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Paraquat depresses the activity of angiotensin converting enzyme expressed by human umbilical vein endothelial cells in culture
CellBiology
International
Reports,
205
Vol. IS, No. 3, 9997
PARAQUATDEPRESSESTIIE ACTIVITY OF ANGIOTENSIN CONVERTINGENZYME EXPRESSED BY HUMANUMBILICAL VEIN ENDOTBELIALCELLS IN CULTURE Kentaro Watanabe+, Noriko Tamaru and Minoru Yoshida Second Department of Internal Medicine, University, Fukuoka, 814-o-1, Japan *Corresponding
School of Medicine,
Fukuoka
author
ABSTRACT The activity of angiotensin converting enzyme (ACE) in cell lysate of cultured human umbilical vein end thelial cells (HUVEC) after a 24-hour incubation with low3 and 10 -2 M of paraquat (PQ) was decreased. However, LDH released into the culture medium of HUVEC during the 24-hour incubation with PQ was not increased. Many investigators show that the change in serum ACE activity reflects of vascular endothelial cells. We showed in this the impairment report that ACE was decreased even at an early stage of endothelial injury induced by PQ, when LDff release is not yet increased.
INTRODUCTION used herbicide, is known to cause Paraquat (PQ) , a widely permeability edema, leading eventually to pulmonary fibrosis. demonstrated that plasma and lung lymph Shibamoto et a1.(1986) concentrations prostacyclin and thromboxane increased intravenous in*? usion of PQ in awake sheep. significantly after lntraperitoneal administration of PQ to mice produced a linear doseresponse increase in serum angiotensin converting enzyme and decrease in total lung ACE (Hollinger et al., 1980). Whether pulmonary vascular endothelial cells as well as alveolar epithelial are the critical sites or not in PQ poisoning has been a cells subject of controversy. In order to test the direct effect of PQ on cells, we cultured vascular endothelial human umbilical vein cells (HUVEC) and examined ACE activity expressed by endothelial HUVECand LDH release from HUVEC.
MATERIALSAND METBODS Culture of HUVEC HUVEC were obtained from human umbilical cord ve ins by digestion with 0.1% collagcnase as previously described (Jaffe et 1973; Jaffe, 1984). The isolated HUVECwere cultured in T75 al., flasks or 35 mmpetri-dishes (all precoated with 0.02% gelatin) in rledium 199 (M 199) containing 25 mM HEPESbuffered saline (HBS), (1 .6 penicillin (80 units/ml), streptomycin (80 ug/ml), L-glutamine 0309-I
651/91/030205-6/$03.00/O
0 1991 Academic
Press Ltd
206
Cell Biology International
Reports, Vol. 75, No. 3, 199 1
rell mM), amphotericin B (2 pg/ml), heparin (90 ug/ml), endothelial growth factor (ECGF) (20 vg/ml) and 4% human serum and 16% fetal calf serum (FCS) at 37’C under 5% C02. ACE activity in cell lysate of IIUVEC ACE activity was measured according to a modification of the fluorimetric method (Friedland et al., 1976; Strittmattcr ct al., 1984 ; Watanabe et al., 1990). HUVECin passage 2 were grown to in 35 mmpetri-dishes. Cells were fed with fresh medium confluency containing 4% human serum and 16% FCS with or without PQ. After 24 hours monolayers were washed 3 times with HBS, scraped by a rubber policeman, and frozen until assay. On the day of the assay, the samples were thawed and sonicated prior to assay. Twenty ul of t,h c sonicated samples were mixed with 80 ~1 of 5 mM hippuryl-histidylleucine (H-H-L) in HBS and incubated for 3 hours at 37’C. After 3 hours the reaction was terminated by the addition of 1.4 ml of’ 0.5 N NaOH. Then, 100 11 of 10 mg/ml of 0-phthaldialdehyde in methanol was added to the reaction mixture, followed by the addition of 250 ul of 6 N HCl after a 5 min interval. Fluorescence of