Inhibitory effect of FK 506 and cyclosporin A on nitric oxide production by LPS-treated cultured rat macrophages

We analyzed the effect of FK 506 on the production of nitric oxide by macrophages. Isolated rat peritoneal macrophages were cultured for 24 h with or without lipopolysaccharide (LPS) (5 microg/ml) and in the absence or presence of FK 506 (0.1 and 1 microg/ml). The concentration of NO2- in culture supernatants was taken as a measure of nitric oxide production. FK 506 (0.1 and 1 microg/ml) reduced the LPS-induced increase of NO2- levels by 68% and 81%, respectively. The impact of cyclosporin A (CsA) was studied in order to compare their effects. CsA (0.1 and 1 microg/ml) decreased the levels of nitrites by 39% and 69%, respectively. The results obtained suggest that both immunosuppressive drugs exhibit a dose-dependent inhibitory effect on nitric oxide production and that FK 506 is a more potent agent than CsA in this respect.     

Nitric oxide enhances PGI(2)production by human pulmonary artery smooth muscle cells

To evaluate the effect of exogenous nitric oxide (NO) and endogenous NO on the production of prostacyclin (PGI(2)) by cultured human pulmonary artery smooth muscle cells (HPASMC) treated with lipopolysaccharide (LPS), interleukin-1(beta)(IL-1(beta)), tumor necrosis factor alpha (TNF(alpha)) or interferon gamma (IFN(gamma)), HPASMC were treated with LPS and cytokines together with or without sodium nitroprusside (SNP), NO donor, N(G)-monomethyl-L-arginine (L-NMMA), NO synthetase inhibitor, and methylene blue (MeB), an inhibitor of the soluble guanylate cyclase. After incubation for 24 h, the postculture media were collected for the assay of nitrite by chemiluminescence method and the assay of PGI(2)by radioimmunoassay. The incubation of HPASMC with various concentrations of LPS, IL-1(beta)or TNF(alpha)for 24 h caused a significant increase in nitrite release and PGI(2)production. However, IFN(gamma)slightly increased the release of nitrite and had little effect on PGI(2)production. Although the incubation of these cells for 24 h with SNP did not cause a significant increase in PGI(2)production, the incubation of HPASMC with SNP and 10 microg/ml LPS, or with SNP and 100 U/ml IL-1(beta)further increase PGI(2)production and this enhancement was closely related to the concentration of SNP. However, stimulatory effect of SNP on PGI(2)production was not found in TNF(alpha)- and IFN(gamma)- treated HPASMC. Addition of L-NMMA to a medium containing LPS or IL-1(beta)reduced nitrite release and attenuated the stimulatory effect of those agents on PGI(2)production. MeB significantly suppressed the production of PGI(2)by HPASMC treated with or without LPS or IL-1(beta). The addition of SNP partly reversed the inhibitory effect of MeB on PGI(2)production by HPASMC. These experimental results suggest that NO might stimulate PGI(2)production by HPASMC. Exogenous NO together with endogenous NO induced by LPS or cytokines from smooth muscle cells might synergetically enhance PGI(2)production by these cells, possibly in clinical disorders such as sepsis and acute respiratory distress syndrome.     

Inhibitory effect of vitamin E on proinflammatory cytokines-and endotoxin-induced nitric oxide release in alveolar macrophages

Nitric oxide is thought to be an important modulator of various functions in normal and inflamed airways. In the present study, we evaluated the effects of high vitamin E (250 mg and 1250 mg alpha-tocopheryl acetate (TA)/kg diet/10 days) on nitric oxide (NO(.)) release by alveolar macrophages (AMs) in response to lipopolysaccharide (LPS), interleukin-1beta (IL-1beta) and tumor necrosis factor (TNF-alpha). LPS and IL-1beta treatment (1-10 microg/ml) enhanced NO(.) release in AMs from control animals fed on 50 mg vitamin E/kg diet in a concentration dependent manner. However, this enhancement of NO(.) was attenuated in the AMs of animals fed with 250 mg or 1250 mg vitamin E/kg diet. TNF-alpha had no effect in eliciting the release of NO(.) in AMs obtained either from control or from hyper vitamin E fed animals. Further, LPS (1-10 microg/ml) enhanced the inducible nitric oxide synthase (iNOS) activity of AMs of control group and TA-fed animals almost to equal extent. Similarly, LPS-induced formation of N-nitrosamine (N-nitroso-L-[(14)C]-proline) in AMs of control and TA-supplemented animals were not different statistically. On the other hand, in vitro addition of vitamin E (200 microM) in AMs of control animals, when triggered with 10 microg LPS/ml, caused a significant decrease in N-nitroso-L-[(14)C]-proline formation. It seems that high doses of TA in diet may play a role in reducing the lipopolysaccharide and proinflammatory cytokines-induced NO(.)-mediated damage by AMs.     

