Biomedical subjects

Y M Kim

Publications and source records attributed to Y M Kim.

At least 91 records · Page 5Linked to original sources

PubMed2000

Acetaminophen inhibits iNOS gene expression in RAW 264.7 macrophages: differential regulation of NF-kappaB by acetaminophen and salicylates.

Acetaminophen is a widely used analgesic and anti-inflammatory drug that is considered a good alternative to salicylates for individuals who cannot tolerate salicylates. Nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been implicated as a mediator of inflammation. Recent evidence suggests that anti-inflammatory effect of salicylates lies in the inhibition of iNOS, but nothing has been reported about the direct effect of iNOS expression by acetaminophen. The present study was designed to elucidate sequentially the action mechanisms of acetaminophen and salicylates (aspirin and sodium salicylate) on lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma)-induced iNOS expression in RAW 264.7 macrophages. Both acetaminophen and salicylates inhibited NO production and iNOS protein expression in a dose dependent manner. Acetaminophen inhibited iNOS mRNA expression, promoter activity of iNOS gene and nuclear factor-kappa B (NF-kappaB) binding activity induced by LPS plus IFN-gamma, whereas salicylates did not show any effect on them. In addition, salicylates did not affect on iNOS mRNA stability induced by LPS plus IFN-gamma. Furthermore, the inhibition of iNOS protein expression and NO production by salicylates was disappeared when salicylates were added for only 5 h to inhibit the early event of iNOS expression. Aspirin also dose dependently inhibited iNOS enzyme activity in cell-free extracts, whereas no significant differences were observed in extracts treated with sodium salicylate or acetaminophen. These findings suggest that acetaminophen may exert analgesic or anti-inflammatory effect by inhibiting iNOS expression induced by LPS plus IFN-gamma at transcriptional level by suppression of NF-kappaB binding activity, whereas salicylates exert its effect by inhibiting iNOS expression at the translational or posttranslational level.

Acetaminophen↗

PubMed2000

Endogenous interleukin-18 modulates immune escape of murine melanoma cells by regulating the expression of Fas ligand and reactive oxygen intermediates.

It has been known that melanoma cells can suppress the immune system by the Fas ligand. The present study investigated whether interleukin (IL)-18, which can enhance Fas ligand expression, is produced by B16F10 melanoma cells and is involved in immune escape of tumor cells. Immunohistology, reverse transcription-PCR, intracellular fluorescence-activated cell-sorting analysis, and immunoblotting demonstrated that melanoma cells express IL-18. C57BL/6 splenocytes cultured with culture supernatants of B16F10 melanoma cells enhanced IFN-gamma production, which was blocked by anti-IL-18 antibody, indicating that IL-18 in the culture supernatants is functional. In addition to IL-18, the IL-18 receptor was also detected in B16F10 melanoma cells, suggesting a role of this cytokine in regulating the functions of B16F10 melanoma cells. The functional effect of IL-18 on B16F10 melanoma cells was shown by reduction of Fas ligand expression in cells treated with anti-IL-18 antibody or transfected with IL-18 antisense cDNA. In addition, the same treatments decreased intracellular reactive oxygen intermediate levels in B16F10 melanoma cells, indicating that IL-18 regulates reactive oxygen intermediate production, which is involved in Fas ligand expression. Furthermore, transfection of IL-18 antisense cDNA into melanoma cells increased the susceptibility of tumor cells to natural killer cells in vitro. When IL-18 antisense transfectants were implanted into syngeneic mice, severe reduction of tumor cell growth was observed with concomitant infiltrated natural killer cells in the tumor area. Taken together, these results demonstrate that IL-18 has a critical role as a survival factor for B16F10 melanoma cells.

Animals↗

PubMed2000

Cellular non-heme iron content is a determinant of nitric oxide-mediated apoptosis, necrosis, and caspase inhibition.

