Bcl-2 and Bcl-X(L) block thapsigargin-induced nitric oxide generation, c-Jun NH(2)-terminal kinase activity, and apoptosis.

The proteins Bcl-2 and Bcl-X(L) prevent apoptosis, but their mechanism of action is unclear. We examined the role of Bcl-2 and Bcl-X(L) in the regulation of cytosolic Ca(2+), nitric oxide production (NO), c-Jun NH(2)-terminal kinase (JNK) activation, and apoptosis in Jurkat T cells. Thapsigargin (TG), an inhibitor of the endoplasmic reticulum-associated Ca(2+) ATPase, was used to disrupt Ca(2+) homeostasis. TG acutely elevated intracellular free Ca(2+) and mitochondrial Ca(2+) levels and induced NO production and apoptosis in Jurkat cells transfected with vector (JT/Neo). Buffering of this Ca(2+) response with 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) or inhibiting NO synthase activity with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) blocked TG-induced NO production and apoptosis in JT/Neo cells. By contrast, while TG produced comparable early changes in the Ca(2+) level (i.e., within 3 h) in Jurkat cells overexpressing Bcl-2 and Bcl-X(L) (JT/Bcl-2 or JT/Bcl-X(L)), NO production, late (36-h) Ca(2+) accumulation, and apoptosis were dramatically reduced compared to those in JT/Neo cells. Exposure of JT/Bcl-2 and JT/Bcl-X(L) cells to the NO donor, S-nitroso-N-acetylpenacillamine (SNAP) resulted in apoptosis comparable to that seen in JT/Neo cells. TG also activated the JNK pathway, which was blocked by L-NAME. Transient expression of a dominant negative mutant SEK1 (Lys-->Arg), an upstream kinase of JNK, prevented both TG-induced JNK activation and apoptosis. A dominant negative c-Jun mutant also reduced TG-induced apoptosis. Overexpression of Bcl-2 or Bcl-X(L) inhibited TG-induced loss in mitochondrial membrane potential, release of cytochrome c, and activation of caspase-3 and JNK. Inhibition of caspase-3 activation blocked TG-induced JNK activation, suggesting that JNK activation occurred downstream of caspase-3. Thus, TG-induced Ca(2+) release leads to NO generation followed by mitochondrial changes including cytochrome c release and caspase-3 activation. Caspase-3 activation leads to activation of the JNK pathway and apoptosis. In summary, Ca(2+)-dependent activation of NO production mediates apoptosis after TG exposure in JT/Neo cells. JT/Bcl-2 and JT/Bcl-X(L) cells are susceptible to NO-mediated apoptosis, but Bcl-2 and Bcl-X(L) protect the cells against TG-induced apoptosis by negatively regulating Ca(2+)-sensitive NO synthase activity or expression.     

Protein-bound dinitrosyl-iron complexes appearing in blood of rabbit added with a low-molecular dinitrosyl-iron complex: EPR studies.

The formation of protein-bound dinitrosyl-iron complexes (DNIC) in blood plasma and packed red cell fraction has been demonstrated by the EPR method in the experiments on rabbits which were i/v injected with the low-molecular DNIC with thiosulphate. This formation was ensured by transfer of Fe(+)(NO(+))(2) moieties from low-molecular DNIC onto serum albumin or hemoglobin molecules. Protein-bound DNICs appeared immediately after low-molecular DNIC injection followed with gradually decreasing their amounts. The complexes could be detected by EPR technique during more than two days. The addition of water-soluble NO scavenger, the iron complex with N-methyl-d-glucamine dithiocarbamate (MGD) resulted in decomposition of a part of protein-bound DNICs and in effective excretion of secondary products (mainly mononitrosyl-iron complexes with MGD) from the blood flow.     

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.     

Selective delivery of nitric oxide to a cellular target: a pseudosubstrate-coupled dinitrosyl-iron complex inhibits the enteroviral protease 2A.