Effects of ambient air particles on nitric oxide production in macrophage cell lines

We assessed the in vitro toxicity of various particles on three murine macrophage cell lines, (J774A.1, WR19M.1, RAW264.7). The cells were exposed to aqueous suspensions (0-100 microg/30 mm2 well) of urban particulate matter (SRM-1648, SRM-1649, EHC-93), fine particulate matter (PM2.5), titanium dioxide (SRM-154b), and respirable cristobalite (SRM-1879) for 2 h and were then stimulated with lipopolysaccharide (LPS, 100 ng/ml) and recombinant interferon-gamma (IFN, 100 U/ml). After overnight incubation with the particles and LPS/IFN, nitric oxide production was estimated from culture supernatant nitrite. Cell viability was determined by monitoring the rate of AlamarBlue reduction. The dose-effect relationships for nitrite and viability were modeled as a power function (Fold change = [Dose+1]beta), where beta represents the slope of the dose-response curve. Potency was defined as the rate of change in nitrite production corrected for cell viability (beta(POTENCY) = beta(NITRITE) - beta(VIABILITY)). Overall, the urban particles decreased nitric oxide production (beta(POTENCY) < 0), while exposure of the cells to fine particulate matter or cristobalite increased the production of nitric oxide (beta(POTENCY) > 0). Titanium dioxide (TiO2) was essentially inactive (beta(POTENCY) approximately to 0). The decrease in nitric oxide production seen in cells exposed to the urban particles was directly correlated to a decrease in the expression of inducible nitric oxide (iNOS) as determined by Western blot analysis. The results indicate that particles are modulators of nitric oxide production in murine macrophages and may directly disrupt expression of iNOS during concomitant pathogen exposure. Pathways leading to enhanced NO production causing cell injury, and to decreased NO release resulting in lower bacterial clearance, may both be relevant to the health effects of ambient particles.     

NO production by cNOS and iNOS reflects blood pressure changes in LPS-challenged mice

Increased nitric oxide (NO) production is the cause of hypotension and shock during sepsis. In the present experiments, we have measured the contribution of endothelial (e) and inducible (i) nitric oxide synthase (NOS) to systemic NO production in mice under baseline conditions and upon LPS treatment (100 microg/10 g ip LPS). NO synthesis was measured by the rate of conversion of l-[guanidino-15N2]arginine to l-[ureido-15N]citrulline, and the contribution of the specific NOS isoforms was evaluated by comparing NO production in eNOS-deficient [(-/-)] and iNOS(-/-) mice with that in wild-type (WT) mice. Under baseline conditions, NO production was similar in WT and iNOS(-/-) mice but lower in eNOS(-/-) mice [WT: 1.2 +/- 0.2; iNOS(-/-): 1.2 +/- 0.2; eNOS(-/-): 0.6 +/- 0.3 nmol. 10 g body wt-1. min-1]. In response to the challenge with LPS (5 h), systemic NO production increased in WT and eNOS(-/-) mice but fell in iNOS(-/-) mice [WT: 2.7 +/- 0.3; eNOS(-/-): 2.2 +/- 0.6; iNOS(-/-): 0.7 +/- 0.1 nmol. 10 g body wt-1. min-1]. After 5 h of LPS treatment, blood pressure had dropped 14 mmHg in WT but not in iNOS(-/-) mice. The present findings provide firm evidence that, upon treatment with bacterial LPS, the increase of NO production is solely dependent on iNOS, whereas that mediated by cNOS is reduced. Furthermore, the data show that the LPS-induced blood pressure response is dependent on iNOS.     

Effect of curcumin on the production of nitric oxide by cultured rat mammary gland.

