,000 x g for 60 min. MAP in response to big ET-1 at 1 nmol/kg was not signifi- The washed membrane pellet was resuspended in the Tris cantly different from the response to ET-1 at 1 nmol/kg [52 buffer with 3% Tween 20 and stirred overnight. After cen- ± 4 mmHg (n = 10) vs. 57 ± 9 mmHg (n = 8)]. When the trifugation at 100,000 x g for 1 hr, the supernatant was metalloprotease inhibitor phosphoramidon was administered retained. The detergent-solubilized fraction was applied to an at 30 mg/kgjust prior to big ET-1, the pressor response to big Extracti-Gel D column equilibrated in Tris buffer to remove ET-1 was significantly blocked (Fig. 1, middle tracing). excess detergent. Protein was determined by the method of Phosphoramidon given alone at 30 mg/kg had no effect on Lowry et al. (19). MAP (Fig. 1, bottom tracing). Although the pressor response Rat aortic ring bioassay. Aliquots of the soluble enzyme to big ET-1 was completely blocked by phosphoramidon, the fraction (32 ,ug of protein) were incubated with 0.25 nmol of big ET-1 in 125 ,ul (final volume) of 50 mM Tris HCl, pH 200 7.5/10 ,uM thiorphan. Thiorphan at 5-10 ,uM inhibits degradation of ET-1 by neutral endopeptidase 24.11. In some 100 experiments, phosphoramidon or 1,10 phenanthroline (Sig- BIG ET-I ma) was included in the reaction mixture. After incubation at O(I nmol/kg) 37°C for 30 min, the incubates were added to muscle baths containing rat aortic rings for measurement of contractile c, 200 activity. This preparation has been described in detail (10). E Briefly, aortas from male Sprague-Dawley rats (300-400 g) E (00 were cleaned of adherent fat and connective tissue. The vessels were cut into 3-mm ring segments, "rubbed" to 2 0 Phosphoromidon remove endothelium, and mounted in muscle baths for mea- (30 mg/kg) FIG. 1. Representative surement of isometric force in Krebs solution. The prepara- BIG ET-I tracings showing the effect of a tions were equilibrated for 1 hr and then contracted twice 200 bolus i.v. injection ofbig ET-1 at with 50 mM KCl. When incubation mixtures of big ET-1 and 1 nmol/kg of body weight (Top), were 100 phosphoramidon at 30 mg/kg in- solubilized lung membranes with or without inhibitors jected just prior to big ET-1 ad- added to the rings for bioassay, contractile responses were 0 Phosphoromidon,t ministration at 1 nmol/kg (Mid- normalized to the second contraction obtained with 50 mM (30 mg/kg) dle), and phosphoramidon at 30 KCl (% KClmax). mg/kg administered alone (Bot- HPLC analysis ofconversion ofbig ET-J to ET-J. Reverse- 0 10 20 tom) on MAP in a ganglion- phase HPLC was performed with a Waters 600 multisolvent Time, min blocked anesthetized rat. Downloaded by guest on October 1, 2021 Physiology/Pharmacology: McMahon et A Proc. Natl. Acad. Sci. USA 88 (1991) 705

200 100 -

aC 100 U) c 80 0

0) 0n a, E ET-I E (I nmol/kg) 60 < 200 .-0P 0 40- -0~.o Phosphoromidon 2 20- ET-I d1

