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Nitric oxide enhances cytotoxicity of cultured rabbit gastric mucosal cells induced by hydrogen peroxide
BB ELSEVIER
et Biophysica A~ta Biochimica et Biophysica Acta 1290 (1996) 257-260
Nitric oxide enhances cytotoxicity of cultured rabbit gastric mucosal cells induced by hydrogen peroxide Yasuo Hata a,b,,, Shinichi Ota c, Hideaki Hiraishi c, Akira Terano c, Kevin J. Ivey a,b a Department of Medicine, Veterans Affairs Medical Center, Long Beach, CA, USA b University of California, Irr,ine, CA, USA c 2nd Department of Internal Medicine, University of Tokyo, Tokyo, Japan Received 9 June 1995; accepted 14 February 1996
Abstract While NO has been reported to act as a protective factor to gastric mucosa, it has been shown to be cytotoxic to various cells. NO also has been demonstrated to stimulate prostaglandin (PG) release and mucous glycoprotein secretion which could result in the activation of gastric defensive mechanisms. We examined the effect of NO on cytotoxicity induced by hydrogen peroxide, and mucous glycoprotein secretion and PGE 2 release from cultured rabbit gastric mucosal cells. NO enhanced cytotoxicity induced by hydrogen peroxide. Defensive prostaglandin E 2 release and mucous glycoprotein secretion were not altered by NO. Under certain circumstances, NO might behave as an aggressive factor in gastric mucosal injury. Keywords: Nitric oxide; Hydrogen peroxide; Reactive oxygen metabolite; Gastric cytoprotection; Mucous glycoprotein; Prostaglandin E2; (Rabbit gastric mucosal cell)
1. Introduction Nitric oxide (NO) has generally been presumed to be a cytotoxic agent [1]. However, NO was reported to be cytoprotective to gastric mucosa in vivo [2,3]. In vitro studies on lung fibroblasts showed NO protected against cellular damage and cytotoxicity from reactive oxygen species [4]. We previously found that reactive oxygen metabolites (ROM) such as hydrogen peroxide cause gastric cell damage alone [5], and after exposure to alcohol [6] in vitro. The interrelationship between NO and ROM in gastric cells is uncertain. To study this, we employed our in vitro model of cultured rabbit gastric mucosal cells, which excludes vascular, neural, and humoral factors. 1,3Propanediamine, N-[4-[1-(3-aminopropyl)-2-hydroxy-2nitrosohydrazino]butyl] (SPER/NO), S-nitroso-N-acetylD,L-penicillamine (SNAP), or S-nitroso-L-glutathione (GSNO) was used as a source of spontaneous NO release. We examined the effects of NO generator, S P E R / N O ,
* Corresponding author. Present address: 2nd Department of Internal Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 174, Japan. Fax: +81 3 38140021. 0304-4165/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S 0 3 0 4 - 4 1 6 5 ( 9 6 ) 0 0 0 2 4 - 4
SNAP, or GSNO on cytotoxicity caused by hydrogen peroxide. We also examined the effect of S P E R / N O on release of prostaglandin E 2 and mucous glycoprotein, agents which may protect gastric cells.
2. Materials and methods 2.1. Materials
1,3-Propanediamine, N-[4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl] ( S P E R / N O ) was purchased from Cayman (Ann Arbor, MI). S-nitroso-N-acetylD,L-penicillamine (SNAP) and S-nitroso-L-glutathione (GSNO) were obtained from Alexis (San Diego, CA). 2.2. Cell culture
Gastric fundic mucosal cells from adult male New Zealand white rabbits (Charles River, Wilmington, MA) were cultured as previously described [7]. In brief, minced fundic mucosa was incubated for 15 minutes in basal medium Eagle (BME) with 0.35 m g / m l collagenase type B (Boehringer Mannheim, Indianapolis, IN) in a shaker
258
E Hata et al. / Biochimica et Biophysica Acta 1290 (1996) 257-260
bath at 100 cycles/minute at 37°C under 5% CO 2 and 95% O 2 at pH 7.4. After the first incubation, the tissues were incubated for 5 minutes in Earle's balanced salt solution (EBSS) with 1 mM EDTA. The next incubation was performed in BME with collagenase type B (0.35 m g / m l ) for 15 minutes. Final incubation was done in the same solution for 50 minutes. Cells from the final incubation were cultured with Coon's modified Ham's F-12 medium supplemented with 10% heat-inactivated fetal bovine serum (Hycione, Logan, UT), 15 mM Hepes buffer, 100 U / m l penicillin, 100 I,z g / m l streptomycin, and 5 I~g/ml fungizone and inoculated onto 24-well tissue culture plates (Coster, Cambridge, MA).
