Sodium-dependent transport of ascorbic acid in U937 cell mitochondria.

U937 cells exposed to physiological concentrations of ascorbic acid (AA) accumulate the reduced form of the vitamin in the cytosol and even further in their mitochondria. In both circumstances, uptake was dependent on Na(+) -AA-cotransport, with hardly any contribution of hexose transporters, which might be recruited to transport the oxidized form of the vitamin. There was an identical linear relationship between the mitochondrial accumulation of the vitamin and the extramitochondrial AA concentration, regardless of whether detected in experiments using intact cells or isolated mitochondria. Western blot experiments revealed expression of both SVCT1 and 2 in plasma membranes, whereas SVCT2 was the only form of the transporter expressed at appreciable amounts in mitochondria. These results therefore provide the novel demonstration of SVCT2-dependent mitochondrial transport of AA and hence challenge the present view that mitochondria only take up the oxidized form of the vitamin

Effects of L-histidine on hydrogen peroxide-induced DNA damage and cytotoxicity in cultured mammalian cells.

L-Histidine markedly increased the growth- and DNA synthesis-inhibitory effects elicited by hydrogen peroxide in cultured Chinese hamster ovary cells. DNA single-strand breakage was also higher in the presence of the amino acid and, in addition, these breaks were characterized by a slower rate of repair, compared with that of the breaks generated by the oxidant alone. In the presence of L-histidine, hydrogen peroxide also produced DNA double-strand breakage, a lesion that cannot be detected in cells treated with even exceedingly high concentrations of the oxidant alone. Data reported herein suggest that the L-histidine-mediated increase of the cytotoxic response of cultured Chinese hamster ovary cells to hydrogen peroxide may be at least partially dependent on the formation of DNA double-strand break

Loss-of-rescue of Ryr1I4895T-related pathology by the genetic inhibition of the ER stress response mediator CHOP

RYR1 is the gene encoding the ryanodine receptor 1, a calcium release channel of the endo/sarcoplasmic reticulum. I4898T in RYR1 is one of the most common mutations that give rise to central core disease (CCD), with a variable phenotype ranging from mild to severe myopathy to lethal early-onset core-rod myopathy. Mice with the corresponding I4895T mutation in Ryr1 present mild myopathy when the mutation is heterozygous while I4895T homozygous is perinatal-lethal. Here we show that skeletal muscles of I4895T homozygous mice at birth present signs of stress of the endoplasmic reticulum (ER stress) and of the related unfolded protein response (UPR) with increased levels of the maladaptive mediators CHOP and ERO1. To gain information on the role of CHOP in the pathogenesis of RYR1I4895T-related myopathy, we generated compound Ryr1I4895T, Chop knock-out (-/-) mice. However, the genetic deletion of Chop, although it attenuates ER stress in the skeletal muscle of the newborns, does not rescue any phenotypic or functional features of Ryr1I4895T in mice: neither the perinatal-lethal phenotype nor the inability of Ryr1I4895T to respond to its agonist caffeine, but protects from ER stress-induced apoptosis. These findings suggest that genetic deletion of the ER stress response mediator CHOP is not sufficient to counteract the pathological Ryr1I4895T phenotype

Pivotal role of superoxides generated in the mitochondrial respiratory chain in peroxynitrite-dependent activation of phospholipase A2.

Exposure of PC12 cells to reagent peroxynitrite promotes the release of arachidonic acid (AA) mediated by activation of phospholipase A(2) [Guidarelli, Palomba and Cantoni (2000) Br. J. Pharmacol. 129, 1539-1542]. We now present experimental evidence consistent with the notion that this response is not directly triggered by peroxynitrite but, rather, by reactive oxygen species generated at the level of complex III of the mitochondrial respiratory chain. In particular, superoxide (and not hydrogen peroxide) has a pivotal role in peroxynitrite-dependent activation of phospholipase A(2). This observation was confirmed by results showing that superoxide, or peroxynitrite, promotes release of AA in isolated mitochondria. Consistently, the release of AA elicited by either peroxynitrite or A23187 in intact cells was shown to be calcium-dependent and differentially affected by phospholipase A(2) inhibitors with different levels of specificity. In particular, the effects of peroxynitrite, unlike those of A23187, were both sensitive to low concentrations of two general phospholipase A(2) inhibitors and insensitive to arachidonyltrifluoromethyl ketone, which shows some selectivity towards cytosolic phospholipase A(2). In addition, peroxynitrite and A23187 synergistically enhanced the release of AA. Collectively, the above results demonstrate that peroxynitrite causes inhibition of complex III, followed by enforced formation of superoxides that stimulate the activity of a calcium-dependent PLA(2) isoform, probably localized in the mitochondria

Mitochondrial Uptake and Accumulation of Vitamin C: What Can We Learn From Cell Cultures Studies?