the samples was measured by spectrofluorimeter with emission at 495 nm and with excitation at 365 nm. The standard curve was obtained using known concentrations of histidyl-leucine. Enzyme activity was exprcsscd as nanomoles of substrate hydrolyzed per hour p‘r dish. In another group, PQ was added to 10-5 M into the sonicatcd samples without pretreatment of PQ before mixing with H-II-I, in order to examine the direct inhibitory effect of PQ on ACE. Then the samples were assayed for ACE activity in tho same manner as mentioned above. Mcsurement of LDII release The supernatants after the incubations of HUVEC with various concentrations of PQ for 24 hours were assayed for LDlI by a spectrophotometric assay based on NADHoxidation using a commcrci al kit(Sigma). The LDH measurements were corrected for the LDll activity measured in the pre-culture medium. Maximal LDH relcasc was determined by scraping and sonicating non-PQ-treated ccl Is and measuring LDH in the supernatant. RESUI.TS Effect of PQ on ACE activity in cell lysate of IIUVEC Figure shows ACE activity in cell lysate of fIUVECafter a 24 hour-incubation with or without PQ. Analysis of thcsc data by Jonckheere’s test (Jonckheere, 1954) shows that relationship between ACE activity and concentrations of PQ is statsistically significant for HUVEC(p ’ 0.01). In the group of HUVECinto which PQ was added to 10-3X af’tcr sonication, ACE activity was not significantly changed (99.0 * 6.7%, n=5), compared to the HUVECwithout addition of PQ. To confirm no direct inhibitory effect of PQ on ACE, human :lClf was purchased from PROTOGEN AG-, Switzerland. One hundred, 10 and 1 ng of ACE solutions were assayed for the activity by our method with
Cell Biology International
Reports, Vol. 75, No. 3, 7997
207
or without addition of 1O-3 or 10e4M PQ, but the addition the human ACE solutions had no effect on ACE activity.
of PQ into
Effect of PQ on LDH released from IHJVEC LDH released from HUVECincubated Kith PQ (0 - 10m3M) for 24 changed between controland PQ-treated hours was not. significantly cells. In a dition, incubation of confluent HUVECfor 24 hour with detachment or morphological PQ (O- lo- 4M) did not cause cellular change when assessed by phase contrast microscopy (Table).
Figure ACE activity in cell lysate of AUVJX Confluent HUVEC were incubated with M 199 containing various concentrations of PQ shown in the figure. After 24 hours, the cells were washed, scraped, sonicated and assayed for ACE as mentioned in “Materials and Methods”. Each bar shows mean It S .D. of 6 replicates. -
Concentration
of
PQ
(MI
Table EFFECT OF PQ ON LDH RELEASEFROMHUVEC The medium after the incubation of HUVECwith various concentrations of PQ was measured for LDH. Results are expressed as percentage to maximum LDH release and as the means f S.D. of 3 replicates. Concentration 0 10-6 10-5 10-4 10-3
of PQ (M)
% to maximum release 15.9 15.9 15.3 16.4 15.3
f 0.9 i 1.9 f 0.9 * 1.6 f 2.5
208
Cell Biology International
Reports, Vol. 75, No. 3, 1991
DISCUSSION The lung is one of organs most sensitive to PQ toxicity, but the specificity and the extent of PQ toxicity for individual lung cells the et a1.(1984) demonstrated that is not well-known. Skillrud cells are specific targets of PQ toxicity by epithelial ;;y;pi Cr release from rat type II cells treated with PQ. endothelial cell Brooks (1971), however, showed the focal blebbing, and loosening from the underlying basal lamina swelling, by electron microscopy as the earliest damage to alveolar tissue in 1985) the lungs of PQ-treated mice. In vitro study (Ody et al., revealed that PQ has a direct toxic,effect on cultured porcine from endothelial cells by showing increased LDH release aortic cultured porcine aortic endothelial cell. converting enzyme (ACE) is a dipeptidylAngiotensin carboxypeptidase found in plasma and vascular endothelial cells decapept i dc (Ryan et al., 1976; Ryan et al., 1984). It converts inactivates I to octapeptide angiotensin II and angiotensin bradykinin (Ryan et al., 1976; Ryan et al., 1984). Hollinger et al.