Nitric oxide regulates energy metabolism and Bcl-2 expression in ...

Nitric oxide regulates energy metabolism and Bcl-2 expression in intestinal epithelial cells MANABU NISHIKAWA,1,2 KENTA TAKEDA,1 EISUKE F. SATO,1 TETSO KUROKI,2 AND MASAYASU INOUE1 Departments of 1Biochemistry and 2Internal Medicine, Osaka City University Medical School, Osaka 545, Japan Nishikawa, Manabu, Kenta Takeda, Eisuke F. Sato, Tetso Kuroki, and Masayasu Inoue. Nitric oxide regulates energy metabolism and Bcl-2 expression in intestinal epithelial cells. Am. J. Physiol. 274 (Gastrointest. Liver Physiol. 37): G797–G801, 1998.—Nitric oxide (NO) inhibits the respiration of mitochondria and enteric bacteria, particularly under low O2 concentration, and induces apoptosis of various types of cells. To gain insight into the molecular role of NO in the intestine, we examined its effects on the respiration, Ca21 status, and expression of Bcl-2 in cultured intestinal epithelial cells (IEC-6). NO reversibly inhibited the respiration of IEC-6 cells, especially under physiologically low O2 concentration. Although NO elevated cytosolic Ca21 as determined by the fura 2 method, the cells were fairly resistant to NO. Kinetic analysis revealed that prolonged exposure to NO elevated the levels of Bcl-2 and suppressed the NO-induced changes in Ca21 status of the cells. Because Bcl-2 possesses antiapoptotic function, toxic NO effects might appear minimally in enterocytes enriched with Bcl-2. Thus NO might effectively exhibit its antibacterial action in anaerobic intestinal lumen without inducing apoptosis of Bcl-2-enriched mucosal cells. mitochondria; respiration; apoptosis

NITRIC OXIDE (NO) plays important roles in various biological processes, including relaxation of smooth muscle, neurotransmission, and host defense mechanisms (5, 28, 30). Activated macrophages and neutrophils produce substantial amounts of NO that exhibit cytotoxic activity to mammalian cells and microorganisms (28). Recent studies (6, 11, 17, 32, 40, 43) have revealed that NO reversibly interacts with cytochrome-c oxidase and other components in mitochondria and inhibits respiration of normal cells as well as of cancer cells. Under physiological conditions, substantial amounts of NO occur in the lumen of stomach and intestine (7, 13, 16, 46). Generation of NO in intestine is markedly enhanced by bacterial lipopolysaccharide (LPS) and inflammatory cytokines (12, 44). The lifetime of NO has been postulated to be extremely short, presumably because of its rapid reaction with O2 (37). However, NO is fairly stable under low O2 concentration (42). Because O2 concentration in the intestinal lumen is extremely low, the metabolic effects of NO might be significantly strong in this compartment. Hence the effects of NO on intestinal bacteria would be stronger than on intestinal cells. We previously reported that the respiration and growth of Escherichia coli and other enteric bacteria were inhibited by NO, particularly under low O2 concentration (49). However, the pathophysiological significance of NO in the metabo-

lism of intestinal cells remains to be elucidated. The present study demonstrates the effects of NO on the energy metabolism, Ca21 homeostasis, and expression of Bcl-2 in intestinal epithelial cells. MATERIALS AND METHODS

