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Stimulation of endothelial cells by protease activity in commercial preparations of xanthine oxidase
THROMBOSIS RESEARCH 35; 43-52, 1984 0049-3848/84 $3.00 + .OO Printed in the USA. Copyright (cl 1984 Pergamon Press Ltd. All rights reserved.
STIMULATION OF ENDOTHELIAL CELLS BY PROTEASE ACTIVITY IPjCOKMERCIAL PREPARATIONS OF XANTHILE OXIDASE
A. Agerl,
D.J. Wenham'
and J.L. Cordon 3
1Dept of Immunology, Manchester University, Manchester x13 9PT 2Unit for piechanisms in Tumour Immunology, MRC Centre, Hills Road, Cambridge CB2 2QQ. 3Section of Vascular Biology, KRC Clinical Research Centre, Watford Road, Harrow, Middx. HA1 3UJ.
(Received 22.2.1984; Accepted in revised form 3.4.1984 by Editor G. de Gaetano) ABSTRACT
The oxygen radical generating system of xanthine oxidase plus xanthine, which has been used as a model for the oxidative burst of activated granulocytes, is known to damage endothelium in vivo and in vitro. We previously observed effects (inhibited by catalase, and thus associated with the formation of H202) on several parameters of endothelial function, using a non-commercial preparation of xanthine oxidase. Our present study demonstrates that xanthine oxidase from two different commercial sources has additional effects on endothelial morphology and ion flux that are substrate-independent (i.e. produced in the absence of added xanthine) and are attributable to the presence of pancreatin (a crude enzyme mixture used in the commercial preparation of xanthine oxidase from milk). These effects ate related to the tryptic activity of pancreatin and extend previous observations on the effects of neutral proteases on endothelial cells. Our results emphasise the practical point that studies on the effects of commercial xanthine oxidase preparations on endothelial cells must take account of their trypsin-like activity as well as their capacity to generate oxygen products.
INTRODUCTIOK Endothelial cell death and denudation in the proximity of activated polymorphonuclear neutrophil leucocytes has been shown both -in vivo (1,2) and _in vitro (3-5). Products of the oxidative burst (oxygen radicals and hydrogen peroxide) and granule proteases have both been implicated in this endothelial cytotoxicity. The action of xanthine oxidase on xanthine, which produces oxygen radicals and hydrogen peroxide, has been used to simulate part of the respiratory burst associated with neutrophil activation and has been shown to damage endothelial cells -in vivo (7,8) and in vitro (3,4,9). -Keywords:
Endothelial
cells, proteases,
43
pancreatin, xanthine
oxidase
44
ENDOTH. CELLS & XANTHINE OXIDASE
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While investigating sensitive means of monitoring changes in endoand [3H]-purine release thelial cell functions we found that S6Rb efflux from pre-labelled pig aortic endothelial cells were markedly stimulated by These effects were inhibited by catalase but xanthine plus xanthine oxidase. not by superoxide dismutase suggesting that H202 was responsible (1C). We that commercial xanthine oxidase preparations had effects found, however, on endothelial cell functions even in the absence of added substrate which could not be inhibited by either superoxide dismutase or catalase. Substrate-independent effects of commercial xanthine oxidase on platelets have been reported (11,12) and the species responsible for these effects has been separated from xanthine oxidase by gel filtration (13). We have now investigated the possibility that this contaminant of commercial xanthine oxidase could be pancreatin, an extract of pancreas which is added during the preparation of xanthine oxidase from milk (14). MATERIALS Commercial xanthine oxidase was obtained from Sigma Chemical Co. (Grade III, 27 units/me protein) and from Boehringer Mannheim GmbH (0.4 Units/mg converted per min at pH 7.0 protein). One enzyme unit = one urn01 substrate Xanthine oxidase was dialysed overnight against 1000 volumes of and 25°C. phosphate buffered saline (16) and stored at 4°C before use. For some the enzyme was heat-inactivated in an autoclave for 15 min at experiments, 121°C. Pancreatin (Type II), trypsin inhibitor (Type l-S, 10,000 BAEE units/mg protein) and benzoyl DL-arginine p-nitroanilide HCl were purchased from Sigma Chemical Co. Trypsin (Type 1:250) was purchased from Difco Laboratories. had the following composition (mM): NaCl, Krebs ’ solution 119; KCl, 3.1; MgS04, 0.6; NaHC03, 25; KH2PO4, 1; CaC12, 1.3; glucose, 11.1; the pH was maintained at 7.4 by gassing with 95% 02:5% CO2. METHODS Cell culture. Porcine aortic endothelial cells from 1-14 day old pigs were cultured as previously described in Dulbecco’s modification of Eagle’s medium containing 20% (v/v) heat-inactivated (56’C, 30 min) foetal bovine serum in a humidified atmosphere of 5% CO2 in air (15). Cells were used for experiments after three to ten passages. Cell morphology. Cell morphology and the uptake of 0.1% trypan blue in phosphate buffered saline were monitored by phase contrast microscopy. Cell numbers were measured by electronic particle counting (Coulter model ZB) after detaching the cells with 0.1% trypsin/0.025% EDTA in phosphatebuffered saline. Cell detachment. Cells were plated into 6 mm diameter wells of 96-well tissue culture plates and used when confluent (* lo4 cells/well). After rinsing with phosphate buffered saline cells were pre-incubated at 37°C for 60 min in static culture with 0.003-1.0 units/ml xanthine oxidase or 0.001-1.0 mg/ml pancreatin in Krebs’ solution (50 ul/well). The supernatants were then removed and the cells rinsed once with phosphate buffered saline. The number of cells remaining attached to the plate was then determined in two ways: by electronic counting as described above or by the uptake of [ 3H]-adenine. For measurements of adenine uptake, 50 ~1 of 0.4 UM [2-3H] adenine (15-25 Ci/mmol, Amersham International, U.K.) in Krebs ’ solution were added (- 400,000 cpm) and the cells were incubated for 30 min; the supernatants were then removed, and the cells were rinsed once with phosphate buffered saline. Cell associated radioactivity was measured as previously described (17).
