Nitroso-hemoglobin-ginger conjugates effects on bacterial growth and color stability in a minced beef model.

This study aims to enhance the color and microbiological qualities of a raw beef using natural ingredients. Nitroso-hemoglobin (NO-Hb) integrated with vitamin C (VC), calcium lactate, and ginger complexation were used as natural inhibitors against the growth of aerobic and pathogenic bacteria, namely (Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Salmonella. NO-Hb inhibited E. coli, S. aureus, and Salmonella, and enhanced the color stability more than nitrite in the minced beef model. After the multiexponential analysis of relaxation decays, the water component (T2b) was analyzed using the low-field NMR. The results indicated that, at the 7th d of cold-storage the third component (T2) was detected. Significant correlations were observed between T21 and T22 relaxation times and water-holding capacity in minced beef, implying that the LF-NMR measurements could be an efficient method for the determination and prediction of beef freshness. NO-Hb- ginger mixture, as a novel ingredient, could be used instead of nitrite in terms of meat safety.

Apoptosis induction of poly-S-nitrosated human serum albumin in resistant solid tumor under hypoxia can be restored by phosphodiesterase 5 inhibition.

Poly-S-nitrosated human serum albumin (Poly-SNO-HSA) delivered and accumulated nitric oxide (NO) in tumors and induces apoptosis. Tumor hypoxia is strongly associated with malignant progression and tumor resistance to therapy. In this study, we examined the cytotoxic effect of Poly-SNO-HSA under hypoxia on the murine colon 26 adenocarcinoma (C26) cells in vitro and in vivo. Under hypoxia, at about 4 times LD50 dose of Poly-SNO-HSA in vitro, the reactive oxygen species production was hindered but apoptotic cells were induced via cGMP pathway as the effect was suppressed by a soluble guanylate cyclase inhibitor, NS2028. The apoptosis induction effect of low dose Poly-SNO-HSA on C26 cells in vitro under hypoxia can be restored by a phosphodiesterase 5 (PDE5) inhibitor, vardenafil. In C26-bearing mice, Poly-SNO-HSA/vardenafil combination treatment significantly suppressed the tumor volume compared with Poly-SNO-HSA or vardenafil treatment alone. Furthermore, the core tumor tissues showed increased expression of caspase-3 than the non-core tissue. The expression of caspase-3 appeared to overlap with the hypoxic zone of tumor tissues. Similar results were also obtained when the experiments were repeated using Epimedium extract, a natural herbal supplement with PDE5 inhibition activity. In conclusion, Poly-SNO-HSA/PDE5 inhibitors combination therapy is a promising approach for enhancing the anticancer therapeutic effects of Poly-SNO-HSA against not only anti-cancer drug resistance but also hypoxic stress related solid tumor resistance.

Signaling Mechanisms in the Nitric Oxide Donor- and Amphetamine-Induced Dopamine Release in Mesencephalic Primary Cultured Neurons.

Previous research has shown that nitric oxide (NO) synthase inhibitors prevent rodents' sensorimotor gating impairments induced by dopamine releasing drugs, such as amphetamine (Amph) and methylphenidate. The mechanisms of this effect have not been entirely understood. In the present work, we investigated some possible mechanisms by which the NO donor, NOC-12 (3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene), influence spontaneous and Amph-induced dopamine release, using rat mesencephalic primary cultured neurons preparations. Our results showed that NOC-12 increased dopamine release in a concentration-dependent manner and potentiated the Amph-induced one. Dopamine release induced by NOC-12 was disrupted by N-acetyl-L-cystein (NAC-a free radical scavenger) and MK-801, a NMDA (N-methyl-D-aspartate) non-competitive antagonist, and was concentration dependently affected by oxadiazolo[4,3]quinoxalin-1-one, an inhibitor of the soluble guanylate cyclase (sGC). In contrast, dopamine released by Amph was facilitated by NAC and by MK-801 and not affected by nifedipine (a L-type-Ca(+2) channel blocker), which enhanced NOC-12-induced dopamine release. The present work demonstrates that DA release induced by NOC-12 is partially dependent on sGC and on NMDA activation, and is modulated by L-type Ca(+2) channel and the antioxidant NAC. This mechanism differs from the Amph-induced one, which appears not to depend on L-type Ca(+2) channel and seems to be facilitated by NMDA channel blocking and by NAC. These results suggest that Amph and NOC-12 induce dopamine release through complementary pathways, which may explain the potentiation of Amph-induced dopamine release by NOC-12. These findings contribute to understand the involvement of NO in dopamine-related neuropsychiatric and neurodegenerative diseases.