In this report, we tested the hypothesis that cellular content of non-heme iron determined whether cytotoxic levels of nitric oxide (NO) resulted in apoptosis versus necrosis. The consequences of NO exposure on cell viability were tested in RAW264.7 cells (a cell type with low non-heme iron levels) and hepatocytes (cells with high non-heme iron content). Whereas micromolar concentrations of the NO donor S-nitroso-N-acetyl-DL-penicillamine induced apoptosis in RAW264.7 cells, millimolar concentrations were required to induce necrosis in hepatocytes. Caspase-3 activation and cytochrome c release were evident in RAW264.7 cells, but only cytochrome c release was detectable in hepatocytes following high dose S-nitroso-N-acetyl-DL-penicillamine exposure. Pretreating RAW264.7 cells with FeSO(4) increased intracellular non-heme iron to levels similar to those measured in hepatocytes and delayed NO-induced cell death, which then occurred in the absence of caspase-3 activation. Iron loading was also associated with the formation of intracellular dinitrosyl-iron complexes (DNIC) upon NO exposure. Cytosolic preparations containing DNIC as well as pure preparations of DNIC suppressed caspase activity. These data suggest that non-heme iron content is a key factor in determining the consequence of NO on cell viability by regulating the chemical fate of NO.

Animals↗

PubMed2000

Detection of cryptic Y chromosome mosaicism by coamplification PCR with archived cytogenetic slides of suspected Turner syndrome.

Turner syndrome is one of the most common cytogenetic abnormalities. It is known that the Y chromosome or Y derived material is present in 6-9% of TS patient and it may develop a high risk of gonadoblastoma in 15-25%. So it is crucial to carry out cyto genetic analysis and Y-specific probe studies for all persons with gonadal dysgenesis to rule out mosaicism with Y-bearing cell line; eg 45,X/46,XY. In this study, 26 archival slides previously analyzed cytogenetically as 45,X, 45,X/46,X,i(X), 45,X/46,X,r(X), and 45,X/46,XX were examined. Coamplification PCR, having the advantage of providing rapid result and confirming PCR failure, was performed with the slide samples in the regions of dystrophin gene in Xp21and DYZ3 in the Y centromeric region. All of archived slides were positive for X-specific gene and one slide of 45,X was found to have the cryptic Y chromosome material. Our result suggests that the archived cytogenetic slides could be applied for the detection of Y chromosome rapidly and efficiently in TS patients.

Biopsy↗

PubMed2000

Sphingosine 1-phosphate stimulates tyrosine phosphorylation of focal adhesion kinase and chemotactic motility of endothelial cells via the G(i) protein-linked phospholipase C pathway.

We have previously shown that sphingosine 1-phosphate (S1P) stimulates motility of human umbilical vein endothelial cells (HUVECs) (O.-H. Lee et al., Biochem. Biophys. Res. Commun. 264, 743-750, 1999). To investigate the molecular mechanisms by which S1P stimulates HUVEC motility, we examined tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)) which is important for cell migration. S1P induces a rapid increase in tyrosine phosphorylation of p125(FAK). Compared with other structurally related lipid metabolites such as sphingosine, C2-ceramide, and lysophosphatidic acid, S1P uniquely stimulated p125(FAK) tyrosine phosphorylation and migration of HUVECs. The effect of S1P on p125(FAK) tyrosine phosphorylation was markedly reduced by treatment with pertussis toxin or U73122, a phospholipase C (PLC) inhibitor. As a downstream signal of PLC, p125(FAK) tyrosine phosphorylation in response to S1P was totally blocked by depletion of the intracellular calcium pool. However, protein kinase C (PKC) inhibitor had no effect on the response to S1P. Finally, chemotaxis assays revealed that inhibition of PLC but not PKC significantly abrogated S1P-stimulated HUVEC migration. These results suggest that the G(i)-coupled receptor-mediated PLC-Ca(2+) signaling pathway may be importantly involved in S1P-stimulated focal adhesion formation and migration of endothelial cells.