o 10 20 0 ' Time, min Big 10 30 100 ET-1 Thiorphanmg/kg FIG. 2. Representative tracings showing the effect of a bolus i.v. alone + big ET-1 injection of ET-1 at 1 nmol/kg (Upper) and the effects of phosphoramidon at 30 mg/kg injected just prior to ET-1 at 1 nmol/kg (Lower) FIG. 4. Effect of thiorphan at 10, 30, and 100 mg/kg on the on MAP in a ganglion-blocked anesthetized rat. pressor response to big ET-1 at 1 nmol/kg in ganglion-blocked anesthetized rats. The increase in MAP with big ET-1 at each dose pressor response to ET-1 was unchanged in the presence of ofthiorphan is expressed as a percentage of the response to big ET-1 (Fig. 2). given alone at 1 nmol/kg in a separate group of rats (n = 4 for each phosphoramidon dose of thiorphan). Inhibition of the pressor response to big ET-1 by phosphoramidon was dose-dependent with an ID50 of -5 mg/kg and this mixture was then bioassayed with rat aortic rings, a (Fig. 3). The metalloprotease inhibitor thiorphan also inhib- large contractile response was observed [102 + 4% KClmax (n ited the pressor response to big ET-1 but was substantially = 5)]. This contraction was "ET-like" in that it was slow in less potent than phosphoramidon (ID50 = 60 mg/kg) (Fig. 4). onset and very difficult to wash out. Moreover, an inactivat- Like phosphoramidon, thiorphan blocked only the pressor ing polyclonal antibody to ET-1 (Peptides International), response to big ET-1; the pressor response to ET-1 at 1 added to the rings prior to addition of the enzyme/big ET-1 nmol/kg was not inhibited by thiorphan [138 + 20% of ET-1 mixture, blocked the contractile response of this mixture response without inhibitor (n = 3)]. (data not shown). When the metalloprotease inhibitors phos- Other protease inhibitors were tested for their ability to phoramidon or 1,10-phenanthroline were included in the block the pressor response to big ET-1 in the ganglion- incubation mixture, the contractile response was inhibited in blocked anesthetized rat. The serine protease inhibitor leu- a concentration-dependent manner (Table 1). However, peptin and the cysteinyl protease inhibitor E-64 were inef- these inhibitors at high concentrations did not block the fective at blocking the pressor response to big ET-1 (Fig. 5). contractile response to ET-1. At 2 nM, ET-1 produced 109 + Likewise, the metalloprotease inhibitors captopril and kela- 9% KClmax (n = 4), whereas in the presence of 500 uM torphan did not block big ET-1 pressor activity (Fig. 5). None phosphoramidon or 500 uM 1,10-phenanthroline, the ET-1 of the inhibitors affected MAP when administered alone. responses were 107 ± 9% (n = 4) and 95 ± 6% KCImax (n = In Vitro Studies. A soluble enzyme fraction from rabbit lung 3), respectively. (32 ug of protein) produced no contractile response when Fig. 6 A-D shows the time-dependent conversion of ad- added to rat aortic rings. Likewise, 50 nM big ET-1 produced ministered porcine big ET-1 to ET-1 by partially purified only a negligible contraction in this preparation [2 ± 1% ECE from rabbit lung as monitored by HPLC. The peak at KClma, (n = 3)]. However, when solubilized rabbit lung 36.8 min (peak 2), which is assigned to ET, increases in membranes were incubated for 30 min at 37°C with big ET-1 intensity concomitantly with a decrease in the big ET-1 peak an reaction mixture run in 100 at 34.3 min (peak 1). In identical the presence of 100 tkM phosphoramidon, the production of ET-1 is inhibited after a 2-hr incubation (Fig. 6 C and E). That

(n 80- peak 2 is indeed ET-1 is supported by several lines of CL0~ evidence. When this peak was collected after an assay period U, aD of 3 hr and bioassayed with rat aortic rings, ET-like contrac- 60 tile activity was observed (data not shown). Furthermore, w .) Table 1. Effect of phosphoramidon and 1,10-phenanthroline on "0 40- 0 ECE activity in solubilized rabbit lung membranes % inhibition of < 20-1 Conc., contractile response Inhibitor AM in the RAR bioassay Phosphoramidon 1 24 ± 5 0- V~ly u vv 10 60 ± 9 ET1 Phosphoramidon, mg/kg 50 82 ± 2 alone + big ET-1 500 88 ± 4 1,10-Phenanthroline 1 11 ± 2 FIG. 3. Effect of phosphoramidon at 1, 10, and 30 mg/kg on the 10 31 ± 8 pressor response to big ET-1 at 1 nmol/kg in ganglion-blocked 50 59 ± 12 anesthetized rats. The increase in MAP with big ET-1 at each dose 500 88 ± 5 of phosphoramidon is expressed as a percentage of the response to big ET-1 given alone in a separate group of rats (n = 4 for each dose Values are means ± SEM for n = 4. Conc., concentration; RAR, of phosphoramidon). rat aortic ring. Downloaded by guest on October 1, 2021 706 Physiology/Pharmacology: McMahon et al. Proc. Natl. Acad. Sci. USA 88 (1991)