Nitric Oxide Increases Arterial Endotheial Permeability through Mediating VE-Cadherin Expression during Arteriogenesis.

Macrophage invasion is an important event during arteriogenesis, but the underlying mechanism is still only partially understood. The present study tested the hypothesis that nitric oxide (NO) and VE-cadherin, two key mediators for vascular permeability, contribute to this event in a rat ischemic hindlimb model. In addition, the effect of NO on expression of VE-caherin and endothelial permeability was also studied in cultured HUVECs. We found that: 1) in normal arteriolar vessels (NAV), eNOS was moderately expressed in endothelial cells (EC) and iNOS was rarely detected. In contrast, in collateral vessels (CVs) induced by simple femoral artery ligation, both eNOS and iNOS were significantly upregulated (P<0.05). Induced iNOS was found mainly in smooth muscle cells, but also in other vascular cells and macrophages; 2) in NAV VE-cadherin was strongly expressed in EC. In CVs, VE-cadherin was significantly downregulated, with a discontinuous and punctate pattern. Administration of nitric oxide donor DETA NONOate (NONOate) further reduced the amounts of Ve-cadherin in CVs, whereas NO synthase inhibitor L-NAME inhibited downregulation of VE-cadherin in CVs; 3) in normal rats Evans blue extravasation (EBE) was low in the musculus gracilis, FITC-dextron leakage was not detected in the vascular wall and few macrophages were observed in perivascular space. In contrast, EBE was significantly increased in femoral artery ligation rats, FITC-dextron leakage and increased amounts of macrophages were detected in CVs, which were further enhanced by administration of NONOate, but inhibited by L-NAME supplement; 4) in vitro experiments confirmed that an increase in NO production reduced VE-cadherin expression, correlated with increases in the permeability of HUVECs. In conclusion, our data for the first time reveal the expression profile of VE-cadherin and alterations of vascular permeability in CVs, suggesting that NO-mediated VE-cadherin pathway may be one important mechanism responsible, at least in part, for macrophage invasion during arteriogenesis.

Nitric oxide-mediated activity in anti-cancer photodynamic therapy.

Cell recurrence in cancer photodynamic therapy (PDT) is an important issue that is poorly understood. It is becoming clear that nitric oxide (NO) is a modulator of PDT. By acting on the NF-κB/Snail/RKIP survival/anti-apoptotic loop, NO can either stimulate or inhibit apoptosis. We found that pheophorbide a/PDT (Pba/PDT) induces the release of NO in B78-H1 murine amelanotic melanoma cells in a concentration-dependent manner. Low-dose PDT induces low NO levels by stimulating the anti-apoptotic nature of the above loop, whereas high-dose PDT stimulates high NO levels inhibiting the loop and activating apoptosis. When B78-H1 cells are treated with low-dose Pba/PDT and DETA/NO, an NO-donor, intracellular NO increases and cell growth is inhibited according to scratch-wound and clonogenic assays. Western blot analyses showed that the combined treatment reduces the expression of the anti-apoptotic NF-κB and Snail gene products and increases the expression of the pro-apoptotic RKIP gene product. The combined effect of Pba and DETA/NO was also tested in C57BL/6 mice bearing a syngeneic B78-H1 melanoma. We used pegylated Pba (mPEG-Pba) due to its better pharmacokinetics compared to free Pba. mPEG-Pba (30 mg/Kg) and DETA/NO (0.4 mg/Kg) were i.p. injected either as a single molecule or in combination. After photoactivation at 660 nM (fluence of 193 J/cm(2)), the combined treatment delays tumor growth more efficiently than each individual treatment (p<0.05). Taken together, our results showed that the efficacy of PDT is strengthened when the photosensitizer is used in combination with an NO donor.

Contribution of either YY1 or BclXL-induced inhibition by the NO-donor DETANONOate in the reversal of drug resistance, both in vitro and in vivo. YY1 and BclXL are overexpressed in prostate cancer.

