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Protective effects of tetrahydrobiopterin against nitric oxide-induced endothelial cell death
Life Sciences, Vol. 63, No. 18, pp. 1585~1592,1998 lzqyright 0 1998 Ekvier science Inc. Printed in the USA. All rights rc.urvcd 0024-3205/98 $19.00 + .oo
PI1 SOOZS-3205(98)00427-S
ELSEVIER
PROTECTIVE
EFFECTS OF TETRAHYDROBIOPTERIN AGAINST INDUCED ENDOTHELIAL CELL DEATH
Shunichi Shimizu”,
NITRIC
OXIDE-
Masakazu Ishii’, Yutaka Kawakami’, Kazutaka Momose’ and Toshinori Yamamoto’
Departments of Pharmacology’ and Clinical Pharmacy2, School of Pharmaceutical Sciences, Showa University, l-S-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. (Received in final form August 13, 1998)
Summary The purpose of this study was to examine whether tetrahydrobiopterin (BH,), one of the cofactors of nitric oxide (NO) synthase, attenuates NO-induced endothelial cell death. S-Nitroso-N-acetyl-DL-penicillamine (SNAP) was used as a NO donor. Endothelial cell death was assessed by the leakage of intracellular lactate dehydrogenase (LDH). Addition of SNAP to endothelial cells time- and concentrationdependently induced endothelial cell death. The SNAP-induced endothelial cell death was strongly reduced by the treatment with carboxy-PTIO, a NO scavenger, or catalase, but not with superoxide dismutase (SOD). Moreover, pretreatment with sepiapterin, a precursor of BH,, increased intracellular BH, content, and strongly reduced the SNAP-induced endothelial cell death. Both the increase in BH, content and the protective effects of sepiapterin were prevented by co-pretreatment with Nacetylserotonin (NAS), an inhibitor of BH, synthesis. These findings suggest that the cytotoxicity of NO released from SNAP involves Hz02 production, and increase in intracellular BH, content attenuates NO-induced endothelial cell death. Scavenging of Hz02 by BH, may be at least one of the mechanisms by which BH, reduces NOinduced endothelial cell death. &Y Words: tetrahydrobiopterin, S-Nitroso-N-acetyl-DL-penicillamine, nitric oxide, cell death, hydrogen peroxide, endothelial cells
Nitric oxide (NO), a nitrogen-centered free radical gas, is an important signaling molecule in the regulation of vascular tone and immune surveillance. NO is synthesized by the conversion of L-arginine to L-citrulline by NO synthase. There are three isoforms of NO synthase, including two constitutive forms from cerebellum and endothelial cells, and an inducible form from macrophages
* To whom correspondence
should be addressed. TEL: +8 l-3-3784-82 12. FAX: +81-3-3784-3232.
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Tetrahydrobiopterin Reduces NO Cytotoxicity
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and smooth muscle cells (1). The latter NO synthase, which is induced by lipopolysaccharide (LPS) and/or cytokines, continuously releases large amounts of NO (2-6). Large amounts of NO induce cell death in various cell types, including endothelial cells, smooth muscle cells and HL-60 cells (7-9). Although the mechanisms of NO-induced cell death are not yet clear, the cytotoxicity of NO is likely to involve a production of H,O, in the cells, since the toxic effect of NO has been shown to be reduced by catalase (8). In fact, Asahi et al. (lo,1 1) have shown that glutathione peroxidase, which is an enzyme involved in intracellular HzOZ scavenging, is inactivated by NO. Therefore, NO may induce to accumulate intracellular HzO, via inhibition of glutathione peroxidase, and may cause cell death. Recently, we showed that tetrahydrobiopterin (BH,), a cofactor of NO synthase, has a protective effect against H,O,-induced endothelial cell injury (12). The protective effect of BH, seems to involve H,O? scavenging activity. Interestingly, increases of BH, synthesis as well as inducible NO synthase have been shown to be induced by LPS and/or cytokines in vascular endothelial cells, smooth muscle cells and neuroblastoma cells (13-16). Although BH, is an essential cofactor for producing NO from NO synthase, it is possible that increased BH, content induced by LPS or cytokines prevents NO cytotoxicity. In the present study, to evaluate our hypothesis that the increase in BH, content that accompanies inducible NO synthase induction by LPS and/or cytokines protects cells against NO cytotoxicity, the effect of BH, on NO donor-induced endothelial cell death was examined.
