Deuterated Arachidonic Acid Ameliorates Lipopolysaccharide-Induced Lung Damage in Mice

Arachidonic acid (ARA) is a major component of lipid bilayers as well as the key substrate for the eicosanoid cascades. ARA is readily oxidized, and its non-enzymatic and enzymatic oxidation products induce inflammatory responses in nearly all tissues, including lung tissues. Deuteration at bis-allylic positions substantially decreases the overall rate of ARA oxidation when hydrogen abstraction is an initiating event. To compare the effects of dosing of arachidonic acid (H-ARA) and its bis-allylic hexadeuterated form (D-ARA) on lungs in conventionally healthy mice and in an acute lung injury model, mice were dosed with H-ARA or D-ARA for six weeks through dietary supplementation and then challenged with intranasal lipopolysaccharide (LPS) for subsequent analysis of bronchoalveolar lavage fluid and lung tissue. Dosing on D-ARA resulted in successful incorporation of D-ARA into various tissues. D-ARA significantly reduced LPS-induced adverse effects on alveolar septal thickness and the bronchoalveolar area. Oral deuterated ARA is taken up efficiently and protects against adverse LPS-induced pathology. This suggests novel therapeutic avenues for reducing lung damage during severe infections and other pathological conditions with inflammation in the pulmonary system and other inflammatory diseases

A Caenorhabditis elegans mutant lacking functional nicotinamide nucleotide transhydrogenase displays increased sensitivity to oxidative stress

Proton-translocating mitochondrial nicotinamide nucleotide transhydrogenase (NNT) was investigated regarding its physiological role in Caenorhabditis elegans. NNT catalyzes the reduction of NADP+ by NADH driven by the electrochemical proton gradient, Delta p, and is thus a potentially important source of mitochondrial NADPH. Mitochondrial detoxification of reactive oxygen species (ROS) by glutathione-dependent peroxidases depends on NADPH for regeneration of reduced glutathione. Transhydrogenase may therefore be directly involved in the defense against oxidative stress. nnt-1 deletion mutants of C elegans, nnt-1(sv34), were isolated and shown to grow essentially as wild type under normal laboratory conditions, but with a strongly lowered GSH/GSSG ratio. Under conditions of oxidative stress, caused by the superoxide-generating agent methyl viologen, growth of worms lacking nnt-1 activity was severely impaired. A similar result was obtained by using RNAi. Reintroducing nnt-1 in the nnt-1(sv34) knockout mutant led to a partial rescue of growth under oxidative stress conditions. These results provide evidence for the first time that nnt-1 is important in the defense against mitochondrial oxidative stress. (c) 2005 Elsevier Inc. All rights reserved

Expression and cellular localization of Na,K-ATPase isoforms in the rat ventral prostate.

OBJECTIVE: To determine the expression and plasma membrane domain location of isoforms of Na,K-ATPase in the rat ventral prostate. MATERIALS AND METHODS: Ventral prostate glands from adult male rats were dissected, cryosectioned (7 micro m) and attached to poly-l-lysine coated glass slides. The sections were then fixed in methanol and subjected to indirect immunofluorescence and immunoperoxidase procedures using a panel of well-characterized monoclonal and polyclonal antibodies raised against known Na,K-ATPase subunit isoforms. Immunofluorescence micrographs were digitally captured and analysed by image analysis software. RESULTS: There was expression of Na,K-ATPase alpha1, beta1, beta2 and beta3 subunit isoforms in the lateral and basolateral plasma membrane domains of prostatic epithelial cells. The alpha1 isoform was abundant but there was no evidence of alpha2, alpha3 or gamma isoform expression in epithelial cells. The alpha3 isoform was not detected, but there was a relatively low level of alpha2 isoform expression in the smooth muscle and stroma. CONCLUSION: Rat prostate Na,K-ATPase consists of alpha1/beta1, alpha1/beta2 and alpha1/beta3 isoenzymes. These isoform proteins were located in the lateral and basolateral plasma membrane domains of ventral prostatic epithelial cells. The distribution and subcellular localization of Na,K-ATPase is different in rodent and human prostate. Basolateral Na,K-ATPase probably contributes to the establishment of transepithelial ionic gradients that are a prerequisite for the uptake of metabolites by secondary active transport mechanisms and active citrate secretion

Expression and cellular localization of Na,K-ATPase isoforms in the rat ventral prostate.

OBJECTIVE: To determine the expression and plasma membrane domain location of isoforms of Na,K-ATPase in the rat ventral prostate. MATERIALS AND METHODS: Ventral prostate glands from adult male rats were dissected, cryosectioned (7 micro m) and attached to poly-l-lysine coated glass slides. The sections were then fixed in methanol and subjected to indirect immunofluorescence and immunoperoxidase procedures using a panel of well-characterized monoclonal and polyclonal antibodies raised against known Na,K-ATPase subunit isoforms. Immunofluorescence micrographs were digitally captured and analysed by image analysis software. RESULTS: There was expression of Na,K-ATPase alpha1, beta1, beta2 and beta3 subunit isoforms in the lateral and basolateral plasma membrane domains of prostatic epithelial cells. The alpha1 isoform was abundant but there was no evidence of alpha2, alpha3 or gamma isoform expression in epithelial cells. The alpha3 isoform was not detected, but there was a relatively low level of alpha2 isoform expression in the smooth muscle and stroma. CONCLUSION: Rat prostate Na,K-ATPase consists of alpha1/beta1, alpha1/beta2 and alpha1/beta3 isoenzymes. These isoform proteins were located in the lateral and basolateral plasma membrane domains of ventral prostatic epithelial cells. The distribution and subcellular localization of Na,K-ATPase is different in rodent and human prostate. Basolateral Na,K-ATPase probably contributes to the establishment of transepithelial ionic gradients that are a prerequisite for the uptake of metabolites by secondary active transport mechanisms and active citrate secretion

The non-gastric H,K-ATPase as a tool to study the ouabain-binding site in Na,K-ATPase.

Contains fulltext : 81222.pdf (publisher's version ) (Closed access)Based on studies with chimeras between (non-)gastric H,K-ATPase and Na,K-ATPase, a model for the ouabain binding site has recently been presented (Qiu et al. J.Biol.Chem. 280 (2005) 32349). In this model, hydrogen bonds between specific amino acid residues of Na,K-ATPase and hydroxyl groups of ouabain play a crucial role. In the present study, a series of ouabain analogues were tested on baculovirus-expressed Na,K-ATPase and an ouabain-sensitive mutant of non-gastric H,K-ATPase (D312E/ S319G/ A778P/ I795L/ F802C). For each analogue, the results obtained by measuring ATPase inhibition and [(3)H]ouabain replacement agreed rather well. In Na,K-ATPase, strophanthidin had a 7-10 times higher and digoxin a 4-12 times lower affinity than ouabain. The results of the non-gastric H,K-ATPase mutant were rather similar to that of Na,K-ATPase with exception of dihydro-ouabain that showed a much lower affinity with the non-gastric H,K-ATPase mutant. Docking studies showed that all analogues bind to the same pocket in Na,K-ATPase. However, the amino acids to which hydrogen bonds were formed differed and depended on the availability of hydroxyl or keto groups in the ouabain analogues