Chemicals. NO and argon gas were obtained from Kinkisanki (Osaka, Japan). Rotenone and antimycin A were obtained from Nakalai Tesque (Kyoto, Japan) and Sigma Chemical (St. Louis, MO), respectively. Fura 2-AM and 2,28-(hydroxynitrosohydrazono)bis-ethanamine (NOC18) were obtained from Dojindo Chemical (Kumamoto, Japan). All other reagents used were of analytical grade. Preparation of NO solution. NO solution was prepared by bubbling NO gas through 50 mM HEPES-NaOH buffer, pH 7.4, as described previously (10). Briefly, two small tubes were fitted with an air-tight septum with glass tubes inserted for delivery and escape of gases, with a first tube containing 5 M KOH and a second tube containing the HEPES-NaOH buffer. Argon was delivered into two tubes at a flow rate of 100 ml/min. After 15 min, argon was replaced with NO at a flow rate of 100 ml/min. After another 15 min, the saturated NO solution (1.9 mM) was kept on ice and used for experiments within 3 h; the concentration of NO in the stock solution remained unchanged during the experiments. NO concentration was determined by using electron spin resonance (ESR) and the NO trapping agent 2-(4-carboxyphenyl)-4,4,5,5tetramethyl-imidazoline-1-oxyl 3-oxide (1, 3). Cell culture. Well-characterized rat intestinal epithelial crypt cells (IEC-6) were obtained from Riken Cell Bank (Tsukuba, Japan). IEC-6 cells were grown to confluence in DMEM (GIBCO, Gaithersburg, MD) containing 5% FCS at 37°C and 5% CO2 and 20% or 5% O2. Analysis of cell respiration. O2 consumption by IEC-6 cells was determined polarographically using a Clark-type O2 electrode fitted to a 2-ml water-jacketed closed chamber at 37°C (34). Cellular respiration was analyzed in Krebs-Ringer phosphate buffer (KRP; pH 7.4) consisting of 50 mM HEPES, 100 mM NaCl, 5 mM KCl, and 1 mM each of MgCl2, NaH2PO4, and CaCl2. The reaction was started by adding 107 cells to the assay mixture. During the experiments, aliquots of NOsaturated solution were added to the reaction mixture. Analysis of DNA fragmentation. Cells (2 3 106 ) were incubated for 10 min in 100 µl ice-cold lysis buffer (10 mM Tris · HCl, pH 7.4, 10 mM EDTA, 0.5% Triton X-100) at 4°C. After incubation for 20 min, the mixture was centrifuged at 10,000 g for 20 min. The supernatant fraction was incubated with 40 µg RNase A at 37°C for 1 h, then with 40 µg proteinase K for 1 h. DNA was precipitated by incubating with 1 volume of isopropanol and 0.2 volume of 5 M NaCl at 220°C for 12 h and centrifuged at 10,000 g for 20 min. Pellets were air dried and dissolved in 10 µl of buffer composed of 10 mM Tris · HCl, pH 8.0, and 1 mM EDTA. The DNA samples thus obtained were subjected to 1.7% agarose gel electrophoresis at 100 V using 40 mM Tris-acetate, pH 8.0, and 1 mM

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NO REGULATES RESPIRATION AND BCL-2 IN IEC-6

EDTA as a running buffer. The gel was stained with 0.1 µg/ml of ethidium bromide and visualized under ultraviolet light. Western blot analysis of Bcl-2. Proteins (10 µg) in IEC-6 cell lysates were separated in 15% SDS-PAGE and blotted onto nitrocellulose sheets using a Pharmacia semidry blot system (2 mA/cm2 for 1 h in 192 mM Tris-glycine buffer). The sheets were incubated in TBS solution (140 mM NaCl, 50 mM Tris · HCl, pH 7.2) containing 0.1% Tween 20 and 5% low-fat milk powder for 12 h at 4°C. Then the sheets were incubated with rabbit anti-rat Bcl-2 antibody (1:1,000 in TBS solution with 0.5% low-fat milk powder) for 12 h at 4°C. The incubated sheets were washed five times to eliminate nonspecific binding of the antibody. After incubation with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:1,000 in TBS with 0.5% low-fat milk powder) at 25°C for 1 h, immunoreactive spots were analyzed by enhanced chemiluminescence (Amersham, Buckinghamshire, UK). Analysis of mRNA. Total RNA was extracted from IEC-6 cells by the acid guanidium-phenol-chloroform method, as described previously (9). Levels of mRNA for b2-microglobulin and bcl-2 were analyzed by RT-PCR method (15). RT was carried out at 42°C for 15 min. PCR was carried out by 1 min denaturation at 95°C, 1 min hybridization at 55°C, and 1.5 min extension at 72°C. The primers used were 58-TCAGATCTGTCCTTCAGCAA-38 and 58-CATGTCTCGGTCCCAGGTGA-38 for b2-microglobulin and 58-TAACCGGGAGATCGTG-38 and 58-ACATCTCTGCAAAGTCGCGA-38 for bcl-2. PCR for b2-microglobulin and bcl-2 mRNA was performed for 35 cycles. PCR products were subjected to 1.7% agarose gel electrophoresis. The gel was stained with 0.1 µg/ml of ethidium bromide and visualized under ultraviolet light. Measurement of intracellular Ca21 level. IEC-6 cells (108 cells/ml) were incubated in KRP containing 1 µM of fura 2-AM at 37°C for 15 min. After washing twice with KRP, cells (5 3 104/ml) were incubated in the same buffer at an O2 concentration of 25 µM. Fura 2 fluorescence was measured in a Hitachi F-2000 fluorescence spectrophotometer with excitation and emission wavelengths of 340/380 and 510 nm, respectively. Cytosolic levels of Ca21 were determined from