Materials and Methods Cell culture Bovine aortic endothelial cells were cultured as previously described (17). Fresh bovine thoracic aortae obtained from an abattoir were kept in ice-cold phosphate-buffered saline solution containing 100 U/ml penicillin and 100 ug/ml streptomycin. Endothelial cells were obtained by scraping the luminal surface with a razor blade and were cultured in minimum essential medium containing 10% fetal bovine serum, 100 U/ml penicillin and 100 ug/ml streptomycin. The cells were finally grown in 24-well plates to measure leakage of lactate dehydrogenase (LDH) or in 6-well plates to measure BH, content. Cells at 4 to 8 passages were used for the experiments. Lactate dehydrogenase release Cell death was estimated by determining LDH release as previously described (17). Confluent endothelial cells in 24-well plates were washed three times with physiological saline solution (PSS, pH 7.4) containing 118.5 mM NaCl, 4.74 mM KCl, 2.5 mM CaCI,, 1.18 mM 1.18 mM KH2P0,, 2.5 mM NaHC03, 11 mM glucose and 10 mM N-2MgSO,, hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES). The washed cells were then treated with various concentrations of SNAP at 37°C in 0.5 ml of PSS. LDH activity was determined in cell supematants and cell fractions of endothelial cells solubilized in 2% Triton X-100 (18). The percentage of the total LDH activity (supematant fraction plus cell fraction) released into the supematant fraction was then calculated. Sepiapterin and N-acetylserotonin (NAS) were added to culture medium for 24-h pretreatments, and washed out with PSS. They were not present throughout the experiments. All drugs used did not affect the measurement of LDH activity. Measurement of intracellular tetrahydrobiopterin content Intracellular BH, content was measured as previously described (19). Confluent endothelial cells in 6-well plates were washed 3 times with PSS. Intracellular BH, levels were determined as
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Tctrahydrohiapterin Rcduccs NO Cytotaxicity
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biopterin. by oxidation in acidic conditions and reverse-phase high performance liquid chromatography with fluorometric detection. as previously reported (20). Cell were treated with sepiapterin and NAS in culture medium for 24-h.
Muteriuls Sepiapterin was obtained from Cayman Chemical Co. (Ann Arbor. MI. U.S.A.). S-NitrosoN-acetyl-DL-penicillamine (SNAP) and carboxy-PTIO were obtained from Dojindo Laboratories (Kumamoto. Japan). N-Acetylserotonin (NAS) and catalase were obtained from Sigma Chemical Co. (St. Louis. MO. U.S.A.). Superoxide dismutase was obtained from Wako Pure Chemicals (Osaka. Japan). All other reagents were of the highest grade commercially available. Stutisticul unu1y.C Data are presented as means + S.E.M. of n observations. The statistical significance of observed differences was determined by analysis of variance followed by Bonferroni’s method. Differences between means were considered significant when P was less than 0.05.
60
control SNAP h
0.5 mM
SNAP
I mM
SNAP
2 mM
time ( hour ) Fig. 1 Time course of LDH release from endothelial cells exposed to various concentrations of SNAP at 37°C. At the indicated times after addition of SNAP, the percentage of LDH released into the medium was determined. Typical results from 3 experiments are shown as the means * S.E.M. of quadruplicate assays.
Results Induction of endothelial cell death by SNAP treatment. We examined whether NO induces endothelial cell death using SNAP, a NO donor (Fig.l). Endothelial cell death was assessed by measuring the release of intracellular LDH. SNAP caused LDH release from endothelial cells in a concentration-dependent manner starting at 8 h after its addition (Fig. 1). The SNAP (2 mM)-induced cell death was strongly attenuated by treatment with carboxy-PTIO. a specific NO scavenger. suggesting that NO released from SNAP is implicated in SNAP-induced cell death (Fig. 2).
Vol. 63, No. 18, 1998
Tetrahydrobiipterio Reduces NO Cytotoxicity
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10
6 &
loo
carboxy-PTIO (pM)
d
Fig. 2 Effect of NO scavenger on SNAP-induced LDH release from endothelial cells. Cells were incubated with SNAP (2 mM) in the presence or absence of carboxy-PTIO (10, 100 pM) for 10 h at 37°C. Typical results from 3 experiments are shown as the means + S.E.M. of quadruplicate assays. *Significant change in comparison to control group (p
B
A - ‘001
_ 601
$
time (hour)
Q
$
#sj?0.1 c' 8
I
10 loo loo
sepiapterin (PM) 7~ (1 mM)
Fig. 3
Effects of sepiapterin and N-acetylserotonin (NAS) on SNAP-induced LDH release from endothelial cells. A; Cells were incubated with sepiapterin (100 pM) for 24 h. The cells were washed with PSS, and then incubated with SNAP (2 mM) at 37°C. At the indicated times after addition of SNAP, the percentage of LDH released into the medium was determined. B; Cells were incubated with sepiapterin (0.1-100 pM) or sepiapterin (100 PM) plus NAS (1 mM) for 24 h. The cells were washed with PSS, and then incubated with SNAP (2 mM) for 10 h at 37°C. Typical results from 3 experiments are shown as the means f S.E.M. of quadruplicate assays. *Significant change in comparison to control group (~~0.05). “Significant change in comparison to sepiapterin (100 PM) alone group (~~0.05).