(1980) reported that serum ACE increased and lung ACE decreased intraperitoneal injection of PQ, and they in the mouse after concluded that this is probably caused by damage to the luminal cells resulting in a membrane of pulmonary vascular endothelial also showed leakage of enzyme into the blood. Many investigators that the change in ACE activity reflects the impairment of vascular endothelial cells by using bleomycin (Lazo et al., 1981; Neuman et 1980), oleic acid (Nukiwa et al., 1982), PQ (Masaoka et al., al., 1989) and others. We previously demonstrated, however, that ACE activity was decreased in HUVEC treated with lipopolysaccharide without increased LDH release (Watanabe et al., 1990). In this study lysate of was decreased in cell we also showed that ACE activity HUVEC treated with PQ for 24 hours without increased LDH release. This result looks contradictory to that of Ody et al (1985). In the their experiments, however, LDH release was not increased until 2nd day from the start of the incubation with PQ and they used cultured porcine aortic endothelial cells which are difffercnt from ours. ACE activity could be decreased even in early stage of endothelial injury, when LDH release is not yet increased. But after a certain period (2 days or more), endothelial cells could be obviously damaged. Abe et a (1988) reported that furaassay is more sensitive than LDH and 51Cr assays for detection of endothelial Fujii et al. (1988) reported that serum ACE level injury. in a patient with PQ poisoning was decreased from the 2nd to 5th day of the disease. Our experimental result could support their clinical observation.
REFERENCES Abe,
M., Morita, I. and Murota, S. (1988). A new in vitro using furafor the quantification of endothelial cell Prostaglandins Luekot Essent Fatty Acids 34: 69-74.
method injury.
Cell Biology International
Reports, Vol. 15, No. 3, 1991
209
of lung lesions produced by Brooks, R.E. (1971). Ultrastructure ingested chemicals. I. Effect of the herbicide paraquat on mouse lung. Lab. Invest. 25: 536-545. J. and Siverstein, E. (1976). A sensitive fluorimetric Friedland, enzyme. Am. .J. Cl in. assay for serum angiotensin-converting Pathol. 66: 416-424. Fujii, Y., Nishiyama, Y., Miyauchi, Y., Maekawa, T. and Takeshita, H. (1988). Angiotensin converting enzyme in paraquat poisoning. Jap. J. Acute Med. 12: 331-332. S.W., Zuckerman, J.E., Hollinger, M.A., Patwell, Gorin, A.B., G. and Giri, S.N. (1980). Effect of paraquat on serum Parsons, converting enzyme. Am. Rev. Respir. Dis. 121: 795angiotensin 798. Nachman R.L., Becker, C.G. and Minick, C.R. (1973). ,Jaffe, E.A., Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin. Invest. 52: 2745-2756. Culture and identification of large vessel Jaffe, E.A. (1984). Biology of endothclial endothelial cells, in: Jaffe, E.A.(ed.). cells. Boston: Martinus-Nijhoff. A.R. (1954). A distribution-free k-sample test against Jonckheere, ordered alternatives. Biometrika 41: 133-145, 1954 Catravas, J.D. and Gillis, C.N. (1981). LilZO, .J.S., Reduction in serum and pulmonary angiotensin rabbit converting enzyme activity after subacute bleomycin treatment. Biochem. Pharmacol. 