Nitric oxide (NO) regulates multiple biological processes. To use NO as a potential therapeutic substance, a more selective modulation of individual NO targets is desirable. Here, we tested whether peptide conjugation of the dinitrosyl-iron complex (DNIC), a potent NO donor, confers targeted NO delivery. As target, we used the protease 2A of Coxsackie-B-viruses (2A(pro)), which can cause dilated cardiomyopathy. Through S-nitrosylation, NO inhibits this protease, which is essential for viral replication. The tetrapeptide Leu-Ser-Thr-Cys (LSTC) (based on the 2A(pro) substrate recognition motif) and DNIC generated LSTC-DNIC in vitro by S-nitrosylation as evidenced by reverse-phase chromatography. In vitro, LSTC-DNIC (IC(50) 510 nM) dose-dependently inhibited purified 2A(pro) 4.7-fold more effectively than DNIC (IC(50) 2.4 microM), whereas LSTC alone had no effect. In intact cells, expression of Coxsackievirus protease 2A by transient transfection led to eIF4G-I-cleavage. LSTC-DNIC (IC(50) 23 microM) dose-dependently inhibited eIF4G cleavage in 2A(pro)-transfected cells 3.8-fold more effectively than DNIC (IC(50) 88 microM). To test the specificity of the DNIC-conjugated LSTC peptide part, we investigated its influence on Caspase-3, a known target for S-nitrosylation. LSTC-DNIC and DNIC inhibited purified Caspase-3 in vitro (IC(50) 3.7 microM) and in intact cells similarly. LSTC conjugation of DNIC enhances its fidelity for inhibition of 2A(pro) in vitro and intracellularly. Peptide-DNIC may be useful to selectively modulate cellular processes by NO, i.e., to enhance its antiviral properties.     

Peptides derived from BH3 domains of Bcl-2 family members: a comparative analysis of inhibition of Bcl-2, Bcl-x(L) and Bax oligomerization,induction of cytochrome c release,and activation of cell death

Overexpression of Bcl-2, an anti-apoptotic oncoprotein, is commonly observed in a variety of human malignancies and is associated with resistance to chemotherapy and radiotherapy. Although the precise mechanism of Bcl-2 action remains elusive, current evidence indicates that Bcl-2 inhibits apoptosis by binding and inhibiting pro-apoptotic molecules such as Bax. Therefore, agents that disrupt the ability of Bcl-2, or other anti-apoptotic molecules, to bind to pro-apoptotic molecules may have therapeutic value. Several studies have shown that the BH3 domains of Bcl-2 and Bax are critically important for Bax/Bcl-2 heterodimerization. In this report, we designed and synthesized peptides based on the BH3 domains of three distinct Bcl-2 family members, Bcl-2, Bax and Bad. In vitro interaction assays were used to compare the abilities of the different peptides to inhibit Bax/Bcl-2 and Bax/Bcl-x(L) heterodimerization, as well as Bcl-2 and Bax homodimerization. Bax BH3 peptide (20-amino acids) potently inhibited both Bax/Bcl-2 and Bax/Bcl-x(L) interactions, exhibiting IC(50) values of 15 and 9.5 microM, respectively. The Bad BH3 peptide (21 amino acids) was slightly more potent than Bax BH3 at inhibiting Bax/Bcl-x(L) but failed to disrupt Bax/Bcl-2. Bcl-2 BH3 peptide (20-amino acids) was inactive toward Bax/Bcl-2 and had only a weak inhibitory effect on Bax/Bcl-x(L) heterodimerization. All three BH3 peptides failed to significantly inhibit homodimerization of Bcl-2 or Bax. Consistent with its ability to disrupt Bax/Bcl-2 heterodimerization, Bax BH3 peptide was able to overcome Bcl-2 overexpression and induce cytochrome c release from mitochondria of Bcl-2-overexpressing Jurkat T leukemic cells. Bad BH3 peptide, while potently inducing cytochrome c release in wild-type Jurkat cells, only partially overcame the effects of Bcl-2 overexpression. Bcl-2 BH3 failed to induce cytochrome c release, even in wild-type cells. Delivery of the Bax BH3 and Bad BH3 peptides into wild-type Jurkat cells induced comparable levels of cell death. In cells overexpressing Bcl-2, the potency of Bax BH3 peptide was similar to that seen in wild-type cells, while the efficacy of Bad BH3 peptide was reduced. By contrast, in Bcl-x(L)-overexpressing cells, Bad BH3 exhibited greater cell-killing activity than Bax BH3. The Bcl-2 BH3 peptide and a mutant Bax BH3 peptide had no appreciable effect on Jurkat cells. Together, our data suggest that agents based on the Bax BH3 domain may have therapeutic value in cancers overexpressing Bcl-2, while agents based on the BH3 domain of Bad may be more useful for tumors overexpressing Bcl-x(L).     