Vol. 63, No. 18, 1998
Tetrahydrobiopterin
0’
control’
0.1
Reduces
1.0 sepiapterin
NO Cytotoxicity
10
1589
100
(CM)
NAS
Fig. 4 Effects of sepiapterin and N-acetylserotonin (NAS) on tetrahydrobiopterin content in endothelial cells. Cells were incubated with sepiapterin (0.1-100 PM) or sepiapterin (100 PM) plus NAS (1 mM) for 24 h. Typical results from 3 experiments are shown as the means + S.E.M. of quadruplicate assays. *Significant change in comparison to control group (~~0.05). #Significant change in comparison to sepiapterin (100 PM) alone group (p(O.05).
Effects of sepiapterin and N-acetylserotonin on SNAP-induced endothelial cell death. Pretreatment with sepiapterin (100 PM), a precursor of BH, synthesis, reduced SNAPinduced endothelial cell death at 8 and 10 h after addition of SNAP, but not at 24 h (Fig. 3A). The protective effect of sepiapterin was concentration dependent (Fig. 3B). The protective effect of sepiapterin was blocked by co-pretreatment with NAS (1 mM), which is an inhibitor of sepiapterin reductase (Fig. 3B). Esfects of sepiapterin and N-acetylserotonin on tetrahydrobiopterin content in endothelial cells. Addition of sepiapterin to endothelial cells concentration-dependently increased BH, content. and the increase in BH, content by sepiapterin (100 PM) was inhibited by NAS (1 mM) (Fig.4). @,&zctsof SOD and catalase on SNAP-induced cell death. To determine whether superoxide and/or H20z are implicated in SNAP-induced cell death, the effects of SOD and catalase were examined. As shown in Fig. 5, SNAP-induced cell death was markedly reduced by treatment with catalase (100, 300, or 500 U/ml). Inactivated catalase by incubated in boiling water for 1 min did not affect SNAP-induced cell death (data not shown). On the other hand, SOD at 50 6r 100 U/ml had no effect on SNAP-induced cell death, and a high concentration of SOD (300 U/ml) increased the cell death.
Discussion In the present study, addition of SNAP to endothelial
cells induced cell death. NO released
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from NO-donors, including SNAP, sodium nitroprusside (SNP) and 3-morpholinosydnonimine-Nethylcarbamide (SIN-l), has been shown to induce cell injury in various cell types (7-9). In the present study, the SNAP-induced endothelial cell death was strongly attenuated by treatment with carboxy-PTIO, a scavenger of NO. Therefore, SNAP-induced endothelial cell death is likely to be mediated by NO released from SNAP. Although the mechanisms of NO-induced cell death remain unclear, the toxicity of SIN- 1, which produces both superoxide and NO, has been shown to be reduced by treatment with catalase. but not by SOD, in rat liver endothelial cells (7). Interestingly, even the toxicity of SNP. which produces NO alone, has been shown to be reduced by the treatment with catalase (8). We also found that SNAP-induced endothelial cell death was attenuated by catalase. but not by SOD. In fact, Asahi et al. (10,ll) have shown that glutathione peroxidase, which is part of the intracellular H,Oz-scavenging system, is inactivated by NO. Intracellular glutathione (GSH) content may also be decreased by reaction of GSH with NO to form S-nitroso-glutathione (21). Therefore, NO may induce to accumulate intracellular Hz02 via inhibition of glutathione peroxidase activity, and may cause cell death. We previously reported that treatment of endothelial cells with a GSH-depleting agent induces cell death (22). Thus, endogenous production of H,Oz caused by the treatment with NO is likely to be implicated in NOinduced cell death. NO has been shown to react with HzO1 to form peroxynitrite and singlet oxygen, which are toxic (23,24). Although there are some reports that NO attenuates H,O,-induced cytotoxicity (25,26), HzO, has also been shown to enhance NO toxicity (27,28). Therefore, H,O, caused by the treatment with NO may increase NO toxicity.
$
,L
SOD (U/ml)
catalase (U/ml)
3
Effects of Cells were or catalase are shown comparison
Fig. 5 SOD and catalase on SNAP-induced LDH release from endothelial cells. incubated with SNAP (2 mM) in the presence of SOD (50, 100, or 300 U/ml) (100, 300, or 500 U/ml) for 10 h at 37°C. Typical results from 3 experiments as the means k S.E.M. of quadruplicate assays. *Significant change in to control group (~‘0.05).