We have hypothesized that one aspect of the antitumor activity of curcumin (diferuloylmethane) during the promotion stage of mammary gland tumorigenesis may be linked to reduction of free radicals (Inano et al., Carcinogenesis, 20: 1011-1018, 1999). Nitric oxide (NO) has been found to inflict damage on important biomolecules, and the overproduction of NO in diseases may be implicated in carcinogenesis and tumor progression. We have reported that the presence of three isoforms of nitric oxide synthases (NOS) and NO generation in the mammary gland correlate with the mammary gland development and mammary carcinogenesis. We, therefore, investigated the inhibitory activity of curcumin for the production of NO in rat mammary glands by using an organ culture system to validate the effectiveness and usefulness of curcumin in the pathophysiology of the mammary gland. A diced mammary gland (approximately 3 mm cubes) from the inguinal part of a female Wistar-MS rat treated with estradiol and progesterone was cultured with 2 ml of 5% FCS/DMEM in the presence or absence of LPS (0.5 microg/ml) for 2-3 days. Curcumin ( approximately 100 microM) was added at the same time to the LPS-treated cultures. In some experiments, curcumin was added to the culture after the LPS had been washed out. The NO production was significantly increased (by almost 20-fold compared to the control) by the addition of LPS to the culture system. This enhancement of NO production by LPS was reduced to 76 and to 56% by addition of 30 and 100 microM curcumin, respectively, to the culture. When LPS was eliminated from the culture after prestimulation for 1 day, the production of NO by the mammary gland dropped off, although some NO was still detectable. Curcumin did not further inhibit the production of NO by the prestimulated mammary gland after the elimination of LPS from the culture. The inducible nitric oxide synthase (iNOS, 122 kDa) and endothelial nitric oxide synthase (eNOS, 152 kDa) isoforms were detected in the mammary gland extracts at the end of the organ culture. The quantity of iNOS was apparently increased in the gland treated with LPS, while the eNOS expression was clearly diminished. Curcumin (100 microM) obviously suppressed the iNOS expression in the mammary glands cultured with LPS, and a recovery in the eNOS expression was observed. On the other hand, curcumin exhibited scavenging activity for the NO released from N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC 12), a NO donor compound, in the coincubation mixture. These results indicate that curcumin has the ability to inhibit iNOS induction by LPS in the mammary gland and to scavenge NO radicals, which might explain, at least partly, its therapeutic properties in inflammation of the mammary gland.     

Overexpression of regucalcin suppresses cell death and apoptosis in cloned rat hepatoma H4-II-E cells induced by lipopolysaccharide, PD 98059, dibucaine, or Bay K 8644

The effect of regucalcin, a regulatory protein in intracellular signaling pathway, on cell death was investigated by using the cloned rat hepatoma H4-II-E cells overexpressing regucalcin. The hepatoma cells (wild-type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured for 12-72 h in medium without FBS containing either vehicle or lipopolysaccharide (LPS; 0.1 or 1.0 microg/ml). The number of wild-type cells was significantly decreased by culture for 24 or 48 h in the presence of LPS (0.1 or 1.0 microg/ml). The effect of LPS (0.1 or 1.0 microg/ml) in decreasing the number of hepatoma cells was significantly prevented in transfectants overexpressing regucalcin. However, the culture with LPS (0.1 or 1.0 microg/ml) for 72 h caused a significant decrease in cell number of transfectants. Ca(2+)/calmodulin-dependent nitric oxide (NO) synthase activity was significantly decreased by culture with LPS (1.0 microg/ml) for 24-72 h of wild-type cells. This decrease was significantly prevented in transfectants. LPS (0.1 or 1.0 microg/ml)-induced decrease in the number of wild-type cells was significantly prevented by culture with caspase-3 inhibitor (10(-8) M). Moreover, the number of wild-type cells was significantly decreased by culture with PD 98059 (10(-6) M), dibucaine (10(-6) M), or staurosporine (10(-6) M), which is an inhibitor of various protein kinases. The effect of PD 98059 or dibucaine on the number of wild-type cells was not observed in transfectants, although the effect of staurosporine was seen in transfectants. Culture with Bay K 8644 (2.5 x 10(-6) M), an agonist of Ca(2+) entry in cells, caused a significant decrease in the number of wild-type cells. Such an effect was not seen in transfectants. The presence of LPS did not significantly decrease the number of wild-type cells in the presence of Bay K 8644. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with Bay K 8644, and this DNA fragmentation was significantly prevented in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death induced by LPS or various intracellular signaling-related factors.     