30: 2577-2584. Masaoka, F., Akahori, F., Ikeda, J. and Arai, S. (1989). Effect of I converting enzyme paraquat on serum and lung angiotensin (AICE) activity in beagle dogs. Vet. Hum. Toxicol. 31: 430-435. Neuman, R.A., Kimberly, P.J., Stuart, J.A. and Kelley, J. (1980). Assessment of bleomycin lung toxicity using angiotensin enzyme in pulmonary lavage. Cancer Res. 40: 3621converting 3626. Y., Arai, T. and Kira, Nukiwa, T., Matsuoka, R., Takagi, fi., Ishii, s. (1982). Responses of serum and lung angiotensin-converting enzyme activities in the early phase of pulmonary damage induced by oleic acid in dogs. Am. Rev. Respir. Dis. 126: 1080-1086. Ody, C. and Junod, A.F. (1985). Direct toxic effect of paraquot and oxygen on cultured endothelial cells. Lab. Invest. 52: 77-84. Ryan, J.W., Whitaker, C. and Chiu, A. (1976). Ryan, U.S., fAocalization of angiotensin converting enzyme (Kininase II). II. Immunocytochemistry and immunofluorescence. Tissue and Cell 8: 125-145. Ryan, U.S. and Ryan, J.W. (1984). Ccli biology of pulmonary cndothelium. Circulation 7O(suppl III): 46-62. Shibamoto, T. and Kobayashi, T. (1986). Acute effect of paraquat on lung fluid balance and prostanoid production in awake sheep. Am. Rev. Respir. Dis. 134: 1252-1257. Ski1 lrud, D.M. and Martin II, W.J. (1984). Paraquot-induced injury injury. of Type II alveolar cells. An in vitro model-of oxidant Am. Rev. Respir. Dis. 129: 995-999. Strittmatter, S.M. and Snyder, S. H. (1984). Angiotensin-converting in the male rat reproductive system: enzyme Autographic
210
Cell Biology International
Reports, Vol. 75, No. 3, 799 1
visualization with [‘Hlcaptopril. Endocrinol. 115: 2332-2342. Watanabe, K., Yoshida, M. and Jaffe, E.A. (1990). Influences of lipopolysaccharide on angiotensin converting enzyme activity expressed by human umbilical vein endothelial cells in culture. Jap. J. Thorac. Dis. 28: 1214-1219.
Paper
received
16.1O.go.
Revised
paper
accepted
7.1 .ql
.
International
Reports,
205
Vol. IS, No. 3, 9997
PARAQUATDEPRESSESTIIE ACTIVITY OF ANGIOTENSIN CONVERTINGENZYME EXPRESSED BY HUMANUMBILICAL VEIN ENDOTBELIALCELLS IN CULTURE Kentaro Watanabe+, Noriko Tamaru and Minoru Yoshida Second Department of Internal Medicine, University, Fukuoka, 814-o-1, Japan *Corresponding
School of Medicine,
Fukuoka
author
ABSTRACT The activity of angiotensin converting enzyme (ACE) in cell lysate of cultured human umbilical vein end thelial cells (HUVEC) after a 24-hour incubation with low3 and 10 -2 M of paraquat (PQ) was decreased. However, LDH released into the culture medium of HUVEC during the 24-hour incubation with PQ was not increased. Many investigators show that the change in serum ACE activity reflects of vascular endothelial cells. We showed in this the impairment report that ACE was decreased even at an early stage of endothelial injury induced by PQ, when LDff release is not yet increased.
INTRODUCTION used herbicide, is known to cause Paraquat (PQ) , a widely permeability edema, leading eventually to pulmonary fibrosis. demonstrated that plasma and lung lymph Shibamoto et a1.(1986) concentrations prostacyclin and thromboxane increased intravenous in*? usion of PQ in awake sheep. significantly after lntraperitoneal administration of PQ to mice produced a linear doseresponse increase in serum angiotensin converting enzyme and decrease in total lung ACE (Hollinger et al., 1980). Whether pulmonary vascular endothelial cells as well as alveolar epithelial are the critical sites or not in PQ poisoning has been a cells subject of controversy. In order to test the direct effect of PQ on cells, we cultured vascular endothelial human umbilical vein cells (HUVEC) and examined ACE activity expressed by endothelial HUVECand LDH release from HUVEC.