Pretreatment with sepiapterin, a precursor of BH, synthesis, increased BH, content and strongly attenuated SNAP-induced endothelial cell death. Both the increase in BH4 content and the protective effect of sepiapterin were prevented by co-pretreatment with NAS, a sepiapterin
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Tetrahydrobiopterin Reduces NO Cytotoxicity
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reductase inhibitor. These findings indicate that an increase in BH, content reduces NO-induced endothelial cell death. We recently reported that the increase in BH, content caused by treatment with sepiapterin enhances H,O, scavenging and markedly reduces H?O,-induced endothelial cell death (12). Pterins, including BH,, have been shown to have scavenging activity for reactive oxygen species and antioxidative activity (29). Therefore, these results strongly suggest that H,O?scavenging activity and/or antioxidative activity of BH, may be one of the mechanisms of the protective effect of sepiapterin. Kojima et al. (9) have reported that SNAP-induced cell injury is inhibited by exogenous BH, at an early time post-treatment with SNAP in HL-60 cells. They speculated that BH, quenches NO, since BH, reacted with l,l-diphenyl-2-picrylhydrazyl, a stable radical, in an ethanol solution. However, that investigation was indirect. Future studies are needed to determine whether BH, reduces NO-induced cell death via scavenging of H,O, or NO, or both. Protective effect of sepiapterin was not observed at 24 h after addition of SNAP. Kojima et al. (9) reported that SNAP-induced HL-60 cell injury at 5 h after addition of it is reduced by BH,. whereas BH, increased the cytotoxicity at 20 h after addition of it. They discussed that a longer incubation of SNAP with BH, may yield more toxic NO-related products such as peroxynitrite. However, it is speculative. More detail studies are needed to determine why BH, does not affect SNAP-induced cytotoxicity in a longer incubation. Inducible NO synthase, which is induced by LPS and/or inflammatory cytokines, produces large amounts of NO, and causes hypotension and circulatory failure in septic shock (30-32). BH, synthesis is also induced by LPS and/or cytokines in many types of cells, including macrophages. smooth muscle cells and endothelial cells (13- 16). Although induction of BH, synthesis is believed to be required for NO production by inducible NO synthase (13,33-35) the role of BH, is not fully understood. In this report, we showed that BH, protects endothelial cells against NO-induced cytotoxicity. It is possible that increase in BH, content plays a role in protecting endothelial cells against death induced by large amounts of NO produced by inducible NO synthase. In conclusion, our findings indicate that NO released from SN.4P induces endothelial cell death, and the SNAP-induced endothelial cell death may be mediated by the endogenous production of H,O,, rather than superoxide. Moreover, increase in BH, content strongly blocks NO-induced endothelial cell injury. The protective effect of BH, is likely to involve scavenging of the H,O, which is produced by treatment of cells with NO.
References 1. 2. 3. 4. 5. 6. 7. 8.
C. NATHAN AND Q.W. XIE, Cell 78 91.5-918 (1994). R. BUSSE AND A. MULSCH, FEBS Lett. 275 87-90 (1990). A.K. NUSSLERAND T.R. BILLIAR, J. Leukoc. Biol. 54 171-178 (1993). M.W. RADOMSKI, R.M.J. PALMER AND S. MONCADA, Proc. Natl. Acad. Sci. U.S.A. 87 10043-10047 (1990). R.M.J. PALMER, L. BRIDGE, N.A. FOXWELL AND S. MONCADA, Br. J. Pharmacol. 105 11-12 (1992). K. KANNO, Y. HIRATA, T. IMAI, M. IWASHINA AND F. MARUMO, Am. J. Physiol. 267 H23 1g-H2324 (1994). T. VOLK, I, IOANNIDIS, M. HENSEL, H. DeGROOT AND W..J. KOX, Biochem. Biophys. Res. Commun. 213 196-203 (1995). G.T. GOBBEL, T.Y.-Y. CHAN AND P.H. CHAN, J. Pharmacol. Exp. Ther. 282 1600-l 607 (1997).
