Cytochrome c-mediated caspase-9 activation triggers apoptosis in Streptococcus pyogenes-infected epithelial cells

Epithelial cells are the initial sites of host invasion by group A Streptococcus pyogenes (GAS), and their infection of epithelial cells has been suggested to induce apoptosis. However, the mechanism responsible for bacteria–host interaction and the induction of apoptosis has not been clearly understood. We demonstrate here that human pharyngeal epithelial HEp-2 cells became apoptotic with DNA fragmentation by invasion of GAS strains JRS4 (M6⁺, F1⁺) and JRS145 (M6⁻, F1⁺ mutant of JRS4), whereas apoptotic cellular changes were not observed in SAM1 (M6⁺, F1⁻ mutant) or SAM2 (M6⁻, F1⁻ mutant) infected HEp-2 cells. Confocal microscopy revealed that Bax translocation to mitochondria and cytochrome c release occurred after 4 h of infection. Western blot analyses showed that the amounts of Bcl-2 and Bcl-x_L were decreased in the mitochondria of infected cells. In addition, we demonstrated that the release of nuclear histone from infected cells was prevented by the addition of caspase-9 inhibitor (Ac-LEHD-CHO). We conclude that the internalization of GAS in epithelial cells is necessary and sufficient for the induction of apoptosis, which is initiated by mitochondrial dysfunction, and the mechanism of GAS-induced apoptosis is clearly different from that induced by other intracellular invasive bacteria, e.g. Shigella and Salmonella species.     

S-nitrosothiol formation in blood of lipopolysaccharide-treated rats

The administration of the gram-negative bacterial cell wall component lipopolysaccharide (LPS) to experimental animals results in the dramatic up-regulation of the inducible form of nitric oxide synthase (iNOS). The resulting sustained overproduction of nitric oxide (NO) is thought to contribute to the septic shock-like state in these animals. Numerous studies have characterized the kinetics and magnitude of expression of iNOS as well as the production of NO-derived nitrite and nitrate. However, little is known regarding the ability of iNOS-derived NO to interact with physiological substrates such as thiols to yield biologically active S-nitrosothiols during endotoxemia. It has been hypothesized that these relatively stable, vaso-active compounds may serve as a storage system for NO and they may thus play an important role in the pathophysiology associated with endotoxemia. In the present study, we demonstrate that 5 h after i.p. administration of LPS in rats, circulating S-nitrosoalbumin was increased by approximately 3. 4-fold over control. S-nitrosohemoglobin was increased by approximately 25-fold over controls and by threefold over S-nitrosoalbumin. No increase in low molecular weight S-nitrosothiols (i.e., S-nitrosoglutathione and S-nitrosocysteine) could be detected under our experimental conditions. Taken together these data demonstrate that endotoxemia dramatically enhances circulating S-nitrosothiol formation.     

Cytochrome c-mediated apoptosis in cells lacking mitochondrial DNA. Signaling pathway involving release and caspase 3 activation is conserved.

Mitochondria serve as a pivotal component of the apoptotic cell death machinery. However, cells that lack mitochondrial DNA (rho(0) cells) retain apparently normal apoptotic signaling. In the present study, we examined mitochondrial mechanisms of apoptosis in rho(0) osteosarcoma cells treated with staurosporine. Immunohistochemistry revealed that rho(0) cells maintained a normal cytochrome c distribution in mitochondria even though these cells were deficient in respiration. Upon staurosporine treatment, cytochrome c was released concomitantly with activation of caspase 3 and loss of mitochondrial membrane potential (Deltapsi(m)). After mitochondrial loss of cytochrome c, rho(0) cells underwent little change in glutathione (GSH) redox potential whereas a dramatic oxidation in GSH/glutathione disulfide (GSSG) pool occurred in parental rho(+) cells. These results show that mitochondrial signaling of apoptosis via cytochrome c release was preserved in cells lacking mtDNA. However, intracellular oxidation that normally accompanies apoptosis was lost, indicating that the mitochondrial respiratory chain provides the major source of redox signaling in apoptosis.     

Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: identification of homo- and heterodimers

Here we report that ferricytochrome c (cyt c(3+)) induces oxidation of hydroethidine (HE) and mitochondria-targeted hydroethidine (Mito-HE or MitoSOX Red) forming highly characteristic homo- and heterodimeric products. Using an HPLC-electrochemical (EC) method, several products were detected from cyt c(3+)-catalyzed oxidation of HE and Mito-HE and characterized by mass spectrometry and NMR techniques as follows: homodimers (HE-HE, E(+)-E(+), Mito-HE-Mito-HE, and Mito-E(+)-Mito-E(+)) and heterodimers (HE-E(+) and Mito-HE-Mito-E(+)), as well as the monomeric ethidium (E(+)) and mito-ethidium (Mito-E(+)). Similar products were detected when HE and Mito-HE were incubated with mitochondria. In contrast, mitochondria depleted of cyt c(3+) were much less effective in oxidizing HE or Mito-HE to corresponding dimeric products. Unlike E(+) or Mito-E(+), the dimeric analogs (E(+)-E(+) and Mito-E(+)-Mito-E(+)) were not fluorescent. Superoxide (O(2)(*-)) or Fremy's salt reacts with Mito-HE to form a product, 2-hydroxy-mito-ethidium (2-OH-Mito-E(+)) that was detected by HPLC. We conclude that HPLC-EC but not the confocal and fluorescence microscopy is a viable technique for measuring superoxide and cyt c(3+)-dependent oxidation products of HE and Mito-HE in cells. Superoxide detection using HE and Mito-HE could be severely compromised due to their propensity to undergo oxidation.     

Mechanism of S-nitrosothiol formation and degradation mediated by copper ions.

Experimental evidence is presented supporting a mechanism of S-nitrosothiol formation and degradation mediated by copper ions using bovine serum albumin, human hemoglobin and glutathione as models. We found that Cu(2+), but not Fe(3+), induces in the presence of NO a fast S-nitrosation of bovine serum albumin and human hemoglobin, and the reaction is prevented by thiol blocking reagents. During the reaction, Cu(+) is accumulated and accounts for destabilization of the S-nitrosothiol formed. In contrast, glutathione rapidly dimerizes in the presence of Cu(2+), the reaction competing with S-nitrosation and therefore preventing the formation of S-nitrosoglutathione. We have combined the presented role of Cu(2+) in S-nitrosothiol formation with the known destabilizing effect of Cu(+), providing a unique simple picture where the redox state of copper determines either the NO release from S-nitrosothiols or the NO scavenging by thiol groups. The reactions described are fast, efficient, and may occur at micromolar concentration of all reactants. We propose that the mechanism presented may provide a general method for in vitro S-nitrosation.     

A novel role for cytochrome c: Efficient catalysis of S-nitrosothiol formation

Although S-nitrosothiols are regarded as important elements of many NO-dependent signal transduction pathways, the physiological mechanism of their formation remains elusive. Here, we demonstrate a novel mechanism by which cytochrome c may represent an efficient catalyst of S-nitrosation in vivo. In this mechanism, initial binding of glutathione to ferric cytochrome c is followed by reaction of NO with this complex, yielding ferrous cytochrome c and S-nitrosoglutathione (GSNO). We show that when submitochondrial particles or cell lysates are exposed to NO in the presence of cytochrome c, there is a robust formation of protein S-nitrosothiols. In the case of submitochondrial particles protein S-nitrosation is paralleled by an inhibition of mitochondrial complex I. These observations raise the possibility that cytochrome c is a mediator of S-nitrosation in biological systems, particularly during hypoxia, and that release of cytochrome c into the cytosol during apoptosis potentially releases a GSNO synthase activity that could modulate apoptotic signaling.     

Alpha-fetoprotein positively regulates cytochrome c-mediated caspase activation and apoptosome complex formation.

Previous results have shown that the oncoembryonic marker alpha-fetoprotein (AFP) is able to induce apoptosis in tumor cells through activation of caspase 3, bypassing Fas-dependent and tumor necrosis factor receptor-dependent signaling. In this study we further investigate the molecular interactions involved in the AFP-mediated signaling of apoptosis. We show that AFP treatment of tumor cells is accompanied by cytosolic translocation of mitochondrial cytochrome c. In a cell-free system, AFP mediates processing and activation of caspases 3 and 9 by synergistic enhancement of the low-dose cytochrome c-mediated signals. AFP was unable to regulate activity of caspase 3 in cell extracts depleted of cytochrome c or caspase 9. Using high-resolution chromatography, we show that AFP positively regulates cytochrome c/dATP-mediated apoptosome complex formation, enhances recruitment of caspases and Apaf-1 into the complex, and stimulates release of the active caspases 3 and 9 from the apoptosome. By using a direct protein-protein interaction assay, we show that pure human AFP almost completely disrupts the association between processed caspases 3 and 9 and the cellular inhibitor of apoptosis protein (cIAP-2), demonstrating its release from the complex. Our data suggest that AFP may regulate cell death by displacing cIAP-2 from the apoptosome, resulting in promotion of caspase 3 activation and its release from the complex.     