Glutathione is a factor of resistance of Jurkat leukemia cells to nitric oxide-mediated apoptosis

We have previously reported that nitric oxide (NO) stimulates apoptosis in different human neoplastic lymphoid cell lines through mitochondrial damage (including degradation of cardiolipin, a major mitochondrial lipid) followed by activation of caspases. Here we demonstrate that Jurkat human leukemia cells which survive after 24 h treatment with NO form subpopulations with higher and lower cardiolipin content (designated as NAO(high) and NAO(low), respectively). Sorted NAO(high) cells were found to survive in culture whereas sorted NAO(low) cells died. Moreover, NAO(high) cells acquired an increased resistance to the exposure to NO donors which remained unchanged during long-term culture. These cells showed a similar cardiolipin content and expressed the same level of anti-apoptotic proteins Bcl-2 and Bcl-x(L) as APO-S unsorted cells but contained significantly higher concentration of the antioxidant glutathione. Depletion of glutathione in these cells with buthionine-sulfoximine (BSO) correlated with a significant stimulation of NO-mediated apoptosis whereas the exposure of NO-sensitive APO-S cells to the glutathione precursor N-acetylcysteine (NAC) resulted in a substantial suppression of this effect. Our data suggest a complex mechanism of the resistence to NO-induced apoptosis in Jurkat human leukemia cells in which glutathione plays an important role.     

Nitric oxide-forming reactions of the water-soluble nitric oxide spin-trapping agent, MGD.

The objective of this study was to elucidate the nitric oxide-forming reactions of the iron-N-methyl-D-glucamine dithiocarbamate (Fe-MGD) complex from the nitrogen-containing compound hydroxyurea. The Fe2+(MGD)2 complex is commonly used in electron paramagnetic resonance (EPR) spectroscopic detection of NO both in vivo and in vitro. The reaction of Fe2+(MGD)2 with NO yields the resultant NO-Fe2+(DETC)2 complex, which has a characteristic triplet EPR signal. It is widely believed that only NO reacts with Fe2+(MGD)2 to form the NO-Fe2+(MGD)2 complex. In this report, the mechanism leading to the formation of NO-Fe2+(MGD)2 was investigated using oxygen-uptake studies in conjunction with the EPR spin-trapping technique. We found that the air oxidation of Fe2+(MGD)2 complex results in the formation of the Fe3+(MGD)3 complex, presumably concomitantly with superoxide (O3*-). Dismutation of superoxide forms hydrogen peroxide, which can subsequently reduce Fe3+(MGD)3 back to Fe2+(MGD)2. The addition of NO to the Fe3+(MGD)3 complex resulted in the formation of the NO-Fe2+(MGD)2 complex. Hydroxyurea is not considered to be a spontaneous NO donor, but has to be oxidized in order to form NO. We present data showing that in the presence of oxygen, Fe2+(MGD)2 can oxidize hydroxyurea to yield the stable NO-Fe2+(MGD)2 complex. These results imply that hydroxyurea can be oxidized by reactive oxygen species that are formed from the air oxidation of the Fe2+(MGD)2 complex. Formation of the NO-Fe2+(MGD)2 complex in this case could erroneously be interpreted as spontaneous formation of NO from hydroxyurea. The chemistry of the Fe2+(MGD)2 complexes in aerobic conditions must be taken into account in order to avoid erroneous conclusions. In addition, the use of these complexes may contribute to the overall oxidative stress of the system under investigation.     