Nitric oxide (NO) donors have been shown to activate or inhibit constitutively-activated survival/anti-apoptotic pathways, such as NF-κB, in cancer cells. We report here that treatment of drug-resistant human prostate carcinoma cell lines with high levels (500-1000 µM) of the NO-donor DETANONOate sensitized the resistant tumor cells to apoptosis by CDDP and the combination was synergistic. We hypothesized that DETANONOate inhibits previously identified NF-κB-regulated resistant factors such as Yin Yang 1 (YY1) and Bcl-2/BclXL. Lysates from tumor cells treated with DETANONOate showed inhibition of YY1 and BclXL expressions. Transfection with either YY1 or BclXL siRNA resulted in the inhibition of both YY1 and BclXL expressions and sensitized the cells to CDDP apoptosis. Mice bearing PC-3 tumor xenografts and treated with the combination of DETANONOate and CDDP resulted in significant inhibition of tumor growth; treatment with single agent alone did not have any effect on tumor growth. Analysis of patients TMA tissues with prostatic cancer revealed higher expression of both YY1 and BclXL as a function of tumor grades and their levels were directly correlated. Thus, both YY1 and BclXL are potential prognostic biomarkers. Overall, the above findings suggest that one mechanism of DETANONOate-induced sensitization of resistant tumor cells to CDDP correlated with the inhibition of NF-κB and its targets YY1 and BclXL. The examination of the combination of NO donors and cytotoxic therapy in the treatment of resistant prostate cancer may be warranted.

Reversal of isoflurane-induced depression of myocardial contraction by nitroxyl via myofilament sensitization to Ca2+.

Isoflurane (ISO) is known to depress cardiac contraction. Here, we hypothesized that decreasing myofilament Ca(2+) responsiveness is central to ISO-induced reduction in cardiac force development. Moreover, we also tested whether the nitroxyl (HNO) donor 1-nitrosocyclohexyl acetate (NCA), acting as a myofilament Ca(2+) sensitizer, restores force in the presence of ISO. Trabeculae from the right ventricles of LBN/F1 rats were superfused with Krebs-Henseleit solution at room temperature, and force and intracellular Ca(2+) ([Ca(2+)](i)) were measured. Steady-state activations were achieved by stimulating the muscles at 10 Hz in the presence of ryanodine. The same muscles were chemically skinned with 1% Triton X-100, and the force-Ca(2+) relation measurements were repeated. ISO depressed force in a dose-dependent manner without significantly altering [Ca(2+)](i). At 1.5%, force was reduced over 50%, whereas [Ca(2+)](i) remained unaffected. At 3%, contraction was decreased by ∼75% with [Ca(2+)](i) reduced by only 15%. During steady-state activation, 1.5% ISO depressed maximal Ca(2+)-activated force (F(max)) and increased the [Ca(2+)](i) required for 50% activation (Ca(50)) without affecting the Hill coefficient. After skinning, the same muscles showed similar decreases in F(max) and increases in Ca(50) in the presence of ISO. NCA restored force in the presence of ISO without affecting [Ca(2+)](i). These results show that 1) ISO depresses cardiac force development by decreasing myofilament Ca(2+) responsiveness, and 2) myofilament Ca(2+) sensitization by NCA can effectively restore force development without further increases in [Ca(2+)](i). The present findings have potential translational value because of the efficiency and efficacy of HNO on ISO-induced myocardial contractile dysfunction.

Dual role of NO donors in the reversal of tumor cell resistance and EMT: Downregulation of the NF-κB/Snail/YY1/RKIP circuitry.

Several studies have implicated the role of Nitric Oxide (NO) in the regulation of tumor cell behavior and have shown that NO either promotes or inhibits tumorigenesis. These conflicting findings have been resolved, in part, by the levels of NO used such that low levels promote tumor growth and high levels inhibit tumor growth. Our studies have focused on the use of high levels of NO provided primarily by the NO donor, DETANONOate. We have shown that treatment of resistant tumor cells with DETANONOate sensitizes them to apoptosis by both chemotherapeutic drugs and cytotoxic immunotherapeutic ligands. The underlying mechanisms by which NO sensitizes tumor cells to apoptosis were shown to be regulated, in part, by NO-mediated inhibition of the NF-κB survival/anti-apoptotic pathways and downstream of NF-κB by inhibition of the transcription factor Yin Yang 1 (YY1). In addition to NO-induced sensitization to apoptosis, we have also shown that NO induced the expression of the metastasis-suppressor/immunosurveillance cancer gene product, Raf-1 kinase inhibitor protein (RKIP). Overexpression of RKIP mimics NO in tumor cells-induced sensitization to apoptosis. The induction of RKIP by NO was the result of the inhibition of the RKIP repressor, Snail, downstream of NF-κB. These findings established the presence of a dysregulated NF-κB/Snail/YY1/ RKIP circuitry in resistance and that treatment with NO modifies this loop in tumor cells in favor of the inhibition of tumor cell survival and the response to cytotoxic drugs. Noteworthy, the NF-κB/Snail/YY1/RKIP loop consists of gene products that regulate the epithelial to mesenchymal transition (EMT) and, thus, tumor metastasis. Hence, we have found that treatment of metastatic cancer cell lines with DETANONOate inhibited the EMT phenotype, through both the inhibition of the metastasis-inducers, NF-κB and Snail and the induction of the metastasis-suppressor, RKIP. Altogether, the above findings establish, for the first time, the dual role of high levels of NO in the sensitization of tumor cells to apoptotic stimuli as well as inhibition of EMT. Hence, NO donors may be considered as novel potential therapeutic agents with dual roles in the treatment of patients with refractory cancer and in the prevention of the initiation of the metastatic cascade via EMT.