Mechanistic studies of S-nitrosothiol formation by NO*/O2 and by NO*/methemoglobin

The mechanisms of formation of S-nitrosothiols under physiological conditions and, in particular, of generation of SNO-Hb (the hemoglobin form in which the cysteine residues beta93 are S-nitrosated) are still not completely understood. In this paper, we investigated whether, in the presence of O2, NO* is more efficient to nitrosate protein-bound thiols such as Cysbeta93 or low molecular weight thiols such as glutathione. Our results show that when substoichiometric amounts of NO* are mixed slowly with the protein solution, NO*, O2, and possibly NO2* and/or N2O3 accumulate in hydrophobic pockets of hemoglobin. Since the environment of the cysteine residue beta93 is rather hydrophobic, these conditions facilitate SNO-Hb production. Moreover, we show that S-nitrosation mediated by reaction of NO* with the iron(III) forms of Hb or Mb is significantly more effective when it can take place intramolecularly, as in metHb. Intermolecular reactions lead to lower S-nitrosothiol yields because of the concurring hydrolysis to nitrite.     

Equol induces apoptosis through cytochrome c-mediated caspases cascade in human breast cancer MDA-MB-453 cells

This study investigated the role of the caspase activation cascade in extrinsic and intrinsic apoptosis induced by equol in human breast cancer MDA-MB cells. First, the antiproliferative effect of equol was determined in cells treated with 1-100 μM equol for 24, 48, and 72 h. Equol significantly inhibited cell proliferation in a dose-dependent manner (p < 0.05). Exposure to 50 or 100 μM equol for 72 h strongly promoted apoptosis. Under the same conditions, remarkable cytochrome c release was observed. Subsequently, caspase-9, which acts in mitochondria-mediated apoptosis, was cleaved by equol at high concentrations, but caspase-8 activation of receptor-mediated apoptosis was not observed. At both equol concentrations, the caspase-8 and -9 activity assays showed similar patterns. In addition, equol treatment activated caspase-3, which is downstream from caspase-9, and this was accompanied by the cleavage of capase-6 and -7. Activation of these caspases leads to increased activation of PARP, lamin, and ICAD. This study suggests that equol induces the intrinsic pathway of apoptosis via caspase-9 and cytochrome c, independent of caspase-8, in human breast cancer MDA-MB-453 cells.     

细胞色素c与细胞凋亡

在哺乳动物细胞凋亡的发生过程中,位于线粒体内膜外侧的细胞色素ｃ被释放进入胞质,作为辅助因子参与死亡蛋白酶－３的激活,后者在凋亡过程中起到重要作用。Ｂｃｌ－２、Ｂｃｌ－ＸＬ,Ｂａｘ可阻止或诱导线粒体细胞色素ｃ释放入胞质,这可能是Ｂｃｌ－２,Ｂｃｌ－ＸＬ,Ｂａｘ调节凋亡的机制之一。     

Mixing artifacts from the bolus addition of nitric oxide to oxymyoglobin: implications for S-nitrosothiol formation

The addition of nitric oxide (NO) solution to oxygenated heme proteins has been used to measure NO concentration and as an experimental model to investigate the biochemical mechanism of NO metabolism. In this paper we demonstrate that bolus addition of NO to oxymyoglobin (oxyMb) results in the artifactual formation of nitrosating intermediates. When NO is added as a bolus, using fully aerated oxyMb solutions, the measured NO concentration is half as much as that when the oxyMb solution is partially degassed (0.86 +/- 0.01 mM vs. 1.61 +/- 0.02 mM, mean +/- SD). Similar results are found when calibrating NO concentration using a nitronyl nitroxide-type NO scavenger. The apparent stoichiometry of NO to oxyMb increases when the solution oxygen concentration increases. A fraction of the added NO generates nitrite or, in the presence of glutathione (GSH), S-nitrosoglutathione (GSNO). When using an NO donor, which slowly releases NO, oxyMb oxidation shows no dependence on the presence of oxygen in solution, and no nitrosating intermediate is formed. Bolus NO addition causes a local high concentration of NO. Kinetic calculations under this condition using known rate constants indicate that both the NO/oxyMb reaction and the NO/O(2) reaction can occur before it is possible fully to mix the solution. Our results suggest that the presence of the NO/O(2) reaction is an artifact of bolus NO addition, and leads to the formation of nitrite, or GSNO in the presence of GSH.     