Cell Adhesion Molecules↗

PubMed2000

Ionizing radiation potentiates the induction of nitric oxide synthase by interferon-gamma (Ifn-gamma) or Ifn-gamma and lipopolysaccharide in bnl cl.2 murine embryonic liver cells: role of hydrogen peroxide.

The effects of ionizing irradiation on the nitric oxide (NO) production in murine embryonic liver cell line, BNL CL.2 cells, were investigated. Various doses (5-40 Gy) of radiation made BNL CL.2 cells responsive to interferon-gamma alone for the production of NO in a dose-dependent manner. Small amounts of lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) synergized with IFN-gamma in the production of NO from irradiated BNL CL.2 cells, even though LPS or TNF-alpha alone did not induce NO production from the same cells. Immunoblots showed parallel induction of inducible nitric oxide synthase (iNOS). NO production in irradiated BNL CL.2 cells by IFN-gamma or IFN-gamma plus LPS was decreased by the addition of catalase, suggesting that H(2)O(2) produced by ionizing irradiation primed the cells to trigger NO production in response to IFN-gamma or IFN-gamma plus LPS. Furthermore, the treatment of nongamma-irradiated BNL CL.2 cells with H(2)O(2) made the cells responsive to IFN-gamma or IFN-gamma plus LPS for the production of NO. This study shows that ionizing irradiation has the ability to induce iNOS gene expression in responsive to IFN-gamma via the formation of H(2)O(2) in BNL CL.2 murine embryonic liver cells.

Animals↗

PubMed2000

Synthesis and in vitro/in vivo evaluation of 5-aminosalicyl-glycine as a colon-specific prodrug of 5-aminosalicylic acid.

A simple synthetic route for the preparation of amino acid conjugate of 5-aminosalicylic acid (5-ASA) was exploited and prepared 5-aminosalicyl-glycine (5-ASA-Gly) in good yield. In vitro and in vivo properties of 5-ASA-Gly as a colon-specific prodrug of 5-ASA were investigated using rats as the test animal. Incubation of 5-ASA-Gly with cecal or colonic contents at 37 degrees C released 5-ASA in 65 or 27% of the dose in 8 h, respectively. No 5-ASA was detected from the incubation of 5-ASA-Gly with the homogenates of stomach or small intestine. Plasma concentration of 5-ASA-Gly decreased rapidly after intravenous administration of 5-ASA-Gly, and no 5-ASA was detected in the blood, which indicated 5-ASA-Gly was not degraded in the plasma. After oral administration of 5-ASA-Gly, about 50% of the administered dose was recovered as 5-ASA and N-acetyl-ASA and 3% as 5-ASA-Gly from feces and 14% as 5-ASA-Gly and 28% as 5-ASA and N-acetyl-ASA from urine in 24 h. These results suggested that a large fraction of 5-ASA-Gly was delivered to the large intestine and activated to liberate 5-ASA. For comparison, total recovery of 5-ASA and N-acetyl-5-ASA from feces after oral administration of 5-ASA-Gly was greater than that from sulfasalazine, which is one of the most commonly prescribed prodrugs of 5-ASA.

Administration, Oral↗

PubMed2000

A broad-spectrum caspase inhibitor blocks concanavalin A-induced hepatitis in mice.