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86Rubidium efflux. Cells were plated onto coverslips, 5 x 15 mm, at confluent density and used the following day. After rinsing with phosphate buffered saline cells were incubated for at least 2 h with 10 uCi/ml 86rubidium chloride (l-12 mCi/mg Rb, Amersham International, U.K.) in growth medium plus 20% foetal calf serum. Two coverslips were then placed back to back in a 3.5 ml perfusion chamber and efflux of the isotope was monitored by perfusing with Krebs' solution at 37°C at a rate of 3.5 ml/min. Fractions were collected every 2 min, radioactivity was measured by gamma counting and a first order desaturation curve was plotted from the results as previously described (18). After a steady efflux rate had been obtained (>20 min) test solutions were applied for 4 min followed by a 16 min wash, with a maximum of two challenges per experiment. The change in efflux rate,4R, was calculated as the difference between the maximal efflux rate and the mean of the five points immediately before the test solution was applied. Trypsin assay. The tryptic activity of pancreatin and of commercial xanthine oxidase was measured with Difco trypsin as a standard and benzoyl DL-arginine p-nitroanilide hydrochloride as substrate. Method II of Erlanger et al. (19) was followed except that 5M sodium formate, pH 3.0, was used to stop the reaction.
RESULTS Effect of xanthine oxidase on cell morphology. Up to 0.03 units/ml Sigma or Boehringer xanthine oxidase were without effect on endothelial cell morphology. One out of five preparations of Sigma xanthine oxidase affected cell morphology at 0.1 unit/ml. At 0.3 to 1.0 units/ml of all commercial xanthine oxidase preparations tested, endothelial cells began to round up and detach from the culture dish within 5 min of adding the stimulus. There was a dose-dependent reduction in the number of cells still attached to the culture dish after 60 min (Fig. 1). Virtually all cells excluded trypan blue (>99%) after such treatment and 6 h after removing the stimulus the cells that had rounded up but not detached had flattened and resumed their normal morphology. Up to 1.0 unit/ml of heat- inactivated enzyme had no effect on cell morphology. Effect of xanthine oxidase on [3H]-adenine uptake: relation to cell detachment. After 30 min incubation with [jH]-adenine 45.7 + 0.3 (+ S.E. n = 12)% of label was cell associated in control wells. The-amount-of 13H-adenine which became cell associated under the same conditions after 60 min pre-incubation with Sigma or Boehringer xanthine oxidase correlated with the number of endothelial cells still attached to the culture dish (Fig. 1). Effects of xanthine oxidase on 86Rb efflux. The first-order rate constant for 86Rb efflux from pig aortic endothelial cells superfused on coverslips reached a steady baseline of 23.8 + 0.9 x 10s3 mine1 (+ S.E., n = 15) after Boehringer and Sigma xanthine oxidase produced a dose~~,~~d~,': ,'~~~,"~~~~,nof 86Rb efflux. Fig. 2A shows the effects of Sigma enzyme at 0.003 units/ml (llR= 8.6 2 2.3), 0.01 units/ml (AR = 18.0 + 1.7) and 0.03 units/ml (s = 37.4 + 4.5). At equivalent enzyme concentrations Boehringer enzyme produced a two-fold greater efflux of 86Rb (3R = 68.8 x 10m3 min-l for 0.03 units/ml). These enzyme preparations were not tested at concentrations above 0.03 units/ml because of their effects on cell detachment.
45
46
ENDOTH. CELLS & XANTHINE
(units/ml)
OXIDASE
Vo1.35, No.1
(mglnl)
FIG. 1 Effect of commercial xanthine oxidase and pancreatin on endothelial cell detachment. Endothelial cells in static monolayer culture were pre-incubated for 60 min with Sigma xanthine oxidase or pancreatin. The number of cells remaining attached after this treatment was determined either by direct cell counting or by [3H]-adenine u take. The ordinate axis shows cell counts (open columns) and [ !HI-adenine uptake (hatched columns) expressed as a percentage of control values + S.E. (n = 4). Control cell count was 14.6 2 1.0 x 103 cells and control [3H]-adenine uptake was 37.7 + 5.5 x lo3 cpm. Dialysis of Sigma or Boehringer xanthine oxidase did not affect the efflux of 86Rb: e.g. in one experiment AR = 70.2 for 0.03 units/ml undialysed Boehringer enzyme and AR = 73.2 for dialysed enzyme. Heat treatment (15 min at 121'C) reduced the response by >80% (see Fig. 2A). Addition of 100 @l xanthine to 0.03 units/ml Sigma enzyme potentiated 86Rb efflux: AR = 37.4 + 5.1 without added substrate and 66.5 + 8.0 with xanthine added (mean vaiues + S.E., n = 8). Neither superoxide dismutase nor catalase alone at 20 pg/ml blocked this potentiation:AR = 67.9 + 9.3 with superoxide dismutase,AR = 63.7 + 5.2 (n = 3) with catalase. Txe addition of both superoxide dismutase-and catalase reduced 86Rb efflux to that seen with xanthine oxidase in the absence of substrate: AR = 39.5 2 5.1 (Fig. 2B).
Effects of pancreatin on pig aortic endothelial cells. Pancreatin (0.3 1.0 ng/ml) caused a dose-dependent rounding and detachment of endothelial cells similar to that seen with commercial xanthine oxidase. The effects on cell detachment, as measured directly by cell counting, were paralleled by a reduction in [3H]-adenine uptake (Fig. 1).
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ENDOTH. CELLS & XANTHINE OXIDASE
47
5
i
4 min
4 nin
T
5
b (3)
FIG.