Cytochrome c-mediated electron transfer between ubiquinol-cytochrome c reductase and cytochrome c oxidase. Kinetic evidence for a mobile cytochrome c pool.

Ubiquinol oxidase has been reconstituted from ubiquinol-cytochrome c reductase (Complex III), cytochrome c and cytochrome c oxidase (Complex IV). The steady-state level of reduction of cytochrome c by ubiquinol-2 varies with the molar ratios of the complexes and with the presence of antimycin in a way that can be quantitatively accounted for by a model in which cytochrome c acts as a freely diffusible pool on the membrane. This model was based on that of Kröger & Klingenberg [(1973) Eur. J. Biochem. 34, 358-368] for ubiquinone-pool behaviour. Further confirmation of the pool model was provided by analysis of ubiquinol oxidase activity as a function of the molar ratio of the complexes and prediction of the degree of inhibition by antimycin.     

Cytochrome P450 mediates dopamine formation in the brain in vivo

The cytochrome P450-mediated synthesis of dopamine from tyramine has been shown in vitro. The aim of the present study was to demonstrate the ability of rat cytochrome P450 (CYP) 2D to synthesize dopamine from tyramine in the brain in vivo. We employed two experimental models using reserpinized rats with a blockade of the classical pathway of dopamine synthesis from tyrosine. Model A estimated dopamine production from endogenous tyramine in brain structures in vivo (ex vivo measurement of a tissue dopamine level), while Model B measured extracellular dopamine produced from exogenous tyramine (an in vivo microdialysis). In Model A, quinine (a CYP2D inhibitor) given intraperitoneally caused a significant decrease in dopamine level in the striatum and nucleus accumbens and tended to fall in the substantia nigra and frontal cortex. In Model B, an increase in extracellular dopamine level was observed after tyramine given intrastructurally (the striatum). After joint administration of tyramine and quinine, the amount of the dopamine formed was significantly lower compared to the group receiving tyramine only. The results of the two complementary experimental models indicate that the hydroxylation of tyramine to dopamine may take place in rat brain in vivo, and that CYP2D catalyzes this reaction.     

Gastric S-nitrosothiol formation drives the antihypertensive effects of oral sodium nitrite and nitrate in a rat model of renovascular hypertension

Many effects of nitrite and nitrate are attributed to increased circulating concentrations of nitrite, ultimately converted into nitric oxide (NO^•) in the circulation or in tissues by mechanisms associated with nitrite reductase activity. However, nitrite generates NO^• , nitrous anhydride, and other nitrosating species at low pH, and these reactions promote S-nitrosothiol formation when nitrites are in the stomach. We hypothesized that the antihypertensive effects of orally administered nitrite or nitrate involve the formation of S-nitrosothiols, and that those effects depend on gastric pH. The chronic effects of oral nitrite or nitrate were studied in two-kidney, one-clip (2K1C) hypertensive rats treated with omeprazole (or vehicle). Oral nitrite lowered blood pressure and increased plasma S-nitrosothiol concentrations independently of circulating nitrite levels. Increasing gastric pH with omeprazole did not affect the increases in plasma nitrite and nitrate levels found after treatment with nitrite. However, treatment with omeprazole severely attenuated the increases in plasma S-nitrosothiol concentrations and completely blunted the antihypertensive effects of nitrite. Confirming these findings, very similar results were found with oral nitrate. To further confirm the role of gastric S-nitrosothiol formation, we studied the effects of oral nitrite in hypertensive rats treated with the glutathione synthase inhibitor buthionine sulfoximine (BSO) to induce partial thiol depletion. BSO treatment attenuated the increases in S-nitrosothiol concentrations and antihypertensive effects of oral nitrite. These data show that gastric S-nitrosothiol formation drives the antihypertensive effects of oral nitrite or nitrate and has major implications, particularly to patients taking proton pump inhibitors.     

