Sustained CaMKII activity mediates transient oxidative stress-induced long-term facilitation of L-type Ca(2+) current in cardiomyocytes

Oxidative stress remodels Ca²⁺ signaling in cardiomyocytes, which promotes altered heart function in various heart diseases. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) was shown to be activated by oxidation, but whether and how CaMKII links oxidative stress to pathophysiological long-term changes in Ca²⁺ signaling remain unknown. Here, we present evidence demonstrating the role of CaMKII in transient oxidative stress-induced long-term facilitation (LTF) of L-type Ca²⁺ current (I_Ca,L) in rat cardiomyocytes. A 5-min exposure of 1 mM H₂O₂ induced an increase in I_Ca,L, and this increase was sustained for ~ 1 h. The CaMKII inhibitor KN-93 fully reversed H₂O₂-induced LTF of I_Ca,L, indicating that sustained CaMKII activity underlies this oxidative stress-induced memory. Simultaneous inhibition of oxidation and autophosphorylation of CaMKII prevented the maintenance of LTF, suggesting that both mechanisms contribute to sustained CaMKII activity. We further found that sarcoplasmic reticulum Ca²⁺ release and mitochondrial ROS generation have critical roles in sustaining CaMKII activity via autophosphorylation- and oxidation-dependent mechanisms. Finally, we show that long-term remodeling of the cardiac action potential is induced by H₂O₂ via CaMKII. In conclusion, CaMKII and mitochondria confer oxidative stress-induced pathological cellular memory that leads to cardiac arrhythmia.     

Phenylarsine oxide interferes with the death inducing signaling complex and inhibits tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced apoptosis

The mechanism by which tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces death is the subject of intense scrutiny due to its preferential targeting of transformed cells for deletion. Based on recent findings that the TRAIL-dependent death inducing signaling complex (DISC) forms and signals at the plasma membrane without being internalized, we investigated the possibility that agents that prevent endocytosis may stabilize the surface bound DISC and thereby enhance TRAIL-dependent signaling. We utilized phenylarsine oxide (PAO), a trivalent arsenical that has been reported to inhibit endocytosis and to induce mitochondrial permeability transition. Therefore PAO could, by two separate and independent activities, enhance TRAIL-induced killing. Paradoxically, we found that rather than synergizing with TRAIL, PAO was an effective inhibitor of TRAIL-induced killing. Recruitment of FADD and caspase-8 to the TRAIL-dependent DISC was diminished in a concentration-dependent manner in cells exposed to PAO. The effects of PAO could not be reversed by washing cells under non-reducing conditions, suggesting covalent linkage of PAO with its cellular target(s); however, 2,3-dimercaptoethanol effectively overcame the inhibitory action of PAO and restored sensitivity to TRAIL-induced apoptosis. PAO inhibited formation of the TRAIL-dependent DISC and therefore prevented all subsequent apoptotic events.     

Domain movement of iron sulfur protein in cytochrome bc1 complex is facilitated by the electron transfer from cytochrome b(L) to b(H)

The key step of the "protonmotive Q-cycle" mechanism for cytochrome bc1 complex is the bifurcated oxidation of ubiquinol at the Qp site. ISP is reduced when its head domain is at the b-position and subsequent move to the c1 position, to reduce cytochrome c1, upon protein conformational changes caused by the electron transfer from cytochrome b(L) to b(H). Results of analyses of the inhibitory efficacy and the binding affinity, determined by isothermal titration calorimetry, of Pm and Pf, on different redox states of cytochrome bc1 complexes, confirm this speculation. Pm inhibitor has a higher affinity and better efficacy with the cytochrome b(H) reduced complex and Pf binds better and has a higher efficacy with the ISP reduced complex.     

