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178,
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Pages 899-905
15, 1991
CONVERSION OF BIG ENDOTHELIN-1 TO ENDOTHELIN-1 BY TWOTYPES OF METALLOPROTEINASES OF CULTURED PORCINE VASCULAR SMOOTH MUSCLE CELLS
Yasuo Matsumura,
Ruriko
of Pharmacology,
Department
2-10-65
Received
June
12,
Yaeko Tsukahara, and Shiro Morimoto Ikegawa,
Kawai,
Masanori
Osaka University of Pharmaceutical Matsubara, Osaka 580, Japan
Takaoka,
Sciences
1991
SUMMARY: Incubation of big endothelin-1 (big ET-l, l-39) with the membranefraction obtained from cultured vascular smooth muscle cells (VSMCs) resulted in an increase in immunoreactive-ET (IR-ET), which was inhibited by EDTA but not by phosphoramidon,a metalloproteinase inhibitor. When the incubation was performed in the presence of Nethylmaleimide (NEM), the generation of IR-ET was markedly augmented and this augmentationwas abolishedby phosphoramidon. The pH profile for IR-ET generationin the presenceof NEM was apparentlydistinct from that observedin the absenceof NEM. Reversephase HPLC of the incubation mixture with or without NEM revealed one major IR-ET componentcorrespondingta the elution position of synthetic ET- 1 (l-2 1). When the cultured VSMCs were incubated with big ET-l, a conversion to the mature ET-l was observed. This ET- 1 generationfrom exogenouslyappliedbig ET- 1 wasmarkedly inhibited by the addition of phosphoramidon,although the inhibitor did not influence the basal secretion of ET-l-like materials. These results suggest the presenceof two types of metalloproteinases,which can generate ET-l, in VSMCs. The possibility that ET-I functions in an autocrine manner to control the cardiovascularsystemwarrantsfurther attention. 0 1991Academic we**, Inc. Endothelin-1 (ET-l) is a 21-amino acid vasoconstrictor peptide first isolated from the culture supematantof vascular endothelial cells (ECs) (1). Basedon the amino acid sequence of the prepro-form, ET-l is consideredto be producedfrom a 39-aminoacid intermediateform, termed big ET-I, through an unusual proteolytic processingat the Trp21-Va122 bond by a putative ET-converting enzyme (ECE) (1). Using cultured ECs, two types of proteinaseshave been proposedto be a possiblecandidate for ECE. First, we noted that a mature ET-l was generatedfrom big ET- 1 via a singlecleavagebetweenTrp21 and Va122by pepstatin-sensitive aspartic proteinasein ECs (2,3). Subsequently, Sawamura et al. (4) demonstratedthat the pepstatin-sensitiveconverting activities were due to cathepsinD. Since this enzyme is active only at acidic pH and since cathepsinD from bovine spleencleaves the Asn18-Ile19 bond in addition to the Trp21-Va122 bond of big ET-I (5,6), the physiological relevance of the conversion by cathepsinD is unclear. On the other hand, we (7) and others (8) have recently found usingcultured vascularECs that a neutral metalloproteinasesensitiveto phosphoramidon can specifically cleave Trp21-Va122 bond of big ET-l. We reported that the intravenous administration of phosphoramidonto anesthetizedrats markedly suppressedthe big ET-linduced hypertensive effect without affecting the hypertension induced by ET-l (9). Similar
899
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results were subsequently obtained by Fukuroda et al. (10) and McMahon et al. (11). Furthermore, we noted that phosphoramidon caused a decrease in ET-l secretion and an increase in big ET-l secretion from cultured ECs, probably resulting from the inhibition of ECE by phosphoramidon ( 12). These findings strongly suggest that phosphoramidon-sensitive metalloproteinase is responsible for the conversion of big ET- 1 in vascular ECs. Resink et al. (13) demonstrated that cultured vascular smooth muscle cells (VSMCs)
can
express ETmRNA and secrete ET-l-like materials and they proposed an autocrine regulatory mechanism of the action for ET in addition to the known paracrine function of ET in the vasculature.
We report
here that cultured
VSMCs
contain
two
types
of neutral
metalloproteinases which convert big ET- 1 to ET-l.