Fig. 1. Effect of nitric oxide (NO) on O2 consumption. Intestinal epithelial cells (IEC-6 cells) (5 3 106 cells/ml) were incubated in a closed chamber containing 2 ml of Krebs-Ringer phosphate buffer at 37°C. At the indicated times (arrows), NO was added to give a final concentration of 2 µM. During incubation, O2 concentration in the medium was monitored as described in text. Experiments were repeated 5 times with similar results.

Fig. 2. Effect of NO on DNA fragmentation. IEC-6 cells were cultured without (lanes 1 and 5) or with NOC18 (10 µM, lanes 2 and 6; 100 µM, lanes 3 and 7; 1 mM, lanes 4 and 8) under O2 concentration of 20% (lanes 1–4) or 5% (lanes 5–8) for 24 h. C, control (cells before treatment). DNA was extracted and subjected to agarose gel electrophoresis as described in text. Experiments were repeated 3 times with similar results.

the ratio of fura 2 fluorescence intensities at excitation of 340 and 380 nm (18). The data were processed with a computer fitted to a Hitachi F-2000 fluorescence spectrophotometer, and estimated intracellular Ca21 concentration was recorded continuously. RESULTS

Effect of NO on respiration of IEC-6 cells. IEC-6 cells revealed a marked O2 consumption without adding respiratory substrates (Fig. 1). Because O2 consumption by IEC-6 cells was enhanced by dinitrophenol and completely inhibited by potassium cyanide (data not shown), it fully reflected the respiration of their mitochondria. The respiration was transiently inhibited by a fairly low concentration of NO and recovered after certain periods of incubation. The inhibitory effect of NO increased with a concomitant decrease in O2 concentration. DNA fragmentation. Some compounds that inhibit mitochondrial electron transfer have been shown to induce apoptosis of cells (47). Fragmentation of inter-

Fig. 3. Regulation of bcl-2 gene expression by NO. IEC-6 cells were cultured in the presence of 100 µM NOC18 under O2 concentration of 20% or 5% for 4 and 24 h. mRNAs for bcl-2 and b2-microglobulin (b2-MG) were analyzed with these cells by RT-PCR. PCR for bcl-2 and b2-microglobulin was performed for 35 cycles each. Experiments were repeated 3 times with similar results.


Fig. 4. Regulation of Bcl-2 protein expression by NO. IEC-6 cells were cultured in the presence of 100 µM NOC18 under O2 concentration of 20% or 5% for 4, 8, 12, and 24 h. Western blot of 10 µg protein of cell lysate developed with specific antibody demonstrates Bcl-2 protein. Experiments were repeated 3 times with similar results.