Hyperoxia-mediated oxidative stress increases expression of UCP3 mRNA and protein in skeletal muscle

The uncoupling protein-3 (UCP3) is a mitochondrial protein expressed mainly in skeletal muscle. Among several hypotheses for its physiological function, UCP3 has been proposed to prevent excessive production of reactive oxygen species. In the present study, we evaluated the effect of an oxidative stress induced by hyperoxia on UCP3 expression in mouse skeletal muscle and C2C12 myotubes. We found that the hyperoxia-mediated oxidative stress was associated with a 5-fold and 3-fold increase of UCP3 mRNA and protein levels, respectively, in mouse muscle. Hyperoxia also enhanced reactive oxygen species production and UCP3 mRNA expression in C2C12 myotubes. Our findings support the view that both in vivo and in vitro UCP3 may modulate reactive oxygen species production in response to an oxidative stress.     

Functional expression of plant alternative oxidase decreases antimycin A-induced reactive oxygen species production in human cells

Alternative oxidase (AOX) plays a pivotal role in cyanide-resistance respiration in the mitochondria of plants, fungi and some protists. Here we show that AOX from thermogenic skunk cabbage successfully conferred cyanide resistance to human cells. In galactose medium, HeLa cells with mitochondria-targeted AOX proteins were found to have significantly less reactive oxygen species production in response to antimycin-A exposure, a specific inhibitor of respiratory complex III. These results suggest that skunk cabbage AOX can be used to create an alternative respiration pathway, which might be important for therapy against various mitochondrial diseases.     

Occurrence, biosynthesis and function of isoprenoid quinones

Isoprenoid quinones are one of the most important groups of compounds occurring in membranes of living organisms. These compounds are composed of a hydrophilic head group and an apolar isoprenoid side chain, giving the molecules a lipid-soluble character. Isoprenoid quinones function mainly as electron and proton carriers in photosynthetic and respiratory electron transport chains and these compounds show also additional functions, such as antioxidant function. Most of naturally occurring isoprenoid quinones belong to naphthoquinones or evolutionary younger benzoquinones. Among benzoquinones, the most widespread and important are ubiquinones and plastoquinones. Menaquinones, belonging to naphthoquinones, function in respiratory and photosynthetic electron transport chains of bacteria. Phylloquinone K₁, a phytyl naphthoquinone, functions in the photosynthetic electron transport in photosystem I. Ubiquinones participate in respiratory chains of eukaryotic mitochondria and some bacteria. Plastoquinones are components of photosynthetic electron transport chains of cyanobacteria and plant chloroplasts. Biosynthetic pathway of isoprenoid quinones has been described, as well as their additional, recently recognized, diverse functions in bacterial, plant and animal metabolism.     

Aquaporin-facilitated transmembrane diffusion of hydrogen peroxide

Background

Hydrogen peroxide (H₂O₂) is an important signaling compound that has recently been identified as a new substrate for several members of the aquaporin superfamily in various organisms. Evidence is emerging about the physiological significance of aquaporin-facilitated H₂O₂ diffusion.

Scope of review

This review summarizes current knowledge about aquaporin-facilitated H₂O₂ diffusion across cellular membranes. It focuses on physicochemical and experimental evidence demonstrating the involvement of aquaporins in the transport of this redox signaling compound and discusses the regulation and structural prerequisites of these channels to transmit this signal. It also provides perspectives about the potential importance of aquaporin-facilitated H₂O₂ diffusion processes and places this knowledge in the context of the current understanding of transmembrane redox signaling processes.

Major conclusions

Specific aquaporin isoforms facilitate the passive diffusion of H₂O₂ across biological membranes and control H₂O₂ membrane permeability and signaling in living organisms.

General significance

Redox signaling is a very important process regulating the physiology of cells and organisms in a similar way to the well-characterized hormonal and calcium signaling pathways. Efficient transmembrane diffusion of H₂O₂, a key molecule in the redox signaling network, requires aquaporins and makes these channels important players in this signaling process. Channel-mediated membrane transport allows the fine adjustment of H₂O₂ levels in the cytoplasm, intracellular organelles, the apoplast, and the extracellular space, which are essential for it to function as a signal molecule. This article is part of a Special Issue entitled Aquaporins.     