MATERIALS
AND METHODS
Cell Culture and Preparation of Membrane Fraction: Aortas were obtained from freshly killed pigs. The outer adventitial layer and the endothelial layer were removed. VSMCs were cultured from medial explants and used between the 5th and 10th passages. To obtain the membrane fraction of VSMCs, the confluent cells were scraped with a Cell Lifter (Costar, MA). After washing with phosphate buffered saline, the cells were homogenized in ice-cold 20 mM Tris-HCI buffer (pH 8.0) containing 30 mM KCI, then the preparation was centrifuged at 105,000 x g for 30 min. The pellet was washed with the Tris-HC1 buffer and resuspended in the same buffer, then the preparation was used as the membrane fraction. Measurement of ET Converting Activity: Fifty pl of the membrane fraction (derived from l06cells) and 0.05 ml of enzyme inhibitor solution were mixed with 0.35 ml of 50 mM sodium phosphate buffer (pH 6.0-7.5). After preincubation at 37°C for 30 min, 0.05 ml of porcine big ET-1 solution (final concentration: 100 ng big ET-l/ml) was added to the mixture and the preparation was incubated at 37°C for 0.5-24 hr. The reaction was stopped by boiling for 10 min. The samples were neutralized and centrifuged at 8,000 x g for 5 min. The resulting supernatant was used for the radioimmunoassay (RIA) and reverse-phase (RP)HPLC. Radioimmunoassay (RIA): RIAs for ET and the C-terminal fragment (CTF, 22-39) of big ET-1 were performed as described (2,3). In RIA for ET, the cross-reactivity with big ET-l was lower than O.l%, whereas no cross-reactivity was observed with ET-l in RIA for CTF. The antiserum used in the latter had a 100% cross-reactivity with big ET-l. Reverse-Phase High Performance Liquid Chromatography (RP-HPLC): RPHPLC was performed using a Capcell-Pak 5C18-SG300 column (4.6 x 250 mm, Shiseido, Tokyo, Japan) eluted with a linear gradient from 0% to 35% CH3CN in 0.02% trifluoroacetic acid (TFA) for 15 min, followed by isocratic elution at 35% CH3CN in 0.02% TFA for 15 min and a linear gradient from 35% to 63% CH3CN in 0.02% TFA for 15 min. The flow rate was 0.5 ml/min. Each fraction was evaporated and assayed for immunoreactive (IR)-ET and IRCTF, using RIA. Peptides: Synthetic porcine ET-l (1-21) and big ET-l (l-39) were obtained from Peptide Institute Inc. (Osaka, Japan). The CTF (22-39) was prepared by solid phase synthesis, The homogeneity was confirmed by RP-HPLC and by amino acid analysis.
RESULTS
AND
DISCUSSION
When big ET-l was incubated with the membrane fraction of cultured VSMCs at pH 6.5 for 6 hr, the IR-ET content in the reaction mixture increased from 0.21 f 0.01 (the value 900
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NEM , phosphoramidon NM EDTA captoprir thiorphan phosphoramldon bestatin p-APMSF chymostattn
no addition 0
100
Increase
200
in IR-ET
300
(%)
of IR-ET inducedby the Fieure 1, Effects of variousagentson the increasedresponses membrane fraction of culturedVSMCs. Valuesrepresentthe meanf S.E. from four to six separateexperiments. E-64 ( lO-4M), chymostatin(2 x lO-6M), p-APMSF (5 x IO-4M), bestatin(4 x 10ms M), phosphoramidon (lo-4 M), thiorphan( lO-5M), captopril( lO-4M), EDTA (lo-3 M), NEM (lo-3 M).
without the membrane fraction) to 1.14 f 0.14 ng IR-ET/ml.
Although no appreciable
alteration in the IR-ET generationwas observedby enzyme inhibitors such as E-64 (lo-4 M), chymostatin (2 x lo-6 M), p-APMSF (5 x 10e4M), bestatin (4 x 10-s M), phophoramidon (10-J M), thiorphan (lo-3 M) and captopril (10m4M), the metal chelator EDTA (lo-3 M) completely inhibited the IR-ET generation by the membrane fraction.