nucleosomal DNA has been used as a marker of apoptosis of various cell types. To test the possible occurrence of apoptosis of NO-treated IEC-6 cells, their DNA samples were isolated and analyzed by agarose gel electrophoresis. When cells were cultured for 24 h under 20% O2 in the presence of ,100 µM NOC18, no appreciable fragmentation of DNA was found to occur (Fig. 2). When O2 concentration was decreased to 5%, NOC18 slightly enhanced the fragmentation of cellular DNA. The presence of extremely high concentrations of NO (1 mM) strongly induced DNA fragmentation; the fragmentation was more apparent at an O2 concentration of 5% than at 20%. Under identical conditions, both rotenone (1 µM) and antimycin A (0.1 µM), specific inhibitors of mitochondrial electron transport, failed to induce DNA fragmentation and to affect the viability of IEC-6 cells (data not shown). The concentrations of the inhibitors used in the experiments effectively inhibited the respiration of IEC-6 cells. Thus, unlike with other types of cells, inhibition of cellular respiration did not induce apoptosis of IEC-6 cells. Effect of NO on Bcl-2 expression. Bcl-2 exhibits antiapoptotic activity by stabilizing membrane potential of mitochondria (39). To elucidate the mechanism by which IEC-6 cells showed strong resistance to NO, the effect of NOC18 on the expression of bcl-2 in IEC-6 cells was studied. At 20% O2, NOC18 enhanced the expression of bcl-2 gene only slightly (Fig. 3). However, the expression of bcl-2 gene was strongly enhanced under low O2 concentration (5%), particularly at 24 h of incubation. NO did not affect the expression of Bcl-2 protein under 20% O2 but increased it under 5% O2 (Fig. 4). After 24 h of incubation under 5% O2, Bcl-2 levels increased by about fivefold. Under the standard culture conditions (20% O2-5% CO2 ), the enhancing effect of NOC18 also depended on its dose; 100 µM of

Fig. 5. Dose-dependent effect of NO on Bcl-2 protein expression. IEC-6 cells were cultured without (lanes 1 and 5) or with NOC18 (10 µM, lanes 2 and 6; 100 µM, lanes 3 and 7; 1 mM, lanes 4 and 8) under O2 concentration of 20% (lanes 1–4) or 5% (lanes 5–8) for 24 h. Western blot of 10 µg protein of cell lysate developed with specific antibody demonstrates Bcl-2 protein. Experiments were repeated 3 times with similar results.

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Fig. 6. Effect of mitochondrial electron transport inhibitors on Bcl-2 protein expression. IEC-6 cells were cultured in the presence of 1 µM rotenone or 0.1 µM antimycin A for 24 h. Western blot of 10 µg protein of cell lysate developed with monoclonal antibody demonstrates Bcl-2 protein. Control, cells before treatment. Experiments were repeated 3 times with similar results.

NOC18 increased Bcl-2 levels by ,50% (Fig. 5). However, doses of NOC18 .1 mM strongly inhibited the expression of Bcl-2, especially under low O2 concentration. Effect of specific inhibitors of mitochondrial electron transport on Bcl-2 expression. Western blot analysis revealed that both rotenone and antimycin A markedly enhanced cellular levels of Bcl-2 in IEC-6 cells (Fig. 6). Effect of NO on cellular Ca21 status. Mitochondria have been known to regulate cellular Ca21 homeostasis by an ATP-dependent mechanism (19). Because NO inhibited the mitochondrial respiration of IEC-6 cells, its effect on the cytosolic levels of Ca21 was determined. Cytosolic levels of Ca21 were markedly elevated by NO in a concentration-dependent manner (Fig. 7). The elevating effect of NO was stronger in control cells expressing low levels of Bcl-2 than in NOC18-pretreated cells with enhanced expression of this protein. DISCUSSION

The present study demonstrates that NO reversibly inhibits the respiration of intestinal epithelial cells and

Fig. 7. Effect of NO on cytosolic Ca21 levels. Changes in intracellular Ca21 concentration ([Ca21]i ) in IEC-6 cells were determined with control (A) or Bcl-2-overexpressed cells, which were pretreated with 100 µM NOC18 under 5% O2 for 24 h (B), by the fura 2-AM method at an O2 concentration of 25 µM. At the indicated times (arrows), NO solution was added to reaction mixture to make final concentrations of 10 (2), 20 (3) and 50 µM (4). 1, Experiments performed in the absence of NO. Experiments were repeated 5 times with similar results.