200 -

a) cn °0 150- val L big ET-1 (Inmol/kg) | 0 Kelatorphan (100 mg/kg) + big ET-1 LA FIG. 5. Effect of protease inhibitors on the 0) 100 - 1E _ Captopril (10 mglkg) + big ET-1 pressor response to big ET-1 administered at 1 0 nmol/kg. The inhibitors were given at the doses 0 E-64 (30 mg/kg) + big ET-1 indicated just prior to big ET-1 in ganglion-blocked L- 50- anesthetized rats. The increase in MAP with big Cl Leupeptin (10 mg/kg) + big ET-1 ET-1 in the presence of the inhibitors is expressed -1 as a percentage of the response to big ET-1 given alone at 1 nmol/kg in a separate group of rats (n = I 0 .. 3-6).

when the assay mixture containing phosphoramidon was big ET-1 in vivo is due to conversion ofthis precursor to ET-1 spiked with 62 pmol of ET-1, the peak at 36.8 increased in by ECE. This conversion most likely occurs in tissues rather intensity in an amount consistent with that predicted based than plasma, since we have shown that big ET-1 is not on the molar extinction coefficient calculated for ET-1 at 215 converted to ET-1 when incubated with plasma (unpublished nm (Fig. 6F). observations) and the concentrations of big ET-1 and ET-1 are approximately equimolar in circulating blood (26, 27). DISCUSSION Phosphoramidon and thiorphan were the only metallopro- Although big ET-1 has only 1/100-1/150th the contractile tease inhibitors that blocked the pressor response to big activity of ET-1 in vitro, when injected intravenously into ET-1, and neither ofthese agents blocked the pressor activity ganglion-blocked anesthetized rats, big ET-1 produces a large ofET-1. This lack ofblockade ofET-1 response confirms that pressor response. Presumably this pressor activity is due to activation of big ET-1 is the process blocked by these conversion of big ET-1 to ET-1 by ECE, although the inhibitors and not the coupling between ET-1 and blood response to big ET-1 differs somewhat from the response to pressure elevation. Phosphoramidon was originally identified ET-1 itself. In the ganglion-blocked anesthetized rat, the as a potent inhibitor of the bacterial metalloendopeptidase pressor response to ET-1 is preceded by a transient (15-20 thermolysin (28). It is known that phosphoramidon also sec) depressor response, whereas this transient fall in MAP inhibits other metalloproteases, most notably endoprotease is not present when big ET-1 is administered (Figs. 1 and 2). 3.4.24.11 [neutral endopeptidase (NEP) 24.11, or enkephali- The absence of this transient depressor response with injec- nase], the enzyme responsible for the degradation of small tion ofbig ET-1 is not well understood, primarily because the peptides such as atriopeptin, bradykinin, enkephalins, and mechanisms underlying the initial fall in MAP in response to substance P (29). The mechanism of phosphoramidon inhi- ET-1 are not known (20-23). Perhaps when big ET-1 is bition of metalloenzymes has been shown to be coordination injected intravenously into the ganglion-blocked anesthe- of the catalytic Zn atom in the active site by the phosphor- tized rat, the conversion of this precursor into ET-1 occurs amidate group (30). Accordingly, we suggest that ECE is a with a slowed time course such that the depressor response metalloprotease because only metalloprotease inhibitors to the ET-1 formed is masked. Alternatively, the pressor were able to block its activity both in vitro and in vivo. activity of big ET-1 could be the result of conversion of this Analogs ofphosphoramidon must be synthesized to elucidate precursor to some C-terminal extended form of ET-1 that is the mechanism of phosphoramidon-mediated inhibition of devoid ofdepressor activity. This seems unlikely because big ECE. However, the specific nature of this inhibition will ET-1-(1-22) has only 1/400th the activity of ET-1 in rat aorta probably remain unknown until ECE is isolated and purified. (24) and big ET-1-(1-25) is only 1/50th as potent as ET-1 in Thiorphan, originally developed as an enkephalinase in- isolated porcine coronary artery (25). Despite these uncer- hibitor, was found in this study to be a very weak ECE tainties, it is reasonable to assume that the pressor activity of inhibitor. On the other hand kelatorphan, a potent inhibitor