Fulminant hepatic failure (FHF) is a clinical syndrome resulting from massive death of liver cells or sudden and severe impairment of liver function. The causes of FHF are diverse and the overall mortality is very high. Recently, it became clear that apoptosis of hepatocytes is the critical cause of acute hepatic failure in FHF. It is well known that a family of cysteine proteases called caspase is one of the key mediators of the apoptotic pathway. Thus, caspases are attractive potential targets for the treatment of disorders resulting from excessive apoptosis. In this report, we examined the activity of a new caspase inhibitor, Xyz 033 mp. This nonpeptide inhibitor showed broad-spectrum caspase-inhibiting activity and protected primary rat hepatocytes from apoptotic death. In a mouse model of FHF induced by concavalin A (Con A), Xyz 033 mp suppressed elevated AST and ALT and specifically reduced IL-1 beta concentration. Also, Xyz 033 mp rescued mice from lethal experimental hepatitis induced by Con A. In addition, histological examinations indicated that Xyz 033 mp protected hepatocytes from the fatal apoptogenic effect of Con A. These results suggest that Xyz 033 mp may be a candidate therapeutic agent for FHF caused by massive apoptotic death of hepatocytes.

Animals↗

PubMed2000

Pathological fracture of the femoral neck as the first manifestation of osteonecrosis of the femoral head.

We describe two cases of pathological fracture of the femoral neck occurring as the first manifestation of osteonecrosis of the femoral head (ONFH). No abnormal findings suggestive of ONFH were identified on the radiographs for either of the patients, and the fractures occurred like spontaneous fractures without any trauma or unusually increased activity. The patients' medical history, age, and good bone quality suggested ONFH as a possible underlying cause of the fractures. If we had not suspected ONFH as a predisposing condition, these minimally displaced fractures might have been fixed internally with multiple pins, and this would have led to nonunion or collapse of the femoral head. To avoid inappropriate treatment, ONFH should be considered as a predisposing factor in pathological fractures of the femoral neck.

Femoral Neck Fractures↗

PubMed2000

Development of pulmonary arteriovenous fistulas after bidirectional cavopulmonary shunt.

BACKGROUND: A high incidence of pulmonary arteriovenous fistulas (PAVF) has been reported after bidirectional cavopulmonary shunt (BCPS) or total cavopulmonary shunt (TCPS; BCPS in patients with interrupted inferior vena cava). However, the definite diagnostic criteria or standard diagnostic modality of PAVF has not yet been defined. The goal of this study was to evaluate the diagnostic modalities and the prevalence of PAVF. METHODS: We selected 10 patients with TCPS and 27 patients with BCPS. Lung perfusion scan, contrast echocardiogram, and pulmonary angiogram were performed. The results were compared among groups of patients and among each diagnostic modality. RESULTS: All 10 patients with TCPS and 16 and 13 patients with BCPS showed positive results on contrast echocardiograms and lung scans, respectively. Six patients with TCPS and 4 patients with BCPS showed positive results on pulmonary angiograms. All patients with TCPS developed subclinical or clinical PAVF and 19 patients with BCPS developed subclinical PAVF and none of them had clinical PAVF during the short-term follow-up. CONCLUSIONS: Most patients with bidirectional cavopulmonary anastomosis have subclinical evidence of right-to-left intrapulmonary shunting. This problem can be demonstrated with various diagnostic modalities.

Arteriovenous Fistula↗

PubMed2000

Pentoxifylline potentiates nitric oxide production in interleukin-1beta-stimulated vascular smooth muscle cells through cyclic AMP-dependent protein kinase A pathway.

In the present study, we observed that pentoxifylline (PTX) significantly augmented the nitric oxide (NO) production and the iNOS gene expression by interleukin-1beta (IL-1beta)-stimulated vascular smooth muscle cells (VSMCs). The enhancing effects of PTX on the IL-1beta-induced NO production was associated with an increased intracellular cyclic AMP (cAMP) levels, and the synergistic effects of PTX on the IL-1beta-induced NO production was blocked by cAMP-dependent protein kinase A (PKA) inhibitors. PKA inhibitors, KT5720 and H89, markedly decreased the augmented expression of iNOS gene whereas ODQ, a soluble guanylate cyclase inhibitor, did not affect the enhancing effect. In addition, the pretreatment with KT5720 or H89 abolished the increased translocation of the p65 subunit of NF-kappaB into the nucleus by PTX in the IL-1beta-stimulated VSMCs. These results suggest that enhancing effects of PTX on the iNOS gene expression in the IL-1beta-stimulated VSMCs is mediated predominantly through the activation of NF-kappaB via cAMP-dependent PKA pathway.