Cu,Zn-superoxide dismutase-dependent apoptosis induced by nitric oxide in neuronal cells

Nitric oxide (NO) challenge to human neuroblastoma cells (SH-SY5Y) ultimately results in apoptosis. Tumor suppressor protein p53 and cell cycle inhibitor p21 accumulate as an early sign of S-nitrosoglutathione-mediated toxicity. Cytochrome c release from mitochondria and caspase 3 activation also occurred. Cells transfected with either wild type (WT) or mutant (G93A) Cu, Zn-superoxide dismutase (Cu,Zn-SOD) produced comparable amounts of nitrite/nitrate but showed different degree of apoptosis. G93A cells were the most affected and WT cells the most protected; however, Cu, Zn-SOD content of these two cell lines was 2-fold the SH-SY5Y cells under both resting and treated conditions. We linked decreased susceptibility of the WT cells to higher and more stable Bcl-2 and decreased reactive oxygen species. Conversely, we linked G93A susceptibility to increased reactive oxygen species production since simultaneous administration of S-nitrosoglutathione and copper chelators protects from apoptosis. Furthermore, G93A cells showed a significant decrease of Bcl-2 expression and, as target of NO-derived radicals, showed lower cytochrome c oxidase activity. These results demonstrate that resistance to NO-mediated apoptosis is strictly related to the level and integrity of Cu,Zn-SOD and that the balance between reactive nitrogen and reactive oxygen species regulates neuroblastoma apoptosis.     

The source of non-heme iron that binds nitric oxide in cultivated macrophages.

In cultured macrophages (J 774 line) a decrease in iron-sulfur centers (ISC) was not observed after 5 min treatment with nitric oxide (NO) (10(-7) M NO/10(7) cells). The content of these centers was measured by electron spin resonance (ESR) spectroscopy at 16-60 K. However, the appearance of a characteristic ESR signal at g(av) = 2.03 indicated the formation of dinitrosyl iron complex (DNIC) in these cells. These findings suggest that loosely bound non-heme iron (free iron) but not iron from ISC is mainly involved in DNIC formation. ISC might release iron for DNIC formation after their destruction induced by the products of NO oxidation (NO2, N2O3, etc).     

Bcl-xL does not inhibit the function of Apaf-1

Bcl-2 and its relative, Bcl-xL, inhibit apoptotic cell death primarily by controlling the activation of caspase proteases. Previous reports have suggested at least two distinct mechanisms: Bcl-2 and Bcl-xL may inhibit either the formation of the cytochrome c/Apaf-1/caspase-9 apoptosome complex (by preventing cytochrome c release from mitochondria) or the function of this apoptosome (through a direct interaction of Bcl-2 or Bcl-xL with Apaf-1). To evaluate this latter possibility, we added recombinant Bcl-xL protein to cell-free apoptotic systems derived from Jurkat cells and Xenopus eggs. At low concentrations (50 nM), Bcl-xL was able to block the release of cytochrome c from mitochondria. However, although Bcl-xL did associate with Apaf-1, it was unable to inhibit caspase activation induced by the addition of cytochrome c, even at much higher concentrations (1-5 microM). These observations, together with previous results obtained with Bcl-2, argue that Bcl-xL and Bcl-2 cannot block the apoptosome-mediated activation of caspase-9.     