5
t 16)
5
d (3)
Li
a 16)
5
b (81
h5
c (3)
2 Effect of commercial xanthine oxidase on 86Rb efflux from endothelial cells: potentiation by xanthine and inhibition by superoxide dismutase plus catalase. Endothelial cells on coverslips were superfused as described in the text and challenged for 4 min with Sigma xanthine oxidase. Fig. 2A shows the responses to: (a) 0.003 units/ml; (b) 0.01 units/ml; (c) 0.03 units/ml; (d) 0.03 units/ml after heat inactivation for 15 min at 121°C. Fig. 28 shows the effects of 0.03 units/ml (a) alone; (b) plus 100 uM xanthine; (c) plus 100 uM xanthine, 20 ug/ml superoxide dismutase and 20 ug/ml catalase. Each point is the mean first-order rate constant (R) for 86Rb efflux from pre-labelled cells, in units of min-l, in 2 min fractions of perfusate. The first point shown ( ) is the mean basal 86Rb efflux rate + S.E. of 15 observations. Error bars on the peaks show the S.E.The number of observations is given in parentheses below each peak.
ancreatin (0.001-0.1 mg/ml) also produced a dose-dependent stimulation of 8! Rb efflux from endothelial cells. Fig. 3 shows the effect of increasing doses of pancreatin in one experiment on cells of the same strain. Sigma xanthine oxidase (0.03 units/ml) was tested during the same experiments; R = 13.2 at 0.01 mg/ml pancreatin, 22.4 at 0.1 mg/ml pancreatin and 20.5 at 0.03 units/ml Sigma xanthine oxidase. Higher doses of pancreatin were not tested because of their effects on cell morphology and detachment. Trypsin activity in pancreatin and commercial xanthine oxidase: relation to effects on endothelial cells. The effects of pancreatin and commercial xanthine oxidase on cell morphology resembled those observed during routine subculture of endothelial cells using Difco trypsin, and the trypsin assay revealed that 1 mg pancreatin contained activity equivalent to 42 pg of 1 unit of Sigma xanthine oxidase was equivalent to 35 ug of Difco trypsin, trypsin and 1 unit of Boehringer enzyme to 90 ug of trypsin.
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ENDOTH. CELLS & XANTHINE OXIDASE
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4 min
a
FIG. 3
b
C
d
Stimulation of 86Rb efflux from pig aortic endothelial cells by pancreatin and Sigma xanthine oxidase. Endothelial cells were superfused as described in the text and challenged for 4 min with pancreatin (M) or Sigma xanthine oxidase (O-0). Each point is the first-order rate constant (AR) in units of min-l, in 2 min fractions of perfusate, measured from single observations on cells of the same strain (a) 0.001 mg/ml pancreatin; (b) 0.01 mg/ml pancreatin; (c) 0.1 mg/ml pancreatin; (d) 0.03 units/ml Sigma xanthine oxidase. The first point shown (m) is the mean basal 86Rb efflux rate + S.E. of 15 observations.
Table 1 shows the effects of commercial preparations of xanthine oxidase, pancreatin and trypsin on cell detachment and [3H]-adenine uptake, in relation to their trypsin activities. The magnitude of the effects on both parameters was consistent with the levels of trypsin activity measured. Soybean trypsin inhibitor (5 mg/ml) virtually abolished the effects of all preparations (Table 1). TABLE 1 Cell detachment induced by commercial xanthine oxidase, pancreatin and trypsin: relation to trypsin activity. Endothelial cells in static monolayer culture were preincubated for 60 min with Sigma xanthine oxidase, pancreatin or trypsin (Difco), with or without 5 mg/ml soybean trypsin inhibitor. The number of cells remaining attached after this treatment was determined either by direct cell counting or by [3H]-adenine uptake. The results are expressed as the mean percentage of control values + S.E. (n = 4) and related to the amount of trypsin activity measured in each preparation. Trypsin activity (ug/ml) Sigma xanthine oxidase Boehringer xanthine oxidase Pancreatin Trypsin
3 30 80 100
Cell numbers [3H]-adenine uptake + + no no inhibitor inhibitor inhibitor inhibitor 64 + - 3
93 + 3
76 + 2
96 + - 3
56 17 10
95 + 4 91 +6 98 T 2
53 + 2 18 : 2
84 + 7 94 T 3 96 T - 1
+ 2 T2 T - 1
13 T - 1
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49
The effects of Difco trypsin on 86Rb efflux from superfused endothelial cells were determined and compared with responses to xanthine oxidase and pancreatin. Difco trypsin induced a dose-dependent stimulation of 86Rb efflux, with a minimum effective concentration <1 ug/ml. Pancreatin and xanthine oxidase (Sigma and Boehringer at equivalent levels of trypsin activity had comparable effects on B6Rb efflux (Table 2).
TABLE 2 66Rb Efflux xanthine
from
oxidase,
pig
aortic
pancreatin
endothelial
cells
induced
relation
and trypsin:
to
by commercial trypsin
activity
Endothelial cells were superfused as described in the text and challenged or with trypsin. The change in 86Rb efflux, R, calculated as described in the text is related to the amount of trypsin activity measured in each preparation. Trypsin (vg/ml)
Xanthine
oxidase
(Sigma)
Xanthine
oxidase
(Boehringer)
Pancreatin
AR x 10-3 nin -1
1
16
10
71
8
82
Trypsin
(Difco)
1
21
Trypsin
(Difco)
10
69
DISCUSSION
Commercial preparations of xanthine oxidase, in the absence of added substrate, produced a dose-dependent stimulation of 86Rb efflux from pig aortic endothelial cells in culture and (over a higher concentration range) dose-dependent cell detachment from the culture dish. These effects were caused by a non-dialysable, heat-labile component of the enzyme preparation. They were not inhibited by superoxide dismutase or catalase (i.e. were independent of the oxidative properties of xanthine oxidase). Pancreatin, an extract of pancreas which is added during the commercial had dose-dependent effects on preparation of xanthine oxidase from milk, endothelial cells which were similar to those of commercial xanthine oxidase.
