The stimulation of glutamine hydrolysis in isolated rat liver mitochondria by Mg2+ depletion and hypo-osmotic incubation conditions.
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ABSTRACT:
SUBMITTER: Joseph SK
PROVIDER: S-EPMC1162714 | biostudies-other | 1981 Jan
REPOSITORIES: biostudies-other
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Project description:In our previous work, we found that perfusion of the rat cerebral cortex with hypo-osmotic medium triggers massive release of the excitatory amino acid L-glutamate but decreases extracellular levels of L-glutamine (R. E. Haskew-Layton et al., PLoS ONE, 3: e3543). The release of glutamate was linked to activation of volume-regulated anion channels, whereas mechanism(s) responsible for alterations in extracellular glutamine remained unclear. When mannitol was added to the hypo-osmotic medium to reverse reductions in osmolarity, changes in microdialysate levels of glutamine were prevented, indicating an involvement of cellular swelling. As the main source of brain glutamine is astrocytic synthesis and export, we explored the impact of hypo-osmotic medium on glutamine synthesis and transport in rat primary astrocyte cultures. In astrocytes, a 40% reduction in medium osmolarity moderately stimulated the release of L-[(3) H]glutamine by ∼twofold and produced no changes in L-[(3) H]glutamine uptake. In comparison, hypo-osmotic medium stimulated the release of glutamate (traced with D-[(3) H]aspartate) by more than 20-fold. In whole-cell enzymatic assays, we discovered that hypo-osmotic medium caused a 20% inhibition of astrocytic conversion of L-[(3) H]glutamate into L-[(3) H]glutamine by glutamine synthetase. Using an HPLC assay, we further found a 35% reduction in intracellular levels of endogenous glutamine. Overall, our findings suggest that cellular swelling (i) inhibits astrocytic glutamine synthetase activity, and (ii) reduces substrate availability for this enzyme because of the activation of volume-regulated anion channels. These combined effects likely lead to reductions in astrocytic glutamine export in vivo and may partially explain occurrence of hyperexcitability and seizures in human hyponatremia.
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2003-04-15 | GSE407 | GEO
Project description:Environmental salinity creates a key barrier to limit the distribution of most aquatic organisms. Adaptation to osmotic fluctuation is believed to be a factor facilitating species diversification. Adaptive evolution often involves beneficial mutations at more than one locus. Bivalves hold great interest, with numerous species living in waters, as osmoconformers, who maintain the osmotic pressure balance mostly by free amino acids. In this study, 107,076,589 reads from two groups of Crassostrea hongkongensis were produced and the assembled into 130,629 contigs. Transcripts putatively involved in stress-response, innate immunity and cell processes were identified according to Gene ontology and KEGG pathway analyses. Comparing with the transcriptome of C. gigas to characterize the diversity of transcripts between species with osmotic divergence, we identified 182,806 high-quality single nucleotide polymorphisms (SNPs) for C. hongkongensis, and 196,779 SNPs for C. gigas. Comparison of 11,602 pairs of putative orthologs allowed for identification of 14 protein-coding genes that experienced strong positive selection (Ka/Ks>1). In addition, 45 genes that may show signs of moderate positive selection (1 ≥ Ka/Ks>0.5) were also identified. Based on Ks ratios and divergence time between the two species published previously, we estimated a neutral transcriptome-wide substitution mutation rate of 1.39 × 10(-9) per site per year. Several genes were differentially expressed across the control and treated groups of each species. This is the first time to sequence the transcriptome of C. hongkongensis and provide the most comprehensive transcriptomic resource available for it. The increasing amount of transcriptome data on Crassostrea provides an excellent resource for phylogenetic analysis. A large number of SNPs identified in this work are expected to provide valuable resources for future marker and genotyping assay development. The analysis of natural selection provides an innovative view on the adaptation within species and sets the basis for future genetic and evolutionary studies.
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Project description:Aneuploidy, which refers to unbalanced chromosome numbers, represents a class of genetic variation that is associated with cancer, birth defects and eukaryotic micro-organisms1-4. Whereas it is known that each aneuploid chromosome stoichiometry can give rise to a distinct pattern of gene expression and phenotypic profile4,5, it remains a fundamental question as to whether there are common cellular defects that are associated with aneuploidy. Here we show the existence in budding yeast of a common aneuploidy gene-expression signature that is suggestive of hypo-osmotic stress, using a strategy that enables the observation of common transcriptome changes of aneuploidy by averaging out karyotype-specific dosage effects in aneuploid yeast-cell populations with random and diverse chromosome stoichiometry. Consistently, aneuploid yeast exhibited increased plasma-membrane stress that led to impaired endocytosis, and this defect was also observed in aneuploid human cells. Thermodynamic modelling showed that hypo-osmotic-like stress is a general outcome of the proteome imbalance that is caused by aneuploidy, and also predicted a relationship between ploidy and cell size that was observed in yeast and aneuploid cancer cells. A genome-wide screen uncovered a general dependency of aneuploid cells on a pathway of ubiquitin-mediated endocytic recycling of nutrient transporters. Loss of this pathway, coupled with the endocytic defect inherent to aneuploidy, leads to a marked alteration of intracellular nutrient homeostasis.