The mitochondrial fraction of L-ascorbic acid (AA) is of critical importance for the regulation of the redox status of these organelles and for cell survival. Recent Advances: Most cell types take up AA by high affinity sodium-dependent vitamin C transporter 2 (SVCT2), sensitive to inhibition by dehydroascorbic acid (DHA). DHA can also be taken up by glucose transporters (GLUTs) and then reduced back to AA. DHA concentrations, normally very low in biological fluids, may only become significant next to superoxide releasing cells. Very little is known on the mechanisms mediating the mitochondrial transport of the vitamin

Low Concentrations of Arsenite Target the Intraluminal Inositol 1, 4, 5-Trisphosphate Receptor/Ryanodine Receptor Crosstalk to Significantly Elevate Intracellular Ca2

Arsenite is an established human carcinogen inducing cyto- and genotoxic effects through poorly defined mechanisms involving the formation of reactive oxygen species (ROS) and deregulated Ca2+ homeostasis. We used variants of the U937 cell line to address the central issue of the mechanism whereby arsenite affects Ca2+ homeostasis. We found that a 6 h exposure to the metalloid (2.5 μM), while not associated to an immediate or delayed toxicity, causes a significant increase in the intracellular Ca2+ concentration ([Ca2+]i) through a mechanism characterized by the following components: i) it was not affected by ROS produced under the same conditions; ii) a small amount of Ca2+ was mobilized from the inositol-1,4,5-trisphosphate receptor (IP3R). This response was not further augmented by greater concentrations of the metalloid; iii) large amounts of Ca2+ were instead dose-dependently mobilized from the ryanodine receptor (RyR) in response to IP3R stimulation; iv) the cells maintained an intact responsiveness to agonist-stimulated Ca2+ mobilization from both channels; v) arsenite, even at 5-10 μM, failed to directly mobilize Ca2+ from the RyR; vi) arsenite failed to enhance Ca2+ release from the RyR under conditions in which the [Ca2+]i was increased by either RyR agonists or ionophore-stimulated Ca2+ uptake. We therefore conclude that arsenite elevates the [Ca2+]i by directly targeting the IP3R and its intraluminal crosstalk with the RyR. This mechanism likely mediates mitochondrial superoxide formation, downstream damage on various biomolecules, including genomic DNA, and mitochondrial dysfunction/apoptosis eventually occurring after longer incubation to, or exposure to greater concentrations of, arsenite

Enhancing effects of intracellular ascorbic acid on peroxynitrite-induced U937 cell death are mediated by mitochondrial events resulting in enhanced sensitivity to peroxynitrite-dependent inhibition of complex III and formation of hydrogen peroxide.

A short-term pre-exposure to dehydroascorbic acid (DHA) promotes U937 cell death upon exposure to otherwise non-toxic levels of peroxynitrite (ONOO-). Toxicity is mediated by a saturable mechanism and cell death takes place as a consequence of mitochondrial permeability transition. The following lines of evidence are consistent with the notion that the enhancing effects of DHA were related to mitochondrial events resulting in inhibition of complex III upon exposure to otherwise inactive concentrations of ONOO-. First, DHA, as well as bona fide complex III inhibitors, similarly enhanced toxicity and subsequent formation of H2O2 induced by ONOO- via a rotenone- or catalase-sensitive mechanism. Secondly, bona fide complex III inhibitors were ineffective in DHA-pre-loaded cells. In addition, respiration-deficient cells were resistant to toxicity elicited by ONOO- and their supplementation with increasing concentrations of DHA, although resulting in the accumulation of vitamin C levels identical with those observed in respiration-proficient cells, failed to affect ONOO- toxicity. Finally, oxygen-consumption experiments demonstrated that pre-exposure to DHA promotes the ONOO--dependent inhibition of complex III. In conclusion, the above results collectively demonstrate that increasing the intracellular accumulation of vitamin C promotes mitochondrial events leading to ONOO--dependent formation of H2O2 and resulting in a rapid necrotic response

Inhibition of activity/expression, or genetic deletion, of ERO1α blunts arsenite geno- and cyto-toxicity

Our recent studies suggest that arsenite stimulates the crosstalk between the inositol 1, 4, 5-triphosphate receptor (IP3R) and the ryanodine receptor (RyR) via a mechanism dependent on endoplasmic reticulum (ER) oxidoreductin1α (ERO1α) up-regulation. Under these conditions, the fraction of Ca2+ released by the RyR via an ERO1α-dependent mechanism was promptly cleared by the mitochondria and critically mediated O2-. formation, responsible for the triggering of time-dependent events associated with strand scission of genomic DNA and delayed mitochondrial apoptosis. We herein report that, in differentiated C2C12 cells, this sequence of events can be intercepted by genetic deletion of ERO1α as well as by EN460, an inhibitor of ERO1α activity. Similar results were obtained for the early effects mediated by arsenite in proliferating U937 cells, in which however the long-term studies were hampered by the intrinsic toxicity of the inhibitor. It was then interesting to observe that ISRIB, an inhibitor of p-eIF2 alpha, was in both cell types devoid of intrinsic toxicity and able to suppress ERO1α expression and the resulting downstream effects leading to arsenite geno- and cyto-toxicity. We therefore conclude that pharmacological inhibition of ERO1α activity, or expression, effectively counteracts the deleterious effects induced by the metalloid via a mechanism associated with prevention of mitochondrial O2-. formation