Soluble endothelin degradation enzyme activities in various rat tissues YILUNDENG'
AND
ARCOY.
JENG'
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Research Department, Pharmaceuticals Division, CIBA-GEIGY Corporation, Summit, NJ 07901, U.S.A. Received April 23, 1992 DENG,Y., and JENG,A. Y. 1992. Soluble endothelin degradation enzyme activities in various rat tissues. Biochem. Cell Biol. 70: 1385-1389. From soluble extract of rat kidney we have previously identified an endothelin degradation enzyme that rapidly and specifically cleaves off the C-terminal tryptophan of endothelin-1, resulting in a peptide that is three orders of magnitude weaker in potency than endothelin-1 in causing smooh muscle contraction. The tissue distribution of this enzyme was examined, and the soluble extracts of rat kidney were found to contain the highest enzyme activity, followed by the spleen and the liver. In contrast, no enzyme activity was detected in the soluble extracts of brain, heart, and lung. The biochemical properties of the partially purified enzyme from kidney were further investigated. The optimal pH of the enzyme was between 5 and 7. The endothelin degrading activity was effectively blocked by thiol protease inhibitors such as benzyloxycarbonyl-Phe-Ala-diazomethyl ketone and p-hydroxymercuribenzoic acid, as well as by phenylmethylsulfonyl fluoride, but not by metalloprotease and other serine protease inhibitors. This enzyme displayed a clear difference in substrate specificity when compared with other thio! proteases such as cathepsin B, cathepsin H, and cathepsin L, known to be present in the kidney. These results susest that a novel protease with endothelin degrading activity is widely distributed in a number of tissues. Key words: endothelin, endothelin degradation enzyme, thiol protease, carboxypeptidase. DENG,Y., et JENG, A. Y. 1992. Soluble endothelin degradation enzyme activities in various rat tissues. Biochem. Cell Biol. 70 : 1385-1389. Nous avons prkalablement identifit dans l'extrait soluble de rein de rat une degradation enzymatique de l'endotheline qui clive rapidement et specifiquement le thryptophane C-terminal de I'endothCline-1 donnant un peptide dont la puissance est de trois ordres de grandeur plus faible que celle de I'endotheline-1 pour provoquer la contraction des muscles lisses. Nous avons examine la distribution tissulaire de cette enzyme; les extraits solubles de rein de rat renferment I'activite enzymatique la plus Clevee et viennent ensuite la rate et le foie. En revanche, nous n'avons dCtecte aucune activite enzymatique dans les extraits solubles de cerveau, de coeur et de poumon. N w s a w n s examid les propri&tksbiochimiques de l'enzyme renale partiellement purifiee. Le pH optimum de I'enzymc est entre 5 et 7. L'activitC de degradation de l'endothaine est efficacement bloquee par des inhibiteurs de la thiol prottase tels que le benzyloxycarbonyl-Phe-Aladiazomethyl cCtone et l'acide p-hydroxymercuribenzoi'que de mCme que par le phenylmtthyfsulfonyl fluorure, mais non par les inhibiteurs de la metalloprotCase et d'autres shine prottases. Cette enzyme montre une nette difrkrence dans la specificit6 & I'egard du substrat lorsque comparCe avec d'autrer thioI protiases cornme la cathepsine B, la cathepsine H et la cathepsine L, toutes presentes dans le rein. Ces risultats suggerent qu'une nouvelle prottase capable de degrader IYendothClineest Iargement distribuke dans plusieurs tissus. Mots clPs : endotheline, degradation enzymatique de l'endotheline, thiol protease, carboxypeptidase. [Traduit par la redaction]
Introduction Endothelin-1 is a potent peptidic vasoconstrictor which causes many cardiac, hemodynamic, and renal effects (Masaki et al. 1991; Randall 1991; Simonson and Dunn 1991). In addition, ET-1 exerts a long-lasting action (Yanagisawa et al. 1988; Kasuya et al. 1989)' which is prob-
ably related to the nearly irreversible binding of ET-I to various tissue preparations (Jeng et at. 1990). These properties of ET-I suggest that its levels must be well regulated, presumably through biosynthesis and clearance. The biosynthesis of ET-1 has been studied by many investigators (for a review, see Masaki et al. 1991), but the clearance of ET-I is not we11 understood. It has been shown ABBREVIATIONS: ET- 1, endothelin-1 ; NEP, neutral endothat the bound ET-I is subjected to internalization into the peptidase 24-11 /EC 3.4.24.1 1); EDE, endothetin degradation cytoplasmic compartment of vascular smooth muscle cells enzyme; ET-1-desTrp, ET-I minus the C-terminaI tryptophan; (Hirata er ol, 1988) and is cleared rapidly in the circulation PM SF, phenytrnethy[sulfonyi fluoride; E-64, trans-epoxysuccinyl(Sirvio eta!. 1990; Hemsen et al. 1991). Furthermore, it has L-leucylamido(4-guanidino)butane; pHMB. p-hydroxymercuribeen reported that, during intravenous infusion of ET-1into benzoic acid; phosphoramidon. N-(a-rhamnopyranosylthe pig, the imrnunoreactive ET-I levels obtained from the oxyhydraxyphosp hinyl-t-leucyl-L-tryptophan TES, N-trisrenal and splenic veins are 92 and 82%, respectively, lower (hydroxymethyl)rnethyI-2-amin0ethanesuIFonic acid; Z-PheAlaCHN,, benzyloxycarbonyi-Phe-Alaaiazomerhyl ketone; than that measured in the systematic artery (Pernow et 01. ConA, concanavalin A; TFA, trifluoroacetic acid; HPLC, high 1989). The precise mechanism of this tissue-specific clearance pressure liquid chromatography; Z-Arg-Arg-AFC, benzyloxyof ET-1 is not certain. It is possible that proteolytic degradacarbonyl-Arg-Arg-7-arnino-4-trifluoromethylcoumar; Arg-AFC, tion of ET-1 may play a role. Arg-7-amino-4-trifluoromethylcoumarin; Z-Phe-Arg-AFC, Severd proteolytic enzymes have been demonstrated to benzyloxycarbonyl-Phe-Arg-7-amino-4-trifluoromethylcoumarin; degrade ET-1 in vifro. The phosphoramidon-sensitive NEP ANP, atrial natriuretic peptide. purified from rat kidneys has been shown to cleave ET-1 'present address: Department of Medicine, Medical College of nonspecifically at multiple sites (Vijayaraghavan el 01. 1990). Ohio, C.S. 10008, Toledo, OH 43699, U.S.A. Moreover, it hydrolyzes the three endothelins at different ' ~ u t h o r to whom all correspondence should be sent at the positions. In a separate study, ET-I has also be demonfollowing address: V-132, CIBA-GEIGY Corporation, 556 Morris Ave., Summit, N.J. 07901, U.S.A. strated to be rapidly inactivated at multiple positions by
Prinled In Canada / Irn~rirneau Canada
BIOCHEM. CELL BIOL. VOL. 70.