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ENDOTH. CELLS & XANTHINE OXIDASE
Vo1.35, No.1
The effects of pancreatin and commercial xanthine oxidase on cell and the amounts of tryptic morphology were similar to those of trypsin, activity present in commercial xanthine oxidase and pancreatin were consistent with the effects of these agents on cell detachment (as measured by cell counting and [3H]-adenine uptake) and on 86Rb efflux. Protease activity in xanthine oxidase prepared using pancreatin has been which led to the development of a method for xanthine previously noted, oxidase preparation that avoided the use of pancreatin (20), but apparently there have been no previous attempts to determine whether this residual protease activity had biologically significant effects on intact cells. One unit of commercial xanthine oxidase could contain as much as 4 mg which is more than enough to induce the effects on of pancreatin (14), endothelial cells that we observed. These calculations, in conjunction with our own observations, lead us to conclude that the substrate-independent effects of commercial xanthine oxidase on pig aortic endothelial cell function can be explained by the presence of pancreatin in these preparations. Further, the species responsible for these effects appears to be a trypsin-like enzyme. There have been reports of commercial preparations of xanthine oxidase exerting substrate-independent effects on platelets, which are independent of the oxidative properties of the enzyme (11,12). Ishii et al. (13) separated this activity from xanthine oxidase by gel filtration and found it to be a protein of 17,000 molecular weight, which stimulated platelet aggregation at concentrations as low as 1 ug/ml. Trypsin stimulates platelet aggregation at comparable concentrations (21) and it therefore appears likely that the substrate-independent effects of commercial xanthine oxidase on platelets, as on endothelial cells, are caused wholly or in part by the tryptic activity of these preparations. It is important to emphasise that these effects of trypsin on endothelial cells are reversible: treated cells still exclude trypan blue, and endothelial cells (in common with many others) are routinely sub-cultured by trypsin treatment with no significant cell loss, even though the concentration usually employed is 1 mg/ml - over lOOO-fold greater than the *6Rb efflux. minimum concentration that stimulates We have recently shown that chymotrypsin and cathepsin G (the major neutral protease of human ranulocytes) are, like trypsin (22), powerful 86 Rb efflux stimulants of endothelial and prostaglandin production may be important in vivo when endothelial cells are in (23) - Such effects close proximity to activated neutrophils (l), which secrete neutral proteases as well as oxygen products; both these classes of neutrophil secretory products affect endothelial function (3,4,5,8-10,22,23). In addition, our present results indicate that when the effects of commercial xanthine oxidase preparations on endothelial cells (and perhaps other cells also) are being studied, it should not be assumed that oxygen products are responsible - indeed, unless proteolytic activity has been eliminated, this is likely to play a major role.
ACKNOWLEDGEMENT Ann Ager was supported
by a Fellowship
from
Ciba-Geigy.
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REFERENCES 1.
STEWART, G.J., RITCHIE, W.G.M. and LYNCH, P.R. Venous endothelial damage produced by massive sticking and migration of leucocytes. Am. J. Pathol. 74, 507-521, 1974.
2.
TILL, G.O., JOHNSON, K.J., KUNKEL, R. and WARD, P.A. Intravascular activation of complement and acute lung injury. J. Clin. Invest. 9_, 1126-1135, 1982.
3.
HARLAN, J.M., KILLEN, P.D., HARKER, L.A., STRIKER, G.E. and WRIGHT, D.G. Seutrophil-mediated endothelial injury -in vitro. J. Clin. Invest. 68, 1394-1403, 1981.
4.
SACKS, T., MOLDOW, C.F., CRADDOCK, P.R., BOWERS, T.K. and JACOB, H.S. Oxygen radicals mediate endothelial cell damage by complementstimulated granulocytes. J. Clin. Invest. 61, 1161-1167, 1978.
5.
WEISS, S.J., YOUNG, J., LOBUGLIO, A.F., SLIVKA, A. and NIIIEH, N.F. Role of hydrogen peroxide in neutrophil-mediated destruction of cultured endothelial cells. J. Clin. Invest. 68, 714-721, 1981.
6.
ROSEN, H. and KLEBANOFF, S.J. Bactericidal activity of a superoxide anion-generating system. A model for the polymorphonuclear leucocyte. J. Exp. Med. 149, 27-39, 1979.
7.
DEL MAESTRO, R.F., BJORK, J. and ARFORS, K.E. Increase in microvascular permeability induced by enzymatically generated free I. -In vivo study. Microvasc. Res. 2, radicals. 239-254, 1981.
8.
JOHNSON, K.J., FANTONE, J.C., KAPLAN, J. and WARD, P.A. In vivo damage of rat lungs by oxygen metabolites. J. Clin. Invest. 67, 983-993, 1981.
9.
STEINBERG, H., GREENWALD, R.A., SCIUBBA, J. and DAS, D.K. The effect of oxygen-derived free radicals on pulmonary endothelial cell function in the isolated perfused rat lung. Exp. Lung Res. 3, 163-173, 1982.
10.
AGER, A. and GORDON, J.L. Differential effects of hydrogen peroxide J. Exp. Med. ljs, 592-603, on indices of endothelial cell function. 1984.
11.
PATSCHEKE, H., PASCHEN, W. and WORNER, P. Superoxide-independent Hoppe-Seyler's Z. Physiol. platelet response to xanthine oxidase. Chem. 359, 933-937, 1978.
12.
LEVINE, P.H., SLADDIN, D.G. and KRINSKY, N.I. Superoxide, xanthine Further studies on mechanisms by oxidase and platelet reactions: which oxidants influence platelets. Thrombos. Haemostas. 45, 290-293, 1981.
13.
ISHII, H., HIRAISHI, S. and KAZAMA, M. A platelet aggregating substance contained in xanthine oxidase preparation from cow's milk. Thromb. Research. 28, 85-92, 1982.
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OXIDASE
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14.
GILEERT, D.A. and BERGEL, F. The chemistry of xanthine oxidase. 9. An Biochem. J. 90, improved method of preparing the bovine milk enzyme. 350-353, 1964.
15.
PEARSON, J.D., CARLETON, J.S., HDTCHINGS, A. and GORDON, J.L. Uptake and metabolism of adenosine by pig aortic endothelial and smooth Biochem. J. 170, 265-271, 1978. muscle cells in culture.
16.
DLJLBECCO, R. and VOGT, M. Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 2, 167-182, 1954.
17.
DE BONO, D.P., MACINTYRE, D.E., WHITE, J.G. and GORDON, J.L. Endothelial adenine uptake as an assay for cell- or complementmediated cytotoxicity. Immunology, 32, 321-326, 1977.
18.
MARTIN, W. and'GORDON, J.L. Differential calcium dependence of contractile responses and 86Rb efflux from the rabbit aorta induced by vasoactive stimuli. J. Cell. Physiol. 115, 46-52, 1983.
