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613-618

THE BIOSYNTHESIS OF ENDOTHELIN-1 BY HUMAN POLYMORPHONUCLEAR LEUKOCYTES William

C. Sessa*, Semiko Kaw, Markus

Hecker

and John R.Vane

The William Harvey Research Institute, St. Bartholomew’s Hospital Medical College, Charterhouse Square, London EClM 6BQ, United Kingdom Received

December

7, 1990

Human polymorphonuclear leukocytes (PMNs) converted human big endothelin @ET; 2 LIM) to an endothelin-1 (ET-l) like contractile factor, as assessedby bioassay. The generation of this ET-1 like activity was rapid (minutes), time-dependent and more pronounced in non-activated cells, suggestinga yla;tial degradation b;ptiv;;$ PMNs. Ph?sphor;m&ljF (54;hp,!ttlta formatlon inhibited contractile ’ phenylmethylsulfonylfluoride (PMSF; 25 pg/ml), pepstatin A (i /(g/ml) 0; epoxysuccinyl-L-leucylamido-(guanidino)butane (E-64; 10 pug/ml) did not. Incubations of activated PMNs with PMSF significantly potentiated the generation of ET-1 like activity and selectively inhibited the degradation of [lZ51]ET-1 by activated PMNs. These findings indicate that human PMNs contain and/or release neutral proteases, which can both rapidly produce and degrade ET-l, an observation which may have important (patho)physiologic implications. cc1991Academic me**, Inc. Summary.

The 21-amino acid peptide endothelin-1 (ET-l)

is the most potent

vasoconstrictor known to date. It is formed by cultured endothelial cells from a 3%amino acid precursor, big endothelin (bET), via a proteolytic cleavage between Trp2’ and Va122 (1). Since its discovery, this rather unusual processing of bET has been a matter of intense investigation. Hitherto, a cathepsin D-like aspartic protease, which generates ET-l

from bET at acid pH, has been

characterized in porcine and bovine aortic cultured endothelial cells (2,3), bovine adrenal medullary

cells (4) and rat lung (5). In

addition,

a

metalloprotease, which is inhibited by phosphoramidon, has been implicated in the conversion of bET to ET-l by bovine or porcine aortic endothelial cells (6,7). Interestingly, phosphoramidon also prevents the pressor effects of bET in anaesthetized rats without affecting responsesto ET-l (8). Polymorphonuclear leukocytes (PMNs) are a rich source of proteases and, hence, have the capacity to convert inactive peptides, such as angiotensinogen or kininogen, to biologically active products, ie. angiotensin II

*To whom correspondence should be addressed. 0006-291X/91

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(10). Proteases of the cathepsin family, in particular,

cathepsin

G (ll), are abundant in PMNs and the secretion of cathepsin G is an index of PMN activation in vitro (12). We have, therefore, investigated whether freshly isolated human PMNs can convert bET to ET-l or, alternatively, degrade ET-l, and attempted to characterize MATERIALS

the enzyme(s)

involved therein.