Addition of the
sulfhydryl blocking reagentN-ethylmeleimide (NEM, 10-3M) markedly augmentedthe IR-ET generation, and interestingly this augmentation was abolished in the presence of the metalloproteinase inhibitor phosphoramidon (Fig. 1). We further evaluated the effect of phosphoramidonon IR-ET generationin the absenceor presenceof NEM. Figure 2 showsthe time courseof IR-ET generationduring incubation of big ET- 1 with the membranefraction. In the absenceof NEM, phosphoramidoncausedno remarkablechangeon the IR-ET generation, whereasthis metalloproteinaseinhibitor produced 70-90%inhibition on the increasedresponse of IR-ET in the presenceof NEM. As shown in Fig. 3, the pH profiles of the increased responsesof IR-ET in the absenceor presenceof NEM exhibited a highly contrastingpattern. The optimum pH of the former and the latter was 7.0 and 6.5, respectively. Phosphoramidon markedly suppressedonly the increasedresponsesin the caseof NEM treatment. From these results, we assumethat different types of metalloproteinasesare responsiblefor the IR-ET generationin the absenceand presenceof NEM. We reportedthat porcine cultured ECs contain two types of metalloproteinases(one is phosphoramidon-sensitive and membrane-boundform, and the other is phosphoramidon-insensitive and solubleform) which have big ET-l converting activity (7). Furthermore, we noted that the phosphoramidon-insensitive converting activity is markedly suppressedby NEM and that enzymatic degradationof ET-l and big ET-l during incubation with the membranefraction of cultured ECs is inhibited by NEM (Matsumura et al., 901
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Figure 2. Time course of the increase in IR-ET during incubation of big ET-l with the membrane fraction of cultured VSMCs. Values represent the mean f S.E. from four separate experiments. 3. Effectsof pH on the increase in IR-ET duringincubationof big ET-I with the membrane fractionof culturedVSMCs. Valuesrepresent the mean+ S.E.from four separate experiments. Figure
submitted). Taken together, it seemsreasonableto considerthat in the caseof the incubationof big ET-l with the membrane fraction of cultured VSMCs, phosphoramidon-sensitive converting activities are maskedby thoseinsensitiveto phosphoramidonand the degradationof peptides,and the NEM treatment appearsto unmaskthe net converting activities sensitive to phosphoramidon. The RP-HPLC profiles of the reactionmixture of big ET-l with the membranefraction in the presenceof NEM revealed one major IR-ET component corresponding to the elution position of synthetic ET-1 (Fig. 4A), whereasIR-CTF consistedof one major and one minor component corresponding to elution position of synthetic porcine big ET-l and CTF, respectively (Fig. 4B). Qualitatively similar results were obtained in case of the reaction mixture without NEM (data not shown), although recovery of the big ET-l added to the reactionmixture waslower than that observedin the presenceof NEM (60-70% in the presence of NEM vs. 3040% in the absenceof NEM). This difference may be due to a nonspecific degradationof big ET- 1 by thiol-dependentproteinases,asindicated by Okada et al. (8) who useda membranefraction of cultured bovine ECsand p-chloromercuriphenylsulfonicacid. The addition of phosphoramidon to the reaction mixture in the presenceof NEM resulted in decreasesin ET-l- and CTF-like materials accompaniedby an increase in big ET-l -like materials(Fig. 4A and B). In the presentstudy, when the reactionmixture of big ET-l with the membranefraction of VSMCs was analysed by RP-HPLC, the yield of CTF-like materialswas considerably lower than that of ET-l-like materials. In caseof the reactionmixture with the cultured ECs, amounts of CTF- and ET-l-like materials were equally yielded on a molar basis (3,7). Thus, the 902
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40
Retention
time
50
(min)
Figure 4. RP-HPLC profiles of IR-ET (A) and IR-CTF (B) in the reaction mixture of big ET-1 with the membrane fraction of cultured VSMCs. Incubation was carried out at pH 6.5 for 6 hr with 1O-3M NEM, in the absence (0) or presence (0) of 1O-4 M phosphoramidon. Arrows indicate the elution positions of ET-l (l), CTF (2) and big ET-l (3).