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9. S. KOJIMA, K. NIMURA, H. KOMATSU, T. TAGUCHI AND H. IIZUKA, Anticancer Res. 17 929-938 (1997). 10. M. ASAHI, J. FUJII, K. SUZUKI, H. G. SEO, T. KUZUYA, M. HORI, M. TADA, S. FUJI1 AND N. TANIGUCHI, J. Biol. Chem. 270 2 1035-2 1039 (1995). 11. M. ASAHI, J. FUJII, T. TAKAO, T. KUZUYA, M. HORI, Y. SHIMONISHI AND N. TANIGUCHI, J. Biol. Chem. 272 19152-19157 (1997). 12. M. ISHII, S. SHIMIZU, K. MOMOSE AND T. YAMAMOTO, Pharmacol. Toxicol. 82 280286 (1998). 13. S.S. GROSS AND R. LEVI, J. Biol. Chem. 267 25722-25729 (1992). 14. G. SCHOEDON, N. BLAU, M. SCHNEEMANN, G. FLURY AND A. SCHAFFNER, Biochem. Biophys. Res. Commun. 199 504-5 10 (1994). 15. Y. HATTORI, N. NAKANISHI, K. KASAI, Y. MURAKAMI AND S. SHIMODA, Exp. Physiol. 81 665-671 (1996). 16. A. OTA, S. YOSHIDA, T. NOMURA, S. MATSUI, Y. HAGINO, K. UMEZAWA, S. KATOH AND T. NAGATSU, J. Neurochem. 67 2540-2548 (1996). 17. S. SHIMIZU, M. NOMOTO, T. YAMAMOTO AND K. MOMOSE, Br. J. Pharmacol. 113, 564-568 (1994). 18. R.L. THIES AND A.P. AUTOR, Arch. Biochem. Biophys. 286 353-363 (1991). 19. M. ISHII, S. SHIMIZU, T. YAMAMOTO, K. MOMOSE AND Y. KUROIWA, Life Sci. 61 739-747 (1997). 20. T. FUKUSHIMA AND J.C. NIXON, Anal. Biochem. 102 176-l 88 (1980). 21. R.M. CLANCY, D. LEVARTOVSKY, J. LESZCZYNSKA-PIZIAK, J. YEGUDIN AND S.B. ABRAMSON, Proc. Natl. Acad. Sci. U.S.A. 91 3680-3684 (1994). 22. M. ISHII, T. YAMAMOTO, S. SHIMIZU, A. SANO, K. MOMOSE AND Y. KUROIWA, Pharmacol. Toxicol. 80 191-196 (1997). 23. A.A. NORONHA-DUTRA, M. M. EPPERLEIN AND N. WOOLF, FEBS Lett. 321 59-62 (1993). 24. J.S. STAMLER, D.J. SINGEL AND J. LOSCALZO, Science 258 1898-1902 (1992). 25. M.P. GUPTA, V. EVANOFF AND C.M. HART, Am. J. Physiol. 272 L1133-L1141 (1997). 26. J. CHANG, N.V. RAO, B.A. MARKEWITZ, J.R. HOIDAL AND J.R. MICHAEL, Am. J. Physiol. 270 L93 1-L940 (1996). 27. Y. HATA, S. OTA, H. HIRAISHI, A. TERANO AND K. J. IVERY, Biochim. Biophys. Acta 1290 257-260 (1996). 28. I. IOANNIDIS, T. VOLK AND H. DeGROOT, Adv. Exp. Med. Biol. 387 25-30 (1996). 29. S. KOJIMA, S. ONA, I. IIZUKA, T. ARAI, H. MORI AND K. KUBOTA, Free Radic. Res. 23 419-430 (1995). 30. E. NAVA, R.M.J. PALMER AND S. MONCADA, Lancet 338 1555-1557 (1991). 3 1. A. PETROS, D. BENNETT AND P. VALLANCE, Lancet 338 1557- 1558 (1991). 32. S. ENDO, K. INADA, Y. YAMADA, T. TAKAKUWA, H. NAKAE, T. KASAI, S. KOIKE. Y. INOUE, M. NIIMI, G. WAKABAYASHI AND S. TANIGUCHI, Res. Commun. Mol. Pathol. Pharmacol. 94 23-38 (1996). 33. K. SCHMIDT, E. R. WERNER, B. MAYER, H. WACHTER AND W. R. KUKOVETZ, Biochem. J. 281297-300 (1992). 34. D.K. NAKAYAMA, D. A. GELLER, M. D. SILVIO, G. BLOOMGARDEN, P. DAVIES, B. R. PITT, K. HATAKEYAMA. H. KAGAMIYAMA, R. L. SIMMONS AND T. R. BILLIAR, Am. J. Physiol. 266 L455-L460 (1994). 35. G. SCHOEDON, N. BLAU, M. SCHNEEMANN, G. FLURY AND A. SCHAFFNER, Biochem. Biophys. Res. Commun. 199 504-5 10 (1994).
PI1 SOOZS-3205(98)00427-S
ELSEVIER
PROTECTIVE
EFFECTS OF TETRAHYDROBIOPTERIN AGAINST INDUCED ENDOTHELIAL CELL DEATH
Shunichi Shimizu”,
NITRIC
OXIDE-
Masakazu Ishii’, Yutaka Kawakami’, Kazutaka Momose’ and Toshinori Yamamoto’
Departments of Pharmacology’ and Clinical Pharmacy2, School of Pharmaceutical Sciences, Showa University, l-S-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. (Received in final form August 13, 1998)
Summary The purpose of this study was to examine whether tetrahydrobiopterin (BH,), one of the cofactors of nitric oxide (NO) synthase, attenuates NO-induced endothelial cell death. S-Nitroso-N-acetyl-DL-penicillamine (SNAP) was used as a NO donor. Endothelial cell death was assessed by the leakage of intracellular lactate dehydrogenase (LDH). Addition of SNAP to endothelial cells time- and concentrationdependently induced endothelial cell death. The SNAP-induced endothelial cell death was strongly reduced by the treatment with carboxy-PTIO, a NO scavenger, or catalase, but not with superoxide dismutase (SOD). Moreover, pretreatment with sepiapterin, a precursor of BH,, increased intracellular BH, content, and strongly reduced the SNAP-induced endothelial cell death. Both the increase in BH, content and the protective effects of sepiapterin were prevented by co-pretreatment with Nacetylserotonin (NAS), an inhibitor of BH, synthesis. These findings suggest that the cytotoxicity of NO released from SNAP involves Hz02 production, and increase in intracellular BH, content attenuates NO-induced endothelial cell death. Scavenging of Hz02 by BH, may be at least one of the mechanisms by which BH, reduces NOinduced endothelial cell death. &Y Words: tetrahydrobiopterin, S-Nitroso-N-acetyl-DL-penicillamine, nitric oxide, cell death, hydrogen peroxide, endothelial cells
Nitric oxide (NO), a nitrogen-centered free radical gas, is an important signaling molecule in the regulation of vascular tone and immune surveillance. NO is synthesized by the conversion of L-arginine to L-citrulline by NO synthase. There are three isoforms of NO synthase, including two constitutive forms from cerebellum and endothelial cells, and an inducible form from macrophages
* To whom correspondence
should be addressed. TEL: +8 l-3-3784-82 12. FAX: +81-3-3784-3232.