AND METHODS

Materials. Synthetic human bET and ET-l were purchased from the Peptide Research Institute (Osaka, Japan). [Tyr’3-‘2SI]ET-1 (specific activity 2000 Ci/mmol) was obtained from Amersham International plc (Amersham, U.K.). Phenylmethylsulfonylfluoride (PMSF), pepstatin A, phosphoramidon, epoxysuccinyl-L-leucylamido-( uanidino)butane (E-64), N-formyl-L-methionyl-Lleucyl-L-phenylalanine (fMLP f , cathepsin D, human leukocyte cathepsin G, thermolysin, cytochalasin B, indomethacin, mepyramine, methysergide and prazosin were purchased from Sigma Chemical Co. (Poole, U.K.). Bioassay. Male New Zealand white rabbits (2.0-2.5 kg) were sacrificed by an intravenous injection of sodium pentobarbital (60 mg/kg). The jugular vein (RbJV) was removed, trimmed of periadventitial fat, cut into a helical strip and mounted in a superfusion bioassay cascade (13) under 1.0 g pre-load. The tissue was superfused at 5 ml/min with warmed (37’C), oxygenated (95% 0,/5% CO,) Krebs’ solution containing 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH,PO,, 1.27 mM MgSO;7H,O, 2.5 mM CaCl*6H,O, 25 mM NaHCO, and 5.6 mM glucose. In order to block the actions of various vasoactive substances released from PMNs, the following mixture of antagonists was also added to the Krebs’ solution: mepyramine (1 PM), prazosin (1 PM), methysergide (0.1 PM), and indomethacin (5.6 PM). The lengths of the RbJV was monitored with auxotonic levers attached to a Harvard isotonic transducer and displayed on a Graphtec WR3101 recorder. Preparation and incubation of human PMNs. Human PMNs were prepared from titrated blood as previously described for rabbit PMNs (14). Cell suspensions contained 2.5-4.0 x lo7 cells/ml and were greater than 97% viable, as assessed by trypan blue exclusion. PMNs (4 x lo6 cells/ml) were incubated in phosphate-buffered saline containing glucose (5.6 PM), CaCl .6H,O (1 mM), MgCl, (0.5 mM) and bET (2 nmol/mk) or ET-l (0.1-l nmo!l/ml’) in a final volume of 250 ~1 for O-60 min at 37 C. To activate the cells, PMNs were incubated with fMLP (100 nM) and cytochalasin B (5 pg/ml). In some experiments one of the following protease inhibitors was added to the incubation mixture: PMSF (25 pg/ml), pepstatin A (1 pg/ml), phosphoramidon (54 pug/ml) or E-64 (10 pg/ml). At the end of each incubation period, the cells were centrifuged for 2 min in an Eppendorf microfuge, and aliquots of the cell-free supernatants were injected directly over the RbJV to assess ET-l-like constricting activity. HPLC analysis of ET-l degradation. PMNs (4 x lo6 cells/ml) were incubated with 0.1 pmol [Tyr’3-‘251]ET-1 for O-60 min as described previously. The incubations were terminated by acidification of the samples to pH 3.0 with trifluoroacetic acid (TFA) and addition of 10 mM EDTA (final concentration) to remove any residual calcium. After 10 min at 0-4’C, the samples were centrifuged at 10,000 x 6 for 10 min followed by solid-phase extraction of the supernatant with octadecylsilica cartridges (TechElut). The extraction solvent (methanol) was evaporated under a stream of nitrogen and the residues dissolved in 100~1 of 20% acetonitrile in 0.05% TFA. The processedsample was then applied to a TOSOH TSK gel ODS-120T reversed phase HPLC column (250x4.6(i.d.)mm from Anachem, Luton, U.K.) fitted with a SynChropak RP SOx4.6(i.d.)mm guard column (from FSA Laboratory Supplies, Loughborough, U.K.). The column was eluted isocratically at 1 ml/min with 614

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acetonitrile/TFA/water 35:0.05:65 (v/v) for 20 min followed by 100% methanol for 10 min. Radioactivity in the eluate was monitored with a Berthold LB 506 AT on-line radioactivity monitoring system. [‘251]-labelled ET-l eluted at 12.5 min.

RESULTS Bolus injections of ET-l

(l-5 pmol) over the RbJV elicited contractions

of the

tissue which were rapid in onset (ca. 30 set) and long in duration (ca. 15 min). Human bET evoked similar contractions but was 250 to 500 times less potent than ET-l. Within 2 min at 37’C and pH 7.4, both non-activated and fMLPactivated PMNs (4 x lo6 cells/ml) generated ET-l like activity from bET (2 nmol/ml). The appearance of this activity was time-dependent in non-activated cells, but followed a “bell-shaped” pattern in activated cells (ie. a time-dependent increase at 2-5 min follwed by a time-dependent decrease in production at 15-30 min; n =3). As shown in Fig. lA, after 30 min non-activated PMNs generated an ET-l like activty from bET which was not detected with cells or bET alone

ET-1 (1 pm4

(100

pmol)

PMNstfMLP

(100

nM)

PMNstbETtfMLP t PMSF

(25 pg/ml)

_. ET-l in PBS (1 pmol)

F&J.

ET-l activated

t PMNs

Al activated t PMSF

+ PMNs (25 pg/ml)

PMNs generate ET-I like activity from bET (A) and degrade ET-I (B). The figure shows the contractions of a superfused RbJV denuded of endothelium. A) Bolus injection of ET-l (1 pmol) elicited a contraction of the RbJV similar to that seen with cell-free supernatants from PMNs (4 x 106/ml) incubated (30 min, 37’C) with bET (2 nmol/ml). Cells alone or bET incubated for the same length of time did not yield ET-l like activity. Incubation of fMLP (100 nM)-activated PMNs with bET did not yield ET-1 like activity unless PMSF (25 pg/ml) was added to the incubation. This trace is representative of 5 experiments. B) Incubation of ET-1 (30 min, 37’C) with fMLP-activated PMNs (4 x 10h/ml) resulted in the loss of its biologic activity. This loss of activity was partially prevented by co-incubation with PMSF (25 @g/ml). Similar results were obtained in 5 other experiments. 615