The nitric oxide prodrug, V-PYRRO/NO, protects against cadmium toxicity and apoptosis at the cellular level.

The liver is an important target tissue of cadmium. The compound O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2 diolate (V-PYRRO/NO) is a liver-selective nitric oxide (NO) prodrug that is metabolized by hepatic P450 enzymes to release NO in hepatocytes. In vivo, V-PYRRO/NO can protect against the toxicity of various hepatotoxicants, including cadmium. Since NO is an effective vasodilator, whether this protective effect against cadmium toxicity is at the level of the hepatic vascular system or actually within the liver cells has not been defined. Thus, we studied the effects of V-PYRRO/NO pretreatment on cadmium-induced toxicity and apoptosis in cultured rat liver epithelial (TRL 1215) cells. Cells were pretreated with V-PYRRO/NO at 500 or 1000 microM for up to 24 h, then exposed to cadmium (as CdCl2) for additional 24 h and cytotoxicity was measured. Cadmium was significantly less cytotoxic in V-PYRRO/NO (1000 microM) pretreated cells (LC50=6.1+/-0.6 microM) compared to control cells (LC50=3.5+/-0.4 microM). TRL 1215 cells acted upon the prodrug to release NO, producing nitrite levels in the extracellular media after 24 h of exposure to 500 or 1000 microM V-PYRRO/NO measured at 87.0+/-4.2 and 324+/-14.8 microM, respectively, compared to basal levels of 7.70+/-0.46 microM. V-PYRRO/NO alone produced small increases in metallothionein (MT), a metal-binding protein associated with cadmium tolerance. However, V-PYRRO/NO pretreatment greatly enhanced cadmium induction of MT. V-PYRRO/NO pretreatment also markedly reduced apoptotic cell death induced by cadmium (5 microM), apparently by blocking cadmium-induced activation of the c-Jun N-terminal kinase (JNK) pathway. Thus, the prodrug, V-PYRRO/NO, protects against the adverse effects of cadmium directly within rat liver cells apparently through generation of NO and, at least in part, by facilitation of cadmium-induced MT synthesis.     

The mechanisms of S-nitrosothiol decomposition catalyzed by iron.

The mechanisms of S-nitrosothiol transformation into paramagnetic dinitrosyl iron complexes (DNICs) with thiol- or non-thiol ligands or mononitrosyl iron complex (MNICs) with N-methyl-D-glucamine dithiocarbamate catalyzed by iron(II) ions under anaerobic conditions were studied by monitoring EPR or optical features of the complexes and S-nitrosothiols. The kinetic investigations demonstrated the appearance of short-living paramagnetic mononitrosyl-iron complex with L-cysteine prior to the formation of stable dinitrosyl-iron complex with cysteine in the solution of iron(II)-citrate complex (50-100 microM), S-nitrosocysteine (400 microM), and L-cysteine (20 mM) in 100 mM Hepes buffer (pH 7.4). The addition of deoxyhemoglobin (100 microM) did not influence the process, which points to a direct interaction between S-nitrosocysteine and iron(II) ions to yield DNIC. The reaction of DNIC-cysteine formation is first- and second-order in iron and S-nitrosocysteine, respectively. The third-order rate constant is (1.0 +/- 0.2) x 10(5) M(-2) s(-1) (estimated from EPR results) or (2.0 +/- 0.1) x 10(4) M(-2) s(-1) (estimated by optical method). A similar process of DNIC-cysteine formation was observed in a solution of iron(II)-citrate complex, L-cysteine, and NO-proline (200 microM) as a NO* donor. The appearance of a less stable dinitrosyl-iron complex with phosphate was detected when solutions of iron(II)-citrate containing 100 mM phosphate buffer (pH 7.4) were mixed with S-nitrosocysteine or NO-proline. The rapid formation of DNIC with phosphate was followed by its decay. When the concentration of L-cysteine in solutions was reduced from 20 to 1 mM, the life-time of the DNIC-cysteine diminished notably; this was caused by consumption of L-cysteine in the process of DNIC-cysteine formation from S-nitrosocysteine and iron. Thus, L-cysteine is consumed. Formation of DNIC with glutathione was also observed in a solution of glutathione (20 mM), S-nitrosoglutathione (400 microM), and iron(II) complex (800 microM) in 100 mM Hepes buffer (pH 7.4), but the rate of formation was about 10 times slower than the formation of the DNIC-cysteine. The rate of MNIC-MGD formation from iron(II)-MGD complexes and S-nitrosocysteine was first-order in both reactants. The second-order rate constant for this reaction, estimated from EPR measurements, was 30 +/- 5 M(-1) s(-1). Rate constants of MNIC-MGD formation from iron(II)-MGD and the more stable S-nitrosoglutathione and S-nitroso-D,L-penicillamine were equal to 3.0 +/- 0.3 and 0.3 +/- 0.05 M(-1) s(-1), respectively. Thus, the concerted mechanism of DNIC and MNIC formation from S-nitrosothiols and iron(II) ions can be suggested to be predominant.     