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TABLE1. Comparison of EDE activity in different rat tissues Tissue
Purification step
Proteina (Yo)
Activity (nmol/(mg.min))
Recovery (%)
Fold purification
-
Brain
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Heart Kidney Liver Lung Spleen
Soluble DE52 ConA Soluble DE52 ConA Soluble DE52 ConA Soluble DE52 ConA Soluble DE52 ConA Soluble DE52 ConA
extract extract extract extract extract extract
'Protein loaded varied from 400 to 1570 mg for kidney and from 110 to 320 mg for other tissues. b ~ e t e c t i o nlimit. %lean + SE (n = 9) for kidney and mean ? range (n = 2) for other tissues. ET-I (500 pmol) and the enzyme preparation at different stages of purification was incubated for 5-10 rnin at 37°C. The enzyme activity was calculated from the rate of disappearance of ET-I by HPLC analysis. d ~ o determined. t
polymorphonuclear leukocyte-derived (Patrignani et al. 1991).
serine proteases
We have previously identified and partiaIly purified a soIuble EDE from rat kidney @eng ef ul. 1992). This enzyme rapidly a n d specifically cleaves off the C-terminal tryptophan of ET-I, resulting in a peptide (ET-I-desTrp) that is three orders of magnitude weaker in potency than ET-I in causing s m o o t h muscle contraction. F u r t h e r m o r e , ET-1-desTrp causes a transient contraction rather than the long-lasting effects observed with authentic ET-1 (Kimura et al. 1988). Thus, proteolytic degradation of ET-1 by EDE seems t o provide a n efficient mechanism in the reversal of ET-1 -induced vasoconstriction. I n this report, the EDE activities in various rat tissues were examined a n d the biochemical properties of EDE partially purified from the kidney were further studied.
Materials and methods Materials ET-I was purchased from American Peptide (Sunnyvale. CaIif .). Soybean trypsin inhibitor, PMSF,o-phenanthroline, E-64, pHMB, phosphoramidon, trypsin-chymotrypsin inhibitor, iodoacetic acid, iodoacetamide, leupeptin, cyst atin, meth yl mannopyranoside, and TES were products of Sigma (St. Louis. Mo.). Z-Phe-AiaCNN,. cathepsin B, cathepsin If, cathepsin L, and enzyme overlay membranes were purchased from Enzyme Systems Products (Livmore, Calif.). DE52 anion-exchange resin and ConA-Sepharose were obtained rrom Wha~man (Hillsborw, Oreg.) and Pharmacia (Pismaway, N.J.), respectively. Partial purification of EDE Ten-week-old male Sprague-Dawley rats (TaeN(SD)fBR) from Taconic Farms (Germantawn, N.Y.) were euthanatized by carbon dioxide inhalation and decapitated. The brain, heart, kidney, liver, lung, and spleen were removed and separately homogenized at 4°C in a buffer containing 4 m M EDTA, 4 mM EGTA, and SO rnM TES (pH 7.0) at a ratio of 1 g tissue/4 mL buffer. The homogenates were centrifuged at 150 000 x g for 45 min and the supernatant from each tissue was loaded onto separate DE52 anion-
exchange matrices (2.4 x 5.5 cm). The matrix was washed with 10 bed volumes of 50 mM NaCl in 50 mM TES (pH 7.0) and proteins were eluted by 0.2 M NaCl in 50 mM TES (pH 7.0). The eluted enzyme was concentrated and desalted by Amicon stirred cells (Danvers, Mass.) using PM30 membranes, and was then loaded onto a ConA-Sepharose matrix (0.7 x 1.3 cm). This matrix was washed with 10 bed volumes of 0.5 M NaCl in 20 mM TES (pH 7.4) and the enzyme was eluted with a buffer containing 0.5 M NaCl and 0.5 M methyl mannopyranoside in 20 mM TES (pH 7.0). This enzyme preparation was concentrated, washed with 50 mM TES (pH 7.0) and stored at -80°C. Protein was determined according to a published method (Bradford 1976). ET-I degradation assay Depending on the experiments, the assay for EDE activity was carried out by incubating 500 pmol of ET-1 with 1-30 pg of protein in 50 mM TES (pH 7.0), at 37°C for 10 min in a total volume of 15 uL. The reaction was terminated bv the addition of 95 uL of 2840 acetonitrile in 0.09% TFA. peptides were separated by HPLC (Waters, Milford, Mass.) using a 3.9 x 150 mm Nova-Pack C,, column (Waters). The solvent system used was a 10 min 22-35% gradient from buffer A (0.09% TFA) to buffer B (90% acetonitrile in 0.09% TFA), followed by a 20-min isocratic elution with 35% buffer B at a flow rate of 1.1 mL/min. The absorbance of the peptides was monitored at 215 nm. EDE activity was calculated from the rate of disappearance of ET-1. Effects of protease inhibitors on EDE activity Protease inhibitors at various concentrations were preincubated for 15 min at room temperature with 1 pg of EDE partially purified from rat kidney using DE52 and ConA-Sepharose matrices. ET-1 (500 pmol) was then added to the mixture and incubated further for 5 min at 37°C and pH 6.0 in a total volume of 15 pL. The reaction was terminated by the same protocol as described in the ET-1 degradation assay and each sample was analyzed by HPLC. Comparison of substrate specificity of thiol proteases The substrate specificity of cathepsin B, cathepsin H, and cathepsin L were compared with that of EDE using enzyme overlay membranes impregnated with different substrates: Z-Arg-Arg-AFC for cathepsin B, Arg-AFC for cathepsin H , and Z-Phe-Arg-AFC for
NOTES
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TABLE 2. Effects of protease inhibitors on EDE activity from rat kidney Inhibitor
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EDTA
o-Phenanthroline Phosphoramidon Soybean trypsin inhibitor Trypsin-chymotrypsin inhibitor PMSF Z-Phe-AlaCHN, Leupeptin E-64
pHMB Iodoacetic acid Iodoacetamide Cystatin
Concentration VO inhibition 10 mM 100 mM 100 pM 100 pg/mL 250 pg/mL I mM I mM
I
I PM 1 mM I mM I mM I mM 160 pg/mL
NOTE:Phosphoramidon, 2-Phe-AlaCHNz, E-64, iodoacetic acid, and iodoacetamide were dissolved at 100 mM in dimethyl sulfoxide; o-phenanthroline and pHMB were dissolved at 1 M in methanol; and PMSF was dissolved at 100 mM in ethanol. The solvents used did not have significant effects on EDE activity. All other inhibitors were dissolved in the assay buffer at concentrations at least lMfold higher than those shown in the table. Inhibitors were preincubated at room temperature for 15 min with 1 pg protein of the partially purified rat kidney soluble extract. ET-1 (500 pmol) was then added to the mixture and incubated further for 5 min at 37°C and pH 6.0 in a total volume of 15 pL. The reaction was terminated and each sample was analyzed by HPLC. The values shown are means + SEM (n = 3-5).