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(n=5). However, the yield of activity was less in cell-free supernatants from activated cells than in those from non-activated cells. The formation of the contractile

factor

by both non-activated

and activated

cells was inhibited

by

phosphoramidon (54 pg/ml), but not by pepstatin A (1 yg/ml) or E-64 (10 pug/ml); n=3 for each inhibitor). Incubations of activated PMNs with PMSF (25 ,ug/ml) increased the apparent conversion

of bET to ET-l,

of the ET-1 produced had been subsequently The contractile Krebs’

buffer

activity of ET-l,

suggesting that some

degraded (n=5; Fig.lA).

which was not affected by incubation

for 30 min, was substantially

activated PMNs, indicative of its degradation ET-l was prevented by PMSF (25 pg/ml;

reduced

when

incubated

in with

(n=5; Fig. 1B). The metabolism

n=5), but not by phosphoramidon

of (54

pg/ml), pepstatin A (1 pg/ml) or E-64 (10 pug/ml; n=3 for each inhibitor). In addition, supernatants prepared from activated PMNs also degraded ET-1 and this metabolism

was

inhibited

[1251]ET-1 was metabolized at least 5 different

by PMSF

(n=3).

In separate

experiments,

by activated PMNs in a time-dependent

products,

as determined

by HPLC

analysis (65.6 +. 6.4 and

72.9 +. 6.8% metabolism

after 30 and 60 min, respectively;

metabolism of [12j IlET54.5 f_ 14.8% inhibition

at 30 and 60 min, respectively;

was prevented

phosphoramidon (54 pg/ml), PMNs metabolized [125 IlET-

manner to

n=3; Fig. 2). This

by PMSF (25 pg/ml;

79.3 + 9.3 and

n=3;),

but not by

thus confirming the bioassay results. Moreover, to products which were similar to those generated

[‘251]ET-l v

I

60 min

0

5

10

15

20

Time (min)

m.

The PMSF.

degradation

of /12’ IlET-

by fMLP-activated

PMNs

is inhibited

by

The figure shows typical HPLC chromatograms representative of 3 experiments with PMNs from different blood donors. In the top trace, [12 IlETwas added to the cells and immediately extracted for HPLC analysis. Similar results were obtained when [‘2SI]ET-1 was incubated in PBS for 60 min. In the middle and bottom traces, PMNs were incubated with [“SI]ET-l (60 min, 37’C) in the absence or presence of PMSF (25 pg/ml), and samples prepared for HPLC analysis as described in Materials a/zd

Methods.

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by thermolysin

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(15), cathepsin D or cathepsin G (5 pug/ml of each). In addition,

phosphoramidon whereas

AND

inhibited

the metabolism

of [‘251]ET-1

by thermolysin,

PMSF inhibited the degradation by cathepsin G (data not shown).

DISCUSSION This study demonstrates inactivate ET-l

that PMNs can convert

ET-1 by two distinct enzymatic activities.

like activity occurs rapidly in both non-activated

inhibited

by phosphoramidon,

aspartate

proteinases.

Endothelial

to ET-l pressor

(6,7). Moreover, effects

endothelium

participates

metalloendopeptidase

in anaesthetized

which

suggesting

bET

inhibits

pathway,

the

it is possible

to the pressor

HPLC analysis clearly demonstrates by a phosphoramidon

observation),

bET is cleaved by PMN-derived

converts

that the vascular

of bET to ET-1 by PMNs may also contribute

et rrl., unpublished

or

of bET in vivo (8). As PMNs convert

in the conversion

effects of this peptide in vivo. Although PMNs convert [1251]bET to [125 IlET(Hecker

cysteine

rats, phosphoramidon

activity via a phosphoramidon-sensitive

that the conversion

of serine,

cells cultured from porcine or bovine aorta

of bET, but not those of ET-l,

bET to ET-l-like

and also of bET to

and activated PMNs and is

but not by inhibitors

contain a phosphoramidon-sensitive

bET to ET-l

The conversion

that

sensitive cleavage

we can not rule out the possibility

that

cathepsin G to a peptide chromatographically

distinct from ET-1 but with similar biological activity (see 17). The time-dependent

decrease in the generation

activity from bET in fMLP-activated

of ET-l

like contractile

PMNs suggested that the ET-l formed was

subsequently degraded. Indeed this was the case, for PMSF potentiated formation of ET-l like activity from bET and selectively inhibited degradation HPLC ET-l.

of ET-l

by activated PMNs, as demonstrated

the the

by both bioassay and

analysis. Relatively little is known about proteases capable of degrading Recent data indicate that a phosphoramidon-sensitive neutral

endopeptidase

prepared from rat kidney or lung can degrade ET-1 (15,16). The

enzyme responsible

for ET-1 metabolism

by human PMNs is clearly different,

for

PMSF and not phosphoramidon, inhibits the metabolism of ET-1 by these cells. As PMSF and other serine proteinase inhibitors block the activities of leukocytederived cathepsin G (l&19)

or elastase (20), it is likely that either one or both of

these enzymes is responsible

for the degradation of ET-l.