19.
ERLANGER, B.F., KOKOWSKY, N. and COHEN, W. The preparation and properties of two new chromogenic substrates of trypsin. Arch. Biochim. Biophys. 95, 271-278, 1961.
20.
WAUD, W.R., BRADY, F.O., WILEY, R.D. and RAJAGOPALAN, K.V. A new purification procedure for bovine milk xanthine oxidase: effect of proteolysis on the subunit structure. Arch. Biochem. Biophys. 169, 695-701, 1975.
21.
HASLAM, R.J. Role of adenosine diphosphate in the aggregation of human blood platelets by thrombin and by fatty acids. Nature 202, 765-768, 1964.
22.
WEKSLER, B.B., LEY, C.W. and JAFFE, E.A. Stimulation of endothelial cell prostacyclin production by thrombin, trypsin and the ionophore A23187. J. Clin. Invest. 62, 923-930, 1978.
23.
LEROY, E.C., AGER, A. and GORDON, J.L. Effects of neutrophil elastase and other proteases on porcine aortic endothelial prostaglandin 12 production, adenine nucleotide release and responses to vasoactive agents. Submitted for publication.
STIMULATION OF ENDOTHELIAL CELLS BY PROTEASE ACTIVITY IPjCOKMERCIAL PREPARATIONS OF XANTHILE OXIDASE
A. Agerl,
D.J. Wenham'
and J.L. Cordon 3
1Dept of Immunology, Manchester University, Manchester x13 9PT 2Unit for piechanisms in Tumour Immunology, MRC Centre, Hills Road, Cambridge CB2 2QQ. 3Section of Vascular Biology, KRC Clinical Research Centre, Watford Road, Harrow, Middx. HA1 3UJ.
(Received 22.2.1984; Accepted in revised form 3.4.1984 by Editor G. de Gaetano) ABSTRACT
The oxygen radical generating system of xanthine oxidase plus xanthine, which has been used as a model for the oxidative burst of activated granulocytes, is known to damage endothelium in vivo and in vitro. We previously observed effects (inhibited by catalase, and thus associated with the formation of H202) on several parameters of endothelial function, using a non-commercial preparation of xanthine oxidase. Our present study demonstrates that xanthine oxidase from two different commercial sources has additional effects on endothelial morphology and ion flux that are substrate-independent (i.e. produced in the absence of added xanthine) and are attributable to the presence of pancreatin (a crude enzyme mixture used in the commercial preparation of xanthine oxidase from milk). These effects ate related to the tryptic activity of pancreatin and extend previous observations on the effects of neutral proteases on endothelial cells. Our results emphasise the practical point that studies on the effects of commercial xanthine oxidase preparations on endothelial cells must take account of their trypsin-like activity as well as their capacity to generate oxygen products.
INTRODUCTIOK Endothelial cell death and denudation in the proximity of activated polymorphonuclear neutrophil leucocytes has been shown both -in vivo (1,2) and _in vitro (3-5). Products of the oxidative burst (oxygen radicals and hydrogen peroxide) and granule proteases have both been implicated in this endothelial cytotoxicity. The action of xanthine oxidase on xanthine, which produces oxygen radicals and hydrogen peroxide, has been used to simulate part of the respiratory burst associated with neutrophil activation and has been shown to damage endothelial cells -in vivo (7,8) and in vitro (3,4,9). -Keywords:
Endothelial
cells, proteases,
43
pancreatin, xanthine
oxidase
44
ENDOTH. CELLS & XANTHINE OXIDASE
Vo1.35, No.1
While investigating sensitive means of monitoring changes in endoand [3H]-purine release thelial cell functions we found that S6Rb efflux from pre-labelled pig aortic endothelial cells were markedly stimulated by These effects were inhibited by catalase but xanthine plus xanthine oxidase. not by superoxide dismutase suggesting that H202 was responsible (1C). We that commercial xanthine oxidase preparations had effects found, however, on endothelial cell functions even in the absence of added substrate which could not be inhibited by either superoxide dismutase or catalase. Substrate-independent effects of commercial xanthine oxidase on platelets have been reported (11,12) and the species responsible for these effects has been separated from xanthine oxidase by gel filtration (13). We have now investigated the possibility that this contaminant of commercial xanthine oxidase could be pancreatin, an extract of pancreas which is added during the preparation of xanthine oxidase from milk (14). MATERIALS Commercial xanthine oxidase was obtained from Sigma Chemical Co. (Grade III, 27 units/me protein) and from Boehringer Mannheim GmbH (0.4 Units/mg converted per min at pH 7.0 protein). One enzyme unit = one urn01 substrate Xanthine oxidase was dialysed overnight against 1000 volumes of and 25°C. phosphate buffered saline (16) and stored at 4°C before use. For some the enzyme was heat-inactivated in an autoclave for 15 min at experiments, 121°C. Pancreatin (Type II), trypsin inhibitor (Type l-S, 10,000 BAEE units/mg protein) and benzoyl DL-arginine p-nitroanilide HCl were purchased from Sigma Chemical Co. Trypsin (Type 1:250) was purchased from Difco Laboratories. had the following composition (mM): NaCl, Krebs ’ solution 119; KCl, 3.1; MgS04, 0.6; NaHC03, 25; KH2PO4, 1; CaC12, 1.3; glucose, 11.1; the pH was maintained at 7.4 by gassing with 95% 02:5% CO2. METHODS Cell culture. Porcine aortic endothelial cells from 1-14 day old pigs were cultured as previously described in Dulbecco’s modification of Eagle’s medium containing 20% (v/v) heat-inactivated (56’C, 30 min) foetal bovine serum in a humidified atmosphere of 5% CO2 in air (15). Cells were used for experiments after three to ten passages. Cell morphology. Cell morphology and the uptake of 0.1% trypan blue in phosphate buffered saline were monitored by phase contrast microscopy. Cell numbers were measured by electronic particle counting (Coulter model ZB) after detaching the cells with 0.1% trypsin/0.025% EDTA in phosphatebuffered saline. Cell detachment. Cells were plated into 6 mm diameter wells of 96-well tissue culture plates and used when confluent (* lo4 cells/well). After rinsing with phosphate buffered saline cells were pre-incubated at 37°C for 60 min in static culture with 0.003-1.0 units/ml xanthine oxidase or 0.001-1.0 mg/ml pancreatin in Krebs’ solution (50 ul/well). The supernatants were then removed and the cells rinsed once with phosphate buffered saline. The number of cells remaining attached to the plate was then determined in two ways: by electronic counting as described above or by the uptake of [ 3H]-adenine. For measurements of adenine uptake, 50 ~1 of 0.4 UM [2-3H] adenine (15-25 Ci/mmol, Amersham International, U.K.) in Krebs ’ solution were added (- 400,000 cpm) and the cells were incubated for 30 min; the supernatants were then removed, and the cells were rinsed once with phosphate buffered saline. Cell associated radioactivity was measured as previously described (17).