cathepsin L. These membranes were pretreated according to manufacturer's instructions. To initiate reactions, 0.1 U of cathepsin B, cathepsin H, or cathepsin L in 1 pL was spotted onto the membranes. The reactions were allowed to proceed for 10 min and recorded as bright spots under illumination of a UV transilluminator. For comparison, 1-5 pg of protein, obtained from rat kidneys at various stages of purification, was used. Results and discussion Tissue distribution of EDE We previously reported the partial purification of EDE from soluble extracts of rat kidneys using DE52 anionexchange and ConA-Sepharose matrices (Deng et al. 1992). These matrices were chosen because EDE was found to be negatively charged and glycosylated in preliminary experiments. The same purification scheme was used to compare EDE activities in various rat tissues. After purification by DE52 and ConA-Sepharose matrices, EDE activity in the kidney preparations was 40 + 6 nmol/(mg - min) (mean k SE, n = 9), which represented a 57-fold purification (Table 1). Following the same purification procedure, the specific activity and fold purification of EDE from spleen were 26 k 5 nmol/(mg.min) (mean range, n = 2) and 43-fold, respectively, whereas the corresponding values for EDE from liver were 16 + 4 nmol/(mg.min) (n = 2) and 32-fold, respectively. The enzyme activities in the brain, lung, and heart were lower than 0.03 nmol/ (mg -min), the detection limit of EDE assay (Table 1). These findings correlate with the results obtained from a study on the clearance of intravenously infused ET-1 into the pig, where the immunoreactive ET-1 levels in the renal and splenic veins are 92 and 82070, respectively, lower than that measured in the arteries (Pernow et al. 1989). The same study needs to be further extended to other organs such as liver, lung, and heart to substantiate the involvement of EDE in the degradation of ET-1. Although the specific activity of EDE in the kidney was slightly higher than that of the spleen, the total enzyme +_
FIG. 1. Comparison of substrate specificity between EDE and cathepsins. The substrate strips for cathepsin B (B), cathepsin H (H), and cathepsin L (L) were used. Various enzymes in 1 pL were spotted onto the membranes and the reactions were allowed to proceed for 10 min prior to visualization under a UV transilluminator. Lane 1 , O . 1 U of cathepsin B; lane 2,O. 1 U of cathepsin H; lane 3, 0.1 U of cathepsin L; lane 4, 5 pg of the soluble kidney extract; lane 5, 5 pg of the combined flow through and 0.05 M NaCl wash fractions from a DE52 matrix; lane 6, 5 pg of the 0.2 M NaCl eluted fraction from a DE52 matrix; lane 7, 5 pg of the flow-through fraction from a ConA-Sepharose matrix; lane 8, 1 pg of the 0.5 M methyl mannopyranoside eluted fraction from a ConA-Sepharose matrix.
activity in the former was significantly greater. Therefore, the partially purified enzyme from the kidney was utilized for further biochemical studies.
pH dependence of EDE The renal EDE activity was determined under different pH conditions. The enzyme showed a broad pH optimum; it ranged between pH 5 and 7. No enzyme activity was observed at pH values below 3 or above 7.5 (results not shown). Thus, characterization of EDE was carried out at pH 6.0 in subsequent experiments. Effects of protease inhibitors on EDE activity The effects of various protease inhibitors on EDE activity were examined. When measured at pH 4.0, pepstatin A, an aspartylprotease inhibitor, did not show a significant effect on EDE activity (results not shown). At pH 6.0, EDTA and phosphoramidon (metalloprotease inhibitors), as well as soybean trypsin inhibitor and trypsin-chymotrypsin inhibitor (serine protease inhibitors), did not have any effect on EDE activity (Table 2). However, o-phenanthroline, another metalloprotease inhibitor, showed a moderate inhibition only at concentrations 10-100 times greater than the recommended effective concentrations (Beynon and Salvesen 1989). PMSF, a serinekhiol protease inhibitor (Dunn 1989), and Z-Phe-AIaCHN2, a thiol protease inhibitor (Watanabe et al. 1979), were the most effective; they inhibited EDE activity completely at 1 mM (Table 2). Other thiol protease inhibitors were also tested. While leupeptin, E-64, and cystatin were inactive, pHMB and iodoacetamide at 1 mM inhibited EDE activity by 78 and 59070, respectively. Interestingly, iodoacetic acid did not have any effect at the same concentration. Since iodoacetamide is uncharged and iodoacetic acid is negatively charged under the assay conditions, these results suggest that the active site of EDE is either uncharged or negatively charged.
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I
ET-I
FIG. 2. Comparison of EDE activity at different stages of purification. ET-I (500 pmol) was incubated with buffer alone (A), with 5 pg of the 0.2 M NaCl eluted fraction from a DE52 matrix (B), or with 1 pg of the 0.5 M methyl mannopyranoside eluted fraction from a ConA-Sepharose matrix (C) in a total volume of 15 pL at 37OC for 10 min prior to analysis by HPLC. The arrows indicate the elution positions of authentic ET-1 and the degradation product of ET-1 (labeled as "1").