membranefraction of cultured VSMCs may containdegradingenzymesof CTF. Alternatively, the generationof ET- 1 by the membranefraction of VSMCs may be mediated not only by the singlecleavagebetweenTrp21 andVa122but alsoby other cleavages. As shown in Fig. 5, the amount of IR-ET accumulating in the serum-free culture supematantof VSMCs was 0.11 + 0.01 pmol/106cells/24hr and this wasnot influenced by the presenceof phosphoramidon. On the other hand, an increase in IR-ET observed with the exogenous application of big ET-l (35 pmoI/l06cells) was markedly suppressed by
big ET-1 + phosphoramuion
big ET-1
phosphoramidon
no addition
00
0.2
04
IR-ET
(ptnol/106
06
06
1.0
cells124hr)
Fieure 5, Changes in IR-ET content in the culture medium of VSMCs after incubation for 24 hr. The cells grown in 60-mm Petri dishes (about 2 x 106 cells) were incubated with 3 ml of serum-free Dulbecco’s modified Eagle’s medium containing 0.01% heat-inactivated bovine serum albumin, in the absence or presence of phosphoramidon ( 1O-4 M) and big ET-l (70 pmol). Values represent the mean f S.E. from six to ten separate experiments. 903
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phosphoramidon. Theseresultssuggestthat two types of enzymes may function to generate ET-l in VSMCs. We have recently demonstratedusing cultured ECs that phosphoramidon markedly decreasesthe amountof ET-l secretedendogenouslyby inhibiting the conversion of big ET-l (12). Further, we noted that phosphoramidonabolishedthe increasedgenerationof ET-l observed with the exogenous
application of big ET-I (unpublishedobservation). Thus,
the conversion of exogenously applied big ET-I in VSMCs appears to be due to a phosphoramidon-sensitive enzyme similar to that in ECs. Since the amountof IR-ET secreted endogenously from cultured VSMCs was only about 3% of that from cultured ECs, the physiological significanceof this phenomenoninsensitiveto phosphoramidonis unclear. Noncultured vascular smoothmusclehomogenatesof bovine carotid arteriesconvert big ET-l to ET-l at both acid and neutral pH and conversion at the latter is inhibited by phosphoramidon or EDTA (14). In addition, Fukuroda et al. (10) demonstrated that phosphoramidonblocked the vasoconstrictionof isolated, non-endotheliumporcine coronary arteries caused by big ET-l, without affecting the ET-l-induced vasoconstriction, thereby suggestingthat the conversion of big ET-I by phosphoramidon-sensitive metalloproteinasein vascularsmoothmuscleis involved in the peptide-inducedvasoconstriction. Our resultssuggestthat cultured VSMCs contain two types of metalloproteinases,which convert big ET- 1 to the matureET- 1. One is phosphoramidon-sensitive and is at leastlikely to function to generate ET-l from exogenously applied big ET-I in the vasculature. Further studiesare required to clarify whether or not the other type is involved in the physiological conversionof big ET-l.
Acknowledgments We are grateful to Drs. H. Yoshizumi and T.Tanaka, Institute for FundamentalResearch, Suntory Ltd., for providing facilities to synthesizeCTF, and to M. Ohara for critical comments. This study was supportedin part by a Grant-in-Aid for Encouragementof Young Scientists from the Ministry of Education, ScienceandCulture of Japan.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K. and Masaki, T. (1988) Nature 332,411-415. Matsumura, Y., Ikegawa, R., Takaoka, M. and Morimoto, S. (1990) Biochem. Biophys. Res. Commun. 167, 203-210. Ikegawa, R., Matsumura, Y., Takaoka, M. and Morimoto, S. (1990) Biochem. Biophys. Res. Commun. 167, 860-866. Sawamura, T., Kimura, S., Shinmi, O., Sugita, Y., Kobayashi, M., Mitsui, Y., Yanagisawa, M., Goto, K. and Masaki, T. (1990) Biochem. Biophys. Res. Commun. 169, 1138-1144. Sawamura,T., Shinmi, O., Kishi, N., Sugita, Y., Yanagisawa, M., Goto, K., Masaki, T. and Kimura, S. (1990) Biochem. Biophys. Res. Commun. 172, 883-889. Takaoka, M., Hukumori, Y., Shiragami, K., Ikegawa, R., Matsumura, Y. and Morimoto, S. (1990) Biochem. Biophys. Res.Commun. 173, 1218-1223. Matsumura, Y., Ikegawa, R., Tsukahara, Y., Takaoka, M. and Morimoto, S. (1990) FEBS Lett. 272, 166-170. Okada, K., Miyazaki, Y., Takada, J., Matsuyama, K., Yamaki, T. and Yano, M. (1990) Biochem. Biophys. Res. Commun. 171, 1192-l 198. 904