Big ET-1 C AlI E 1

0 35 40 45 50 3 ,0 35 40 45 50 v30 35 40 45 50 FIG. 6. Time course of production of ET-1 (peak 2) from big - ET-1 (peak 1) by partially purified B Big-Er-1 ECE from rabbit lung membranes in the absence (A-D) and presence (E and F) of 100 utM phosphoramidon. Peaks 1 and 2 were mon- 0 1 0 -I itored by HPLC at times (A), hr 2 (B), 2 hr (C, E, and F), and 3 hr J- (D). F differs from E by the addition of a spike of 62 pmol of ET-1. Arrows indicate the elution posi- 30 35 40 45 50 tions of big ET-1 and ET-1 stan- Time, min dards. Downloaded by guest on October 1, 2021 Physiology/Pharmacology: McMahon et al. Proc. Natl. Acad. Sci. USA 88 (1991) 707 of enkephalinase or NEP, did not block the pressor response 8. Masoaka, H., Suzuki, R., Hirata, Y., Emori, T., Marumo, F. to big ET-1, even when tested at 100 mg/kg. This lack of an & Hirakawa, K. (1989) Lancet ii, 1402. inhibitory effect of kelatorphan on ECE indicates that ECE 9. Harris, R. B. (1989) Arch. Biochem. Biophys. 275, 315-333. 10. McMahon, E. G., Fok, K. F., Moore, W. M., Smith, C. E., is distinct from NEP, since phosphoramidon, kelatorphan, Siegel, N. R. & Trapani, A. J. (1989) Biochem. Biophys. Res. and thiorphan are virtually equipotent as inhibitors of NEP Commun. 161, 406-413. (31, 32). Furthermore, Vijayaraghavan and coworkers (33) 11. Ohlstein, E. H., Arleth, A., Ezekiel, M., Horohonich, S., Ator, showed that purified NEP from rat kidneys inactivates en- M. A., Caltabiano, M. M. & Sung, C. (1990) Life Sci. 46, dothelin by cleavage at bonds between Ser-5 and Leu-6 and 181-188. 12. Takaoka, M., Takenobu, Y., Miyata, Y., Ikegawa, R., Matsu- between Asp-18 and Ile-19. This report has recently been mura, Y. & Morimoto, S. (1990) Biochem. Biophys. Res. confirmed by Sokolovsky et al. (34) using NEP from bovine Commun. 166, 436-442. kidneys. However, it is not known whether NEP-mediated 13. Matsumura, Y., Ikegawa, R., Takaoka, M. & Morimoto, S. degradation of ET has any important physiological role. In (1990) Biochem. Biophys. Res. Commun. 167, 203-210. our study, in vivo responses to ET-1 were not consistently 14. Nichols, J., Wiseman, J., Hassman, F., Rimele, T., Queen, K. potentiated in the presence ofNEP inhibitors, suggesting that & Berman, J. (1990) FASEB J. 4, A959 (abstr.). other mechanisms of inactivation may be more relevant. 15. Ohnaka, K., Takayanagi, R., Yamauchi, T., Okazaki, H., Ohashi, M., Umeda, F. & Nawata, H. (1990) Biochem. Bio- Our hypothesis that the pressor activity of big ET-1 in vivo phys. Res. Commun. 168, 1128-1136. is due to conversion of big ET-1 to ET-1 by an unusual 16. Bindra, J. S. (1982) U.S. Patent 4,329,495. metalloprotease is supported by our in vitro findings. Solu- 17. Roques, J. S., Schwartz, J. C. & Lecomte, J. M. (1985) U.S. bilized rabbit lung membranes incubated with porcine big Patent 4,513,009. ET-1 produce a large contractile response when bioassayed 18. Fournie-Zaluski, M. C., Lucas, E., Waksman, G. & Roques, with rat aortic rings, and this contraction is blocked in a B. P. (1984) Eur. J. Biochem. 