Comparison of EDE activity with those of cathepsin B, cathepsin H, and cathepsin L Since EDE activity was effectively inhibited by Z-PheAlaCHN2, a known inhibitor of the lysosomal cysteine proteases (Leary and Shaw 1977; Barrett and Kirschke 1981), the substrate specificity of EDE and these lysosomal enzymes were compared utilizing enzyme overlay membranes impregnated with fluorogenic substrates specific for cathepsin B, cathepsin H, and cathepsin L. As shown in Fig. 1, cathepsin H (lane 2) and cathepsin L (lane 3) recognized their respective substrates specifically, while cathepsin B showed moderate activity toward the substrate for cathepsin L in addition to its own substrate (lane 1). The soluble extract of rat kidney contained enzyme activities similar to the three cathepsins (lane 4). However, most of these activities were found in the combined flow through and the 0.05 M NaCl wash fractions from the DE52 matrix (lane 5). Only cathepsin-H-like activity remained in the 0.2 M NaCl eluted fraction, where the majority of EDE activity was recovered (lane 6). In a subsequent purification step using a ConASepharose matrix, the majority of cathepsin-H-like activity was recovered in the flow-through fraction (lane 7) and not in the 0.5 M methyl mannopyranoside eluted fraction (lane 8). Therefore, this last fraction contained very low lysosomal cysteine-protease-likeactivities. In contrast, it had high EDE activity; 1 pg protein of this fraction could cleave off the C-terminal tryptophan residue from at least 500 pmol ET-1 within 10 min at 37°C (Fig. 2C). For comparison, the
rate of cleavage was about half as much, even when 5 pg protein of the 0.2 M NaCl eluted fraction from the DE52 matrix was used (Fig. 2B). The above results suggest that the enzyme described here is a novel protease with endothelin degrading activity. Other proteolytic enzymes such as NEP have been implicated in the degradation of ET-1 (Vijayaraghavan et al. 1990; Fagny et al. 1991). This enzyme has been shown previously to cleave ANP in vitro (Sonnenberg et al. 1988). The fact that NEP is a physiologically relevant ANP-degrading enzyme has been demonstrated using UK-69,578 (Danilewicz et al. 1989) and sinorphan and retorphan (Lecomte et al. 1990), specific inhibitors of NEP. Increases in plasma ANP levels along with natriuretic and diuretic responses have been observed in mice administered with UK-69,578 intravenously (Danilewicz et al. 1989). Similar results have also been obtained in humans and mice after oral administration of sinorphan and retorphan (Lecomte et al. 1990). In contrast, treatment with phosphoramidon, another potent inhibitor of NEP, does not prolong the half-life of plasma ET-1 upon intravenous, bolus injection of the peptide into the pig (Modin et al. 1991), nor does it elevate the mean arterial pressure in the rat as ET-1 would (McMahon et al. 1991). Thus, NEP may not be a physiologically relevant ET-1 degradation enzyme. Likewise, whether or not EDE is responsible for the degradation of ET-1 in vivo needs to be evaluated when potent inhibitors of EDE are available. Acknowledgments We thank Drs. Richard Kramer and Joseph Balwierczak for critical reading of this manuscript. Barrett, A. J., and Kirschke, H. 1981. Cathepsin B, cathepsin H, and cathepsin L. Methods Enzymol. 80: 535-561. Beynon, R.J., and Salvesen, G. 1989. Commercially available protease inhibitors. Edited by R.J. Beynon and J.S. Bond. IRL Press, Oxford, England. pp. 241-249. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. Danilewicz, J.C., Barclay, P.L., Barnish, I.T., Brown, D., Campbell, S.F., James, K., Samuels, G.M.R., Terrett, N.K., and Wythes, M.J. 1989. UK-69,578, a novel inhibitor of EC 3.4.24.11 which increases endogenous ANF levels and is natriuretic and diuretic. Biochem. Biophys. Res. Commun. 164: 58-65. Deng, Y., Martin, L.L., DelGrande, D., and Jeng, A.Y. 1992. A soluble protease identified from rat kidney degrades endothelin-1 but not proendothelin-1. J. Biochem. (Tokyo). 112: 168-172. Dunn, B.M. 1989. Determination of protease mechanism. Edited by R.J. Beynon and J.S. Bond. IRL Press, Oxford, England. pp. 57-81. Fagny, C., Michel, A., Leonard, I., Berkenboom, G., Fontaine, J., and Deschodt-Lanckman, M. 1991. In vitro degradation of endothelin- 1 by endopeptidase 24.11 (enkephalinase). Peptides (Fayetteville, N.Y.), 12: 773-778. Hemsen, A., Pernow, J., and Lundberg, J.M. 1991. Regional extraction of endothelins and conversion of big endothelin to endothelin-1 in the pig. Acta Physiol. Scand. 141: 325-334. Hirata, Y., Yoshimi, H., Takaichi, S., Yanagisawa, M., and Masaki, T. 1988. Binding and receptor down-regulation of a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells. FEBS Lett. 239: 13-17. Jeng, A.Y., Savage, P., Soriano, A., and Balwierczak, J.L. 1990. Different affinities and selectivities of endothelin-1 and
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endothelin-3 binding to various rat tissues. Biochem. Int. 22: 669-676. Kasuya, Y., Ishikawa, T., Yanagisawa, M., Kimura, S., Goto, K., and Masaki, T. 1989. Mechanism of contraction to endothelin in isolated porcine coronary artery. Am. J. Physiol. 257: H1828-H1835. Kimura, S., Kasuya. Y., Sawamura, T., Shinmi, O., Sugita, Y., Yanagisawa, M., Goto, K., and Masaki, T. 1988. Structureactivity relationships of endothelin: importance of the C-terminal moiety. Biochem. Biophys. Res. Commun. 156: 1182-1 186. Leary, R., and Shaw, E. 1977. Inactivation of cathepsin B, by diazomethyl ketones. Biochem. Biophys. Res. Commun. 79: 926-93 1. Lecomte, J.-M., Baumer, P., Lim, C., Duchier, J., Cournot, A., Dussaule, J.-C., Ardaillou, R., Gros, C., Chaignon, B., Souque, A., and Schwartz, J.-C. 1990. Stereoselective protection of exogenous and endogenous atrial natriuretic factor by enkephalinaseinhibitors in mice and humans. Eur. J. Pharmacol. 179: 65-73. Masaki, T., Kimura, S., Yanagisawa, M., and Goto, K. 1991. Molecular and cellular mechanism of endothelin regulation. Implications for vascular function. Circulation, 84: 1457-1468. McMahon, E.G., Palomo, M.A., Moore, W.M., McDonald, J.F., and Stern, M.K. 1991. 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. Proc. Natl. Acad. Sci. U.S.A. 88: 703-707. Modin, A., Pernow, J., and Lundberg, J.M. 1991. Phosphoramidon inhibits the vasoconstrictor effects evoked by big endothelin-1 but not the elevation of plasma endothelin-1 in vivo. Life Sci. 49: 1619-1625. Patrignani, P., Del Maschio, A., Bazzoni, G., Daffonchio, L., Hernandez, A., Modica, R., Montesanti, L., Volpi, D., Patrono,
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C., and Dejana, E. 1991. Inactivation of endothelin by polymorphonuclear leukocyte-derived lytic enzymes. Blood, 78: 2715-2720. Pernow, J., Hemsen, A., and Lundberg, J.M. 1989. Tissue specific distribution, clearance and vascular effects of endothelin in the pig. Biochem. Biophys. Res. Commun. 161: 647-653. Randall, M.D. 1991. Vascular activities of the endothelins. Pharmacol. Ther. 50: 73-93. Simonson, M.S., and Dunn, M. J. 1991. Endothelins: a family of regulatory peptides. Hypertension (Suppl.), 17: 856-863. Sirvio, M.-L., Metsarinne, K., Saijonmaa, O., and Fyhrquist, F. 1990. Tissue distribution and half-life of '251-endothelinin the rat: importance of pulmonary clearance. Biochem. Biophys. Res. Commun. 167: 1191-1 195. Sonnenberg, J.L., Sakane, Y., Jeng, A.Y., Koehn, J.A., Ansell, J.A., Wennogle, L.P., and Ghai, R.D. 1988. Identification of protease 3.4.24.11 as the major atrial natriuretic factor degrading enzyme in the rat kidney. Peptides (Fayetteville, N.Y.), 9: 173-180. Vijayaraghavan, J., Scicli, A.G., Carretero., O.A., Slaughter, C., Moomaw, C., and Hersh, L.B. 1990. The hydrolysis of endothelins by neutral endopeptidase 24.11 (enkephalinase). J. Biol. Chem. 265: 14 150 - 14 155. Watanabe, H., Green, G.D. J., and Shaw, E. 1979. A comparison of the behavior of chymotrypsin and cathepsin B towards peptidyl diazomethyl ketones. Biochem. Biophys. Res. Comrnun. 89: 1354-1360. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T. 1988. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature (London), 332: 41 1-415.