Role of sodium in mitochondrial membrane depolarization induced by P2X7 receptor activation in submandibular glands

The effect of ATP on mitochondrial membrane depolarization in rat submandibular glands was investigated. Exposure of the cell suspension to high concentrations of ATP induced a sustained depolarization of mitochondrial membrane. This effect was blocked in the presence of magnesium and reproduced by low concentrations of 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP), suggesting the implication of the P2X(7) purinergic receptor. This point was confirmed by comparison of the response to ATP by wild-type and P2X(7) knock-out (P2X(7)R(-/-)) mice. Mitochondria took up calcium after ATP stimulation but the depolarization of the mitochondrial membrane by ATP was not affected by the removal of calcium from the extracellular medium. It was nearly fully suppressed in the absence of sodium and partially blocked by the mitochondrial Na/Ca exchanger inhibitor 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP-37157). Both ATP and monensin increased the uptake of extracellular sodium (as shown by the depolarization of the plasma membrane) but the sodium ionophore did not affect the mitochondrial membrane potential. It is concluded that the activation of P2X(7) receptors depolarizes the mitochondrial membrane. The uptake of extracellular sodium is necessary but not sufficient to induce this response.     

Antioxidant activity of isocoumarins isolated from Paepalanthus bromelioides on mitochondria

The isocoumarins (1-50 microM) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin isolated from Paepalanthus bromelioides, were assessed for antioxidant activity using isolated rat liver mitochondria and non-mitochondrial systems, and compared with the flavonoid quercetin. The paepalantine and paepalantine dimers, but not vioxanthin, were effective at scavenging both 1,1-diphenyl-2-picrylhydrazyl (DPPH(*)) and superoxide (O(2)(-)) radicals in non-mitochondrial systems, and protected mitochondria from tert-butylhydroperoxide-induced H(2)O(2) accumulation and Fe(2+)-citrate-mediated mitochondrial membrane lipid peroxidation, with almost the same potency as quercetin. These results point towards paepalantine, followed by paepalantine dimer, as being a powerful agent affording protection, apparently via O(2)(-) scavenging, from oxidative stress conditions imposed on mitochondria, the main intracellular source and target of those reactive oxygen species. This strong antioxidant action of paepalantine was reproduced in HepG2 cells exposed to oxidative stress condition induced by H(2)O(2).     

Occult hepatitis C virus elicits mitochondrial oxidative stress in lymphocytes and triggers PI3-kinase-mediated DNA damage response

Occult hepatitis C viral infection (OHCI) is a newly reported pathological entity associated with increased risk of developing hepatocellular carcinoma and lymphoproliferative disorders. Although hepatocytes are the primary sites of viral replication, hepatitis C virus is potentially lymphotropic, invading and propagating in cells of the immune system. Lymphocytes, the extrahepatic viral reservoirs, are differentially implicated in the occult and the active forms of the disease. This study aimed to elucidate the implications of mitochondrial oxidative stress on the immune pathophysiological mechanisms of OHCI. We herein report that OHCI induces mitochondrial oxidative stress, leading to DNA double-strand breaks and elicitation of a phosphoinositol 3-kinase-mediated cellular response in peripheral blood lymphocytes. Compared to controls, OHCI subjects showed higher accumulation of pATM, pATR, γH2AX, and p-p53, along with active recruitment of repair proteins (Mre11, Rad50, and Nbs1) and altered mitochondrial DNA content. Increased mitochondrial membrane depolarization and circulating nucleosome levels along with chromatid-type aberrations and decreased T-cell proliferative index observed in the OHCI group further indicated that this damage might lead to Bax-triggered mitochondria-mediated cellular apoptosis. Together our results provide the mechanistic underpinnings of mitochondrial dysfunction in OHCI, a previously unknown paradigm, for explaining the immune pathogenesis in a redox-dependent manner.     