Animals↗

PubMed2000

Differences in counseling men and women: family planning in Kenya.

A comparison of family planning sessions with male and female clients in Kenya found distinct gender differences. Most men came for information, while women wanted to adopt, continue, or change contraceptive methods. Consultations with men and couples were more than twice as long as consultations with women. Men communicated actively (for example, by volunteering extra information, asking questions, and expressing worries) during 66% of their turns to speak, compared with 27% for women. Providers offered men more detailed information than women, asked them fewer questions, issued fewer instructions, and responded more supportively. These communication patterns may be seen as a reflection of Kenyan gender roles and men's and women's different reasons for seeking family planning services. Kenyan providers need to improve the quality of their interactions with women. They also need to anticipate men's outspokenness and understand the male agenda if they are to counsel men effectively.

Adult↗

PubMed2000

Expression of cyclin D1, cyclin E, cdk4 and loss of heterozygosity of 8p, 13q, 17p in hepatocellular carcinoma: comparison study of childhood and adult hepatocellular carcinoma.

AIMS/BACKGROUND: In hepatitis B virus-related hepatocellular carcinoma (HCC), at least 20-40 years of continuous necro-inflammation is necessary for the hepato-carcinogenesis to occur. However, HCC in childhood shows an unusually short latent period and rapid progression. In our previous report, mutation of c-met was found only in childhood HCC, but not in adult HCC. In order to determine the specific biological tumorous features of childhood HCC, a comparison study of childhood and adult HCC was performed. METHODS: Eighteen cases of HBV positive HCC (nine children and nine adults aged more than 40 years) were selected. The expression of G1 phase regulatory proteins (cyclin D1, cyclin E and cdk4) was studied using immunohistochemical methods. Loss of heterozygosity (LOH) on chromosomal arms 8p, 13q and 17p was analyzed. RESULTS: Cyclin D1 expression was significantly lower in childhood HCC than in adult HCC (cases of cyclin D1 expression under 3+: childhood 5/9 vs. adult 1/9, p=0.046). No difference in cyclin E and cdk4 expression was found between childhood and adult HCC. LOH frequency on 13q was relatively higher in childhood than in adult HCC (66.7% vs. 22.2%, p=0.058). LOH frequency on 8p and 17p was 44.4% and 33.3% in childhood HCC and 44.4% and 75% in adult HCC with no statistical significance between the two groups. CONCLUSION: Our data suggest that childhood HCC may be less dependent on cyclin D1 protein for tumor growth and progression than adult HCC, and that the LOH on 13q may be an important feature of childhood HCC.

Adolescent↗

PubMed2000

Cyclic adenosine monophosphate inhibits ursolic acid-induced apoptosis via activation of protein kinase A in human leukaemic HL-60 cells.

This study was designed to investigate the effect of cAMP on ursolic acid-induced apoptosis of HL-60 cells. Ursolic acid decreased the viability of the cells in a dose-dependent manner, which was revealed as an apototic process characterized by ladder-pattern DNA fragmentation in agarose gel electrophoresis and segmented nuclei in DAPI-sulpharhodamin 101 staining. Ursolic acid-induced apoptosis of the cells was markedly inhibited by the addition of cAMP-elevating agents including DB-cAMP, CPT-cAMP, 8-Br-cAMP and forskolin. These results were further evidenced by the fact that inhibitors of cAMP-dependent protein kinase including H89 and KT5720 completely inhibited the cAMP-mediated rescue of HL-60 cells from ursolic acid-induced apoptosis. In addition, differentiating agents of the cells such as dimethyl sulfoxide and retinoic acid did not affect the ursolic acid-induced apoptosis of HL-60 cells. These results suggest that signaling pathway of cAMP-dependent activation of protein kinase A may affect the responsiveness of tumor cells upon ursolic acid.