MATERIALSAND METBODS Culture of HUVEC HUVEC were obtained from human umbilical cord ve ins by digestion with 0.1% collagcnase as previously described (Jaffe et 1973; Jaffe, 1984). The isolated HUVECwere cultured in T75 al., flasks or 35 mmpetri-dishes (all precoated with 0.02% gelatin) in rledium 199 (M 199) containing 25 mM HEPESbuffered saline (HBS), (1 .6 penicillin (80 units/ml), streptomycin (80 ug/ml), L-glutamine 0309-I
651/91/030205-6/$03.00/O
0 1991 Academic
Press Ltd
206
Cell Biology International
Reports, Vol. 75, No. 3, 199 1
rell mM), amphotericin B (2 pg/ml), heparin (90 ug/ml), endothelial growth factor (ECGF) (20 vg/ml) and 4% human serum and 16% fetal calf serum (FCS) at 37’C under 5% C02. ACE activity in cell lysate of IIUVEC ACE activity was measured according to a modification of the fluorimetric method (Friedland et al., 1976; Strittmattcr ct al., 1984 ; Watanabe et al., 1990). HUVECin passage 2 were grown to in 35 mmpetri-dishes. Cells were fed with fresh medium confluency containing 4% human serum and 16% FCS with or without PQ. After 24 hours monolayers were washed 3 times with HBS, scraped by a rubber policeman, and frozen until assay. On the day of the assay, the samples were thawed and sonicated prior to assay. Twenty ul of t,h c sonicated samples were mixed with 80 ~1 of 5 mM hippuryl-histidylleucine (H-H-L) in HBS and incubated for 3 hours at 37’C. After 3 hours the reaction was terminated by the addition of 1.4 ml of’ 0.5 N NaOH. Then, 100 11 of 10 mg/ml of 0-phthaldialdehyde in methanol was added to the reaction mixture, followed by the addition of 250 ul of 6 N HCl after a 5 min interval. Fluorescence of the samples was measured by spectrofluorimeter with emission at 495 nm and with excitation at 365 nm. The standard curve was obtained using known concentrations of histidyl-leucine. Enzyme activity was exprcsscd as nanomoles of substrate hydrolyzed per hour p‘r dish. In another group, PQ was added to 10-5 M into the sonicatcd samples without pretreatment of PQ before mixing with H-II-I, in order to examine the direct inhibitory effect of PQ on ACE. Then the samples were assayed for ACE activity in tho same manner as mentioned above. Mcsurement of LDII release The supernatants after the incubations of HUVEC with various concentrations of PQ for 24 hours were assayed for LDlI by a spectrophotometric assay based on NADHoxidation using a commcrci al kit(Sigma). The LDH measurements were corrected for the LDll activity measured in the pre-culture medium. Maximal LDH relcasc was determined by scraping and sonicating non-PQ-treated ccl Is and measuring LDH in the supernatant. RESUI.TS Effect of PQ on ACE activity in cell lysate of IIUVEC Figure shows ACE activity in cell lysate of fIUVECafter a 24 hour-incubation with or without PQ. Analysis of thcsc data by Jonckheere’s test (Jonckheere, 1954) shows that relationship between ACE activity and concentrations of PQ is statsistically significant for HUVEC(p ’ 0.01). In the group of HUVECinto which PQ was added to 10-3X af’tcr sonication, ACE activity was not significantly changed (99.0 * 6.7%, n=5), compared to the HUVECwithout addition of PQ. To confirm no direct inhibitory effect of PQ on ACE, human :lClf was purchased from PROTOGEN AG-, Switzerland. One hundred, 10 and 1 ng of ACE solutions were assayed for the activity by our method with
Cell Biology International
Reports, Vol. 75, No. 3, 7997
207
or without addition of 1O-3 or 10e4M PQ, but the addition the human ACE solutions had no effect on ACE activity.
of PQ into
Effect of PQ on LDH released from IHJVEC LDH released from HUVECincubated Kith PQ (0 - 10m3M) for 24 changed between controland PQ-treated hours was not. significantly cells. In a dition, incubation of confluent HUVECfor 24 hour with detachment or morphological PQ (O- lo- 4M) did not cause cellular change when assessed by phase contrast microscopy (Table).