Increased stability of S-nitrosothiol solutions via pH modulations

S-nitrosothiol (RSNO) solutions represent a valuable source of nitric oxide and could be used as topical vasodilators, but their fast decomposition rate poses a serious obstacle to their potentially widespread therapeutic use. Our aim was to characterize and quantify the effect of pH on S-nitrosothiol formation and decomposition in simple aqueous solutions of S-nitrosoglutathione (GSNO), S-nitroso-N-acetylcysteine (SNAC) and S-nitroso-3-mercaptopropionic acid (SN3MPA). Furthermore, we investigated the effect of storage pH on the stability of GSNO incorporated in poly(ethylene glycol)/ poly(vinyl alcohol) matrices. S-nitrosothiol concentrations were measured spectrophotometrically and laser Doppler scanning method was used to assess dermal blood flow. GSH and NAC solutions reached a complete transformation to nitrosothiols when synthesized using acidic NaNO(2) solution. The initial concentration of all investigated RSNOs decreased more slowly with pH adjusted to mildly basic values (8.4-8.8) for the storage period. Polymer gels of PVA/PEG compositions at mildly basic storage pH further reduced the decomposition rate succeeding to contain 46.8% of the initial GSNO concentration for 25 days. This amount of topically administered GSNO was still capable of increasing the dermal blood flow over 200% in human subjects.     

Increased stability of S-nitrosothiol solutions via pH modulations

S-nitrosothiol (RSNO) solutions represent a valuable source of nitric oxide and could be used as topical vasodilators, but their fast decomposition rate poses a serious obstacle to their potentially widespread therapeutic use. Our aim was to characterize and quantify the effect of pH on S-nitrosothiol formation and decomposition in simple aqueous solutions of S-nitrosoglutathione (GSNO), S-nitroso-N-acetylcysteine (SNAC) and S-nitroso-3-mercaptopropionic acid (SN3MPA). Furthermore, we investigated the effect of storage pH on the stability of GSNO incorporated in poly(ethylene glycol)/ poly(vinyl alcohol) matrices. S-nitrosothiol concentrations were measured spectrophotometrically and laser Doppler scanning method was used to assess dermal blood flow. GSH and NAC solutions reached a complete transformation to nitrosothiols when synthesized using acidic NaNO₂ solution. The initial concentration of all investigated RSNOs decreased more slowly with pH adjusted to mildly basic values (8.4–8.8) for the storage period. Polymer gels of PVA/PEG compositions at mildly basic storage pH further reduced the decomposition rate succeeding to contain 46.8% of the initial GSNO concentration for 25 days. This amount of topically administered GSNO was still capable of increasing the dermal blood flow over 200% in human subjects.     

Role of S-nitrosothiol transport in the cardioprotective effects of S-nitrosocysteine in rat hearts

The objective of this study was to determine if prior exposure of rat hearts to S-nitrosocysteine (CysNO) was able to provide protection against reperfusion injury. We probed NO release using the extracellular NO scavenger oxyhemoglobin (oxyHb), and we examined the involvement of the amino acid transport system L (L-AT), a known transporter of CysNO, using the L-AT competitor, L-leucine (L-Leu). Isolated (9- to 12-week-old Wistar male) rat hearts (six to eight per group) were perfused with CysNO (10 microM) for 30 min with or without the L-AT competitor L-Leu (1 mM) before 30 min of ischemia. Cardiac function was assessed before, during, and after treatment and during 120 min of reperfusion after ischemia. Functional recovery (rate-pressure product) was significantly improved in the CysNO group compared to hearts in the CysNO+L-Leu group and the control group (p<0.05). Necrosis, measured by triphenyltetrazolium chloride staining, was significantly reduced in CysNO hearts (p<0.05) and this improvement was reversed by L-Leu. The NO scavenger oxyHb (20 microM) was perfused either concomitant with CysNO or just before ischemia. In neither case did oxyHb affect the cardioprotection afforded by CysNO. OxyHb alone, given in either time window, did not alter the course of ischemia-reperfusion injury. When nitrite was used in place of CysNO, no protective effects were observed. Perfusion with CysNO increased tissue S-nitrosothiol (RSNO) levels from an unmeasurable background to a value of about 15.7+/-4.1 pmol RSNO/mg protein, as measured by triiodide-based chemiluminescence in the presence and absence of mercury(II) chloride. In the presence of L-Leu, this value dropped to 0.4+/-0.3 pmol RSNO/mg protein. This study demonstrates that exposure to CysNO before ischemia increases tissue S-nitrosothiol levels, improves postischemic contractile dysfunction, and attenuates necrosis. The mechanism of cardioprotection requires the uptake of CysNO via the L-AT and does not seem to involve NO release either during CysNO exposure or during ischemia. This suggests that the protective effects of CysNO are mediated through the posttranslational modification of cellular proteins through an NO-independent transnitrosation mechanism.