The source of the iron binding with nitric oxide in activated macrophages]

No decrease in iron-sulphur centers was found in cultured macrophage cells (J774) after the treatment with nitric oxide (10(-7) M NO/10(7) cells) during 5 min. The center content was controlled by the electron spin resonance (ESR) method. The macrophages pretreated with dithionite + methyl viologen showed the formation of dinitrosyl iron complexes (DNIC) with a characteristic ESR signal at g approximately 2.03. The data suggest that loosely bound nonheme iron (free iron) mostly contributes to the formation of these complexes. Iron from iron-containing proteins does not release from these centers under the direct action of nitric oxide. The iron-sulphur centers can be destroyed by the products of nitric oxide oxidation (NO2, N2O3, etc.) as oxidizing and acid agents.     

Evidence for involvement of c-Src in the anti-apoptotic action of nitric oxide in serum-deprived RINm5F cells

The mechanism by which nitric oxide (NO) protects from apoptosis is a matter of debate. We have shown previously that phosphorylation of tyrosine residues participates in the protection from apoptosis in insulin-producing RINm5F cells (Inorg. Chem. Commun. 3 (2000) 32). Since NO has been reported to activate the tyrosine kinase c-Src and this kinase is involved in the activation of protein kinase G (PKG) in some cell systems, we aimed at studying the contribution of c-Src and PKG systems in anti-apoptotic actions of NO in serum-deprived RINm5F cells. Here we report that exposure of serum-deprived cells to 10 microM DETA/NO results in protection from degradation of the anti-apoptotic protein Bcl-2, together with a reduction of cytochrome c release from mitochondria and caspase-3 inhibition. Studies with the inhibitors ODQ and KT-5823 revealed that these actions are dependent on both activation of guanylate cyclase and PKG. DETA/NO was also able to induce autophosphorylation and activation c-Src protein both in vivo and in vitro and active c-Src was able to induce tyrosine phosphorylation of Bcl-2 in vitro. The c-Src kinase inhibitor PP1 abrogated the actions of DETA/NO on cGMP formation, PKG activation, caspase activation, cytochrome c release from mitochondria, and Bcl-2 phosphorylation and degradation in serum-deprived cells. We thus propose that activation of c-Src is an early step in the chain of events that signal cGMP-dependent anti-apoptotic actions of NO in mitocohondria.     