2.3. 51Cr release assay Cytotoxicity was quantified by measuring 5~Cr release from prelabeled cells as described previously [8]. In brieL culture medium with 5 i~Ci/ml 51Cr (sodium chromate) (ICN, Irvine, CA) was added to confluent monolayers. After overnight prelabeling, cells were washed and incubated in 0.5 ml EBSS with or without test agent(s). 0.25 ml of supernatant buffer was removed for determination of 5lCr release. Maximum 51Cr release was determined by incubation in 1 N NaOH. The percentage of 51Cr release per sample was expressed as cpm of supernatant × 100/cpm of maximal 51Cr release %. 51Cr radioactivity was counted with a gamma counting system, Beckman 7000 (Beckman).
2.4. Mucous glycoprotein secretion Mucous glycoprotein secretion was quantified as previously described [9]. In brief, the cultured cells at the confluent state were incubated for 16 h at 37°C in culture medium with [3H]glucosamine hydrochloride (2 ~ C i / m l medium) (New England Nuclear, Boston, MA). The prelabeled cells were then rinsed and reincubated in EBSS with or without S P E R / N O for the test period. After incubation, the buffer was removed and the cells were rinsed. The buffer and rinsing fluid from each well were combined with 0.5 ml 50% trichloroacetic acid and 5% phosphotungstic acid and placed on ice. After standing at 4°C for 12 h, the precipitated proteins were collected by centrifugation (I0000 × g for 30 min), washed and dissolved in 1 ml of I N NaOH. Aliquots of the NaOH-solubilized secreted proteins were neutralized with 1 N HCI and counted in a scintillation counter (LS5801, Beckman, Fullerton, CA).
2.5. Assay of PGE: Media contents of PGE 2 were measured by radioimmunoassay (RIA). Cells were washed and incubated in
EBSS with or without SPER/NO. PGE 2 contents of the media were assayed directly with PGE 2 RIA kit (New England Nuclear, Boston, MA). 125I radioactivity was counted with a gamma counting system (Iso-Data 2 0 / 2 0 series, Beckman). Cells were scraped and protein was determined with the dye binding assay [10]. In preliminary experiments possible interference between the agents used and RIA were tested. No significant interference was observed.
2.6. Statistical analysis Data are presented as mean _+ standard error of the mean (S.E.M.). Student's t-test was used to assess significance: P < 0.05 was considered significant.
3. Results
3. I. Cell culture 90% of cultured gastric cells were mucous neck cells, 4% were surface mucous cells, 3% were parietal cells, and 2% were chief cells as previously described [7].
3.2. Effect of SPER / NO on hydrogen peroxide-induced 5/ Cr release from prelabeled cells Hydrogen peroxide (15 mM) increased 5~Cr release from prelabeled cells without S P E R / N O for 1 hour. S P E R / N O (2.0 mM) did not stimulate 51Cr release by itself compared with control, however, S P E R / N O (0.5-2 raM) enhanced 51Cr release (Fig. 1).
15-
7v <
c3 g u.
5-
c,l
Control
SPER/NO 2ram
1t202 15ram
H202 15mM + SPER/NO O.SmM
11202 15raM + SPER/NO t.OmM
11202 15ram + SPER/NO 2.0raM
I=ig. 1. Cells were incubated in EBSS alone (control), with 2 mM S P E R / N O , with 15 mM hydrogen peroxide (H20~), or with 0 . 5 - 2 mM S P E R / N O and 15 mM hydrogen peroxide for 1 hour. Significant differences were compared with values with 15 mM hydrogen peroxide alone. Values represent means _+S.E.M. from 12 cultures. * P < 0.05, * * P < 0.01.