Modulation of the activity of sea bass (Dicentrarchus labrax) head-kidney macrophages by macrophage activating factor(s) and lipopolysaccharide

The aim of this study was to establish the requirements for macrophage activating factor (MAF) production by sea bass head-kidney leucocytes and the kinetics of macrophage activation when exposed to MAF-containing supernatants and/or lipopolysaccharide (LPS), a known macrophage stimulant. MAF activity was found in culture supernatants of total head-kidney leucocytes pulsed with 5 microg ml(-1)Con A, 5 or 10 ng ml(-1)PMA and 100 ng ml(-1)calcium ionophore, or 10 microg ml(-1)Con A alone, as assessed by the capacity to prime macrophages for enhanced production of reactive oxygen intermediates (ROI). Mixed leucocyte cultures from two or eight fish showed higher MAF activity after stimulation, indicating that a mixed leucocyte reaction was also important for MAF production. MAF-induced activation of macrophage cultures was highest at 18 h of exposure and was lost by 72 h except for MAF induced by Con A-stimulation alone. LPS primed macrophages for increased ROI production at early incubation times and down-regulated ROI production after 24 h. LPS had no effect in further stimulating the MAF-induced priming effect on production of ROI and down-regulated the MAF-priming by 48 h. Sea bass head-kidney macrophages did not show increased nitrite production when exposed to MAF and/or LPS, which may be related to their differentiation status.     

Hypoxia enhances gene expression of inducible nitric oxide synthase and matrix metalloproteinase-9 in ras/myc-transformed serum-free mouse embryo cells under simulated inflammatory and infectious conditions

Ras/myc-transformed serum-free mouse embryo (ras/myc SFME) cells were treated with interferon-gamma (IFN-gamma; 100 U/ml) and/or lipopolysaccharide (LPS; 0.5 microg/ml) for 24 h to simulate inflammatory and infectious conditions and investigate their effects on the expression of inducible nitric oxide synthase (iNOS) mRNA, nitric oxide (NO) and matrix metalloproteinase-9 (MMP-9). In addition, aminoguanidine (AG; 1 mM), a NOS inhibitor, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 10-200 microM), an NO donor or (+/-)-N-[(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine carboxamide (NOR4; 10-200 microM), an NO donor, were added to analyze possible associations of NO with MMP-9. Tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 were also measured to analyze possible relationships of NO with the MMP-9/TIMP balance. Furthermore, the cells were treated with 1% O2 under the simulated inflammatory and infectious conditions and the mRNA expressions of iNOS and MMP-9 were analyzed to investigate the possible effects of hypoxia on the expression of genes involved in tumor malignant progression and distant metastasis. Co-treatment with IFN-gamma and LPS increased the expression levels of iNOS mRNA, NO and MMP-9, but NO may not be directly associated with MMP-9 or the MMP-9/TIMP balance. Treatment with 1% O2 markedly increased the gene expression levels of iNOS and MMP-9, indicating that ras/myc SFME cells alter the expression levels of tumor-associated genes and possibly enhance their malignancy as cancer cells under inflammatory, infectious and hypoxic conditions.     

Overexpression of regucalcin suppresses apoptotic cell death in cloned normal rat kidney proximal tubular epithelial NRK52E cells: change in apoptosis-related gene expression