Nitric oxide regulates stretch-induced proliferation in C2C12 myoblasts.

Mechanical stretch of skeletal muscle activates nitric oxide (NO) production and is an important stimulator of satellite cell proliferation. Further, cyclooxygenase (COX) activity has been shown to promote satellite cell proliferation in response to stretch. Since COX-2 expression in skeletal muscle can be regulated by NO we sought to determine if NO is required for stretch-induced myoblast proliferation and whether supplemental NO can counter the effects of COX-2 and NF-kappaB inhibitors. C2C12 myoblasts were cultured for 24 h, then switched to medium containing either the NOS inhibitor, L-NAME (200 microM), the COX-2 specific inhibitor NS-398 (100 microM), the NF-kappaB inhibiting antioxidant, PDTC (5 mM), the nitric oxide donor, DETA-NONOate (10-100 microM) or no supplement (control) for 24 h. Subgroups of each treatment were exposed to 1 h of 15% cyclic stretch (1 Hz), and were then allowed to proliferate for 24 h before fixing. Proliferation was measured by BrdU incorporation during the last hour before fixing, and DAPI stain. Stretch induced a twofold increase in nuclear number compared to control, and this effect was completely inhibited by L-NAME, NS-398 or PDTC (P < 0.05). Although DETA-NONOate (10 microM) did not affect basal proliferation, the NO-donor augmented the stretch-induced increase in proliferation and rescued stretch-induced proliferation in NS-398-treated cells, but not in PDTC-treated cells. In conclusion, NO, COX-2, and NF-kappaB are necessary for stretch-induced proliferation of myoblasts. Although COX-2 and NF-kappaB are both involved in basal proliferation, NO does not affect basal growth. Thus, NO requires the synergistic effect of stretch in order to induce muscle cell proliferation.

Nitric oxide sensitizes tumor cells to TRAIL-induced apoptosis via inhibition of the DR5 transcription repressor Yin Yang 1.

Treatment of TRAIL-resistant tumor cells with the nitric oxide donor DETANONOate sensitizes the tumor cells to TRAIL-induced apoptosis concomitantly with DR5 upregulation. The mechanism of sensitization was examined based on the hypothesis that DETANONOate inhibits a transcription repressor Yin Yang 1 (YY1) that negatively regulates DR5 transcription. Treatment of the prostate carcinoma cell lines with DETANONOate inhibited both NF-kappaB and YY1 DNA-binding activities concomitantly with upregulation of DR5 expression. The direct role of YY1 in the regulation of TRAIL resistance was demonstrated in cells treated with YY1 siRNA resulting in TRAIL-induced apoptosis. The role of YY1 in the transcriptional regulation of DR5 was examined in cells treated with a DR5 luciferase reporter system (pDR5) and two constructs, namely, the pDR5/-605 construct with a deletion of the putative YY1 DNA-binding region (-1224 to -605) and a construct pDR5-YY1 with a mutation of the YY1 DNA-binding site. A significant (3-fold) augmentation of luciferase activity over baseline transfection with pDR5 was observed in cells transfected with the modified constructs. ChIP analysis corroborated the YY1 binding to the DR5 promoter. In vivo, tissues from nude mice bearing the PC-3 xenograft and treated with DETANONOate showed inhibition of YY1 and upregulation of DR5. The present findings demonstrate that YY1 negatively regulates DR5 transcription and expression and these correlated with resistance to TRAIL-induced apoptosis. DETANONOate inhibits both NF-kappaB and YY1 and in combination with TRAIL reverses tumor cell resistance to TRAIL apoptosis.

Nitrosyl-cobinamide, a new and direct nitric oxide releasing drug effective in vivo.