Y. Hata et al. / Biochimica et Biophysica Acta 1290 (1996) 257-260 15-
0"-'0
H202 15mM + SPER/NO 1ram
259
3.6. Effect of SPER / NO on PGE 2 release
o - - - o ~ o 2 lsn, u v
PGE 2 release after 1 hour incubation with SPER/NO (2.0 mM) was 1.27 + 0.34 n g / m g protein, and was not significantly different from 1.22 + 0.16 n g / m g protein in control (n = 12) determinations.
ul
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o ,~o uI
5-
a.
4. D i s c u s s i o n
I
I
I
15
30
45
/
60
(minutes)
Fig. 2. Cells were incubated in EBSS containing 15 m M hydrogen peroxide ( H 2 0 2 ) with or without 1 mM S P E R / N O over 1 hour. Significant differences were compared to values with 15 m M hydrogen peroxide alone. Values represent m e a n s + S . E . M , from 12 cultures. * P < 0 . 0 5 , • * P < 0.01.
3.3. Time-course of enhancement of SPER / NO on 51Cr release
SPER/NO (1 mM) increased 51Cr release induced by 15 mM hydrogen peroxide over 1 hour (Fig. 2). 3.4. Effect of SNAP or GSNO on hydrogen peroxide-induced 5~Cr release from prelabeled cells
Hydrogen peroxide (15 mM) increased 5~Cr release from prelabeled cells without SPER/NO for 1 hour. SNAP (2.0 raM) or GSNO (2.0 mM) did not stimulate 5]Cr release by itself compared with control, however, SNAP (2.0 mM) or GSNO (2.0 mM) enhanced 51Cr release induced by hydrogen peroxide (15 mM) (Table 1). 3.5. Effect of SPER / NO on mucous glycoprotein secretion
Mucous glycoprotein secretion after cells were incubated with SPER/NO (2.0 mM) for 1 hour was 102.2 ___ 7.1% and was not significantly different from 100 + 8.0% in control (n = 12) determinations.
Table 1 Test agent Control H 2 0 2 (15 mM) SNAP (2.0 mM) SNAP (2.0 m M ) + H 2 0 2 (15 mM) GSNO (2.0 mM) GSNO (2.0 m M ) + H 2 0 2 (15 mM)
Cr release (%)
31
8.28 -I- 0.45 9.79+0.54 7.72 + 0.50 12.66+0.53 * * * 7.03 + 0 . 9 5 12.66+0.53 * * *
Cells were incubated in EBSS alone (control) or with test agent(s) for 1 hour. Significant differences were compared with values with 15 m M hydrogen peroxide alone. Values represent m e a n s + S . E . M , from 12 cultures. * * * P < 0.001.