1586
Tetrahydrobiopterin Reduces NO Cytotoxicity
Vol. 63, No. 18, 1998
and smooth muscle cells (1). The latter NO synthase, which is induced by lipopolysaccharide (LPS) and/or cytokines, continuously releases large amounts of NO (2-6). Large amounts of NO induce cell death in various cell types, including endothelial cells, smooth muscle cells and HL-60 cells (7-9). Although the mechanisms of NO-induced cell death are not yet clear, the cytotoxicity of NO is likely to involve a production of H,O, in the cells, since the toxic effect of NO has been shown to be reduced by catalase (8). In fact, Asahi et al. (lo,1 1) have shown that glutathione peroxidase, which is an enzyme involved in intracellular HzOZ scavenging, is inactivated by NO. Therefore, NO may induce to accumulate intracellular HzO, via inhibition of glutathione peroxidase, and may cause cell death. Recently, we showed that tetrahydrobiopterin (BH,), a cofactor of NO synthase, has a protective effect against H,O,-induced endothelial cell injury (12). The protective effect of BH, seems to involve H,O? scavenging activity. Interestingly, increases of BH, synthesis as well as inducible NO synthase have been shown to be induced by LPS and/or cytokines in vascular endothelial cells, smooth muscle cells and neuroblastoma cells (13-16). Although BH, is an essential cofactor for producing NO from NO synthase, it is possible that increased BH, content induced by LPS or cytokines prevents NO cytotoxicity. In the present study, to evaluate our hypothesis that the increase in BH, content that accompanies inducible NO synthase induction by LPS and/or cytokines protects cells against NO cytotoxicity, the effect of BH, on NO donor-induced endothelial cell death was examined.
Materials and Methods Cell culture Bovine aortic endothelial cells were cultured as previously described (17). Fresh bovine thoracic aortae obtained from an abattoir were kept in ice-cold phosphate-buffered saline solution containing 100 U/ml penicillin and 100 ug/ml streptomycin. Endothelial cells were obtained by scraping the luminal surface with a razor blade and were cultured in minimum essential medium containing 10% fetal bovine serum, 100 U/ml penicillin and 100 ug/ml streptomycin. The cells were finally grown in 24-well plates to measure leakage of lactate dehydrogenase (LDH) or in 6-well plates to measure BH, content. Cells at 4 to 8 passages were used for the experiments. Lactate dehydrogenase release Cell death was estimated by determining LDH release as previously described (17). Confluent endothelial cells in 24-well plates were washed three times with physiological saline solution (PSS, pH 7.4) containing 118.5 mM NaCl, 4.74 mM KCl, 2.5 mM CaCI,, 1.18 mM 1.18 mM KH2P0,, 2.5 mM NaHC03, 11 mM glucose and 10 mM N-2MgSO,, hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES). The washed cells were then treated with various concentrations of SNAP at 37°C in 0.5 ml of PSS. LDH activity was determined in cell supematants and cell fractions of endothelial cells solubilized in 2% Triton X-100 (18). The percentage of the total LDH activity (supematant fraction plus cell fraction) released into the supematant fraction was then calculated. Sepiapterin and N-acetylserotonin (NAS) were added to culture medium for 24-h pretreatments, and washed out with PSS. They were not present throughout the experiments. All drugs used did not affect the measurement of LDH activity. Measurement of intracellular tetrahydrobiopterin content Intracellular BH, content was measured as previously described (19). Confluent endothelial cells in 6-well plates were washed 3 times with PSS. Intracellular BH, levels were determined as
Vol. 63, No. 18, 1998
Tctrahydrohiapterin Rcduccs NO Cytotaxicity
1587
biopterin. by oxidation in acidic conditions and reverse-phase high performance liquid chromatography with fluorometric detection. as previously reported (20). Cell were treated with sepiapterin and NAS in culture medium for 24-h.
Muteriuls Sepiapterin was obtained from Cayman Chemical Co. (Ann Arbor. MI. U.S.A.). S-NitrosoN-acetyl-DL-penicillamine (SNAP) and carboxy-PTIO were obtained from Dojindo Laboratories (Kumamoto. Japan). N-Acetylserotonin (NAS) and catalase were obtained from Sigma Chemical Co. (St. Louis. MO. U.S.A.). Superoxide dismutase was obtained from Wako Pure Chemicals (Osaka. Japan). All other reagents were of the highest grade commercially available. Stutisticul unu1y.C Data are presented as means + S.E.M. of n observations. The statistical significance of observed differences was determined by analysis of variance followed by Bonferroni’s method. Differences between means were considered significant when P was less than 0.05.