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enhances the expression of Bcl-2 in dose- and O2 concentration-dependent manners. Because NO reacts with molecular O2 (k 5 6 3 106 M22s21 ), it is fairly stable under low O2 concentration. NO reversibly binds to cytochrome c-oxidase and increases its apparent Michaelis constant value for O2 (6). Hence the effects of NO on the metabolism of enterocytes facing the anaerobic lumen might be stronger than those expected from in vitro experiments performed under air atmospheric conditions. NO exhibits cytotoxic effects on target cells and induces DNA fragmentation (14, 23–25, 41). Fairly high concentrations of NO (,100 µM NOC18) failed to induce DNA fragmentation and apoptosis of IEC-6 cells even under low O2 concentration. At an NOC18 concentration of 100 µM, ,0.6 µM/min of NO would be released under the present experimental conditions by an O2-independent mechanism. A protooncogene product, Bcl-2 localizes in biological membranes, such as endoplasmic reticulum, plasma membranes, mitochondrial inner membranes, and nuclear membranes, and prevents various cells from apoptotic death (8, 20). Although protective effects of Bcl-2 against hazardous stimuli leading to apoptosis have been studied extensively (2, 24–27, 45), the molecular mechanism of its regulation remains to be elucidated. Based on its preferential association with mitochondrial membranes, the antiapoptotic effect of Bcl-2 has been postulated to be enclosed in mitochondrial functions (29, 39, 50). The present study finds that NO inhibits mitochondrial respiration and enhances expression of Bcl-2 gene and protein, which prevents the fragmentation of cellular DNA. Because both rotenone (1 µM) and antimycin A (0.1 µM) markedly increased cellular levels of Bcl-2 by some O2-independent mechanism, the expression of Bcl-2 might be regulated principally by mitochondrial electron flow. Mitochondria play a critical role in the regulation of Ca21 homeostasis (36, 38). NO deenergized mitochondria and elevated cytosolic levels of Ca21 in IEC-6 cells, particularly under physiologically low O2 concentration. Sustained increase in cellular Ca21 levels by a variety of agents often enhances the process of apoptotic cell death (31). Ca21 has been found to activate endonuclease in nuclei (21) and to stimulate apoptosis of thymocytes (48). Thus elevation of cytosolic Ca21 by NO might induce DNA fragmentation in intestinal epithelial cells, especially under physiologically low O2 concentration. It should be noted that NO enhanced the expression of Bcl-2 and suppressed the NO-induced changes in Ca21 status. Bcl-2 enhances mitochondrial Ca21 uptake and exhibits antiapoptotic activity by stabilizing membrane potential of mitochondria (22, 29). Thus Bcl-2 might stabilize membrane potential of mitochondria and Ca21 status in IEC-6 cells and prevent DNA fragmentation. When mitochondrial respiration was inhibited by various inhibitors, including NO, expression of Bcl-2 was enhanced in IEC-6 cells. Therefore Bcl-2 and mitochondrial energy metabolism are closely linked. The molecular mechanism by which Bcl-2 affects the energy metabolism in intestinal epithelial cells should be studied further.