Antineoplastic Agents, Phytogenic↗

PubMed2000

Nitric oxide protects murine embryonic liver cells (BNL CL.2) from cytotoxicity induced by glucose deprivation.

We investigated the protective effects of nitric oxide on cell death of murine embryonic liver cells (BNL CL.2) after glucose deprivation. Endogenous nitric oxide production by BNL CL.2 cells was induced by 6 hr pretreatment with interferon-gamma and lipopolysaccharide. We used sodium nitroprusside and S-nitroso-L-glutathione as exogenous nitric oxide-generating compounds. All agents were used at doses that did not show direct cytotoxicity as measured by crystal violet staining assay. In the BNL CL.2 cells, the viability dropped very steeply after 24 hr incubation with glucose-free media. Endogenous nitric oxide produced by treatment of the cells with interferon-gamma and lipopolysaccharide protected the cells from glucose deprivation-induced cytotoxicity, but did not protect them in the presence of the nitric oxide synthesis inhibitor, N(G)-monomethyl-L-arginine. Exogenous nitric oxide protected the cells from glucose deprivation-induced cytotoxicity in a concentration-dependent manner. Cytoprotection by nitric oxide donors was abolished by the use of nitric oxide scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazole, but not by the soluble guanosine cyclase inhibitor, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. In addition, cytoprotective effects comparable to endogenous or exogenous nitric oxide were not observed when the cells were incubated with dibutyl guanosine 3',5'-cyclic monophosphate. Based upon these results, we suggest that nitric oxide may enhance the cell survival of BNL CL.2 cells after glucose deprivation via a guanosine 3',5'-cyclic monophosphate-independent pathway.

Acetylcysteine↗

PubMed2000

In vivo cytokine responses to interleukin-2 immunotherapy after autologous stem cell transplantation in children with solid tumors.

The potent immunostimulatory cytokine interleukin-2 (IL-2) has been extensively investigated for its potential to induce anti-tumor immunity in a number of tumor models. Only recently the complex interplay of mutually suppressive or supportive cytokines of the IL-2-induced network of cytokines has been better characterized. The aim of this study was to assess which of these in vitro findings are reproducible in vivo in recipients of stem cell transplants (SCT), since in these patients long- lasting impairments in cytokine inducibility have been described. We have therefore studied the kinetics of putative modulators and mediators of IL-2-induced immune activation, namely IL-1beta, IL-4, IL-5, IL-10, IL-12, soluble Fas ligand (sFasL), and GM-CSF during IL-2 therapy. All patients were children or adolescents suffering from solid tumors with poor prognosis who received three 5-day courses of high-dose intravenous IL-2 as an adjuvant to their radio-chemotherapy and autologous SCT. While IL-1beta, IL-4 and IL-12 were not, and sFasL was only mildly affected by the IL-2 therapy, we observed a consistent and early rise of IL-10, IL-5, and GM-CSF. These increases were rapidly reversible after discontinuation of IL-2 therapy. The inducibility of IL-10, IL-5 and GM-CSF was more pronounced with increasing time from the SCT, and in the third cycle reached an order of magnitude as in high-dose IL-2 patients without SCT. Together with the abundant in vitro data, these findings may help devise a combination immunotherapy permitting stronger anti-tumor effects, but lesser adverse effects.

Adolescent↗

PubMed2000

Regulation of interleukin-2 induced interleukin-5 and interleukin-13 production in human peripheral blood mononuclear cells.