60
control SNAP h
0.5 mM
SNAP
I mM
SNAP
2 mM
time ( hour ) Fig. 1 Time course of LDH release from endothelial cells exposed to various concentrations of SNAP at 37°C. At the indicated times after addition of SNAP, the percentage of LDH released into the medium was determined. Typical results from 3 experiments are shown as the means * S.E.M. of quadruplicate assays.
Results Induction of endothelial cell death by SNAP treatment. We examined whether NO induces endothelial cell death using SNAP, a NO donor (Fig.l). Endothelial cell death was assessed by measuring the release of intracellular LDH. SNAP caused LDH release from endothelial cells in a concentration-dependent manner starting at 8 h after its addition (Fig. 1). The SNAP (2 mM)-induced cell death was strongly attenuated by treatment with carboxy-PTIO. a specific NO scavenger. suggesting that NO released from SNAP is implicated in SNAP-induced cell death (Fig. 2).
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Tetrahydrobiipterio Reduces NO Cytotoxicity
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10
6 &
loo
carboxy-PTIO (pM)
d
Fig. 2 Effect of NO scavenger on SNAP-induced LDH release from endothelial cells. Cells were incubated with SNAP (2 mM) in the presence or absence of carboxy-PTIO (10, 100 pM) for 10 h at 37°C. Typical results from 3 experiments are shown as the means + S.E.M. of quadruplicate assays. *Significant change in comparison to control group (p
B
A - ‘001
_ 601
$
time (hour)
Q
$
#sj?0.1 c' 8
I
10 loo loo
sepiapterin (PM) 7~ (1 mM)
Fig. 3
Effects of sepiapterin and N-acetylserotonin (NAS) on SNAP-induced LDH release from endothelial cells. A; Cells were incubated with sepiapterin (100 pM) for 24 h. The cells were washed with PSS, and then incubated with SNAP (2 mM) at 37°C. At the indicated times after addition of SNAP, the percentage of LDH released into the medium was determined. B; Cells were incubated with sepiapterin (0.1-100 pM) or sepiapterin (100 PM) plus NAS (1 mM) for 24 h. The cells were washed with PSS, and then incubated with SNAP (2 mM) for 10 h at 37°C. Typical results from 3 experiments are shown as the means f S.E.M. of quadruplicate assays. *Significant change in comparison to control group (~~0.05). “Significant change in comparison to sepiapterin (100 PM) alone group (~~0.05).
Vol. 63, No. 18, 1998
Tetrahydrobiopterin
0’
control’
0.1
Reduces
1.0 sepiapterin
NO Cytotoxicity
10
1589
100
(CM)
NAS
Fig. 4 Effects of sepiapterin and N-acetylserotonin (NAS) on tetrahydrobiopterin content in endothelial cells. Cells were incubated with sepiapterin (0.1-100 PM) or sepiapterin (100 PM) plus NAS (1 mM) for 24 h. Typical results from 3 experiments are shown as the means + S.E.M. of quadruplicate assays. *Significant change in comparison to control group (~~0.05). #Significant change in comparison to sepiapterin (100 PM) alone group (p(O.05).
Effects of sepiapterin and N-acetylserotonin on SNAP-induced endothelial cell death. Pretreatment with sepiapterin (100 PM), a precursor of BH, synthesis, reduced SNAPinduced endothelial cell death at 8 and 10 h after addition of SNAP, but not at 24 h (Fig. 3A). The protective effect of sepiapterin was concentration dependent (Fig. 3B). The protective effect of sepiapterin was blocked by co-pretreatment with NAS (1 mM), which is an inhibitor of sepiapterin reductase (Fig. 3B). Esfects of sepiapterin and N-acetylserotonin on tetrahydrobiopterin content in endothelial cells. Addition of sepiapterin to endothelial cells concentration-dependently increased BH, content. and the increase in BH, content by sepiapterin (100 PM) was inhibited by NAS (1 mM) (Fig.4). @,&zctsof SOD and catalase on SNAP-induced cell death. To determine whether superoxide and/or H20z are implicated in SNAP-induced cell death, the effects of SOD and catalase were examined. As shown in Fig. 5, SNAP-induced cell death was markedly reduced by treatment with catalase (100, 300, or 500 U/ml). Inactivated catalase by incubated in boiling water for 1 min did not affect SNAP-induced cell death (data not shown). On the other hand, SOD at 50 6r 100 U/ml had no effect on SNAP-induced cell death, and a high concentration of SOD (300 U/ml) increased the cell death.