A limited number of nitric oxide (NO)-generating drugs are available for clinical use for acute and chronic conditions. Most of these agents are organic nitrates, which do not directly release NO; tolerance to the drugs develops, in part, as a consequence of their conversion to NO. We synthesized nitrosyl-cobinamide (NO-Cbi) from cobinamide, a structural analog of cobalamin (vitamin B12). NO-Cbi is a direct NO-releasing agent that we found was stable in water, but under physiologic conditions, it released NO with a half-life of 30 mins to 1 h. We show in five different biological systems that NO-Cbi is an effective NO-releasing drug. First, in cultured rat vascular smooth muscle cells, NO-Cbi induced phosphorylation of vasodilator-stimulated phosphoprotein, a downstream target of cGMP and cGMP-dependent protein kinase. Second, in isolated Drosophila melanogaster Malpighian tubules, NO-Cbi-stimulated fluid secretion was similar to that stimulated by Deta-NONOate and a cGMP analog. Third, in isolated mouse hearts, NO-Cbi increased coronary flow much more potently than nitroglycerin. Fourth, in contracted mouse aortic rings, NO-Cbi induced relaxation, albeit to a lesser extent than sodium nitroprusside. Fifth, in intact mice, a single NO-Cbi injection rapidly reduced blood pressure, and blood pressure returned to normal after 45 mins; repeated NO-Cbi injections induced the expected fall in blood pressure. These studies indicate that NO-Cbi is a useful NO donor that can be used experimentally in the laboratory; moreover, it could be developed into a vasodilating drug for treating hypertension and potentially other diseases such as angina and congestive heart failure.

S-nitrosothiols signal hypoxia-mimetic vascular pathology.

NO transfer reactions between protein and peptide cysteines have been proposed to represent regulated signaling processes. We used the pharmaceutical antioxidant N-acetylcysteine (NAC) as a bait reactant to measure NO transfer reactions in blood and to study the vascular effects of these reactions in vivo. NAC was converted to S-nitroso-N-acetylcysteine (SNOAC), decreasing erythrocytic S-nitrosothiol content, both during whole-blood deoxygenation ex vivo and during a 3-week protocol in which mice received high-dose NAC in vivo. Strikingly, the NAC-treated mice developed pulmonary arterial hypertension (PAH) that mimicked the effects of chronic hypoxia. Moreover, systemic SNOAC administration recapitulated effects of both NAC and hypoxia. eNOS-deficient mice were protected from the effects of NAC but not SNOAC, suggesting that conversion of NAC to SNOAC was necessary for the development of PAH. These data reveal an unanticipated adverse effect of chronic NAC administration and introduce a new animal model of PAH. Moreover, evidence that conversion of NAC to SNOAC during blood deoxygenation is necessary for the development of PAH in this model challenges conventional views of oxygen sensing and of NO signaling.

Excellent absorption enhancing characteristics of NO donors for improving the intestinal absorption of poorly absorbable compound compared with conventional absorption enhancers.

The characteristics of NO donors, NOC5 [3-(2-hydroxy-1-(1-methylethyl-2-nitrosohydrazino)-1-propanamine), NOC12 [N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine] and SNAP [S-nitroso-N-acetyl-DL-penicillamine] as absorption enhancers for poorly absorbable drugs were examined in rats using an in situ closed loop method. They were compared with a group of conventional absorption enhancers including sodium glycocholate (NaGC), sodium caprate (NaCap), sodium salicylate (NaSal) and n-dodecyl-beta-D-maltopyranoside (LM). 5(6)-carboxyfluorescein (CF) was used as a model drug to investigate effectiveness, site-dependency, and concentration-dependency of the tested enhancers. Overall, the NO donors can improve the intestinal absorption of CF at low concentration (5 mM), whereas higher concentration was required for the conventional absorption enhancers to elicit the absorption enhancing effect. In the small intestine, SNAP was the most effective absorption enhancers, although its concentration (5 mM) was lower than the conventional absorption enhancers (20 mM). On the other hand, LM and NaCap as well as the three NO donors were effective to improve the colonic absorption of CF. In the regional difference in the absorption enhancing effects, the NO donors showed significant effects in all intestinal regions, whereas we observed a regional difference in the absorption enhancing effect of the other conventional absorption enhancers. In the conventional enhancers, the absorption enhancing effects were generally greater in the large intestine than those in the small intestine. LM and NaCap were ineffective in the jejunum, although they were effective for improving the absorption of CF in the colon. NaSal was ineffective in both the jejunum and the colon. The absorption enhancement produced by NO donors was greatly affected by increasing the enhancer concentration from 3 to 5 mM, but only a slight increase was obtained when the concentration was raised to 10 mM. Similar results were obtained for the other enhancers over the range of 10 to 20 mM, but the absorption enhancing effects of these enhancers were almost saturated above these concentrations. These results suggest that NO donors possess excellent effectiveness as absorption enhancers for poorly absorbable drugs compared with the conventional enhancers. They can enhance intestinal absorption of CF from all intestinal regions and they are effective at very low concentrations.

S-nitroso human serum albumin attenuates ischemia/reperfusion injury after cardioplegic arrest in isolated rabbit hearts.