Intermonomer electron transfer in the bc1 complex dimer is controlled by the energized state and by impaired electron transfer between low and high potential hemes

The cytochrome bc(1) complex (commonly called Complex III) is the central enzyme of respiratory and photosynthetic electron transfer chains. X-ray structures have revealed the bc(1) complex to be a dimer, and show that the distance between low potential (b(L)) and high potential (b(H)) hemes, is similar to the distance between low potential hemes in different monomers. This suggests that electron transfer between monomers should occur at the level of the b(L) hemes. Here, we show that although the rate constant for b(L)-->b(L) electron transfer is substantial, it is slow compared to the forward rate from b(L) to b(H), and the intermonomer transfer only occurs after equilibration within the first monomer. The effective rate of intermonomer transfer is about 2-orders of magnitude slower than the direct intermonomer electron transfer.     

Encapsulation of the flavonoid quercetin with an arsenic chelator into nanocapsules enables the simultaneous delivery of hydrophobic and hydrophilic drugs with a synergistic effect against chronic arsenic accumulation and oxidative stress

Chronic arsenic exposure causes oxidative stress and mitochondrial dysfunction in the liver and brain. The ideal treatment would be to chelate arsenic and prevent oxidative stress. meso-2,3-Dimercaptosuccinic acid (DMSA) is used to chelate arsenic but its hydrophilicity makes it membrane-impermeative. Conversely, quercetin (QC) is a good antioxidant with limited clinical application because of its hydrophobic nature and limited bioavailability, and it is not possible to solubilize these two compounds in a single nontoxic solvent. Nanocapsules have emerged as a potent drug delivery system and make it feasible to incorporate both hydrophilic and lipophilic compounds. Nanoencapsulated formulations with QC and DMSA either alone or coencapsulated in polylactide-co-glycolide [N(QC+DMSA)] were synthesized to explore their therapeutic application in a rat model of chronic arsenic toxicity. These treatments were compared to administration of quercetin or DMSA alone using conventional delivery methods. Both nanoencapsulated quercetin and nanoencapsulated DMSA were more effective at decreasing oxidative injury in liver or brain compared to conventional delivery methods, but coencapsulation of quercetin and DMSA into nanoparticles had a marked synergistic effect, decreasing liver and brain arsenic levels from 9.5 and 4.8μg/g to 2.2 and 1.5μg/g, respectively. Likewise, administration of coencapsulated quercetin and DMSA virtually normalized changes in mitochondrial function, formation of reactive oxygen species, and liver injury. We conclude that coencapsulation of quercetin and DMSA may provide a more effective therapeutic strategy in the management of arsenic toxicity and also presents a novel way of combining hydrophilic and hydrophobic drugs into a single delivery system.     

Conformation-driven and semiquinone-gated proton-pump mechanism in the NADH-ubiquinone oxidoreductase (complex I)

A novel mechanism for proton/electron transfer is proposed for NADH-quinone oxidoreductase (complex I) based on the following findings: (1) EPR signals of the protein-bound fast-relaxing semiquinone anion radicals (abbreviated as Q(Nf)-) are observable only in the presence of proton-transmembrane electrochemical potential; (2) Iron-sulfur cluster N2 and Q(Nf)- are directly spin-coupled; and (3) The projection of the interspin vector extends only 5A along the membrane normal [Yano, T., Dunham, W.R. and Ohnishi, T. (2005) Biochemistry, 44, 1744-1754]. We propose that the proton pump is operated by redox-driven conformational changes of the quinone binding protein. In the input state, semiquinone is reduced to quinol, acquiring two protons from the N (matrix) side of the mitochondrial inner membrane and an electron from the low potential (NADH) side of the respiratory chain. A conformational change brings the protons into position for release at the P (inter-membrane space) side of the membrane via a proton-well. Concomitantly, an electron is donated to the quinone pool at the high potential side of the coupling site. The system then returns to the original state to repeat the cycle. This hypothesis provides a useful frame work for further investigation of the mechanism of proton translocation in complex I.