Sodium Myo-inositol Transporter Research Articles

Inositol transport in mouse oocytes and preimplantation embryos: effects of mouse strain, embryo stage, sodium and the hexose transport inhibitor, phloridzin

The uptake of myo-inositol by mouse oocytes and preimplantation embryos of a crossbred (DBA x C57BL/6) and a purebred outbred strain (MF1) was measured using [2-(3)H]myo-inositol. Uptake in crossbred embryos increased about 15-fold between the one- and two-cell stages and increased again by about sixfold at the blastocyst stage compared with the morula stage. Uptake in purebred embryos increased about 42-fold between the one- and two-cell stages and increased more than threefold at the blastocyst stage compared with the morula stage. In all stages examined, except two-cell crossbred embryos, inositol uptake was, depending on the stage, either largely or partly sodium dependent and could be inhibited by the sodium-dependent hexose transport inhibitor, phloridzin. This is consistent with the hypothesis that transport occurs via a sodium myo-inositol transporter (SMIT) protein. In addition, there was strong evidence that a sodium-independent mechanism of uptake, possibly a channel, was switched on at the two-cell stage coincident with zygotic gene activation which resulted in 141-fold and 71-fold increases in sodium-independent uptake from the one-cell to two-cell stages in crossbred and purebred embryos, respectively. This mechanism was either abolished or drastically downregulated at the blastocyst stage, whereas sodium-dependent uptake was markedly upregulated. In two-cell crossbred embryos, there was a complete abolition of sodium-dependent uptake, again possibly regulated by zygotic gene activation. The hypothesis that the changes in mechanism of inositol uptake at about the two-cell stage are due to zygotic gene activation was supported by the finding that these changes did not occur in parthenogenetic two-cell embryos.

Compatible osmolytes modulate the response of porcine endothelial cells to hypertonicity and protect them from apoptosis.

Porcine pulmonary arterial endothelial cells accumulated myo-inositol and taurine, as well as betaine, during adaptation to hypertonic stress. The cells grew and maintained their normal morphology during culture in hypertonic (0.5 osmol (kg H(2)O)(-1)) medium that contained osmolytes such as betaine, myo-inositol or taurine at concentrations close to reported physiological values. The cells did not grow well in hypertonic medium depleted of potential compatible osmolytes. After a few days, cell density decreased by about 50 % and many cells rounded up and detached from the plates, their nuclei showing clear apoptotic morphology. The caspase-3 activity of the cells also increased dramatically under these conditions, but remained negligibly low when betaine and myo-inositol were added to the medium. Addition of betaine and myo-inositol to hypertonic medium depleted of compatible osmolytes increased the number of colonies remaining after 12 days of culture; with each solute at 30-100 micromol l(-1) the number increased about sixfold. In the absence of compatible osmolytes, increased mRNA levels and corresponding activities of betaine/gamma-aminobutyric acid transporter (BGT1) and sodium/myo-inositol transporter (SMIT) induced by hypertonicity remained high after 72 h incubation, whereas they were down regulated in the presence of betaine and myo-inositol. Similarly, the down regulation of the amino acid System A transporter (ATA2) was markedly slowed in the absence of compatible osmolytes. We conclude that these compatible osmolytes at concentrations close to physiological values enable the endothelial cells to adapt to hypertonic stress, protecting them from apoptosis, and also modulate the adaptation process.

Structure, chromosomal location, and analysis of the canine Cu/Zn superoxide dismutase (SOD1) gene.

Mutations in Cu/Zn superoxide dismutase (SOD1), a major cytosolic antioxidant enzyme in eukaryotic cells, have been reported in approximately 20% of familial amyotrophic lateral sclerosis (FALS) patients. Hereditary canine spinal muscular atrophy (HCSMA), a fatal inherited motor neuron disease in Brittany spaniels, shares many clinical and pathological features with human motor neuron disease, including FALS. The SOD1 coding region has been sequenced and cloned from several animal species, but not from the dog. We have mapped the chromosomal location, sequenced, and characterized the canine SOD1 gene. Extending this analysis, we have evaluated SOD1 as a candidate for HCSMA. The 462 bp SOD1 coding region in the dog encodes 153 amino acid residues and exhibits more than 83% and 79% sequence identity to other mammalian homologues at both the nucleotide and amino acid levels, respectively. The canine SOD1 gene maps to CFA31 close to syntenic group 13 on the radiation hybrid (RH) map in the vicinity of sodium myo/inositol transporter (SMIT) gene. The human orthologous SOD1 and SMIT genes have been localized on HSA 21q22.1 and HSA 21q21, respectively, confirming the conservation of synteny between dog syntenic group 13 and HSA 21. Direct sequencing of SOD1 cDNA from six dogs with HCSMA revealed no mutations. Northern analysis indicated no differences in steady-state levels of SOD1 mRNA.

Gene expression of the taurine transporter and taurine biosynthetic enzymes in rat kidney after antidiuresis and salt loading.

Taurine is thought to be an osmolyte in the kidney medulla. We have investigated the gene expression of the taurine transporter (TauT) and the enzymes of taurine biosynthesis, cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSD). We achieved this by measuring their mRNA levels using reverse transcriptase polymerase chain reaction (RT-PCR) in five kidney regions of rats in various hydration states; namely, normal hydration, after 2 days of antidiuresis following chronic diuresis and finally after acute salt loading. The mRNA levels of the well-established tonicity-sensitive genes coding for the aldose reductase (AR), the sodium myo-inositol transporter (SMIT) and the betaine transporter (BGT1) were also determined for the sake of comparison. In normally hydrated rats, TauT-, CDO-, and CSD-mRNA were enriched in the outer stripe of the outer medulla (OS). Following antidiuresis, the mRNA levels of TauT, CDO, CSD, SMIT, BGT1 and AR were all similarly increased in the papilla when compared with levels in rats submitted to a chronic diuresis. After acute salt loading, the mRNA level of TauT, like that of SMIT and BGT1, was overexpressed in OS whereas the mRNA levels of CDO and CSD remained unchanged. Like SMIT, BGT1 and AR genes, TauT, CDO and CSD genes appear to be tonicity-sensitive genes which can be activated in vivo by hypertonicity in the rat kidney. However, tonicity-induced activation of the TauT gene is more sensitive than that of CDO and CSD genes.