BACKGROUND: Depletion of nitric oxide (NO) is associated with ischemia/reperfusion injury. The novel NO donor, S-nitroso human serum albumin (S-NO-HSA), could bridge NO depletion during reperfusion in cardiac transplantation and minimize ischemia/reperfusion injury. METHODS: In an isolated erythrocyte-perfused working heart model, rabbit hearts were randomly assigned after assessment of hemodynamic baseline values to receive S-NO-HSA (0.2 micromol/100 ml, n = 8), L-arginine (10 mmol/100 ml, n = 8) or albumin (control) (0.2 micromol/100 ml, n = 8). After 20 minutes of infusion, the hearts were arrested and stored in Celsior (4 degrees C) enriched with respective drugs for 6 hours, followed by 75 minutes of reperfusion. Hemodynamic values were assessed and biopsy specimens were taken to determine calcium-ionophore stimulated release of NO and superoxide. RESULTS: During early reperfusion, recovery of cardiac output (75% +/- 6% vs 49% +/- 5%, p < 0.05) and coronary flow (99% +/- 8% vs 70% +/- 5%, p < 0.05) were higher, and myocardial oxygen consumption was reduced in the S-NO-HSA Group compared with Control (4.08 +/- 0.46 ml/min/0.1 kg vs 6.78 +/- 0.38 ml/min/0.1 kg, p < 0.01). At the end of the experiment cardiac output (53% +/- 5% vs 27% +/- 5%, p < 0.01) was higher and left atrial pressure (115% +/- 9% vs 150% +/- 8%, p < 0.05) was lower in the S-NO-HSA Group compared with Control. NO release was increased (1,040 +/- 50 nmol/liter and 1,070 +/- 60 nmol/liter vs 860 +/- 10 nmol/liter, p < 0.01) and superoxide release diminished (31 +/- 5 nmol/liter and 38 +/- 5 nmol/liter vs 64 +/- 5 nmol/liter, p < .01) in the S-NO-HSA and L-arginine Groups compared with Control. CONCLUSION: S-NO-HSA improved hemodynamic functions after prolonged hypothermic cardiac arrest by supplementing NO and thereby decreasing ischemia/reperfusion injury.

Rituximab-induced inhibition of YY1 and Bcl-xL expression in Ramos non-Hodgkin's lymphoma cell line via inhibition of NF-kappa B activity: role of YY1 and Bcl-xL in Fas resistance and chemoresistance, respectively.

Rituximab treatment of B non-Hodgkin's lymphoma (NHL) cell lines inhibits the constitutive NF-kappaB activity and results in the sensitization of tumor cells to both chemotherapy and Fas-induced apoptosis. Cells expressing dominant active IkappaB or treated with NF-kappaB-specific inhibitors were sensitive to both drugs and Fas agonist mAb (CH-11)-induced apoptosis. Down-regulation of Bcl-xL expression via inhibition of NF-kappaB activity correlated with chemosensitivity. The direct role of Bcl-xL in chemoresistance was demonstrated by the use of Bcl-xL-overexpressing Ramos cells, Ramos hemagglutinin (HA)-Bcl-x, which were not sensitized by rituximab to drug-induced apoptosis. However, inhibition of Bcl-xL in Ramos HA-Bcl-x resulted in sensitization to drug-induced apoptosis. The role of Bcl-xL expression in the regulation of Fas resistance was not apparent; Ramos HA-Bcl-x cells were as sensitive as the wild type to CH-11-induced apoptosis. Several lines of evidence support the direct role of the transcription repressor yin-yang 1 (YY1) in the regulation of resistance to CH-11-induced apoptosis. Inhibition of YY1 activity by either rituximab or the NO donor DETANONOate or after transfection with YY1 small interfering RNA resulted in up-regulation of Fas expression and sensitization to CH-11-induced apoptosis. These findings suggest two mechanisms underlying the chemosensitization and immunosensitization of B-NHL cells by rituximab via inhibition of NF-kappaB. The regulation of chemoresistance by NF-kappaB is mediated via Bcl-xL expression, whereas the regulation of Fas resistance by NF-kappaB is mediated via YY1 expression and activity. The potential clinical significance of these findings is discussed.

Dual effects of nitric oxide on meiotic maturation of mouse cumulus cell-enclosed oocytes in vitro.