Thus, PMNs express the dual capacity to form and to degrade ET-l by at least two different neutral proteases. As PMNs adhere and accumulate at discrete sites within the circulation

in pathologic conditons such as myocardial

infarction,

hypertension, endotoxin shock, acute inflammation or renal failure, the balance between the conversion of bET to ET-l and the subsequent metabolism of ET-1 by PMNs may contribute to the pathogenesis of such diseases. 617

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ACKNOWLEDGMENTS The authors thank Mr. Timothy Desmond incubations Parke-Davis

Walsh for his help with the cell

and the HPLC analysis. This work was supported by a grant from Pharmaceutical Research Division of Warner-Lambert Company.

REFERENCES 1. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazake, Y, Goto, K. & Masaki, T. (1988) Nature 332,411-415. 2. Matsumura, Y., Ikegawa, R., Takakoa, M. & Morimoto, S. (1990) Biochem. Biophys. Res. Commun. 167,203-210. 3. Sawamura, T., Kimura, S., Shinmi, O., Sugita, Y., Kobayashi, M., Mitsui, Y., Yanagisawa, M., Goto. K. & Masaki, T. (1990) Biochem. Biophys. Res. Commun. 169, 1138-l 144. 4. Sawamura, T., Kimura, S., Shinmi, O., Sugita, Y., Yanagisawa, M., Goto, K. & Masaki, T. (1990). B&hem. Biophys. Res. Commun. 168, 1230-1236. 5. Wu-Wong, JR., Budzik, G.P., Devine, E.M. & Opgenworth, T. (1990) Biochem. Biophys. Res. Commun. 174,1291-1296. 6. Ikegawa, R., Matsumura, Y., Tsukahara, Y., Takakoa, M. & Morimoto, S. (1990) Biochem. Biophys. Res. Commun. 171,669-675. 7. Okada, K., Miyzaki, Y., Takada, J., Matsuyama, K., Yamaki, T. & Yano, M. (1990). Biochem. Biophys. Res. Commun. 171,1192-1198.2. 8. Matsumura, Y., Hisaki, K., Takakoa, M. & Morimoto, S. (1990) Eur. J. Pharmacol. 185, 103-106. 9. Wintroub, B.U., Klickstein, L.B., Dzau, V.J. & Watt, K.W. (1984) Biochemistry 23, 227-232. 10. Greenbaum, L.M. & Kim, K.S. (1967) Br. J. Pharmacol. 29,613-623. 11. Bagglioni, M. & Dewald, B. (1985) In: Regulation of Leukocyte Function (Snyderman, R, ed); 21-46, Plenum Press, N.Y. 12. Heck, L.W., Rostrand, K.S., Hunter, F.A. & Bhoun, A. (1986) Anal. Biochem. 158,217-227. 13. Vane, J.R. (1964) Br. J. Pharmacol. Chemother. 23,360-373. 14. Sessa, W.C. & Mullane, K.M. (1990) Br. J. Pharmacol. 99,553-559. 15. Vijayaraghavan, J., Scicli, A.G., Carretaro, O.A., Slaughter, C., Moomaw, C. & Hersh, L. (1990) J. Biol. Chem. 265,14150-14155. 16. Scicli, A.G., Vijayaraghavan, J., Hersh, L.B. & Carretaro, O.A. (1989) Hypertension 14,353-3.59. 17. Patterson, K., Macnauf, R., Rubanyi, G.M. & Parker-Botelho, L.H. (1990) FASEB J. 4, A909. 18. Selak, M.A., Chignard, M. & Smith, J.B. (1988) Biochem. J. 251, 293-299. 19. Ferrer-Lopez, P., Renesto, P., Schattner, M., Bassot, S., Laurent, P. & Chignard, M. (1990) Am. J. Physiol. 258, CllOO-C1107. 20. Travis, J. (1988) Am. J. Med. 84,37-42.

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The biosynthesis of endothelin-1 by human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMNs) converted human big endothelin (bET; 2 microM) to an endothelin-1 (ET-1) like contractile factor, as assesse...
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