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Matsumura, Y., Hisaki, K., Takaoka, M. and Morimoto, S. (1990) Eur. J. Pharmacol. 18.5, 103-106. 10. Fukuroda, T., Noguchi, K., Tsuchida, S., Nishikibe, M., Ikemoto, F., Okada, K. and Yano, M. (1990) Biochem. Biophys. Res. Commun. 172,390-395. 11. McMahon, E.G., Palomo, M.A., Moore, W.M., McDonald, J.F. and Stern, M.K. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 703-707. 12. Ikegawa, R., Matsumura, Y., Tsukahara, Y., Takaoka, M. and Morimoto, S. (1990) Biochem. Biophys. Res. Commun. 171, 669-675. 13. Resink, T.J., Hahn, A.W.A., Scott-Burden, T., Powell, J., Weber, E. and Btihler, F.R. (1990) Biochem. Biophys. Res. Commun. 168, 1303-1310. 14. Hioki, Y., Okada, K., Ito, H., Matsuyama, K. and Yano, M. (1991) Biochem. Biophys. Res. Commun. 174, 446-451. 9.
905
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Pages 899-905
15, 1991
CONVERSION OF BIG ENDOTHELIN-1 TO ENDOTHELIN-1 BY TWOTYPES OF METALLOPROTEINASES OF CULTURED PORCINE VASCULAR SMOOTH MUSCLE CELLS
Yasuo Matsumura,
Ruriko
of Pharmacology,
Department
2-10-65
Received
June
12,
Yaeko Tsukahara, and Shiro Morimoto Ikegawa,
Kawai,
Masanori
Osaka University of Pharmaceutical Matsubara, Osaka 580, Japan
Takaoka,
Sciences
1991
SUMMARY: Incubation of big endothelin-1 (big ET-l, l-39) with the membranefraction obtained from cultured vascular smooth muscle cells (VSMCs) resulted in an increase in immunoreactive-ET (IR-ET), which was inhibited by EDTA but not by phosphoramidon,a metalloproteinase inhibitor. When the incubation was performed in the presence of Nethylmaleimide (NEM), the generation of IR-ET was markedly augmented and this augmentationwas abolishedby phosphoramidon. The pH profile for IR-ET generationin the presenceof NEM was apparentlydistinct from that observedin the absenceof NEM. Reversephase HPLC of the incubation mixture with or without NEM revealed one major IR-ET componentcorrespondingta the elution position of synthetic ET- 1 (l-2 1). When the cultured VSMCs were incubated with big ET-l, a conversion to the mature ET-l was observed. This ET- 1 generationfrom exogenouslyappliedbig ET- 1 wasmarkedly inhibited by the addition of phosphoramidon,although the inhibitor did not influence the basal secretion of ET-l-like materials. These results suggest the presenceof two types of metalloproteinases,which can generate ET-l, in VSMCs. The possibility that ET-I functions in an autocrine manner to control the cardiovascularsystemwarrantsfurther attention. 0 1991Academic we**, Inc. Endothelin-1 (ET-l) is a 21-amino acid vasoconstrictor peptide first isolated from the culture supematantof vascular endothelial cells (ECs) (1). Basedon the amino acid sequence of the prepro-form, ET-l is consideredto be producedfrom a 39-aminoacid intermediateform, termed big ET-I, through an unusual proteolytic processingat the Trp21-Va122 bond by a putative ET-converting enzyme (ECE) (1). Using cultured ECs, two types of proteinaseshave been proposedto be a possiblecandidate for ECE. First, we noted that a mature ET-l was generatedfrom big ET- 1 via a singlecleavagebetweenTrp21 and Va122by pepstatin-sensitive aspartic proteinasein ECs (2,3). Subsequently, Sawamura et al. (4) demonstratedthat the pepstatin-sensitiveconverting activities were due to cathepsinD. Since this enzyme is active only at acidic pH and since cathepsinD from bovine spleencleaves the Asn18-Ile19 bond in addition to the Trp21-Va122 bond of big ET-I (5,6), the physiological relevance of the conversion by cathepsinD is unclear. On the other hand, we (7) and others (8) have recently found usingcultured vascularECs that a neutral metalloproteinasesensitiveto phosphoramidon can specifically cleave Trp21-Va122 bond of big ET-l. We reported that the intravenous administration of phosphoramidonto anesthetizedrats markedly suppressedthe big ET-linduced hypertensive effect without affecting the hypertension induced by ET-l (9). Similar
899
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results were subsequently obtained by Fukuroda et al. (10) and McMahon et al. (11). Furthermore, we noted that phosphoramidon caused a decrease in ET-l secretion and an increase in big ET-l secretion from cultured ECs, probably resulting from the inhibition of ECE by phosphoramidon ( 12). These findings strongly suggest that phosphoramidon-sensitive metalloproteinase is responsible for the conversion of big ET- 1 in vascular ECs. Resink et al. (13) demonstrated that cultured vascular smooth muscle cells (VSMCs)
can
express ETmRNA and secrete ET-l-like materials and they proposed an autocrine regulatory mechanism of the action for ET in addition to the known paracrine function of ET in the vasculature.