The major findings of the present study were that the NO generators, SPER/NO, SNAP, or GSNO, while by itself not inducing cytotoxicity, enhanced cytotoxicity caused by hydrogen peroxide in cultured rabbit gastric mucosal cells, and that neither prostaglandin E 2 release nor mucous glycoprotein secretion was affected by SPER/NO. Gastric epithelium is continuously exposed to oxygen radicals that are generated within the lumen. Potential sources of luminal oxidants include ingested food, catalase-negative bacteria, desquamated mucosal cells, and cigarette smoke and tar [11-13]. Despite the exposure to these luminal oxidants, however, the gastric surface epithelium is mostly unaffected under ordinary circumstances. We reported in a preliminary form that cultured rabbit gastric mucosal cells contained high amouts of catalase which detoxified hydrogen peroxide [14]. Therefore, cultured rabbit gastric mucosal cells which we used are hardy, requiring high concentrations of hydrogen peroxide to cause cytotoxicity. Gastric epithelial cells are reported to contain high levels of constitutive NO synthase which converts Larginine to NO, suggesting that NO might have a role in the regulation of epithelial cell function [ 15]. In vivo, nitric oxide (NO), regulates gastric blood flow [16,17]. Topical mucosal application of a NO solution or glyceryl trinitrate or nitroprusside which releases NO, reduces the severity of ethanol-induced hemorrhagic mucosal damage [2]. The NO system may produce this gastroprotective effect through enhancing mucosal circulation [3]. However, NO and hydrogen peroxide acted co-operatively to increase tumoricidal activity in F5 rat hepatoma cells [18]. This result is compatible with our results. Chemical interplay might be involved in this enhancement of cytotoxicity between NO and hydrogen peroxide. NO is postulated to react with hydrogen peroxide to produce potentially cytotoxic singlet oxygen [19]. Alternatively, NO and hydrogen peroxide may target different areas in gastric cells. It is likely that gastric cytoprotection by NO is related to enhancing mucosal circulation in vivo, while in vitro, reaction of NO with hydrogen peroxide intensifies the cytotoxicity of hydrogen peroxide. NO was suggested to mediate prostaglandin production [20] in a macrophage cell line. Our gastric mucosal cells are epithelial cells, while macrophages are endothelial
260
Y. Hata et al. / Biochimica et Biophysica Acta 1290 (1996) 257-260
cells. Therefore, it is possible that only endothelial cells have receptors for NO to stimulate prostaglandin release. While NO was reported to increase mucous gel thickness in vivo [21], this might result from enhanced circulation. NO was also reported to stimulate mucous secretion by isolated gastric mucosal cells [22]. Our cultured ceils like in vivo gastric mucosal cells are polarized, and NO was administered only from the apical side, while isolated cells have no polarity. This might affect results. In conclusion, in cultured gastric mucosal cells, neither PGE 2 release nor mucous glycoprotein secretion were altered by NO. NO enhanced cytotoxicity induced by hydrogen peroxide. Thus, NO might act as an aggressive factor to gastric epithelial cells at the cellular level.
References [1] Hibbs, J.B., Jr., Taintor, R.R. Vavrin, Z. and Rachlin, E.M. (1988) Biochem. Biophys. Res. Commun. 157, 87-94. [2] MacNaughton, W.K., Cirino, G. and Wallace, J.L. (1989) Life Sci. 45, 1869-1876. [3] Konturek, S.J., Brozowski, T., Majka, J., Szlachcic, A. and Czamobilski, K. (1994) Dig. Dis. Sci. 39, 593-600. [4] Wink, D.A., Ingeborg, H., Krishna, M.C., DeGraff, W. and Gamson, J. (1993) Proc. Natl. Acad. Sci. USA 90, 9813-9817. [5] Hiraishi, H., Terano, A., Ota, S., Ivey, K.J. and Sugimoto, T. (1987) Am. J. Physiol. 253, G40-48.