AND
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JENG'
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Research Department, Pharmaceuticals Division, CIBA-GEIGY Corporation, Summit, NJ 07901, U.S.A. Received April 23, 1992 DENG,Y., and JENG,A. Y. 1992. Soluble endothelin degradation enzyme activities in various rat tissues. Biochem. Cell Biol. 70: 1385-1389. From soluble extract of rat kidney we have previously identified an endothelin degradation enzyme that rapidly and specifically cleaves off the C-terminal tryptophan of endothelin-1, resulting in a peptide that is three orders of magnitude weaker in potency than endothelin-1 in causing smooh muscle contraction. The tissue distribution of this enzyme was examined, and the soluble extracts of rat kidney were found to contain the highest enzyme activity, followed by the spleen and the liver. In contrast, no enzyme activity was detected in the soluble extracts of brain, heart, and lung. The biochemical properties of the partially purified enzyme from kidney were further investigated. The optimal pH of the enzyme was between 5 and 7. The endothelin degrading activity was effectively blocked by thiol protease inhibitors such as benzyloxycarbonyl-Phe-Ala-diazomethyl ketone and p-hydroxymercuribenzoic acid, as well as by phenylmethylsulfonyl fluoride, but not by metalloprotease and other serine protease inhibitors. This enzyme displayed a clear difference in substrate specificity when compared with other thio! proteases such as cathepsin B, cathepsin H, and cathepsin L, known to be present in the kidney. These results susest that a novel protease with endothelin degrading activity is widely distributed in a number of tissues. Key words: endothelin, endothelin degradation enzyme, thiol protease, carboxypeptidase. DENG,Y., et JENG, A. Y. 1992. Soluble endothelin degradation enzyme activities in various rat tissues. Biochem. Cell Biol. 70 : 1385-1389. Nous avons prkalablement identifit dans l'extrait soluble de rein de rat une degradation enzymatique de l'endotheline qui clive rapidement et specifiquement le thryptophane C-terminal de I'endothCline-1 donnant un peptide dont la puissance est de trois ordres de grandeur plus faible que celle de I'endotheline-1 pour provoquer la contraction des muscles lisses. Nous avons examine la distribution tissulaire de cette enzyme; les extraits solubles de rein de rat renferment I'activite enzymatique la plus Clevee et viennent ensuite la rate et le foie. En revanche, nous n'avons dCtecte aucune activite enzymatique dans les extraits solubles de cerveau, de coeur et de poumon. N w s a w n s examid les propri&tksbiochimiques de l'enzyme renale partiellement purifiee. Le pH optimum de I'enzymc est entre 5 et 7. L'activitC de degradation de l'endothaine est efficacement bloquee par des inhibiteurs de la thiol prottase tels que le benzyloxycarbonyl-Phe-Aladiazomethyl cCtone et l'acide p-hydroxymercuribenzoi'que de mCme que par le phenylmtthyfsulfonyl fluorure, mais non par les inhibiteurs de la metalloprotCase et d'autres shine prottases. Cette enzyme montre une nette difrkrence dans la specificit6 & I'egard du substrat lorsque comparCe avec d'autrer thioI protiases cornme la cathepsine B, la cathepsine H et la cathepsine L, toutes presentes dans le rein. Ces risultats suggerent qu'une nouvelle prottase capable de degrader IYendothClineest Iargement distribuke dans plusieurs tissus. Mots clPs : endotheline, degradation enzymatique de l'endotheline, thiol protease, carboxypeptidase. [Traduit par la redaction]
Introduction Endothelin-1 is a potent peptidic vasoconstrictor which causes many cardiac, hemodynamic, and renal effects (Masaki et al. 1991; Randall 1991; Simonson and Dunn 1991). In addition, ET-1 exerts a long-lasting action (Yanagisawa et al. 1988; Kasuya et al. 1989)' which is prob-
ably related to the nearly irreversible binding of ET-I to various tissue preparations (Jeng et at. 1990). These properties of ET-I suggest that its levels must be well regulated, presumably through biosynthesis and clearance. The biosynthesis of ET-1 has been studied by many investigators (for a review, see Masaki et al. 1991), but the clearance of ET-I is not we11 understood. It has been shown ABBREVIATIONS: ET- 1, endothelin-1 ; NEP, neutral endothat the bound ET-I is subjected to internalization into the peptidase 24-11 /EC 3.4.24.1 1); EDE, endothetin degradation cytoplasmic compartment of vascular smooth muscle cells enzyme; ET-1-desTrp, ET-I minus the C-terminaI tryptophan; (Hirata er ol, 1988) and is cleared rapidly in the circulation PM SF, phenytrnethy[sulfonyi fluoride; E-64, trans-epoxysuccinyl(Sirvio eta!. 1990; Hemsen et al. 1991). Furthermore, it has L-leucylamido(4-guanidino)butane; pHMB. p-hydroxymercuribeen reported that, during intravenous infusion of ET-1into benzoic acid; phosphoramidon. N-(a-rhamnopyranosylthe pig, the imrnunoreactive ET-I levels obtained from the oxyhydraxyphosp hinyl-t-leucyl-L-tryptophan TES, N-trisrenal and splenic veins are 92 and 82%, respectively, lower (hydroxymethyl)rnethyI-2-amin0ethanesuIFonic acid; Z-PheAlaCHN,, benzyloxycarbonyi-Phe-Alaaiazomerhyl ketone; than that measured in the systematic artery (Pernow et 01. ConA, concanavalin A; TFA, trifluoroacetic acid; HPLC, high 1989). The precise mechanism of this tissue-specific clearance pressure liquid chromatography; Z-Arg-Arg-AFC, benzyloxyof ET-1 is not certain. It is possible that proteolytic degradacarbonyl-Arg-Arg-7-arnino-4-trifluoromethylcoumar; Arg-AFC, tion of ET-1 may play a role. Arg-7-amino-4-trifluoromethylcoumarin; Z-Phe-Arg-AFC, Severd proteolytic enzymes have been demonstrated to benzyloxycarbonyl-Phe-Arg-7-amino-4-trifluoromethylcoumarin; degrade ET-1 in vifro. The phosphoramidon-sensitive NEP ANP, atrial natriuretic peptide. purified from rat kidneys has been shown to cleave ET-1 'present address: Department of Medicine, Medical College of nonspecifically at multiple sites (Vijayaraghavan el 01. 1990). Ohio, C.S. 10008, Toledo, OH 43699, U.S.A. Moreover, it hydrolyzes the three endothelins at different ' ~ u t h o r to whom all correspondence should be sent at the positions. In a separate study, ET-I has also be demonfollowing address: V-132, CIBA-GEIGY Corporation, 556 Morris Ave., Summit, N.J. 07901, U.S.A. strated to be rapidly inactivated at multiple positions by
Prinled In Canada / Irn~rirneau Canada
BIOCHEM. CELL BIOL. VOL. 70.