The present experiment used cultured mouse cumulus cell-enclosed oocytes (CEOs) and denuded oocytes (DOs) to study the function of nitric oxide (NO) in mouse oocyte meiotic maturation. Either positive or negative actions of NO on meiotic maturation has been observed when CEOs or DOs were cultured for 24 h in a medium containing 4 mM hypoxanthine (HX) to maintain meiotic arrest, or in maturation medium (without HX) supplemented with different doses of sodium nitroprusside (SNP, a NO donor), N-omega-nitro-L-arginine methyl ester (L-NAME) or N(w)-nitro-L-arginine (L-NNA) (two inhibitors of NO synthase, NOS), and L-arginine (the only substrate of NOS). Both NOS inhibitors suppressed the formation of first polar body (PB1) of the oocytes in CEOs in a dose dependent manner, but no effect on germinal vesicle break down (GVBD) was observed. An optimal inhibitory effect was observed with either 10(-3) M L-NAME (P<0.01) or 10(-3) M L-NNA (P<0.01) and the inhibition could be reversed by the addition of SNP (10(-5) M). The above mentioned optimal concentration of L-NAME or L-NNA on CEOs exhibited no effect on oocyte meiotic maturation of DOs. Treatments of low concentrations of SNP (10(-7), 10(-6), 10(-5) M) stimulated significantly the oocyte meiotic maturation of CEOs which were inhibited with HX, but had no effect on DOs in the same culture medium. While, the treatment with high concentrations of SNP (0.1-4 mM) during the CEOs cultured in maturation medium resulted in a lower percentage of oocytes at PB1 stage and a higher percentage of atypical oocytes in a dose dependent manner compared with control. A dose of SNP at 1 mM exhibited significant inhibitory effect on the formation of PB1, but without effect on the number of atypical oocytes compared with control, while, this SNP dosage not only inhibited the oocyte PB1 formation but also increased the percentage of dead oocytes in DOs. Although oocytes of all groups underwent GVBD at the end of the culture in the spontaneous maturation medium, the results of the kinetics showed that the treatment of the optimal concentration of SNP (1 mM) could significantly delay GVBD during the first 5 h culture period. The concomitant addition of L-NAME with SNP did not reverse the inhibitory effect of SNP on CEOs. Similarly, neither pre-incubation nor illumination by ultraviolet ray could balance the inhibitory effect of SNP. Finally, when added alone at a concentration of 4 mM, L-arg caused extensive death of both CEOs and DOs. While, administration of 4 mM L-arg and 1 mM L-NAME to both CEOs and DOs simultaneously resulted in markedly reduced CEOs death percentage as compared with L-arg treatment alone, but not in DOs. These data support the idea that NO could act with a dual action (stimulation or inhibition) in mouse meiotic maturation depending on its concentration.

Differential effects of bcl-2 on cell death triggered under ATP-depleting conditions.

The intracellular ATP concentration decides on the onset of either apoptosis or necrosis in Jurkat cells exposed to death stimuli. Bcl-2 can block apoptotic demise, which occurs preferably under conditions of high cellular ATP levels. Here, we investigated the effects of Bcl-2 on the necrotic type of cell demise that prevails under conditions of energy loss. ATP levels were modulated by using mitochondrial inhibitors, such as rotenone or S-nitrosoglutathione, in medium either lacking glucose or supplemented with glucose to stimulate glycolytic ATP generation. Under conditions of ATP depletion, staurosporine (STS) induced >90% necrosis in vector control-transfected cells, whereas bcl-2-transfected cells were protected. Thus, the antiapoptotic protein Bcl-2 can reduce the overall amount of cell death in ATP-depleted cells regardless whether it occurs by apoptosis or necrosis. Cytochrome c release, normally preceding STS-induced necrosis, was also inhibited by Bcl-2. However, Bcl-2 did not prevent an initial STS-induced drop of the mitochondrial membrane potential (DeltaPsi(m)). Therefore, the mechanisms whereby Bcl-2 prevents cell death and favors retention of cytochrome c in the mitochondria require neither the maintenance of mitochondrial DeltaPsi nor the maintenance of normal ATP levels.

Distribution of c-fos mRNA in the brain following intracerebroventricular injection of nitric oxide (NO)-releasing compounds: possible role of NO in central cardiovascular regulation.