[6] Mutoh, H., Hiraishi, H., Ota, S., Ivey, K.J., Terano, A., and Sugimoto, T. (1990) Am. J. Physiol. 258, G603-609. [7] Ota, S., Terano, A., Hiraishi, H., Mutoh, H., Nakada, R., Hata, Y., Shiga, J. and Sugimoto, T. (1990) Gastroenterol. Jpn. 25, 1-6. [8] Terano, A., Mach, T., Stachura, J., Tarnawski, A. and Ivey, K.J. (1984) Gut 25, 19-25. [9] Hiraishi, H., Terano, A., Ota, S., Mutoh, H., Sugimoto, T. and lvey, K.J. (1991) Am. J. Physiol. 261, G662-668. [10] Bradford, M.M. (1976) Biochem. 72~248-254. [11] Cross, C.E., Halliwell, B. and Allen, A. (1984) Lancet 1,1328-1330. [12] Grisham, M.B., Hernandez, L.A. and Granger, D.N. (1986) Am. J. Physiol. 251, G567-574. [13] Olson, C.E., Chen, M.N., Amirian, D.A. and Soil, A.H. (1989) Am. J. Physiol. 256, G925-930. [14] Kawabe, T., Hata, Y., Hiraishi, H., Razandi, M., Terano, A. and Ivey, K.J. (1993) Gastroenterol. 104. A722. [15] Brown, J.F., Tepperman, B.L., Hanson, P.J., Whittle, B.J.R., and Moncada, S. (1992)Biochem. Biophys. Res. Commun. 184,680-685. [16] Pique, J.M., Whiule, B.J.R. and Esplugues, J.V. (1989) Eur. J. Pharmacol. 174, 293-296. [17] Walder, C.E., Hiemermann, C. and Vane, J.R. (1990) Proc. Roy. Soc. Lond. 241, 195-200. [18] Ioannidis, I. and de Groot, H. (1993) Biochem. J. 296, 341-435. [19] Noronha-Dutra, A.A., Epperlein, M.M. and Woolf, N. (1993) FEBS Lett. 321, 59-62. [20] Salvemini, D., Misko, T.P., Masferrer, J.L., Seibert, K., Currie, M.G. and Needleman, P. (1993) Proc. Natl. Acad. Sci. USA 90, 7240-7244. [2l] Brown, J.F., Hanson, P.J. and Whittle, B.J.R. (1992) Eur. J. Pharmacol. 223, 103-104. [22] Brown, J.F., Keates, A.C., Hanson, P.J. and Whittle, B.JR. (1993) Am. J. Physiol. 265, G418-422.
et Biophysica A~ta Biochimica et Biophysica Acta 1290 (1996) 257-260
Nitric oxide enhances cytotoxicity of cultured rabbit gastric mucosal cells induced by hydrogen peroxide Yasuo Hata a,b,,, Shinichi Ota c, Hideaki Hiraishi c, Akira Terano c, Kevin J. Ivey a,b a Department of Medicine, Veterans Affairs Medical Center, Long Beach, CA, USA b University of California, Irr,ine, CA, USA c 2nd Department of Internal Medicine, University of Tokyo, Tokyo, Japan Received 9 June 1995; accepted 14 February 1996
Abstract While NO has been reported to act as a protective factor to gastric mucosa, it has been shown to be cytotoxic to various cells. NO also has been demonstrated to stimulate prostaglandin (PG) release and mucous glycoprotein secretion which could result in the activation of gastric defensive mechanisms. We examined the effect of NO on cytotoxicity induced by hydrogen peroxide, and mucous glycoprotein secretion and PGE 2 release from cultured rabbit gastric mucosal cells. NO enhanced cytotoxicity induced by hydrogen peroxide. Defensive prostaglandin E 2 release and mucous glycoprotein secretion were not altered by NO. Under certain circumstances, NO might behave as an aggressive factor in gastric mucosal injury. Keywords: Nitric oxide; Hydrogen peroxide; Reactive oxygen metabolite; Gastric cytoprotection; Mucous glycoprotein; Prostaglandin E2; (Rabbit gastric mucosal cell)
1. Introduction Nitric oxide (NO) has generally been presumed to be a cytotoxic agent [1]. However, NO was reported to be cytoprotective to gastric mucosa in vivo [2,3]. In vitro studies on lung fibroblasts showed NO protected against cellular damage and cytotoxicity from reactive oxygen species [4]. We previously found that reactive oxygen metabolites (ROM) such as hydrogen peroxide cause gastric cell damage alone [5], and after exposure to alcohol [6] in vitro. The interrelationship between NO and ROM in gastric cells is uncertain. To study this, we employed our in vitro model of cultured rabbit gastric mucosal cells, which excludes vascular, neural, and humoral factors. 1,3Propanediamine, N-[4-[1-(3-aminopropyl)-2-hydroxy-2nitrosohydrazino]butyl] (SPER/NO), S-nitroso-N-acetylD,L-penicillamine (SNAP), or S-nitroso-L-glutathione (GSNO) was used as a source of spontaneous NO release. We examined the effects of NO generator, S P E R / N O ,
* Corresponding author. Present address: 2nd Department of Internal Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 174, Japan. Fax: +81 3 38140021. 0304-4165/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S 0 3 0 4 - 4 1 6 5 ( 9 6 ) 0 0 0 2 4 - 4
SNAP, or GSNO on cytotoxicity caused by hydrogen peroxide. We also examined the effect of S P E R / N O on release of prostaglandin E 2 and mucous glycoprotein, agents which may protect gastric cells.