1992
TABLE1. Comparison of EDE activity in different rat tissues Tissue
Purification step
Proteina (Yo)
Activity (nmol/(mg.min))
Recovery (%)
Fold purification
-
Brain
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Heart Kidney Liver Lung Spleen
Soluble DE52 ConA Soluble DE52 ConA Soluble DE52 ConA Soluble DE52 ConA Soluble DE52 ConA Soluble DE52 ConA
extract extract extract extract extract extract
'Protein loaded varied from 400 to 1570 mg for kidney and from 110 to 320 mg for other tissues. b ~ e t e c t i o nlimit. %lean + SE (n = 9) for kidney and mean ? range (n = 2) for other tissues. ET-I (500 pmol) and the enzyme preparation at different stages of purification was incubated for 5-10 rnin at 37°C. The enzyme activity was calculated from the rate of disappearance of ET-I by HPLC analysis. d ~ o determined. t
polymorphonuclear leukocyte-derived (Patrignani et al. 1991).
serine proteases
We have previously identified and partiaIly purified a soIuble EDE from rat kidney @eng ef ul. 1992). This enzyme rapidly a n d specifically cleaves off the C-terminal tryptophan of ET-I, resulting in a peptide (ET-I-desTrp) that is three orders of magnitude weaker in potency than ET-I in causing s m o o t h muscle contraction. F u r t h e r m o r e , ET-1-desTrp causes a transient contraction rather than the long-lasting effects observed with authentic ET-1 (Kimura et al. 1988). Thus, proteolytic degradation of ET-1 by EDE seems t o provide a n efficient mechanism in the reversal of ET-1 -induced vasoconstriction. I n this report, the EDE activities in various rat tissues were examined a n d the biochemical properties of EDE partially purified from the kidney were further studied.
Materials and methods Materials ET-I was purchased from American Peptide (Sunnyvale. CaIif .). Soybean trypsin inhibitor, PMSF,o-phenanthroline, E-64, pHMB, phosphoramidon, trypsin-chymotrypsin inhibitor, iodoacetic acid, iodoacetamide, leupeptin, cyst atin, meth yl mannopyranoside, and TES were products of Sigma (St. Louis. Mo.). Z-Phe-AiaCNN,. cathepsin B, cathepsin If, cathepsin L, and enzyme overlay membranes were purchased from Enzyme Systems Products (Livmore, Calif.). DE52 anion-exchange resin and ConA-Sepharose were obtained rrom Wha~man (Hillsborw, Oreg.) and Pharmacia (Pismaway, N.J.), respectively. Partial purification of EDE Ten-week-old male Sprague-Dawley rats (TaeN(SD)fBR) from Taconic Farms (Germantawn, N.Y.) were euthanatized by carbon dioxide inhalation and decapitated. The brain, heart, kidney, liver, lung, and spleen were removed and separately homogenized at 4°C in a buffer containing 4 m M EDTA, 4 mM EGTA, and SO rnM TES (pH 7.0) at a ratio of 1 g tissue/4 mL buffer. The homogenates were centrifuged at 150 000 x g for 45 min and the supernatant from each tissue was loaded onto separate DE52 anion-
exchange matrices (2.4 x 5.5 cm). The matrix was washed with 10 bed volumes of 50 mM NaCl in 50 mM TES (pH 7.0) and proteins were eluted by 0.2 M NaCl in 50 mM TES (pH 7.0). The eluted enzyme was concentrated and desalted by Amicon stirred cells (Danvers, Mass.) using PM30 membranes, and was then loaded onto a ConA-Sepharose matrix (0.7 x 1.3 cm). This matrix was washed with 10 bed volumes of 0.5 M NaCl in 20 mM TES (pH 7.4) and the enzyme was eluted with a buffer containing 0.5 M NaCl and 0.5 M methyl mannopyranoside in 20 mM TES (pH 7.0). This enzyme preparation was concentrated, washed with 50 mM TES (pH 7.0) and stored at -80°C. Protein was determined according to a published method (Bradford 1976). ET-I degradation assay Depending on the experiments, the assay for EDE activity was carried out by incubating 500 pmol of ET-1 with 1-30 pg of protein in 50 mM TES (pH 7.0), at 37°C for 10 min in a total volume of 15 uL. The reaction was terminated bv the addition of 95 uL of 2840 acetonitrile in 0.09% TFA. peptides were separated by HPLC (Waters, Milford, Mass.) using a 3.9 x 150 mm Nova-Pack C,, column (Waters). The solvent system used was a 10 min 22-35% gradient from buffer A (0.09% TFA) to buffer B (90% acetonitrile in 0.09% TFA), followed by a 20-min isocratic elution with 35% buffer B at a flow rate of 1.1 mL/min. The absorbance of the peptides was monitored at 215 nm. EDE activity was calculated from the rate of disappearance of ET-1. Effects of protease inhibitors on EDE activity Protease inhibitors at various concentrations were preincubated for 15 min at room temperature with 1 pg of EDE partially purified from rat kidney using DE52 and ConA-Sepharose matrices. ET-1 (500 pmol) was then added to the mixture and incubated further for 5 min at 37°C and pH 6.0 in a total volume of 15 pL. The reaction was terminated by the same protocol as described in the ET-1 degradation assay and each sample was analyzed by HPLC. Comparison of substrate specificity of thiol proteases The substrate specificity of cathepsin B, cathepsin H, and cathepsin L were compared with that of EDE using enzyme overlay membranes impregnated with different substrates: Z-Arg-Arg-AFC for cathepsin B, Arg-AFC for cathepsin H , and Z-Phe-Arg-AFC for
NOTES
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TABLE 2. Effects of protease inhibitors on EDE activity from rat kidney Inhibitor
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EDTA
o-Phenanthroline Phosphoramidon Soybean trypsin inhibitor Trypsin-chymotrypsin inhibitor PMSF Z-Phe-AlaCHN, Leupeptin E-64
pHMB Iodoacetic acid Iodoacetamide Cystatin
Concentration VO inhibition 10 mM 100 mM 100 pM 100 pg/mL 250 pg/mL I mM I mM
I
I PM 1 mM I mM I mM I mM 160 pg/mL
NOTE:Phosphoramidon, 2-Phe-AlaCHNz, E-64, iodoacetic acid, and iodoacetamide were dissolved at 100 mM in dimethyl sulfoxide; o-phenanthroline and pHMB were dissolved at 1 M in methanol; and PMSF was dissolved at 100 mM in ethanol. The solvents used did not have significant effects on EDE activity. All other inhibitors were dissolved in the assay buffer at concentrations at least lMfold higher than those shown in the table. Inhibitors were preincubated at room temperature for 15 min with 1 pg protein of the partially purified rat kidney soluble extract. ET-1 (500 pmol) was then added to the mixture and incubated further for 5 min at 37°C and pH 6.0 in a total volume of 15 pL. The reaction was terminated and each sample was analyzed by HPLC. The values shown are means + SEM (n = 3-5).