Activated neutrophils and macrophages synthesize substantial amounts of NO by inducible NO synthase (iNOS). Increased production of NO might be important for host defense mechanisms. When stimulated by LPS, intestinal epithelial cells also express iNOS and produce NO (12, 44). In fact, patients with inflammatory bowel diseases, such as ulcerative colitis and Crohn’s disease, have high iNOS activity in the large intestine and high nitrite levels in their plasma (4, 35). Because vascular and mucosal permeability increase in inflammation of the intestine, inhibition of bacterial translocation across intestinal walls is critically important. We previously reported (49) that the respiration and growth of E. coli and other enteric bacteria were inhibited by NO, particularly under low O2 concentration. However, when excess amounts of NO were produced locally, NO and/or its reactive metabolites became toxic to both enteric bacteria and host cells (33). Thus, to get selective toxicity to bacteria by NO, intestinal mucosal cells should be more resistant to this gaseous radical than enteric bacteria. In this context, enhanced expression of Bcl-2 in intestinal mucosal cells by NO might be essential in selective inhibition of bacterial growth without injuring intestinal cells. This work was supported by grants from the Ministry for Education, Science, and Culture of Japan and from Osaka City University Research Foundation. Address for reprint requests: M. Nishikawa, Dept. of Biochemistry, Osaka City Univ. Medical School, 1-4-54 Asahimachi, Abeno-ku, Osaka 545, Japan. Received 3 November 1997; accepted in final form 12 January 1998. REFERENCES 1. Akaike, T., M. Yoshida, Y. Miyamoto, K. Sato, M. Kohno, K. Sasamoto, K. Miyazaki, S. Ueda, and H. Maeda. Antagonistic action of imidazolineoxyl N-oxides against endotheliumderived relaxing factor/NO through a radical reaction. Biochemistry 32: 827–832, 1993. 2. Alnemri, E. S., T. F. Fernandes, S. Haldar, C. M. Croce, and G. Litwack. Involvement of Bcl-2 in glucocorticoid-induced apoptosis of human pre-B-leukemias. Cancer Res. 52: 491–495, 1992. 3. Azuma, T., K. Fujii, and O. Yuge. Reaction between imidazolineoxyl N-oxide (carboxy-PTIO) and nitric oxide released from cultured endothelial cells: quantitative measurement of nitric oxide by ESR spectrometry. Life Sci. 54, Suppl.: PL185–PL190, 1994. 4. Boughton-Smith, N. K., S. M. Evans, C. J. Hawkey, A. T. Cole, M. Balsitis, B. J. R. Whittle, and S. Moncada. Nitric oxide synthase activity in ulcerative colitis and Crohn’s disease. Lancet 342: 338, 1993. 5. Bredt, D. S., and S. H. Snyder. Nitric oxide, a novel neuronal messenger. Neuron 8: 3–11, 1992. 6. Brown, G. C. Nitric oxide regulates mitochondrial respiration and functions by inhibiting cytochrome oxidase. FEBS Lett. 369: 136–139, 1995. 7. Chen, K., M. Inoue, and A. Okada. Expression of inducible nitric oxide synthase mRNA in rat digestive tissues after endotoxin and its role in intestinal mucosal injury. Biochem. Biophys. Res. Commun. 224: 703–708, 1996. 8. Chen-Levy, Z., J. Nourse, and M. L. Cleary. The Bcl-2 candidate proto-oncogene product is a 24-kilodalton integralmembrane protein highly expressed in lymphoid cell lines and lymphomas carrying the t(14; 18) translocation. Mol. Cell. Biol. 9: 701–710, 1989. 9. Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156–159, 1987.