2. Materials and methods 2.1. Materials
1,3-Propanediamine, N-[4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl] ( S P E R / N O ) was purchased from Cayman (Ann Arbor, MI). S-nitroso-N-acetylD,L-penicillamine (SNAP) and S-nitroso-L-glutathione (GSNO) were obtained from Alexis (San Diego, CA). 2.2. Cell culture
Gastric fundic mucosal cells from adult male New Zealand white rabbits (Charles River, Wilmington, MA) were cultured as previously described [7]. In brief, minced fundic mucosa was incubated for 15 minutes in basal medium Eagle (BME) with 0.35 m g / m l collagenase type B (Boehringer Mannheim, Indianapolis, IN) in a shaker
258
E Hata et al. / Biochimica et Biophysica Acta 1290 (1996) 257-260
bath at 100 cycles/minute at 37°C under 5% CO 2 and 95% O 2 at pH 7.4. After the first incubation, the tissues were incubated for 5 minutes in Earle's balanced salt solution (EBSS) with 1 mM EDTA. The next incubation was performed in BME with collagenase type B (0.35 m g / m l ) for 15 minutes. Final incubation was done in the same solution for 50 minutes. Cells from the final incubation were cultured with Coon's modified Ham's F-12 medium supplemented with 10% heat-inactivated fetal bovine serum (Hycione, Logan, UT), 15 mM Hepes buffer, 100 U / m l penicillin, 100 I,z g / m l streptomycin, and 5 I~g/ml fungizone and inoculated onto 24-well tissue culture plates (Coster, Cambridge, MA).
2.3. 51Cr release assay Cytotoxicity was quantified by measuring 5~Cr release from prelabeled cells as described previously [8]. In brieL culture medium with 5 i~Ci/ml 51Cr (sodium chromate) (ICN, Irvine, CA) was added to confluent monolayers. After overnight prelabeling, cells were washed and incubated in 0.5 ml EBSS with or without test agent(s). 0.25 ml of supernatant buffer was removed for determination of 5lCr release. Maximum 51Cr release was determined by incubation in 1 N NaOH. The percentage of 51Cr release per sample was expressed as cpm of supernatant × 100/cpm of maximal 51Cr release %. 51Cr radioactivity was counted with a gamma counting system, Beckman 7000 (Beckman).
2.4. Mucous glycoprotein secretion Mucous glycoprotein secretion was quantified as previously described [9]. In brief, the cultured cells at the confluent state were incubated for 16 h at 37°C in culture medium with [3H]glucosamine hydrochloride (2 ~ C i / m l medium) (New England Nuclear, Boston, MA). The prelabeled cells were then rinsed and reincubated in EBSS with or without S P E R / N O for the test period. After incubation, the buffer was removed and the cells were rinsed. The buffer and rinsing fluid from each well were combined with 0.5 ml 50% trichloroacetic acid and 5% phosphotungstic acid and placed on ice. After standing at 4°C for 12 h, the precipitated proteins were collected by centrifugation (I0000 × g for 30 min), washed and dissolved in 1 ml of I N NaOH. Aliquots of the NaOH-solubilized secreted proteins were neutralized with 1 N HCI and counted in a scintillation counter (LS5801, Beckman, Fullerton, CA).
2.5. Assay of PGE: Media contents of PGE 2 were measured by radioimmunoassay (RIA). Cells were washed and incubated in
EBSS with or without SPER/NO. PGE 2 contents of the media were assayed directly with PGE 2 RIA kit (New England Nuclear, Boston, MA). 125I radioactivity was counted with a gamma counting system (Iso-Data 2 0 / 2 0 series, Beckman). Cells were scraped and protein was determined with the dye binding assay [10]. In preliminary experiments possible interference between the agents used and RIA were tested. No significant interference was observed.