139, 267-274. concentration-dependent manner by phosphoramidon and 19. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randell, R. J. (1951) J. Biol. Chem. 193, 265-275. 1,10-phenanthroline. Partially purified ECE from rabbit lung 20. De Nucci, G., Thomas, R., D'Orleans-Juste, P., Antunes, E., cleaves big ET-1 to a molecular species that comigrates on Walder, C., Warner, T. D. & Vane, J. R. (1988) Proc. Nati. HPLC with synthetic ET-1. This conversion is completely Acad. Sci. USA 85, 9797-9800. inhibited by 100 ,AM phosphoramidon. Moreover, the peak 21. Winquist, R. J., Scott, A. L. & Vlasuk, G. P. (1989) Hyper- formed during incubation of big ET-1 with partially purified tension 14, 111-114. rabbit lung ECE produces an ET-like contraction in rat aortic 22. Gardiner, S. M., Compton, A. M., Bennett, T., Palmer, rings. R. M. J. & Moncada, S. (1989) Eur. J. Pharmacol. 171, 237- In results that the 240. conclusion, our indicate physiologically 23. Fukuda, N., Izumi, Y., Soma, M., Watanabe, Y., Watanabe, relevant enzyme responsible for the conversion of big ET-1 M., Hatano, M., Sakuma, I. & Yasuda, H. (1990) Biochem. to ET-1 is a metalloprotease and that this enzyme can be Biophys. Res. Commun. 167, 739-745. blocked by the metalloprotease inhibitors phosphoramidon, 24. Nishikori, K. (1990) Trends Pharmacol. Sci. 11, 341. thiorphan, and 1,10-phenanthroline but not by kelatorphan or 25. Kimura, S., Kasuya, Y., Sawamura, T., Shinmi, O., Sugita, Y., captopril. Therefore, ECE appears to be a previously unre- Yanagisawa, M., Goto, K. & Masaki, T. (1989) J. Cardiovasc. ported metalloendoprotease. Pharmacol. 13, Suppl. 5, S5-S7. After submission of this work for review, two reports 26. Miyauchi, T., Yanagisawa, M., Tomizawa, T., Sugishita, Y., Suzuki, N., Fujino, M., Ajisaka, R., Goto, K. & Masaki, T. appeared in the literature supporting our conclusion that a (1989) Lancet ii, 53-54. phosphoramidon-sensitive metalloprotease is responsible for 27. Suzuki, N., Miyauchi, T., Tomobe, Y., Matsumoto, H., Goto, the conversion of big ET-1 to ET-1 (35, 36). K., Masaki, T. & Masahiko, F. (1990) Biochem. Biophys. Res. Commun. 167, 941-947. The authors thank Kerry Spear and Philippe Bovy for synthesis of 28. Suda, H., Aoyagi, T., Takeuchi, T. & Umezawa, H. (1973) J. the thiorphan and kelatorphan used in these studies. Antiobiot. 26, 621-623. 29. Mumford, R. A., Pierzchala, P. A., Strauss, A. W. & Zimmer- 1. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., man, M. (1981) Proc. Nati. Acad. Sci. USA 78, 6623-6627. Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K. & Masaki, T. 30. Weaver, L. H., Kester, W. R. & Matthews, B. W. (1977) J. (1988) Nature (London) 332, 411-415. Mol. 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