Defective biosynthesis of ascorbic acid in Sod1-deficient mice results in lethal damage to lung tissue.

Superoxide dismutase 1 (Sod1) plays pivotal roles in antioxidation via accelerating the conversion of superoxide anion radicals into hydrogen peroxide, thus inhibiting the subsequent radical chain reactions. While Sod1 deficient cells inevitably undergo death in culture conditions, Sod1-knockout (KO) mice show relatively mild phenotypes and live approximately two years. We hypothesized that the presence of abundant levels of ascorbic acid (AsA), which is naturally produced in mice, contributes to the elimination of reactive oxygen species (ROS) in Sod1-KO mice. To verify this hypothesis, we employed mice with a genetic ablation of aldehyde reductase (Akr1a), an enzyme that is involved in the biosynthesis of AsA, and established double knockout (DKO) mice that lack both Sod1 and Akr1a. Supplementation of AsA (1.5 mg/ml in drinking water) was required for the DKO mice to breed, and, upon terminating the AsA supplementation, they died within approximately two weeks regardless of age or gender. We explored the etiology of the death from pathophysiological standpoints in principal organs of the mice. Marked changes were observed in the lungs in the form of macroscopic damage after the AsA withdrawal. Histological and immunological analyses of the lungs indicated oxidative damage of tissue and activated immune responses. Thus, preferential oxidative injury that occurred in pulmonary tissues appeared to be primary cause of the death in the mice. These collective results suggest that the pivotal function of AsA in coping with ROS in vivo, is largely in pulmonary tissues that are exposed to a hyperoxygenic microenvironment.

Inhibition of thrombin-induced Ca²âº influx in platelets by R59949, an inhibitor of diacylglycerol kinase.

OBJECTIVES: The aim of this study was to determine whether diacylglycerol kinase (DGK) is involved in transplasmalemmal Ca²âº influx of platelets. METHODS: Effects of R59949, an inhibitor of diacylglycerol kinase, on intracellular Ca²âº concentration ([Ca²âº](i) ) and mRNA expression of DGK isozymes were investigated using washed human platelet suspensions. KEY FINDINGS: Thrombin-induced increase in [Ca²âº](i) was significantly inhibited by pretreatment of platelets with R59949, while thapsigargin-induced increase in [Ca²âº](i) was comparable in platelets with and without R59949 pretreatment. Thapsigargin-induced increase in [Ca²âº](i) was markedly attenuated in the presence of SKF-96365. In the presence of SKF-96365, thrombin-induced increase in [Ca²âº](i) was significantly attenuated, and additional treatment with R59949 caused a further decrease in [Ca²âº](i) . Pretreatment of platelets with 1-butanol significantly attenuated thrombin-induced increase in [Ca²âº](i) , while thrombin-induced increase in [Ca²âº](i) was augmented in the presence of propranolol. mRNA expression of DGK-α and DGK-γ, which are known to be inhibited by R59949, in platelets was confirmed by RT-PCR analysis. CONCLUSIONS: R59949 inhibited a store-depletion-insensitive component of transplasmalemmal Ca²âº entry induced by thrombin, while store-operated Ca²âº entry was not affected by R59949. The results of this study suggest that phosphatidic acid is involved in thrombin-induced Ca²âº influx of platelets.