We report
here that cultured
VSMCs
contain
two
types
of neutral
metalloproteinases which convert big ET- 1 to ET-l.
MATERIALS
AND METHODS
Cell Culture and Preparation of Membrane Fraction: Aortas were obtained from freshly killed pigs. The outer adventitial layer and the endothelial layer were removed. VSMCs were cultured from medial explants and used between the 5th and 10th passages. To obtain the membrane fraction of VSMCs, the confluent cells were scraped with a Cell Lifter (Costar, MA). After washing with phosphate buffered saline, the cells were homogenized in ice-cold 20 mM Tris-HCI buffer (pH 8.0) containing 30 mM KCI, then the preparation was centrifuged at 105,000 x g for 30 min. The pellet was washed with the Tris-HC1 buffer and resuspended in the same buffer, then the preparation was used as the membrane fraction. Measurement of ET Converting Activity: Fifty pl of the membrane fraction (derived from l06cells) and 0.05 ml of enzyme inhibitor solution were mixed with 0.35 ml of 50 mM sodium phosphate buffer (pH 6.0-7.5). After preincubation at 37°C for 30 min, 0.05 ml of porcine big ET-1 solution (final concentration: 100 ng big ET-l/ml) was added to the mixture and the preparation was incubated at 37°C for 0.5-24 hr. The reaction was stopped by boiling for 10 min. The samples were neutralized and centrifuged at 8,000 x g for 5 min. The resulting supernatant was used for the radioimmunoassay (RIA) and reverse-phase (RP)HPLC. Radioimmunoassay (RIA): RIAs for ET and the C-terminal fragment (CTF, 22-39) of big ET-1 were performed as described (2,3). In RIA for ET, the cross-reactivity with big ET-l was lower than O.l%, whereas no cross-reactivity was observed with ET-l in RIA for CTF. The antiserum used in the latter had a 100% cross-reactivity with big ET-l. Reverse-Phase High Performance Liquid Chromatography (RP-HPLC): RPHPLC was performed using a Capcell-Pak 5C18-SG300 column (4.6 x 250 mm, Shiseido, Tokyo, Japan) eluted with a linear gradient from 0% to 35% CH3CN in 0.02% trifluoroacetic acid (TFA) for 15 min, followed by isocratic elution at 35% CH3CN in 0.02% TFA for 15 min and a linear gradient from 35% to 63% CH3CN in 0.02% TFA for 15 min. The flow rate was 0.5 ml/min. Each fraction was evaporated and assayed for immunoreactive (IR)-ET and IRCTF, using RIA. Peptides: Synthetic porcine ET-l (1-21) and big ET-l (l-39) were obtained from Peptide Institute Inc. (Osaka, Japan). The CTF (22-39) was prepared by solid phase synthesis, The homogeneity was confirmed by RP-HPLC and by amino acid analysis.