cathepsin L. These membranes were pretreated according to manufacturer's instructions. To initiate reactions, 0.1 U of cathepsin B, cathepsin H, or cathepsin L in 1 pL was spotted onto the membranes. The reactions were allowed to proceed for 10 min and recorded as bright spots under illumination of a UV transilluminator. For comparison, 1-5 pg of protein, obtained from rat kidneys at various stages of purification, was used. Results and discussion Tissue distribution of EDE We previously reported the partial purification of EDE from soluble extracts of rat kidneys using DE52 anionexchange and ConA-Sepharose matrices (Deng et al. 1992). These matrices were chosen because EDE was found to be negatively charged and glycosylated in preliminary experiments. The same purification scheme was used to compare EDE activities in various rat tissues. After purification by DE52 and ConA-Sepharose matrices, EDE activity in the kidney preparations was 40 + 6 nmol/(mg - min) (mean k SE, n = 9), which represented a 57-fold purification (Table 1). Following the same purification procedure, the specific activity and fold purification of EDE from spleen were 26 k 5 nmol/(mg.min) (mean range, n = 2) and 43-fold, respectively, whereas the corresponding values for EDE from liver were 16 + 4 nmol/(mg.min) (n = 2) and 32-fold, respectively. The enzyme activities in the brain, lung, and heart were lower than 0.03 nmol/ (mg -min), the detection limit of EDE assay (Table 1). These findings correlate with the results obtained from a study on the clearance of intravenously infused ET-1 into the pig, where the immunoreactive ET-1 levels in the renal and splenic veins are 92 and 82070, respectively, lower than that measured in the arteries (Pernow et al. 1989). The same study needs to be further extended to other organs such as liver, lung, and heart to substantiate the involvement of EDE in the degradation of ET-1. Although the specific activity of EDE in the kidney was slightly higher than that of the spleen, the total enzyme +_
FIG. 1. Comparison of substrate specificity between EDE and cathepsins. The substrate strips for cathepsin B (B), cathepsin H (H), and cathepsin L (L) were used. Various enzymes in 1 pL were spotted onto the membranes and the reactions were allowed to proceed for 10 min prior to visualization under a UV transilluminator. Lane 1 , O . 1 U of cathepsin B; lane 2,O. 1 U of cathepsin H; lane 3, 0.1 U of cathepsin L; lane 4, 5 pg of the soluble kidney extract; lane 5, 5 pg of the combined flow through and 0.05 M NaCl wash fractions from a DE52 matrix; lane 6, 5 pg of the 0.2 M NaCl eluted fraction from a DE52 matrix; lane 7, 5 pg of the flow-through fraction from a ConA-Sepharose matrix; lane 8, 1 pg of the 0.5 M methyl mannopyranoside eluted fraction from a ConA-Sepharose matrix.
activity in the former was significantly greater. Therefore, the partially purified enzyme from the kidney was utilized for further biochemical studies.
pH dependence of EDE The renal EDE activity was determined under different pH conditions. The enzyme showed a broad pH optimum; it ranged between pH 5 and 7. No enzyme activity was observed at pH values below 3 or above 7.5 (results not shown). Thus, characterization of EDE was carried out at pH 6.0 in subsequent experiments. Effects of protease inhibitors on EDE activity The effects of various protease inhibitors on EDE activity were examined. When measured at pH 4.0, pepstatin A, an aspartylprotease inhibitor, did not show a significant effect on EDE activity (results not shown). At pH 6.0, EDTA and phosphoramidon (metalloprotease inhibitors), as well as soybean trypsin inhibitor and trypsin-chymotrypsin inhibitor (serine protease inhibitors), did not have any effect on EDE activity (Table 2). However, o-phenanthroline, another metalloprotease inhibitor, showed a moderate inhibition only at concentrations 10-100 times greater than the recommended effective concentrations (Beynon and Salvesen 1989). PMSF, a serinekhiol protease inhibitor (Dunn 1989), and Z-Phe-AIaCHN2, a thiol protease inhibitor (Watanabe et al. 1979), were the most effective; they inhibited EDE activity completely at 1 mM (Table 2). Other thiol protease inhibitors were also tested. While leupeptin, E-64, and cystatin were inactive, pHMB and iodoacetamide at 1 mM inhibited EDE activity by 78 and 59070, respectively. Interestingly, iodoacetic acid did not have any effect at the same concentration. Since iodoacetamide is uncharged and iodoacetic acid is negatively charged under the assay conditions, these results suggest that the active site of EDE is either uncharged or negatively charged.
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I
ET-I
FIG. 2. Comparison of EDE activity at different stages of purification. ET-I (500 pmol) was incubated with buffer alone (A), with 5 pg of the 0.2 M NaCl eluted fraction from a DE52 matrix (B), or with 1 pg of the 0.5 M methyl mannopyranoside eluted fraction from a ConA-Sepharose matrix (C) in a total volume of 15 pL at 37OC for 10 min prior to analysis by HPLC. The arrows indicate the elution positions of authentic ET-1 and the degradation product of ET-1 (labeled as "1").