We injected nitric oxide (NO)-releasing compounds and NO synthase (NOS) inhibitors into the brains of conscious, freely moving rats and measured the effects on mean arterial blood pressure (MAP) and heart rate, as well as on the expression of c-fos mRNA, neuronal NOS (nNOS) mRNA and NADPH-diaphorase, an indicator of NOS activity. When administered i.c.v., the NO donor, NOC-18, caused a significant fall in MAP and heart rate, whereas the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), induced a significant rise in MAP. The same dose of NOC-18 or L-NAME when administered i.v. did not affect MAP and heart rate. Centrally administered NOC-18 induced c-fos mRNA expression in several regions of the brain involved in the baroreceptor response, including the nucleus of the solitary tract, the area postrema and the rostral ventrolateral medulla, as well as areas involved in the integration of autonomic, neuroendocrine and behavioural responses, including the medial preoptic area, the organum vasculosum lamina terminalis, the bed nucleus of stria terminalis, the paraventricular nucleus (PVN), the supraoptic nucleus (SON), the central nucleus of amygdala (CeA) and the locus coeruleus. Most of the areas that expressed c-fos also contained nNOS mRNA and/or NADPH-d-positive neurones and fibres. i.c.v. injection of L-NAME induced c-fos mRNA expression in PVN, SON, locus coeruleus and NTS, suggesting a tonic inhibition of neuronal activity by NO or stimulation of neuronal activity by endogenous NO. i.v. injection of NOC-18 or L-NAME did not induce any significant c-fos mRNA expression in rat brain. These results demonstrate that NO acts directly in the brain to reduce the systemic blood pressure, and that the endogenous NO pathway may play a role in cardiovascular and autonomic regulation by modulating neuronal activities in discrete regions of the brain.

Novel mode of nitric oxide neurotransmission mediated via S-nitroso-cysteinyl-glycine.

S-nitroso-cysteinyl-glycine, a novel nitric oxide-adduct thiol compound, can be detected in the brain (2.3+/-0.6 pmol/mg protein), and released following stimulation of sensory afferents to the rat ventrobasal thalamus in vivo (resting conditions 17 nM; stimulation: 186 nM). Iontophoretic application of CysNOGly (20-80 nA) onto thalamic neurons in vivo resulted in enhancements of excitatory responses to either NMDA or AMPA (182+/-13.6% and 244+/-27.8% of control values, n = 15). CysNOGly enhanced responses to stimulation of vibrissal afferents to 132+/-2.2% (n = 7) of control values. In contrast, the dipeptide CysGly reduced responses of ventrobasal neurons to NMDA and AMPA (54+/-8.4% and 55+/-10.8% of control, n = 5). CysNOGly was also a potent activator of soluble guanylate cyclase in vitro. Moreover, we found that NMDA elevated CysNOGly levels in vitro and this stimulatory effect was reduced by inhibitors of the neuronal NO synthase and of the gamma-glutamyl transpeptidase, suggesting that production of NO and CysGly is a prelude to CysNOGly synthesis. These findings suggest that the nitrosothiol CysNOGly plays a role in synaptic transmission in the ventrobasal thalamus. We propose a novel synaptic buffering mechanism where S-nitroso-cysteinyl-glycine serves to restrict the locus of action of nitric oxide and so increase its local availability for target delivery. This could lead to a change in neuronal responses favouring sensory transmission similar to that seen in wakefulness or arousal in order to locally enhance transmission of persistent sensory stimuli.

S-nitrosothiols cause prolonged, nitric oxide-mediated relaxation in human saphenous vein and internal mammary artery: therapeutic potential in bypass surgery.

1. Reduced endothelial nitric oxide (NO) production in conduit vessels for coronary artery bypass grafting (CABG) has been implicated in post-operative complications, including spasm. 2. The brief effects of existing NO donors limits their applicability to improving patency of graft vessels. RIG200 is a novel S-nitrosothiol that might have advantages over conventional drugs because it has sustained effects in areas of endothelial damage. 3. Here we tested the hypothesis that RIG200 and S-nitrosoglutathione (GSNO) have prolonged, NO-mediated effects in human saphenous vein (SV) and internal mammary artery (IMA), compared with glyceryl trinitrate (GTN) and sodium nitroprusside (SNP). 4. 84 SV and 80 IMA rings from 64 patients undergoing CABG were studied in vitro. Rings were precontracted with phenylephrine (EC(80) concentration) and the functional integrity of the endothelium tested with acetylcholine (10 microM). 5. Relaxation of precontracted SV and IMA rings to GTN and SNP (0.01 - 10 microM) generally recovered fully on washout. In contrast, responses to RIG200 and GSNO were sustained during washout (30 min). Sustained relaxation was reversed by the NO scavenger, ferrohaemoglobin (10 microM) but not by the NO synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (100 and 250 microM in SV and IMA respectively). 6. Pretreatment (30 min) of SV with both S-nitrosothiols (10 microM) inhibited phenylephrine-induced contraction for >180 min, compared with <90 min for GTN. In IMA, contractility was suppressed to 49+/-4% (GSNO) and 26+/-4% (RIG200) of baseline after 240 min washout. 7. Pretreatment of bypass conduits with S-nitrosothiols might improve their patency in the early post-operative period.