Figure ACE activity in cell lysate of AUVJX Confluent HUVEC were incubated with M 199 containing various concentrations of PQ shown in the figure. After 24 hours, the cells were washed, scraped, sonicated and assayed for ACE as mentioned in “Materials and Methods”. Each bar shows mean It S .D. of 6 replicates. -
Concentration
of
PQ
(MI
Table EFFECT OF PQ ON LDH RELEASEFROMHUVEC The medium after the incubation of HUVECwith various concentrations of PQ was measured for LDH. Results are expressed as percentage to maximum LDH release and as the means f S.D. of 3 replicates. Concentration 0 10-6 10-5 10-4 10-3
of PQ (M)
% to maximum release 15.9 15.9 15.3 16.4 15.3
f 0.9 i 1.9 f 0.9 * 1.6 f 2.5
208
Cell Biology International
Reports, Vol. 75, No. 3, 1991
DISCUSSION The lung is one of organs most sensitive to PQ toxicity, but the specificity and the extent of PQ toxicity for individual lung cells the et a1.(1984) demonstrated that is not well-known. Skillrud cells are specific targets of PQ toxicity by epithelial ;;y;pi Cr release from rat type II cells treated with PQ. endothelial cell Brooks (1971), however, showed the focal blebbing, and loosening from the underlying basal lamina swelling, by electron microscopy as the earliest damage to alveolar tissue in 1985) the lungs of PQ-treated mice. In vitro study (Ody et al., revealed that PQ has a direct toxic,effect on cultured porcine from endothelial cells by showing increased LDH release aortic cultured porcine aortic endothelial cell. converting enzyme (ACE) is a dipeptidylAngiotensin carboxypeptidase found in plasma and vascular endothelial cells decapept i dc (Ryan et al., 1976; Ryan et al., 1984). It converts inactivates I to octapeptide angiotensin II and angiotensin bradykinin (Ryan et al., 1976; Ryan et al., 1984). Hollinger et al.(1980) reported that serum ACE increased and lung ACE decreased intraperitoneal injection of PQ, and they in the mouse after concluded that this is probably caused by damage to the luminal cells resulting in a membrane of pulmonary vascular endothelial also showed leakage of enzyme into the blood. Many investigators that the change in ACE activity reflects the impairment of vascular endothelial cells by using bleomycin (Lazo et al., 1981; Neuman et 1980), oleic acid (Nukiwa et al., 1982), PQ (Masaoka et al., al., 1989) and others. We previously demonstrated, however, that ACE activity was decreased in HUVEC treated with lipopolysaccharide without increased LDH release (Watanabe et al., 1990). In this study lysate of was decreased in cell we also showed that ACE activity HUVEC treated with PQ for 24 hours without increased LDH release. This result looks contradictory to that of Ody et al (1985). In the their experiments, however, LDH release was not increased until 2nd day from the start of the incubation with PQ and they used cultured porcine aortic endothelial cells which are difffercnt from ours. ACE activity could be decreased even in early stage of endothelial injury, when LDH release is not yet increased. But after a certain period (2 days or more), endothelial cells could be obviously damaged. Abe et a (1988) reported that furaassay is more sensitive than LDH and 51Cr assays for detection of endothelial Fujii et al. (1988) reported that serum ACE level injury. in a patient with PQ poisoning was decreased from the 2nd to 5th day of the disease. Our experimental result could support their clinical observation.
REFERENCES Abe,
M., Morita, I. and Murota, S. (1988). A new in vitro using furafor the quantification of endothelial cell Prostaglandins Luekot Essent Fatty Acids 34: 69-74.
method injury.