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Project description:Mg(2+) plays important roles in numerous cellular functions. Mitochondria take part in intracellular Mg(2+) regulation and the Mg(2+) concentration in mitochondria affects the synthesis of ATP. However, there are few methods to observe Mg(2+) in mitochondria in intact cells. Here, we have developed a novel Mg(2+)-selective fluorescent probe, KMG-301, that is functional in mitochondria. This probe changes its fluorescence properties solely depending on the Mg(2+) concentration in mitochondria under physiologically normal conditions. Simultaneous measurements using this probe together with a probe for cytosolic Mg(2+), KMG-104, enabled us to compare the dynamics of Mg(2+) in the cytosol and in mitochondria. With this method, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP)-induced Mg(2+) mobilization from mitochondria to the cytosol was visualized. Although a FCCP-induced decrease in the Mg(2+) concentration in mitochondria and an increase in the cytosol were observed both in differentiated PC12 cells and in hippocampal neurons, the time-courses of concentration changes varied with cell type. Moreover, the relationship between mitochondrial Mg(2+) and Parkinson's disease was analyzed in a cellular model of Parkinson's disease by using the 1-methyl-4-phenylpyridinium ion (MPP(+)). A gradual decrease in the Mg(2+) concentration in mitochondria was observed in response to MPP(+) in differentiated PC12 cells. These results indicate that KMG-301 is useful for investigating Mg(2+) dynamics in mitochondria. All animal procedures to obtain neurons from Wistar rats were approved by the ethical committee of Keio University (permit number is 09106-(1)).
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Project description:The mosquito Aedes aegypti vectors the arboviral diseases yellow fever, dengue, Zika and chikungunya. Larvae are usually found developing in freshwater; however, more recently they have been increasingly found in brackish water, potential habitats which are traditionally ignored by mosquito control programs. Aedes aegypti larvae are osmo-regulators maintaining their hemolymph osmolarity in a range of ~ 250 to 300 mOsmol l-1. In freshwater, the larvae must excrete excess water while conserving ions while in brackish water, they must alleviate an accumulation of salts. The compensatory physiological mechanisms must involve the transport of ions and water but little is known about the water transport mechanisms in the osmoregulatory organs of these larvae. Water traverses cellular membranes predominantly through transmembrane proteins named aquaporins (AQPs) and Aedes aegypti possesses 6 AQP homologues (AaAQP1 to 6). The objective of this study was to determine if larvae that develop in freshwater or brackish water have differential aquaporin expression in osmoregulatory organs, which could inform us about the relative importance and function of aquaporins to mosquito survival under these different osmotic conditions. We found that AaAQP transcript abundance was similar in organs of freshwater and brackish water mosquito larvae. Furthermore, in the Malpighian tubules and hindgut AaAQP protein abundance was unaffected by the rearing conditions, but in the gastric caeca the protein level of one aquaporin, AaAQP1 was elevated in brackish water. We found that AaAQP1 was expressed apically while AaAQP4 and AaAQP5 were found to be apical and/or basal in the epithelia of osmoregulatory organs. Overall, the results suggest that aquaporin expression in the osmoregulatory organs is mostly consistent between larvae that are developing in freshwater and brackish water. This suggests that aquaporins may not have major roles in adapting to longterm survival in brackish water or that aquaporin function may be regulated by other mechanisms like post-translational modifications.
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Project description:Glutamine (Gln) and glucose (Glc) represent two important nutrients for proliferating cells, consistent with the observations that oncogenic processes are associated with enhanced glycolysis and glutaminolysis. Gln depletion and Glc depletion have been shown to trigger growth arrest and eventually cell death. Solid tumors often outgrow the blood supply, resulting in ischemia, which is associated with hypoxia and nutrient insufficiency. Whereas oxygen-sensing and adaptive mechanisms to hypoxia have been well-studied, how cells directly sense and respond to Gln and Glc insufficiency remains unclear. Using mRNA profiling techniques, we compared the gene expression profiles of acute Gln-depleted cells, Glc-depleted cells and cells adapted to Gln depletion. Here we report the global changes of the gene expression in those cells cultured under the defined nutrient conditions. Analysis of mRNA profiling data revealed that Gln and Glc depletion triggered dramatic gene expression reprogramming. Either Gln or Glc deletion leads to changes of the expression of cell cycle genes, but these conditions have distinctive effects on transcription regulators and gene expression profiles. Moreover, Gln and Glc depletion triggered distinguishable ER-stress responses. The gene expression patterns support that Gln and Glc have distinctive metabolic roles in supporting cell survival and proliferation, and cells use different mechanisms to sense and respond to Gln and Glc insufficiency. Our mRNA profiling database provides a resource for further investigating the nutrient-sensing mechanisms and potential effects of Glc and Gln abundance on the biological behaviors of cells.
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