The effect of regucalcin, a regulatory protein in intracellular signaling pathway, on cell death and apoptosis was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in a medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured for 24-72 h in a medium without BS containing either vehicle, tumor necrosis factor-alpha (TNF-alpha; 0.1 or 1.0 ng/ml of medium), lipopolysaccharide (LPS; 0.1 or 1.0 microg/ml), Bay K 8644 (10(-9)-10(-7) M), or thapsigargin (10(-9)-10(-7) M). The number of wild-type cells was significantly decreased by culture for 42-72 h in the presence of TNF-alpha (0.1 or 1.0 ng/ml), LPS (0.1 or 1.0 microg/ml), Bay K 8644 (10(-7)-10(-5) M), or thapsigargin (10(-8) or 10(-7) M). The effect of TNF-alpha (0.1 or 1.0 ng/ml), LPS (0.1 or 1.0 microg/ml), Bay K 8644 (10(-7)-10(-6) M), or thapsigargin (10(-7) M) in decreasing the number of wild-type cells cultured for 24-72 h was significantly prevented in transfectants overexpressing regucalcin. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with LPS (1.0 microg/ml), Bay K 8644 (10(-7) M), or thapsigargin (10(-8) M) for 24 h, and this DNA fragmentation was significantly suppressed in transfectants. DNA fragmentation in adherent cells was not seen by culture with TNF-alpha (1.0 ng/ml). TNF-alpha-induced decrease in the number of wild-type cells was significantly prevented by culture with caspase-3 inhibitor (10(-8) M), while LPS- or Bay K 8644-induced decrease in cell number was significantly prevented by caspase-3 inhibitor or N omega-nitro-L-arginine methylester (NAME) (10(-5) M), an inhibitor of nitric oxide (NO) synthase. Thapsigargin-induced decrease in cell number was not prevented in the presence of two inhibitors. Bcl-2 and Akt-1 mRNA levels were significantly increased in transfectants cultured for 24 h as compared with those of wild-type cells, while Apaf-1, caspase-3, or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA expressions were not significantly changed in transfectants. Culture with TNF-alpha (1.0 ng/ml), LPS (1.0 microg/ml), Bay K 8644 (l0(-7) M), or thapsigargin (10(-8) M) caused a significant increase in caspase-3 mRNA levels in wild-type cells. LPS (1.0 microg/ml) significantly decreased Bcl-2 mRNA expression in the cells. Their effects on the gene expression of apoptosis-related proteins were not significantly changed in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death and apoptosis induced by various factors which their action are mediated through many intracellular signaling pathways, and that it modulates the gene expression of apoptosis-related proteins.     

白细胞介素—10对血管一氧化氮／一氧化氮合酶系统的影响

为探讨抗炎因子－－白细胞介素－10（IL－10）对大鼠主动脉一氧化氮（NO）／一氧化氮合酶（NOS）系统的影响,应用Griess试剂、^3H－瓜氨酸生成及蛋白免疫印迹杂交等方法,测定IL－10孵育对血管NO释放、NOS活性及表达的影响。结果发现细菌脂多糖（LPS）呈浓度领带性地激活诱导型NOS（iNOS）,促进NO生成。IL－10（10^-10-10^-8g/ml）呈浓度依赖性地上调内皮型NOS（eNOS）蛋白表达及其活性,但对iNOS活性及表达无明显影响,IL－10（10^-9-10^-8g/ml）显著抑制10μg/ml LPS诱导的NO生成和iNOS激活;而高浓度IL－10（10^-7g/ml）则上调iNOS的活性,对eNOS蛋白的表达知活性无明显影响。因此IL－10对NO／NOS系统具有双重影响,一方面可抑制炎症介质诱发的作为炎性物质的iNOS的表达及激活,另一方面可上调内皮源扩血管物质NO的释放。     

Cytokine- and Endotoxin-Induced Nitric Oxide Synthase in Rat Astroglial Cultures: Differential Modulation by Angiotensin II

Abstract: Recent studies have shown that the stimulatory effects of bacterial endotoxin [lipopolysaccharide (LPS)] on inducible nitric oxide (NO) synthase (iNOS) in astroglia are significantly reduced by the peptide angiotensin II (Ang II). In the present study we have compared the modulatory actions of Ang II on cytokine- and LPS-stimulated iNOS in astroglia cultured from adult rat brain. Incubation of astroglia with LPS (100 ng/ml; 24 h) and/or combinations of interleukin-1β (IL-1β; 10 ng/ml, 24 h), interferon-γ (IFN-γ; 100 U/ml, 24 h), or tumor necrosis factor-α (TNF-α; 100 ng/ml, 24 h) resulted in significant increases of iNOS mRNA, iNOS protein, and NO production, with the latter indicated by increased nitrite accumulation. The effects of LPS, IL-1β, and TNF-α were significantly decreased by coincubation with Ang II (100 ng/ml, 24 h). In contrast, Ang II did not alter the stimulation of iNOS mRNA levels and NO production elicited by IFN-γ. Therefore, Ang II differentially modulates the stimulatory actions of LPS and cytokines on iNOS, and subsequently NO production, in astroglia. These data suggest that Ang II may have an important modulatory role in intracerebral immune responses that involve production of NO by astroglia.     