RESULTS
AND
DISCUSSION
When big ET-l was incubated with the membrane fraction of cultured VSMCs at pH 6.5 for 6 hr, the IR-ET content in the reaction mixture increased from 0.21 f 0.01 (the value 900
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178, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
NEM , phosphoramidon NM EDTA captoprir thiorphan phosphoramldon bestatin p-APMSF chymostattn
no addition 0
100
Increase
200
in IR-ET
300
(%)
of IR-ET inducedby the Fieure 1, Effects of variousagentson the increasedresponses membrane fraction of culturedVSMCs. Valuesrepresentthe meanf S.E. from four to six separateexperiments. E-64 ( lO-4M), chymostatin(2 x lO-6M), p-APMSF (5 x IO-4M), bestatin(4 x 10ms M), phosphoramidon (lo-4 M), thiorphan( lO-5M), captopril( lO-4M), EDTA (lo-3 M), NEM (lo-3 M).
without the membrane fraction) to 1.14 f 0.14 ng IR-ET/ml.
Although no appreciable
alteration in the IR-ET generationwas observedby enzyme inhibitors such as E-64 (lo-4 M), chymostatin (2 x lo-6 M), p-APMSF (5 x 10e4M), bestatin (4 x 10-s M), phophoramidon (10-J M), thiorphan (lo-3 M) and captopril (10m4M), the metal chelator EDTA (lo-3 M) completely inhibited the IR-ET generation by the membrane fraction.
Addition of the
sulfhydryl blocking reagentN-ethylmeleimide (NEM, 10-3M) markedly augmentedthe IR-ET generation, and interestingly this augmentation was abolished in the presence of the metalloproteinase inhibitor phosphoramidon (Fig. 1). We further evaluated the effect of phosphoramidonon IR-ET generationin the absenceor presenceof NEM. Figure 2 showsthe time courseof IR-ET generationduring incubation of big ET- 1 with the membranefraction. In the absenceof NEM, phosphoramidoncausedno remarkablechangeon the IR-ET generation, whereasthis metalloproteinaseinhibitor produced 70-90%inhibition on the increasedresponse of IR-ET in the presenceof NEM. As shown in Fig. 3, the pH profiles of the increased responsesof IR-ET in the absenceor presenceof NEM exhibited a highly contrastingpattern. The optimum pH of the former and the latter was 7.0 and 6.5, respectively. Phosphoramidon markedly suppressedonly the increasedresponsesin the caseof NEM treatment. From these results, we assumethat different types of metalloproteinasesare responsiblefor the IR-ET generationin the absenceand presenceof NEM. We reportedthat porcine cultured ECs contain two types of metalloproteinases(one is phosphoramidon-sensitive and membrane-boundform, and the other is phosphoramidon-insensitive and solubleform) which have big ET-l converting activity (7). Furthermore, we noted that the phosphoramidon-insensitive converting activity is markedly suppressedby NEM and that enzymatic degradationof ET-l and big ET-l during incubation with the membranefraction of cultured ECs is inhibited by NEM (Matsumura et al., 901
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(hr)
Figure 2. Time course of the increase in IR-ET during incubation of big ET-l with the membrane fraction of cultured VSMCs. Values represent the mean f S.E. from four separate experiments. 3. Effectsof pH on the increase in IR-ET duringincubationof big ET-I with the membrane fractionof culturedVSMCs. Valuesrepresent the mean+ S.E.from four separate experiments. Figure
submitted). Taken together, it seemsreasonableto considerthat in the caseof the incubationof big ET-l with the membrane fraction of cultured VSMCs, phosphoramidon-sensitive converting activities are maskedby thoseinsensitiveto phosphoramidonand the degradationof peptides,and the NEM treatment appearsto unmaskthe net converting activities sensitive to phosphoramidon. The RP-HPLC profiles of the reactionmixture of big ET-l with the membranefraction in the presenceof NEM revealed one major IR-ET component corresponding to the elution position of synthetic ET-1 (Fig. 4A), whereasIR-CTF consistedof one major and one minor component corresponding to elution position of synthetic porcine big ET-l and CTF, respectively (Fig. 4B). Qualitatively similar results were obtained in case of the reaction mixture without NEM (data not shown), although recovery of the big ET-l added to the reactionmixture waslower than that observedin the presenceof NEM (60-70% in the presence of NEM vs. 3040% in the absenceof NEM). This difference may be due to a nonspecific degradationof big ET- 1 by thiol-dependentproteinases,asindicated by Okada et al. (8) who useda membranefraction of cultured bovine ECsand p-chloromercuriphenylsulfonicacid. The addition of phosphoramidon to the reaction mixture in the presenceof NEM resulted in decreasesin ET-l- and CTF-like materials accompaniedby an increase in big ET-l -like materials(Fig. 4A and B). In the presentstudy, when the reactionmixture of big ET-l with the membranefraction of VSMCs was analysed by RP-HPLC, the yield of CTF-like materialswas considerably lower than that of ET-l-like materials. In caseof the reactionmixture with the cultured ECs, amounts of CTF- and ET-l-like materials were equally yielded on a molar basis (3,7). Thus, the 902
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Figure 4. RP-HPLC profiles of IR-ET (A) and IR-CTF (B) in the reaction mixture of big ET-1 with the membrane fraction of cultured VSMCs. Incubation was carried out at pH 6.5 for 6 hr with 1O-3M NEM, in the absence (0) or presence (0) of 1O-4 M phosphoramidon. Arrows indicate the elution positions of ET-l (l), CTF (2) and big ET-l (3).