Cell Biology International
Reports, Vol. 15, No. 3, 1991
209
of lung lesions produced by Brooks, R.E. (1971). Ultrastructure ingested chemicals. I. Effect of the herbicide paraquat on mouse lung. Lab. Invest. 25: 536-545. J. and Siverstein, E. (1976). A sensitive fluorimetric Friedland, enzyme. Am. .J. Cl in. assay for serum angiotensin-converting Pathol. 66: 416-424. Fujii, Y., Nishiyama, Y., Miyauchi, Y., Maekawa, T. and Takeshita, H. (1988). Angiotensin converting enzyme in paraquat poisoning. Jap. J. Acute Med. 12: 331-332. S.W., Zuckerman, J.E., Hollinger, M.A., Patwell, Gorin, A.B., G. and Giri, S.N. (1980). Effect of paraquat on serum Parsons, converting enzyme. Am. Rev. Respir. Dis. 121: 795angiotensin 798. Nachman R.L., Becker, C.G. and Minick, C.R. (1973). ,Jaffe, E.A., Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin. Invest. 52: 2745-2756. Culture and identification of large vessel Jaffe, E.A. (1984). Biology of endothclial endothelial cells, in: Jaffe, E.A.(ed.). cells. Boston: Martinus-Nijhoff. A.R. (1954). A distribution-free k-sample test against Jonckheere, ordered alternatives. Biometrika 41: 133-145, 1954 Catravas, J.D. and Gillis, C.N. (1981). LilZO, .J.S., Reduction in serum and pulmonary angiotensin rabbit converting enzyme activity after subacute bleomycin treatment. Biochem. Pharmacol. 30: 2577-2584. Masaoka, F., Akahori, F., Ikeda, J. and Arai, S. (1989). Effect of I converting enzyme paraquat on serum and lung angiotensin (AICE) activity in beagle dogs. Vet. Hum. Toxicol. 31: 430-435. Neuman, R.A., Kimberly, P.J., Stuart, J.A. and Kelley, J. (1980). Assessment of bleomycin lung toxicity using angiotensin enzyme in pulmonary lavage. Cancer Res. 40: 3621converting 3626. Y., Arai, T. and Kira, Nukiwa, T., Matsuoka, R., Takagi, fi., Ishii, s. (1982). Responses of serum and lung angiotensin-converting enzyme activities in the early phase of pulmonary damage induced by oleic acid in dogs. Am. Rev. Respir. Dis. 126: 1080-1086. Ody, C. and Junod, A.F. (1985). Direct toxic effect of paraquot and oxygen on cultured endothelial cells. Lab. Invest. 52: 77-84. Ryan, J.W., Whitaker, C. and Chiu, A. (1976). Ryan, U.S., fAocalization of angiotensin converting enzyme (Kininase II). II. Immunocytochemistry and immunofluorescence. Tissue and Cell 8: 125-145. Ryan, U.S. and Ryan, J.W. (1984). Ccli biology of pulmonary cndothelium. Circulation 7O(suppl III): 46-62. Shibamoto, T. and Kobayashi, T. (1986). Acute effect of paraquat on lung fluid balance and prostanoid production in awake sheep. Am. Rev. Respir. Dis. 134: 1252-1257. Ski1 lrud, D.M. and Martin II, W.J. (1984). Paraquot-induced injury injury. of Type II alveolar cells. An in vitro model-of oxidant Am. Rev. Respir. Dis. 129: 995-999. Strittmatter, S.M. and Snyder, S. H. (1984). Angiotensin-converting in the male rat reproductive system: enzyme Autographic
210
Cell Biology International
Reports, Vol. 75, No. 3, 799 1
visualization with [‘Hlcaptopril. Endocrinol. 115: 2332-2342. Watanabe, K., Yoshida, M. and Jaffe, E.A. (1990). Influences of lipopolysaccharide on angiotensin converting enzyme activity expressed by human umbilical vein endothelial cells in culture. Jap. J. Thorac. Dis. 28: 1214-1219.
Paper
received
16.1O.go.
Revised
paper
accepted
7.1 .ql
.