NO REGULATES RESPIRATION AND BCL-2 IN IEC-6 10. Clancy, R. M., Y. Miyazaki, and P. J. Cannon. Use of thionitrobenzoic acid to characterize the stability of nitric oxide in aqueous solutions and in porcine aortic endothelial cell suspension. Anal. Biochem. 191: 138–143, 1990. 11. Cleeter, M. W. J., J. M. Cooper, V. M. Darley-Usmar, S. Moncada, and A. H. V. Schapira. Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. FEBS Lett. 345: 50–54, 1994. 12. Dignass, A. U., D. K. Podolsky, and D. Rachmilewitz. NOx generation by cultured small intestinal epithelial cells. Dig. Dis. Sci. 1859–1865, 1995. 13. Fan, W. Q., J. J. Smolich, J. Wild, V. Y. Yu, and A. M. Walker. Nitric oxide modulates regional blood flow differences in the fetal gastrointestinal tract. Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G598–G604, 1996. 14. Filep, J. G., C. Baron, S. Lachance, C. Perreault, and J. S. Chan. Involvement of nitric oxide in target-cell lysis and DNA fragmentation induced by murine natural killer cells. Blood 87: 5136–5143, 1996. 15. Frohman, M. A., M. K. Dush, and G. R. Martin. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85: 8998–9002, 1988. 16. Furness, J. B., Z. S. Li, H. M. Young, and U. Forstermann. Nitric oxide synthase in the enteric nervous system of the guinea-pig: a quantitative description. Cell Tissue Res. 277: 139–149, 1994. 17. Gassina, A., and R. Radi. Differential inhibitory action of nitric oxide and peroxynitrite on mitochondrial electron transport. Arch. Biochem. Biophys. 328: 309–316, 1996. 18. Grynkiewicz, G., M. Poenie, and R. Y. Tsien. A new generation of Ca21 indicators with greatly improved fluorescence properties. J. Biol. Chem. 260: 3440–3450, 1985. 19. Gunter, T. E., and D. R. Pfeiffer. Mechanism by which mitochondria transport calcium. Am. J. Physiol. 258 (Cell Physiol. 27): C755–C786, 1990. 20. Hockenbery, D., G. Nunez, C. Milliman, R. Schreiber, and S. J. Korsmeyer. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348: 334– 336, 1990. 21. Jones, D. P., D. J. McConkey, P. Nicotera, and S. Orrenius. Calcium-activated DNA fragmentation in rat liver nuclei. J. Biol. Chem. 264: 6398–6403, 1989. 22. Marin, M. C., A. Fernandez, R. J. Bick, S. Brisbay, L. M. Buja, M. Snuggs, D. J. McConkey, A. C. von Eschenbach, M. J. Keating, and T. J. McDonnell. Apoptosis suppression by Bcl-2 is correlated with the regulation of nuclear and cytosolic Ca21. Oncogene 12: 2259–2266, 1996. 23. Messmer, U. K., and B. Brune. Nitric oxide in apoptotic versus necrotic RAW 264.7 macrophage cell death: the role of NO-donor exposure, NAD1 content, and p53 accumulation. Arch. Biochem. Biophys. 327: 1–10, 1996. 24. Messmer, U. K., U. K. Reed, and B. Brune. Bcl-2 protects macrophages from nitric oxide-induced apoptosis. J. Biol. Chem. 271: 20192–20197, 1996. 25. Messmer, U. K., D. M. Reimer, J. C. Reed, and B. Brune. Nitric oxide induced poly(ADP-ribose) polymerase cleavage in RAW 264.7 macrophage apoptosis is blocked by Bcl-2. FEBS Lett. 384: 162–166, 1996. 26. Miyashita, T., and J. C. Reed. Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in human leukemia cell line. Blood 81: 151–157, 1993. 27. Miyashita, T., and J. C. Reed. Bcl-2 gene transfer increases relative resistance of S49.1 and WEH17.2 lymphoid cells to cell death and DNA fragmentation induced by glucocorticoids and multiple chemotherapeutic drugs. Cancer Res. 52: 5407–5411, 1993. 28. Moncada, S., R. M. J. Palmer, and E. A. Higgs. Nitric oxide: physiology, pathology, and pharmacology. Pharmacol. Rev. 43: 109–142, 1991. 29. Murphy, A. N., D. E. Bredesen, G. Cortopassi, E. Wang, and G. Fiskum. Bcl-2 potentiates the maximal calcium uptake

30. 31. 32. 33. 34. 35.

36.

37. 38. 39.

40.

41.

42. 43.

44.

45. 46.

47. 48.

49.

50.