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86Rubidium efflux. Cells were plated onto coverslips, 5 x 15 mm, at confluent density and used the following day. After rinsing with phosphate buffered saline cells were incubated for at least 2 h with 10 uCi/ml 86rubidium chloride (l-12 mCi/mg Rb, Amersham International, U.K.) in growth medium plus 20% foetal calf serum. Two coverslips were then placed back to back in a 3.5 ml perfusion chamber and efflux of the isotope was monitored by perfusing with Krebs' solution at 37°C at a rate of 3.5 ml/min. Fractions were collected every 2 min, radioactivity was measured by gamma counting and a first order desaturation curve was plotted from the results as previously described (18). After a steady efflux rate had been obtained (>20 min) test solutions were applied for 4 min followed by a 16 min wash, with a maximum of two challenges per experiment. The change in efflux rate,4R, was calculated as the difference between the maximal efflux rate and the mean of the five points immediately before the test solution was applied. Trypsin assay. The tryptic activity of pancreatin and of commercial xanthine oxidase was measured with Difco trypsin as a standard and benzoyl DL-arginine p-nitroanilide hydrochloride as substrate. Method II of Erlanger et al. (19) was followed except that 5M sodium formate, pH 3.0, was used to stop the reaction.
RESULTS Effect of xanthine oxidase on cell morphology. Up to 0.03 units/ml Sigma or Boehringer xanthine oxidase were without effect on endothelial cell morphology. One out of five preparations of Sigma xanthine oxidase affected cell morphology at 0.1 unit/ml. At 0.3 to 1.0 units/ml of all commercial xanthine oxidase preparations tested, endothelial cells began to round up and detach from the culture dish within 5 min of adding the stimulus. There was a dose-dependent reduction in the number of cells still attached to the culture dish after 60 min (Fig. 1). Virtually all cells excluded trypan blue (>99%) after such treatment and 6 h after removing the stimulus the cells that had rounded up but not detached had flattened and resumed their normal morphology. Up to 1.0 unit/ml of heat- inactivated enzyme had no effect on cell morphology. Effect of xanthine oxidase on [3H]-adenine uptake: relation to cell detachment. After 30 min incubation with [jH]-adenine 45.7 + 0.3 (+ S.E. n = 12)% of label was cell associated in control wells. The-amount-of 13H-adenine which became cell associated under the same conditions after 60 min pre-incubation with Sigma or Boehringer xanthine oxidase correlated with the number of endothelial cells still attached to the culture dish (Fig. 1). Effects of xanthine oxidase on 86Rb efflux. The first-order rate constant for 86Rb efflux from pig aortic endothelial cells superfused on coverslips reached a steady baseline of 23.8 + 0.9 x 10s3 mine1 (+ S.E., n = 15) after Boehringer and Sigma xanthine oxidase produced a dose~~,~~d~,': ,'~~~,"~~~~,nof 86Rb efflux. Fig. 2A shows the effects of Sigma enzyme at 0.003 units/ml (llR= 8.6 2 2.3), 0.01 units/ml (AR = 18.0 + 1.7) and 0.03 units/ml (s = 37.4 + 4.5). At equivalent enzyme concentrations Boehringer enzyme produced a two-fold greater efflux of 86Rb (3R = 68.8 x 10m3 min-l for 0.03 units/ml). These enzyme preparations were not tested at concentrations above 0.03 units/ml because of their effects on cell detachment.
45
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ENDOTH. CELLS & XANTHINE
(units/ml)
OXIDASE
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(mglnl)
FIG. 1 Effect of commercial xanthine oxidase and pancreatin on endothelial cell detachment. Endothelial cells in static monolayer culture were pre-incubated for 60 min with Sigma xanthine oxidase or pancreatin. The number of cells remaining attached after this treatment was determined either by direct cell counting or by [3H]-adenine u take. The ordinate axis shows cell counts (open columns) and [ !HI-adenine uptake (hatched columns) expressed as a percentage of control values + S.E. (n = 4). Control cell count was 14.6 2 1.0 x 103 cells and control [3H]-adenine uptake was 37.7 + 5.5 x lo3 cpm. Dialysis of Sigma or Boehringer xanthine oxidase did not affect the efflux of 86Rb: e.g. in one experiment AR = 70.2 for 0.03 units/ml undialysed Boehringer enzyme and AR = 73.2 for dialysed enzyme. Heat treatment (15 min at 121'C) reduced the response by >80% (see Fig. 2A). Addition of 100 @l xanthine to 0.03 units/ml Sigma enzyme potentiated 86Rb efflux: AR = 37.4 + 5.1 without added substrate and 66.5 + 8.0 with xanthine added (mean vaiues + S.E., n = 8). Neither superoxide dismutase nor catalase alone at 20 pg/ml blocked this potentiation:AR = 67.9 + 9.3 with superoxide dismutase,AR = 63.7 + 5.2 (n = 3) with catalase. Txe addition of both superoxide dismutase-and catalase reduced 86Rb efflux to that seen with xanthine oxidase in the absence of substrate: AR = 39.5 2 5.1 (Fig. 2B).
Effects of pancreatin on pig aortic endothelial cells. Pancreatin (0.3 1.0 ng/ml) caused a dose-dependent rounding and detachment of endothelial cells similar to that seen with commercial xanthine oxidase. The effects on cell detachment, as measured directly by cell counting, were paralleled by a reduction in [3H]-adenine uptake (Fig. 1).