membranefraction of cultured VSMCs may containdegradingenzymesof CTF. Alternatively, the generationof ET- 1 by the membranefraction of VSMCs may be mediated not only by the singlecleavagebetweenTrp21 andVa122but alsoby other cleavages. As shown in Fig. 5, the amount of IR-ET accumulating in the serum-free culture supematantof VSMCs was 0.11 + 0.01 pmol/106cells/24hr and this wasnot influenced by the presenceof phosphoramidon. On the other hand, an increase in IR-ET observed with the exogenous application of big ET-l (35 pmoI/l06cells) was markedly suppressed by
big ET-1 + phosphoramuion
big ET-1
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00
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04
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Fieure 5, Changes in IR-ET content in the culture medium of VSMCs after incubation for 24 hr. The cells grown in 60-mm Petri dishes (about 2 x 106 cells) were incubated with 3 ml of serum-free Dulbecco’s modified Eagle’s medium containing 0.01% heat-inactivated bovine serum albumin, in the absence or presence of phosphoramidon ( 1O-4 M) and big ET-l (70 pmol). Values represent the mean f S.E. from six to ten separate experiments. 903
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phosphoramidon. Theseresultssuggestthat two types of enzymes may function to generate ET-l in VSMCs. We have recently demonstratedusing cultured ECs that phosphoramidon markedly decreasesthe amountof ET-l secretedendogenouslyby inhibiting the conversion of big ET-l (12). Further, we noted that phosphoramidonabolishedthe increasedgenerationof ET-l observed with the exogenous
application of big ET-I (unpublishedobservation). Thus,
the conversion of exogenously applied big ET-I in VSMCs appears to be due to a phosphoramidon-sensitive enzyme similar to that in ECs. Since the amountof IR-ET secreted endogenously from cultured VSMCs was only about 3% of that from cultured ECs, the physiological significanceof this phenomenoninsensitiveto phosphoramidonis unclear. Noncultured vascular smoothmusclehomogenatesof bovine carotid arteriesconvert big ET-l to ET-l at both acid and neutral pH and conversion at the latter is inhibited by phosphoramidon or EDTA (14). In addition, Fukuroda et al. (10) demonstrated that phosphoramidonblocked the vasoconstrictionof isolated, non-endotheliumporcine coronary arteries caused by big ET-l, without affecting the ET-l-induced vasoconstriction, thereby suggestingthat the conversion of big ET-I by phosphoramidon-sensitive metalloproteinasein vascularsmoothmuscleis involved in the peptide-inducedvasoconstriction. Our resultssuggestthat cultured VSMCs contain two types of metalloproteinases,which convert big ET- 1 to the matureET- 1. One is phosphoramidon-sensitive and is at leastlikely to function to generate ET-l from exogenously applied big ET-I in the vasculature. Further studiesare required to clarify whether or not the other type is involved in the physiological conversionof big ET-l.
Acknowledgments We are grateful to Drs. H. Yoshizumi and T.Tanaka, Institute for FundamentalResearch, Suntory Ltd., for providing facilities to synthesizeCTF, and to M. Ohara for critical comments. This study was supportedin part by a Grant-in-Aid for Encouragementof Young Scientists from the Ministry of Education, ScienceandCulture of Japan.
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