We measured changes in the molar concentration of cytosolic Ca2' ([Ca2+1]) in individual astrocytes in culture produced by the glutamate analog quisqualate (QA) and related substances by using fura-2 digital fluorescence microscopy. In cells cultured from the cortex, hippocampus, and cerebellum, the QA analog a-amino-3-hydroxy-5-methyl-isoxaiole-4-propionate (AMPA; 10 ,uM) produced a slow increase in [Ca2J11 that was modest in amplitude (-200 nM). These effects were completely abolished by 10 ,IM

Ochratoxin A (OTA) is a mycotoxin occurring naturally in a wide range of food commodities. In animals, it has been shown to cause a variety of adverse effects, nephrocarcinogenicity being the most prominent. Because of its high toxic potency and the continuous exposure of the human population, OTA has raised public health concerns. There is significant debate on how to use the rat carcinogenicity data to assess the potential risk to humans. In this context, the question of the mechanism of action of OTA appears of key importance and was studied through the application of a toxicogenomics approach. Male Fischer rats were fed OTA for up to 2 years. Renal tumors were discovered during the last 6 months of the study. The total tumor incidence reached 25% at the end of the study. Gene expression profile was analyzed in groups of animals taken in intervals from 7 days to 12 months. Tissue-specific responses were observed in kidney versus liver. For selected genes, microarray data were confirmed at both mRNA and protein levels. In kidney, several genes known as markers of kidney injury and cell regeneration were significantly modulated by OTA. The expression of genes known to be involved in DNA synthesis and repair, or genes induced as a result of DNA damage, was only marginally modulated. Very little or no effect was found amongst genes associated with apoptosis. Alterations of gene expression indicating effects on calcium homeostasis and a disruption of pathways regulated by the transcription factors hepatocyte nuclear factor 4 alpha (HNF4alpha) and nuclear factor-erythroid 2-related factor 2 (Nrf2) were observed in the kidney but not in the liver. Previous data have suggested that a reduction in HNF4alpha may be associated with nephrocarcinogenicity. Many Nrf2-regulated genes are involved in chemical detoxication and antioxidant defense. The depletion of these genes is likely to impair the defense potential of the cells, resulting in chronic elevation of oxidative stress in the kidney. The inhibition of defense mechanism appears as a highly plausible new mechanism, which could contribute to OTA carcinogenicity.

We examined the effects of different types of glutamate receptor agonists on the intracellular calcium concentration, ([Ca2+]i), in cultured rat cortical glial cells. The cells in these cultures were characterized immunocytochemically using antibodies against glial fibrillary acidic protein, A2B5, and OX-42. The metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3- dicarboxylic acid produced Ca2+ mobilization from intracellular stores in all classes of cells. Agonists at non-NMDA glutamate receptors also produced large increases in [Ca2+]i, primarily in cells of the O-2A lineage. Disruption of intracellular Ca2+ stores with thapsigargin showed that increases in [Ca2+]i produced by activating AMPA/kainate receptors were primarily due to Ca2+ influx rather than Ca(2+)-induced Ca2+ release. Agonists at NMDA receptors were ineffective. Electrophysiological studies revealed that cells of the O-2A lineage exhibited moderate inward currents in response to kainate in Na(+)-containing solutions, but only small inward currents and outward rectification in Na(+)-free solutions. However, in the presence of cyclothiazide, the kainate-induced currents were increased in size and a rightward shift of the reversal potential with increased [Ca2+]o could be demonstrated. Activation of cells by kainate, but not by depolarizing stimuli, stimulated the uptake of Co2+. Polymerase chain reaction studies showed that the glutamate receptor subunits GluR1-4 and GluR6 were all expressed in these cultures, but GluR5 was absent. The nature of the Ca2+ uptake pathway activated by non-NMDA receptor agonists in the O-2A lineage population is discussed. It is considered most likely that the O-2A lineage cells express both non-NMDA receptors that are relatively impermeable to divalent cations, as well as a smaller population that are Ca2+ permeable.

No abstract

Dysregulation of intestinal epithelial cell performance is associated with an array of pathologies whose onset mechanisms are incompletely understood. While whole-genomics approaches have been valuable for studying the molecular basis of several intestinal diseases, a thorough analysis of gene expression along the healthy gastrointestinal tract is still lacking. The aim of this study was to map gene expression in gastrointestinal regions of healthy human adults and to implement a procedure for microarray data analysis that would allow its use as a reference when screening for pathological deviations. We analyzed the gene expression signature of antrum, duodenum, jejunum, ileum, and transverse colon biopsies using a biostatistical method based on a multivariate and univariate approach to identify region-selective genes. One hundred sixty-six genes were found responsible for distinguishing the five regions considered. Nineteen had never been described in the GI tract, including a semaphorin probably implicated in pathogen invasion and six novel genes. Moreover, by crossing these genes with those retrieved from an existing data set of gene expression in the intestine of ulcerative colitis and Crohn's disease patients, we identified genes that might be biomarkers of Crohn's and/or ulcerative colitis in ileum and/or colon. These include CLCA4 and SLC26A2, both implicated in ion transport. This study furnishes the first map of gene expression along the healthy human gastrointestinal tract. Furthermore, the approach implemented here, and validated by retrieving known gene profiles, allowed the identification of promising new leads in both healthy and disease states.