Discussion In the present study, addition of SNAP to endothelial
cells induced cell death. NO released
Tetrahydrobiopterin Reduces NO Cytotoxicity
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from NO-donors, including SNAP, sodium nitroprusside (SNP) and 3-morpholinosydnonimine-Nethylcarbamide (SIN-l), has been shown to induce cell injury in various cell types (7-9). In the present study, the SNAP-induced endothelial cell death was strongly attenuated by treatment with carboxy-PTIO, a scavenger of NO. Therefore, SNAP-induced endothelial cell death is likely to be mediated by NO released from SNAP. Although the mechanisms of NO-induced cell death remain unclear, the toxicity of SIN- 1, which produces both superoxide and NO, has been shown to be reduced by treatment with catalase. but not by SOD, in rat liver endothelial cells (7). Interestingly, even the toxicity of SNP. which produces NO alone, has been shown to be reduced by the treatment with catalase (8). We also found that SNAP-induced endothelial cell death was attenuated by catalase. but not by SOD. In fact, Asahi et al. (10,ll) have shown that glutathione peroxidase, which is part of the intracellular H,Oz-scavenging system, is inactivated by NO. Intracellular glutathione (GSH) content may also be decreased by reaction of GSH with NO to form S-nitroso-glutathione (21). Therefore, NO may induce to accumulate intracellular Hz02 via inhibition of glutathione peroxidase activity, and may cause cell death. We previously reported that treatment of endothelial cells with a GSH-depleting agent induces cell death (22). Thus, endogenous production of H,Oz caused by the treatment with NO is likely to be implicated in NOinduced cell death. NO has been shown to react with HzO1 to form peroxynitrite and singlet oxygen, which are toxic (23,24). Although there are some reports that NO attenuates H,O,-induced cytotoxicity (25,26), HzO, has also been shown to enhance NO toxicity (27,28). Therefore, H,O, caused by the treatment with NO may increase NO toxicity.
$
,L
SOD (U/ml)
catalase (U/ml)
3
Effects of Cells were or catalase are shown comparison
Fig. 5 SOD and catalase on SNAP-induced LDH release from endothelial cells. incubated with SNAP (2 mM) in the presence of SOD (50, 100, or 300 U/ml) (100, 300, or 500 U/ml) for 10 h at 37°C. Typical results from 3 experiments as the means k S.E.M. of quadruplicate assays. *Significant change in to control group (~‘0.05).
Pretreatment with sepiapterin, a precursor of BH, synthesis, increased BH, content and strongly attenuated SNAP-induced endothelial cell death. Both the increase in BH4 content and the protective effect of sepiapterin were prevented by co-pretreatment with NAS, a sepiapterin
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reductase inhibitor. These findings indicate that an increase in BH, content reduces NO-induced endothelial cell death. We recently reported that the increase in BH, content caused by treatment with sepiapterin enhances H,O, scavenging and markedly reduces H?O,-induced endothelial cell death (12). Pterins, including BH,, have been shown to have scavenging activity for reactive oxygen species and antioxidative activity (29). Therefore, these results strongly suggest that H,O?scavenging activity and/or antioxidative activity of BH, may be one of the mechanisms of the protective effect of sepiapterin. Kojima et al. (9) have reported that SNAP-induced cell injury is inhibited by exogenous BH, at an early time post-treatment with SNAP in HL-60 cells. They speculated that BH, quenches NO, since BH, reacted with l,l-diphenyl-2-picrylhydrazyl, a stable radical, in an ethanol solution. However, that investigation was indirect. Future studies are needed to determine whether BH, reduces NO-induced cell death via scavenging of H,O, or NO, or both. Protective effect of sepiapterin was not observed at 24 h after addition of SNAP. Kojima et al. (9) reported that SNAP-induced HL-60 cell injury at 5 h after addition of it is reduced by BH,. whereas BH, increased the cytotoxicity at 20 h after addition of it. They discussed that a longer incubation of SNAP with BH, may yield more toxic NO-related products such as peroxynitrite. However, it is speculative. More detail studies are needed to determine why BH, does not affect SNAP-induced cytotoxicity in a longer incubation. Inducible NO synthase, which is induced by LPS and/or inflammatory cytokines, produces large amounts of NO, and causes hypotension and circulatory failure in septic shock (30-32). BH, synthesis is also induced by LPS and/or cytokines in many types of cells, including macrophages. smooth muscle cells and endothelial cells (13- 16). Although induction of BH, synthesis is believed to be required for NO production by inducible NO synthase (13,33-35) the role of BH, is not fully understood. In this report, we showed that BH, protects endothelial cells against NO-induced cytotoxicity. It is possible that increase in BH, content plays a role in protecting endothelial cells against death induced by large amounts of NO produced by inducible NO synthase. In conclusion, our findings indicate that NO released from SN.4P induces endothelial cell death, and the SNAP-induced endothelial cell death may be mediated by the endogenous production of H,O,, rather than superoxide. Moreover, increase in BH, content strongly blocks NO-induced endothelial cell injury. The protective effect of BH, is likely to involve scavenging of the H,O, which is produced by treatment of cells with NO.
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