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ENDOTH. CELLS & XANTHINE OXIDASE
47
5
i
4 min
4 nin
T
5
b (3)
FIG.
5
t 16)
5
d (3)
Li
a 16)
5
b (81
h5
c (3)
2 Effect of commercial xanthine oxidase on 86Rb efflux from endothelial cells: potentiation by xanthine and inhibition by superoxide dismutase plus catalase. Endothelial cells on coverslips were superfused as described in the text and challenged for 4 min with Sigma xanthine oxidase. Fig. 2A shows the responses to: (a) 0.003 units/ml; (b) 0.01 units/ml; (c) 0.03 units/ml; (d) 0.03 units/ml after heat inactivation for 15 min at 121°C. Fig. 28 shows the effects of 0.03 units/ml (a) alone; (b) plus 100 uM xanthine; (c) plus 100 uM xanthine, 20 ug/ml superoxide dismutase and 20 ug/ml catalase. Each point is the mean first-order rate constant (R) for 86Rb efflux from pre-labelled cells, in units of min-l, in 2 min fractions of perfusate. The first point shown ( ) is the mean basal 86Rb efflux rate + S.E. of 15 observations. Error bars on the peaks show the S.E.The number of observations is given in parentheses below each peak.
ancreatin (0.001-0.1 mg/ml) also produced a dose-dependent stimulation of 8! Rb efflux from endothelial cells. Fig. 3 shows the effect of increasing doses of pancreatin in one experiment on cells of the same strain. Sigma xanthine oxidase (0.03 units/ml) was tested during the same experiments; R = 13.2 at 0.01 mg/ml pancreatin, 22.4 at 0.1 mg/ml pancreatin and 20.5 at 0.03 units/ml Sigma xanthine oxidase. Higher doses of pancreatin were not tested because of their effects on cell morphology and detachment. Trypsin activity in pancreatin and commercial xanthine oxidase: relation to effects on endothelial cells. The effects of pancreatin and commercial xanthine oxidase on cell morphology resembled those observed during routine subculture of endothelial cells using Difco trypsin, and the trypsin assay revealed that 1 mg pancreatin contained activity equivalent to 42 pg of 1 unit of Sigma xanthine oxidase was equivalent to 35 ug of Difco trypsin, trypsin and 1 unit of Boehringer enzyme to 90 ug of trypsin.
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ENDOTH. CELLS & XANTHINE OXIDASE
Vo1.35, No.1
4 min
a
FIG. 3
b
C
d
Stimulation of 86Rb efflux from pig aortic endothelial cells by pancreatin and Sigma xanthine oxidase. Endothelial cells were superfused as described in the text and challenged for 4 min with pancreatin (M) or Sigma xanthine oxidase (O-0). Each point is the first-order rate constant (AR) in units of min-l, in 2 min fractions of perfusate, measured from single observations on cells of the same strain (a) 0.001 mg/ml pancreatin; (b) 0.01 mg/ml pancreatin; (c) 0.1 mg/ml pancreatin; (d) 0.03 units/ml Sigma xanthine oxidase. The first point shown (m) is the mean basal 86Rb efflux rate + S.E. of 15 observations.
Table 1 shows the effects of commercial preparations of xanthine oxidase, pancreatin and trypsin on cell detachment and [3H]-adenine uptake, in relation to their trypsin activities. The magnitude of the effects on both parameters was consistent with the levels of trypsin activity measured. Soybean trypsin inhibitor (5 mg/ml) virtually abolished the effects of all preparations (Table 1). TABLE 1 Cell detachment induced by commercial xanthine oxidase, pancreatin and trypsin: relation to trypsin activity. Endothelial cells in static monolayer culture were preincubated for 60 min with Sigma xanthine oxidase, pancreatin or trypsin (Difco), with or without 5 mg/ml soybean trypsin inhibitor. The number of cells remaining attached after this treatment was determined either by direct cell counting or by [3H]-adenine uptake. The results are expressed as the mean percentage of control values + S.E. (n = 4) and related to the amount of trypsin activity measured in each preparation. Trypsin activity (ug/ml) Sigma xanthine oxidase Boehringer xanthine oxidase Pancreatin Trypsin
3 30 80 100
Cell numbers [3H]-adenine uptake + + no no inhibitor inhibitor inhibitor inhibitor 64 + - 3
93 + 3
76 + 2
96 + - 3
56 17 10
95 + 4 91 +6 98 T 2
53 + 2 18 : 2
84 + 7 94 T 3 96 T - 1
+ 2 T2 T - 1
13 T - 1
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The effects of Difco trypsin on 86Rb efflux from superfused endothelial cells were determined and compared with responses to xanthine oxidase and pancreatin. Difco trypsin induced a dose-dependent stimulation of 86Rb efflux, with a minimum effective concentration <1 ug/ml. Pancreatin and xanthine oxidase (Sigma and Boehringer at equivalent levels of trypsin activity had comparable effects on B6Rb efflux (Table 2).
TABLE 2 66Rb Efflux xanthine
from
oxidase,
pig
aortic
pancreatin
endothelial
cells
induced
relation
and trypsin:
to
by commercial trypsin
activity
Endothelial cells were superfused as described in the text and challenged or with trypsin. The change in 86Rb efflux, R, calculated as described in the text is related to the amount of trypsin activity measured in each preparation. Trypsin (vg/ml)
Xanthine
oxidase
(Sigma)
Xanthine
oxidase
(Boehringer)
Pancreatin
AR x 10-3 nin -1
1
16
10
71
8
82
Trypsin
(Difco)
1
21
Trypsin
(Difco)
10
69
DISCUSSION
Commercial preparations of xanthine oxidase, in the absence of added substrate, produced a dose-dependent stimulation of 86Rb efflux from pig aortic endothelial cells in culture and (over a higher concentration range) dose-dependent cell detachment from the culture dish. These effects were caused by a non-dialysable, heat-labile component of the enzyme preparation. They were not inhibited by superoxide dismutase or catalase (i.e. were independent of the oxidative properties of xanthine oxidase). Pancreatin, an extract of pancreas which is added during the commercial had dose-dependent effects on preparation of xanthine oxidase from milk, endothelial cells which were similar to those of commercial xanthine oxidase.