Adjuvant interleukin (IL)-2 immunotherapy has been used for many years for a variety of malignant and nonmalignant entities. In many cases, a dose escalation might have seemed desirable, but was prevented by the rather severe adverse effects of systemic IL-2 application. Only recently has the regulation of IL-2 induced cytotoxicity been understood better, so that now efforts can be aimed at the design of cytokine cocktails that would selectively induce cytotoxicity but result in as few adverse effects as possible. Previously, induction of IL-5 under systemic IL-2 therapy has been described, and a number of the side-effects have been attributed to this event. We therefore investigated the regulation of IL-2 induced production of IL-5 and IL-13 (which, similarly to IL-5, is a mediator of allergy-like symptoms). At the same time, the effects of regulatory cytokines, such as IL-4, IL-10 and IL-12, on interferon-gamma (IFN-gamma), the major cytotoxic mediator of IL-2 therapy, were studied. All three have been discussed as antitumour immunotherapeutics, either alone or in combination with IL-2. In anti-CD3-treated peripheral blood mononuclear cells, IL-2 induced IL-5 and IL-13 alongside IFN-gamma, IL-10 and IL-12. In the presence of IL-2, inhibition of endogenous IL-12 production further enhanced the IL-5 and IL-13 responses, while IFN-gamma and IL-10 were markedly suppressed. Co-incubation with IL-2 and IL-12 suppressed IL-5/IL-13 below, but enhanced IFN-gamma and IL-10 above, levels induced by IL-2 alone. IL-10 was suppressive on all the investigated cytokines, while IL-4 interfered with IL-2 induced IFN-gamma and IL-12 production, but was additive to IL-2 in its effect on IL-5 and IL-13. These data suggest that the combination of IL-12 with IL-2 would enhance the cytotoxic activity of this regimen, but might reduce its adverse effects.

CD3 Complex↗

PubMed2000

Nitric oxide prevents tumor necrosis factor alpha-induced rat hepatocyte apoptosis by the interruption of mitochondrial apoptotic signaling through S-nitrosylation of caspase-8.

Mitochondrial cytochrome c release plays a critical role in apoptotic signal cascade after the activation of cell surface death receptors. We investigated the role played by nitric oxide (NO) in mitochondrial apoptotic signaling in tumor necrosis factor alpha (TNF-alpha) plus actinomycin D (TNF-alpha/ActD)-induced apoptosis. NO produced either by S-nitroso-N-acetyl-DL-penicillamine (SNAP) or inducible NO synthase (iNOS) prevented TNF-alpha/ActD-induced apoptosis in hepatocytes and also inhibited both caspase-8-like (IETDase) and caspase-3-like protease (DEVDase) activity as well as mitochondrial cytochrome c release. Recombinant human (rh) caspase-8 induced the cleavage of the cytochrome c-effluxing factor Bid and cytochrome c release from purified mitochondria in the reconstitution system with Bid(+/+) cytosol, but not with Bid(-/-) cytosol. The addition of SNAP and the caspase-8 inhibitor Ac-IETD-fmk inhibited caspase-8-dependent Bid cleavage and cytochrome c release. The inhibitory effect of NO on caspase-8 was reversed by dithiothreitol (DTT). Furthermore, rh-caspase-8 was found to be modified by S-nitrosylation with 1.7 moles of NO bound per mole of enzyme. Treatment of hepatocytes with interleukin 1beta (IL-1beta) plus interferon gamma (IFN-gamma), which induced iNOS expression and NO production, suppressed TNF-alpha/ActD-induced Bid cleavage and mitochondrial cytochrome c release. The NOS inhibitor N(G)-monomethyl-L-arginine (NMA) inhibited the protective effects of IL-1beta and IFN-gamma. The liver-specific NO donor V-PYRRO/NO also inhibited in vivo elevation of IETDase activity, Bid cleavage, and mitochondrial cytochrome c release in the livers of rats injected with TNF-alpha plus D-galactosamine. Our results indicate that one mechanism by which NO protects hepatocytes from TNF-alpha/ActD-induced apoptosis is via the interruption of mitochondrial apoptotic signaling through S-nitrosylation of caspase-8.

Animals↗