2.6. Statistical analysis Data are presented as mean _+ standard error of the mean (S.E.M.). Student's t-test was used to assess significance: P < 0.05 was considered significant.
3. Results
3. I. Cell culture 90% of cultured gastric cells were mucous neck cells, 4% were surface mucous cells, 3% were parietal cells, and 2% were chief cells as previously described [7].
3.2. Effect of SPER / NO on hydrogen peroxide-induced 5/ Cr release from prelabeled cells Hydrogen peroxide (15 mM) increased 5~Cr release from prelabeled cells without S P E R / N O for 1 hour. S P E R / N O (2.0 mM) did not stimulate 51Cr release by itself compared with control, however, S P E R / N O (0.5-2 raM) enhanced 51Cr release (Fig. 1).
15-
7v <
c3 g u.
5-
c,l
Control
SPER/NO 2ram
1t202 15ram
H202 15mM + SPER/NO O.SmM
11202 15raM + SPER/NO t.OmM
11202 15ram + SPER/NO 2.0raM
I=ig. 1. Cells were incubated in EBSS alone (control), with 2 mM S P E R / N O , with 15 mM hydrogen peroxide (H20~), or with 0 . 5 - 2 mM S P E R / N O and 15 mM hydrogen peroxide for 1 hour. Significant differences were compared with values with 15 mM hydrogen peroxide alone. Values represent means _+S.E.M. from 12 cultures. * P < 0.05, * * P < 0.01.
Y. Hata et al. / Biochimica et Biophysica Acta 1290 (1996) 257-260 15-
0"-'0
H202 15mM + SPER/NO 1ram
259
3.6. Effect of SPER / NO on PGE 2 release
o - - - o ~ o 2 lsn, u v
PGE 2 release after 1 hour incubation with SPER/NO (2.0 mM) was 1.27 + 0.34 n g / m g protein, and was not significantly different from 1.22 + 0.16 n g / m g protein in control (n = 12) determinations.
ul
..a ul
t0-
el"
o ,~o uI
5-
a.
4. D i s c u s s i o n
I
I
I
15
30
45
/
60
(minutes)
Fig. 2. Cells were incubated in EBSS containing 15 m M hydrogen peroxide ( H 2 0 2 ) with or without 1 mM S P E R / N O over 1 hour. Significant differences were compared to values with 15 m M hydrogen peroxide alone. Values represent m e a n s + S . E . M , from 12 cultures. * P < 0 . 0 5 , • * P < 0.01.
3.3. Time-course of enhancement of SPER / NO on 51Cr release
SPER/NO (1 mM) increased 51Cr release induced by 15 mM hydrogen peroxide over 1 hour (Fig. 2). 3.4. Effect of SNAP or GSNO on hydrogen peroxide-induced 5~Cr release from prelabeled cells
Hydrogen peroxide (15 mM) increased 5~Cr release from prelabeled cells without SPER/NO for 1 hour. SNAP (2.0 raM) or GSNO (2.0 mM) did not stimulate 5]Cr release by itself compared with control, however, SNAP (2.0 mM) or GSNO (2.0 mM) enhanced 51Cr release induced by hydrogen peroxide (15 mM) (Table 1). 3.5. Effect of SPER / NO on mucous glycoprotein secretion
Mucous glycoprotein secretion after cells were incubated with SPER/NO (2.0 mM) for 1 hour was 102.2 ___ 7.1% and was not significantly different from 100 + 8.0% in control (n = 12) determinations.
Table 1 Test agent Control H 2 0 2 (15 mM) SNAP (2.0 mM) SNAP (2.0 m M ) + H 2 0 2 (15 mM) GSNO (2.0 mM) GSNO (2.0 m M ) + H 2 0 2 (15 mM)
Cr release (%)
31
8.28 -I- 0.45 9.79+0.54 7.72 + 0.50 12.66+0.53 * * * 7.03 + 0 . 9 5 12.66+0.53 * * *
Cells were incubated in EBSS alone (control) or with test agent(s) for 1 hour. Significant differences were compared with values with 15 m M hydrogen peroxide alone. Values represent m e a n s + S . E . M , from 12 cultures. * * * P < 0.001.