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The effects of pancreatin and commercial xanthine oxidase on cell and the amounts of tryptic morphology were similar to those of trypsin, activity present in commercial xanthine oxidase and pancreatin were consistent with the effects of these agents on cell detachment (as measured by cell counting and [3H]-adenine uptake) and on 86Rb efflux. Protease activity in xanthine oxidase prepared using pancreatin has been which led to the development of a method for xanthine previously noted, oxidase preparation that avoided the use of pancreatin (20), but apparently there have been no previous attempts to determine whether this residual protease activity had biologically significant effects on intact cells. One unit of commercial xanthine oxidase could contain as much as 4 mg which is more than enough to induce the effects on of pancreatin (14), endothelial cells that we observed. These calculations, in conjunction with our own observations, lead us to conclude that the substrate-independent effects of commercial xanthine oxidase on pig aortic endothelial cell function can be explained by the presence of pancreatin in these preparations. Further, the species responsible for these effects appears to be a trypsin-like enzyme. There have been reports of commercial preparations of xanthine oxidase exerting substrate-independent effects on platelets, which are independent of the oxidative properties of the enzyme (11,12). Ishii et al. (13) separated this activity from xanthine oxidase by gel filtration and found it to be a protein of 17,000 molecular weight, which stimulated platelet aggregation at concentrations as low as 1 ug/ml. Trypsin stimulates platelet aggregation at comparable concentrations (21) and it therefore appears likely that the substrate-independent effects of commercial xanthine oxidase on platelets, as on endothelial cells, are caused wholly or in part by the tryptic activity of these preparations. It is important to emphasise that these effects of trypsin on endothelial cells are reversible: treated cells still exclude trypan blue, and endothelial cells (in common with many others) are routinely sub-cultured by trypsin treatment with no significant cell loss, even though the concentration usually employed is 1 mg/ml - over lOOO-fold greater than the *6Rb efflux. minimum concentration that stimulates We have recently shown that chymotrypsin and cathepsin G (the major neutral protease of human ranulocytes) are, like trypsin (22), powerful 86 Rb efflux stimulants of endothelial and prostaglandin production may be important in vivo when endothelial cells are in (23) - Such effects close proximity to activated neutrophils (l), which secrete neutral proteases as well as oxygen products; both these classes of neutrophil secretory products affect endothelial function (3,4,5,8-10,22,23). In addition, our present results indicate that when the effects of commercial xanthine oxidase preparations on endothelial cells (and perhaps other cells also) are being studied, it should not be assumed that oxygen products are responsible - indeed, unless proteolytic activity has been eliminated, this is likely to play a major role.
ACKNOWLEDGEMENT Ann Ager was supported
by a Fellowship
from
Ciba-Geigy.
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WEISS, S.J., YOUNG, J., LOBUGLIO, A.F., SLIVKA, A. and NIIIEH, N.F. Role of hydrogen peroxide in neutrophil-mediated destruction of cultured endothelial cells. J. Clin. Invest. 68, 714-721, 1981.
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ROSEN, H. and KLEBANOFF, S.J. Bactericidal activity of a superoxide anion-generating system. A model for the polymorphonuclear leucocyte. J. Exp. Med. 149, 27-39, 1979.
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PATSCHEKE, H., PASCHEN, W. and WORNER, P. Superoxide-independent Hoppe-Seyler's Z. Physiol. platelet response to xanthine oxidase. Chem. 359, 933-937, 1978.
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LEVINE, P.H., SLADDIN, D.G. and KRINSKY, N.I. Superoxide, xanthine Further studies on mechanisms by oxidase and platelet reactions: which oxidants influence platelets. Thrombos. Haemostas. 45, 290-293, 1981.
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ISHII, H., HIRAISHI, S. and KAZAMA, M. A platelet aggregating substance contained in xanthine oxidase preparation from cow's milk. Thromb. Research. 28, 85-92, 1982.
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GILEERT, D.A. and BERGEL, F. The chemistry of xanthine oxidase. 9. An Biochem. J. 90, improved method of preparing the bovine milk enzyme. 350-353, 1964.
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PEARSON, J.D., CARLETON, J.S., HDTCHINGS, A. and GORDON, J.L. Uptake and metabolism of adenosine by pig aortic endothelial and smooth Biochem. J. 170, 265-271, 1978. muscle cells in culture.
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DE BONO, D.P., MACINTYRE, D.E., WHITE, J.G. and GORDON, J.L. Endothelial adenine uptake as an assay for cell- or complementmediated cytotoxicity. Immunology, 32, 321-326, 1977.
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MARTIN, W. and'GORDON, J.L. Differential calcium dependence of contractile responses and 86Rb efflux from the rabbit aorta induced by vasoactive stimuli. J. Cell. Physiol. 115, 46-52, 1983.
19.
ERLANGER, B.F., KOKOWSKY, N. and COHEN, W. The preparation and properties of two new chromogenic substrates of trypsin. Arch. Biochim. Biophys. 95, 271-278, 1961.
20.
WAUD, W.R., BRADY, F.O., WILEY, R.D. and RAJAGOPALAN, K.V. A new purification procedure for bovine milk xanthine oxidase: effect of proteolysis on the subunit structure. Arch. Biochem. Biophys. 169, 695-701, 1975.
21.
HASLAM, R.J. Role of adenosine diphosphate in the aggregation of human blood platelets by thrombin and by fatty acids. Nature 202, 765-768, 1964.
22.
WEKSLER, B.B., LEY, C.W. and JAFFE, E.A. Stimulation of endothelial cell prostacyclin production by thrombin, trypsin and the ionophore A23187. J. Clin. Invest. 62, 923-930, 1978.
23.
LEROY, E.C., AGER, A. and GORDON, J.L. Effects of neutrophil elastase and other proteases on porcine aortic endothelial prostaglandin 12 production, adenine nucleotide release and responses to vasoactive agents. Submitted for publication.