Inhibition of nitric-oxide-mediated apoptosis in Jurkat leukemia cells despite cytochrome c release

We have recently shown that nitric-oxide (NO)-induced apoptosis in Jurkat human leukemia cells requires degradation of mitochondria phospholipid cardiolipin, cytochrome c release, and activation of caspase-9 and caspase-3. Moreover, an inhibitor of lipid peroxidation, Trolox, suppressed apoptosis in Jurkat cells induced by NO donor glycerol trinitrate. Here we demonstrate that this antiapoptotic effect of Trolox occurred despite massive release of the mitochondrial protein cytochrome c into the cytosol and mitochondrial damage. Incubation with Trolox caused a profound reduction of intracellular ATP concentration in Jurkat cells treated by NO. Trolox prevented cardiolipin degradation and caused its accumulation in Jurkat cells. Furthermore, Trolox markedly downregulated the NO-mediated activation of caspase-9 and caspase-3. Caspase-9 is known to be activated by released cytochrome c and together with caspase-3 is considered the most proximal to mitochondria. Our results suggest that the targets of the antiapoptotic effect of Trolox are located downstream of the mitochondria and that caspase activation and subsequent apoptosis could be blocked even in the presence of cytochrome c released from the mitochondria.     

Transition metals and nitric oxide production in human endothelial cells

The bioavailability of endothelial nitric oxide (NO) is regulated by transition metals but their mechanisms of action on NO synthesis and degradation are not clearly understood. Using differential pulse amperometry and NO microelectrodes, local NO concentration was measured at the surface of cultured human umbilical vein endothelial cells (HUVECs) stimulated by histamine or thrombin in the presence of transition metal chelators. The agonist-activated NO release required both extracellular Ca2+ and transition metals. In the presence of 1 mM external Ca2+, a low concentration of EGTA (5 microM) inhibited by 40% the NO release from stimulated HUVECs. In the presence of extracellular L-arginine, the inhibitory effect of EGTA was even more marked and, in its absence, it was suppressed by adding exogenous superoxide dismutase. The decrease in NO release induced by the copper chelators, cuprizone and DETC, suggests that extracellular traces of Cu2+ could regulate NO availability.     

Common regulation of apoptosis signaling induced by CD95 and the DNA-damaging stimuli etoposide and gamma-radiation downstream from caspase-8 activation.

The death receptor CD95 (APO-1/Fas), the anticancer drug etoposide, and gamma-radiation induce apoptosis in the human T cell line Jurkat. Variant clones selected for resistance to CD95-induced apoptosis proved cross-resistant to etoposide- and radiation-induced apoptosis, suggesting that the apoptosis pathways induced by these distinct stimuli have critical component(s) in common. The pathways do not converge at the level of CD95 ligation or caspase-8 signaling. Whereas caspase-8 function was required for CD95-mediated cytochrome c release, effector caspase activation, and apoptosis, these responses were unaffected in etoposide-treated and irradiated cells when caspase-8 was inhibited by FLIPL. Both effector caspase processing and cytochrome c release were inhibited in the resistant variant cells as well as in Bcl-2 transfectants, suggesting that, in Jurkat cells, the apoptosis signaling pathways activated by CD95, etoposide, and gamma-radiation are under common mitochondrial control. All three stimuli induced ceramide production in wild-type cells, but not in resistant variant cells. Exogenous ceramide bypassed apoptosis resistance in the variant cells, but not in Bcl-2-transfected cells, suggesting that apoptosis signaling induced by CD95, etoposide, and gamma-radiation is subject to common regulation at a level different from that targeted by Bcl-2.     

Variation of nitric oxide content regulates the development of apoptosis in the retina

It was shown that retinal ischemia entailed apoptosis in the inner layers of the retina. Administration of an NO-synthase inhibitor suppressed the development of ischemic apoptosis. To ascertain whether nitric oxide could induce the retinal apoptosis by itself, a nontoxic NO donor—dinitrosyl iron complex (DNIC) with glutathione—was injected into the vitreous body. DNIC at low concentrations induced apoptosis in the same retinal layers as in ischemia. However, with increasing DNIC doses, the number of apoptotic nuclei decreased markedly. Simultaneous administration of excess glutathione prevented apoptosis at any DNIC dose. The obtained data demonstrate the neurotoxic properties of the excess of nitric oxide in the retina.