The major findings of the present study were that the NO generators, SPER/NO, SNAP, or GSNO, while by itself not inducing cytotoxicity, enhanced cytotoxicity caused by hydrogen peroxide in cultured rabbit gastric mucosal cells, and that neither prostaglandin E 2 release nor mucous glycoprotein secretion was affected by SPER/NO. Gastric epithelium is continuously exposed to oxygen radicals that are generated within the lumen. Potential sources of luminal oxidants include ingested food, catalase-negative bacteria, desquamated mucosal cells, and cigarette smoke and tar [11-13]. Despite the exposure to these luminal oxidants, however, the gastric surface epithelium is mostly unaffected under ordinary circumstances. We reported in a preliminary form that cultured rabbit gastric mucosal cells contained high amouts of catalase which detoxified hydrogen peroxide [14]. Therefore, cultured rabbit gastric mucosal cells which we used are hardy, requiring high concentrations of hydrogen peroxide to cause cytotoxicity. Gastric epithelial cells are reported to contain high levels of constitutive NO synthase which converts Larginine to NO, suggesting that NO might have a role in the regulation of epithelial cell function [ 15]. In vivo, nitric oxide (NO), regulates gastric blood flow [16,17]. Topical mucosal application of a NO solution or glyceryl trinitrate or nitroprusside which releases NO, reduces the severity of ethanol-induced hemorrhagic mucosal damage [2]. The NO system may produce this gastroprotective effect through enhancing mucosal circulation [3]. However, NO and hydrogen peroxide acted co-operatively to increase tumoricidal activity in F5 rat hepatoma cells [18]. This result is compatible with our results. Chemical interplay might be involved in this enhancement of cytotoxicity between NO and hydrogen peroxide. NO is postulated to react with hydrogen peroxide to produce potentially cytotoxic singlet oxygen [19]. Alternatively, NO and hydrogen peroxide may target different areas in gastric cells. It is likely that gastric cytoprotection by NO is related to enhancing mucosal circulation in vivo, while in vitro, reaction of NO with hydrogen peroxide intensifies the cytotoxicity of hydrogen peroxide. NO was suggested to mediate prostaglandin production [20] in a macrophage cell line. Our gastric mucosal cells are epithelial cells, while macrophages are endothelial
260
Y. Hata et al. / Biochimica et Biophysica Acta 1290 (1996) 257-260
cells. Therefore, it is possible that only endothelial cells have receptors for NO to stimulate prostaglandin release. While NO was reported to increase mucous gel thickness in vivo [21], this might result from enhanced circulation. NO was also reported to stimulate mucous secretion by isolated gastric mucosal cells [22]. Our cultured ceils like in vivo gastric mucosal cells are polarized, and NO was administered only from the apical side, while isolated cells have no polarity. This might affect results. In conclusion, in cultured gastric mucosal cells, neither PGE 2 release nor mucous glycoprotein secretion were altered by NO. NO enhanced cytotoxicity induced by hydrogen peroxide. Thus, NO might act as an aggressive factor to gastric epithelial cells at the cellular level.
References [1] Hibbs, J.B., Jr., Taintor, R.R. Vavrin, Z. and Rachlin, E.M. (1988) Biochem. Biophys. Res. Commun. 157, 87-94. [2] MacNaughton, W.K., Cirino, G. and Wallace, J.L. (1989) Life Sci. 45, 1869-1876. [3] Konturek, S.J., Brozowski, T., Majka, J., Szlachcic, A. and Czamobilski, K. (1994) Dig. Dis. Sci. 39, 593-600. [4] Wink, D.A., Ingeborg, H., Krishna, M.C., DeGraff, W. and Gamson, J. (1993) Proc. Natl. Acad. Sci. USA 90, 9813-9817. [5] Hiraishi, H., Terano, A., Ota, S., Ivey, K.J. and Sugimoto, T. (1987) Am. J. Physiol. 253, G40-48.
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