Nitric oxide dependent and independent effects of in vitro incubation or endotoxin on vascular reactivity in rat aorta.

We sought to delineate contributions of nitric oxide (NO) and other mechanisms to impairment of contraction and endothelium-dependent relaxation following prolonged in vitro incubation, endotoxin and interleukin-1 exposure in isolated rat aorta. Responses from freshly-dissected (control) rings +/-endothelium were compared with those from rings incubated in sterile, antibiotic containing medium +/- E. Coli endotoxin (LPS, 100 microg/ml) +/- interleukin-1 (IL-1, 40 ng/ml) at 37 degrees C for 20-24 h. In some experiments, medium included dexamethasone (DEX, 1 microg/ml), cycloheximide (10 microg/ml), or N(G)-nitro-L-arginine (NNLA, 10(-4)M). After incubation, medium nitrite was measured. Incubation alone, without addition of inflammatory mediators, impaired contraction in an agonist-specific manner, by both NO-dependent and NO-independent mechanisms. Either LPS or IL-1 diminished contraction further, in a similarly heterogeneous manner. For example, contractions were changed in LPS-incubated endothelium-intact rings (vs. fresh controls) by -85%, +115%, -15%, -96%, and -37% for phenylephrine (PE), serotonin, prostaglandin F2alpha, angiotensin II, and U46619, respectively. NO synthase inhibition with NNLA either following, or during LPS incubation only partially normalized subsequent PE contractions, an effect which was smaller than that of DEX. Nitrite accumulation was inversely proportional to PE response, even though NO was not the sole mediator of LPS-impaired contraction. LPS and IL-1 nearly abolished ACh-induced relaxation, which was only mildly impaired by incubation alone. We conclude that prolonged incubation impaired vasoconstriction via both NO synthase induction and NO-independent mechanisms. LPS or IL-1 incubation impaired vasoconstriction further, primarily by NO-independent mechanisms. Moreover, vasoconstrictor responses following LPS varied with the agonist's ability to modulate endothelial NO release. These results are in accord with the failure of NO synthase inhibition to fully restore systemic vascular resistance indices in experimental endotoxemia or in hyperdynamic septic patients.     

Endotoxin induces luteal cell apoptosis through the mitochondrial pathway

The effect of endotoxin (lipopolysacharide, LPS) exposure on luteal cells was studied using an in vitro cell culture system. Buffalo luteal cells were isolated from corpora lutea of the late luteal phase (days 14-16 post estrus) and exposed to various LPS doses (5, 10 and 100 microg/ml) for different time periods (6, 12, 18 or 24 h). The cultured cells were subsequently evaluated for oxidative stress (super oxide, nitric oxide, inducible nitric oxide synthase activity, reduced glutathione depletion and lipid peroxidation) and apoptotic markers (mitochondrial membrane potential, DNA fragmentation, apoptotic cells and cell viability). LPS exposure significantly increased the production of super oxide (P<0.05) and nitric oxide (P<0.01) and increased inducible nitric oxide synthase activity (P<0.01). LPS exposure further depleted reduced glutathione (P<0.05) levels and induced lipid peroxidation (P<0.05). LPS exposure also induced the loss of mitochondrial membrane potential (P<0.05), increased DNA fragmentation (P<0.01) and apoptosis (P<0.01) and decreased cell viability (P<0.01). LPS mediated apoptotic pathway in luteal cells was further characterized using a selected LPS dose (10 microg/ml). It was observed that LPS exposure induced mitochondrial translocation of proapoptotic protein Bax, increased the total Bad expression and down regulated the expression of antiapoptotic proteins Bcl2 and BclXL. LPS exposure further induced cytochrome c release and increased Caspase-9 (P<0.01) and Caspase-3 (P<0.01) activities. LPS exposure also inhibited luteal progesterone secretion (P<0.01). It was evident that the LPS mediated apoptotic effects could be prevented by the coincubation of luteal cells with mitochondrial permeability transition pore blocker Cyclosporine A, inducible nitric oxide synthase inhibitor N-[3-(aminomethyl)benzyl]acetamidine and oxidative stress scavenger N-acetyl cysteine. Our study clearly indicates that LPS induces oxidative stress mediated apoptosis in luteal cells through the mitochondrial pathway.