G801

capacity of neural cell mitochondria. Proc. Natl. Acad. Sci. USA 93: 9893–9898, 1996. Nathan, C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 6: 3051–3064, 1992. Nicotera, P., G. Bellomo, and S. Orrenius. Calcium-mediated mechanisms in chemically induced cell death. Annu. Rev. Pharmacol. Toxicol. 32: 449–470, 1992. Nishikawa, M., E. F. Sato, K. Utsumi, and M. Inoue. Oxygendependent regulation of energy metabolism in ascites tumor cells by nitric oxide. Cancer Res. 56: 4535–4540, 1996. Nussler, A. K., and T. R. Billiar. Inflammation, immunoregulation, and inducible nitric oxide synthase. J. Leukoc. Biol. 54: 171–178, 1993. Packer, L., K. Utsumi, and M. G. Mustafa. Oscillatory states of mitochondria I. Arch. Biochem. Biophys. 117: 381–393, 1996. Rachmilewitz, D., J. S. Stamler, D. Bachwich, F. Karmeli, Z. Ackerman, and D. K. Podolsky. Enhanced colonic nitric oxide generation and nitric oxide synthase activity in ulcerative colitis and Crohn’s disease. Gut 36: 718–723, 1995. Richiter, C., V. Gogvadze, R. Schlapbach, M. Schweizer, and J. Schlegel. Nitric oxide kills hepatocytes by mobilizing mitochondrial calcium. Biochem. Biophys. Res. Commun. 205: 1143–1150, 1994. Saran, M., C. Michael, and W. Bros. Reaction of NO and O22, implication for the action of endothelium-derived relaxing factors. Free Radic. Res. Commun. 10: 221–226, 1990. Schweizer, L., and C. Richter. Nitric oxide potently and reversibly deenergizes mitochondria at low oxygen tension. Biochem. Biophys. Res. Commun. 204: 169–175, 1994. Shimizu, S., Y. Eguchi, W. Kamiike, and Y. Tsujimoto. Bcl-2 blocks loss of mitochondrial membrane potential while ICE inhibitors act at a different step during inhibition of death induced by respiratory chain inhibitors. Oncogene 13: 21–29, 1996. Stadler, J., T. R. Billiar, R. D. Curran, D. J. Stuehr, J. B. Ochoa, and R. Simmons. Effect of exogenous and endogenous nitric oxide on mitochondrial respiration of rat hepatocytes. Am. J. Physiol. 260 (Cell Physiol. 29): C910–C916, 1991. Sveinbjornsson, B., R. Olsen, O. M. Seternes, and R. Seljelid. Macrophage cytotoxicity against murine meth A sarcoma involves nitric oxide-mediated apoptosis. Biochem. Biophys. Res. Commun. 223: 643–649, 1996. Taha, Z., F. Kiechle, and T. Malinski. Oxidation of nitric oxide by oxygen in biological systems monitored by porphyrinic sensor. Biochem. Biophys. Res. Commun. 188: 734–739, 1992. Takehara, Y., T. Kanno, T. Yoshioka, M. Inoue, and K. Utsumi. Oxygen-dependent regulation of mitochondrial energy transfer reaction by nitric oxide. Arch. Biochem. Biophys. 323: 27–32, 1995. Tepperman, B. L., J. F. Brown, and B. J. R. Whittle. Nitric oxide synthase induction and intestinal epithelial cell viability in rats. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28): G214–G218, 1993. Tsujimoto, Y. Stress-resistance conferred by high level of Bcl-2 alpha protein in human B lymphoblastoid cell. Oncogene 4: 1331–1336, 1989. Wirthlin, D. J., J. J. Cullen, S. T. Spates, J. L. Conklin, J. Murray, D. K. Caropreso, and K. S. Ephgrave. Gastrointestinal transit during endotoxemia: the role of nitric oxide. J. Surg. Res. 60: 307–311, 1996. Wolvetang, E. J., K. L. Johnson, K. Krauser, S. J. Ralph, and A. W. Linnane. Mitochondrial respiratory chain inhibitors induce apoptosis. FEBS Lett. 339: 40–44, 1994. Wyllie, A. H., R. G. Morris, A. L. Smith, and D. Dunlop. Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J. Pathol. 142: 67–77, 1984. Yu, H., E. F. Sato, K. Nagata, M. Nishikawa, M. Kashiba, T. Arakawa, K. Kobayashi, T. Tamura, and M. Inoue. Oxygendependent regulation of the respiration and growth of Escherichia coli by nitric oxide. FEBS Lett. 409: 161–165, 1997. Zamzami, N., S. A. Susin, P. Marchetti, T. Hirsch, I. GomezMonterrey, M. Castedo, and K. Guido. Mitochondria: control of nuclear apoptosis. J. Exp. Med. 183: 1533–1544, 1996.