Comparison of EDE activity with those of cathepsin B, cathepsin H, and cathepsin L Since EDE activity was effectively inhibited by Z-PheAlaCHN2, a known inhibitor of the lysosomal cysteine proteases (Leary and Shaw 1977; Barrett and Kirschke 1981), the substrate specificity of EDE and these lysosomal enzymes were compared utilizing enzyme overlay membranes impregnated with fluorogenic substrates specific for cathepsin B, cathepsin H, and cathepsin L. As shown in Fig. 1, cathepsin H (lane 2) and cathepsin L (lane 3) recognized their respective substrates specifically, while cathepsin B showed moderate activity toward the substrate for cathepsin L in addition to its own substrate (lane 1). The soluble extract of rat kidney contained enzyme activities similar to the three cathepsins (lane 4). However, most of these activities were found in the combined flow through and the 0.05 M NaCl wash fractions from the DE52 matrix (lane 5). Only cathepsin-H-like activity remained in the 0.2 M NaCl eluted fraction, where the majority of EDE activity was recovered (lane 6). In a subsequent purification step using a ConASepharose matrix, the majority of cathepsin-H-like activity was recovered in the flow-through fraction (lane 7) and not in the 0.5 M methyl mannopyranoside eluted fraction (lane 8). Therefore, this last fraction contained very low lysosomal cysteine-protease-likeactivities. In contrast, it had high EDE activity; 1 pg protein of this fraction could cleave off the C-terminal tryptophan residue from at least 500 pmol ET-1 within 10 min at 37°C (Fig. 2C). For comparison, the
rate of cleavage was about half as much, even when 5 pg protein of the 0.2 M NaCl eluted fraction from the DE52 matrix was used (Fig. 2B). The above results suggest that the enzyme described here is a novel protease with endothelin degrading activity. Other proteolytic enzymes such as NEP have been implicated in the degradation of ET-1 (Vijayaraghavan et al. 1990; Fagny et al. 1991). This enzyme has been shown previously to cleave ANP in vitro (Sonnenberg et al. 1988). The fact that NEP is a physiologically relevant ANP-degrading enzyme has been demonstrated using UK-69,578 (Danilewicz et al. 1989) and sinorphan and retorphan (Lecomte et al. 1990), specific inhibitors of NEP. Increases in plasma ANP levels along with natriuretic and diuretic responses have been observed in mice administered with UK-69,578 intravenously (Danilewicz et al. 1989). Similar results have also been obtained in humans and mice after oral administration of sinorphan and retorphan (Lecomte et al. 1990). In contrast, treatment with phosphoramidon, another potent inhibitor of NEP, does not prolong the half-life of plasma ET-1 upon intravenous, bolus injection of the peptide into the pig (Modin et al. 1991), nor does it elevate the mean arterial pressure in the rat as ET-1 would (McMahon et al. 1991). Thus, NEP may not be a physiologically relevant ET-1 degradation enzyme. Likewise, whether or not EDE is responsible for the degradation of ET-1 in vivo needs to be evaluated when potent inhibitors of EDE are available. Acknowledgments We thank Drs. Richard Kramer and Joseph Balwierczak for critical reading of this manuscript. Barrett, A. J., and Kirschke, H. 1981. Cathepsin B, cathepsin H, and cathepsin L. Methods Enzymol. 80: 535-561. Beynon, R.J., and Salvesen, G. 1989. Commercially available protease inhibitors. Edited by R.J. Beynon and J.S. Bond. IRL Press, Oxford, England. pp. 241-249. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. Danilewicz, J.C., Barclay, P.L., Barnish, I.T., Brown, D., Campbell, S.F., James, K., Samuels, G.M.R., Terrett, N.K., and Wythes, M.J. 1989. UK-69,578, a novel inhibitor of EC 3.4.24.11 which increases endogenous ANF levels and is natriuretic and diuretic. Biochem. Biophys. Res. Commun. 164: 58-65. Deng, Y., Martin, L.L., DelGrande, D., and Jeng, A.Y. 1992. A soluble protease identified from rat kidney degrades endothelin-1 but not proendothelin-1. J. Biochem. (Tokyo). 112: 168-172. Dunn, B.M. 1989. Determination of protease mechanism. Edited by R.J. Beynon and J.S. Bond. IRL Press, Oxford, England. pp. 57-81. Fagny, C., Michel, A., Leonard, I., Berkenboom, G., Fontaine, J., and Deschodt-Lanckman, M. 1991. In vitro degradation of endothelin- 1 by endopeptidase 24.11 (enkephalinase). Peptides (Fayetteville, N.Y.), 12: 773-778. Hemsen, A., Pernow, J., and Lundberg, J.M. 1991. Regional extraction of endothelins and conversion of big endothelin to endothelin-1 in the pig. Acta Physiol. Scand. 141: 325-334. Hirata, Y., Yoshimi, H., Takaichi, S., Yanagisawa, M., and Masaki, T. 1988. Binding and receptor down-regulation of a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells. FEBS Lett. 239: 13-17. Jeng, A.Y., Savage, P., Soriano, A., and Balwierczak, J.L. 1990. Different affinities and selectivities of endothelin-1 and
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NOTES
endothelin-3 binding to various rat tissues. Biochem. Int. 22: 669-676. Kasuya, Y., Ishikawa, T., Yanagisawa, M., Kimura, S., Goto, K., and Masaki, T. 1989. Mechanism of contraction to endothelin in isolated porcine coronary artery. Am. J. Physiol. 257: H1828-H1835. Kimura, S., Kasuya. Y., Sawamura, T., Shinmi, O., Sugita, Y., Yanagisawa, M., Goto, K., and Masaki, T. 1988. Structureactivity relationships of endothelin: importance of the C-terminal moiety. Biochem. Biophys. Res. Commun. 156: 1182-1 186. Leary, R., and Shaw, E. 1977. Inactivation of cathepsin B, by diazomethyl ketones. Biochem. Biophys. Res. Commun. 79: 926-93 1. Lecomte, J.-M., Baumer, P., Lim, C., Duchier, J., Cournot, A., Dussaule, J.-C., Ardaillou, R., Gros, C., Chaignon, B., Souque, A., and Schwartz, J.-C. 1990. Stereoselective protection of exogenous and endogenous atrial natriuretic factor by enkephalinaseinhibitors in mice and humans. Eur. J. Pharmacol. 179: 65-73. Masaki, T., Kimura, S., Yanagisawa, M., and Goto, K. 1991. Molecular and cellular mechanism of endothelin regulation. Implications for vascular function. Circulation, 84: 1457-1468. McMahon, E.G., Palomo, M.A., Moore, W.M., McDonald, J.F., and Stern, M.K. 1991. 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. Proc. Natl. Acad. Sci. U.S.A. 88: 703-707. Modin, A., Pernow, J., and Lundberg, J.M. 1991. Phosphoramidon inhibits the vasoconstrictor effects evoked by big endothelin-1 but not the elevation of plasma endothelin-1 in vivo. Life Sci. 49: 1619-1625. Patrignani, P., Del Maschio, A., Bazzoni, G., Daffonchio, L., Hernandez, A., Modica, R., Montesanti, L., Volpi, D., Patrono,
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C., and Dejana, E. 1991. Inactivation of endothelin by polymorphonuclear leukocyte-derived lytic enzymes. Blood, 78: 2715-2720. Pernow, J., Hemsen, A., and Lundberg, J.M. 1989. Tissue specific distribution, clearance and vascular effects of endothelin in the pig. Biochem. Biophys. Res. Commun. 161: 647-653. Randall, M.D. 1991. Vascular activities of the endothelins. Pharmacol. Ther. 50: 73-93. Simonson, M.S., and Dunn, M. J. 1991. Endothelins: a family of regulatory peptides. Hypertension (Suppl.), 17: 856-863. Sirvio, M.-L., Metsarinne, K., Saijonmaa, O., and Fyhrquist, F. 1990. Tissue distribution and half-life of '251-endothelinin the rat: importance of pulmonary clearance. Biochem. Biophys. Res. Commun. 167: 1191-1 195. Sonnenberg, J.L., Sakane, Y., Jeng, A.Y., Koehn, J.A., Ansell, J.A., Wennogle, L.P., and Ghai, R.D. 1988. Identification of protease 3.4.24.11 as the major atrial natriuretic factor degrading enzyme in the rat kidney. Peptides (Fayetteville, N.Y.), 9: 173-180. Vijayaraghavan, J., Scicli, A.G., Carretero., O.A., Slaughter, C., Moomaw, C., and Hersh, L.B. 1990. The hydrolysis of endothelins by neutral endopeptidase 24.11 (enkephalinase). J. Biol. Chem. 265: 14 150 - 14 155. Watanabe, H., Green, G.D. J., and Shaw, E. 1979. A comparison of the behavior of chymotrypsin and cathepsin B towards peptidyl diazomethyl ketones. Biochem. Biophys. Res. Comrnun. 89: 1354-1360. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T. 1988. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature (London), 332: 41 1-415.
