Manganese induces oxidative impairment in cultured rat astrocytes.
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Excessive free radical formation has been implicated as a causative factor in neurotoxic damage associated with exposures to a variety of metals, including manganese (Mn). It is well established that Mn accumulates in astrocytes, affecting their ability to indirectly induce and/or exacerbate neuronal dysfunction. The present study examined the effects of Mn treatment on the following endpoints in primary astrocyte cultures: (1) oxidative injury, (2) alterations in high-energy phosphate (adenosine 5'-triphosphate, ATP) levels, (3) mitochondrial inner membrane potential, and (4) glutamine uptake and the expression of glutamine transporters. We quantified astrocyte cerebral oxidative damage by measuring F(2)-isoprostanes (F(2)-IsoPs) using stable isotope dilution methods followed by gas chromatography-mass spectrometry with selective ion monitoring. Our data showed a significant (p < 0.01) elevation in F(2)-IsoPs levels at 2 h following exposure to Mn (100 microM, 500 microM, or 1 mM). Consistent with this observation, Mn induced a concentration-dependent reduction in ATP and the inner mitochondrial membrane potential (DeltaPsi(m)), measured by the high pressure liquid chromatography method and the potentiometric dye, tetramethyl rhodamine ethyl ester, respectively. Moreover, 30 min of pretreatment with Mn (100 microM, 500 microM, or 1 mM) inhibited the net uptake of glutamine (GLN) ((3)H-glutamine) measured at 1 and 5 min. Expression of the messenger RNA coding the GLN transporters, SNAT3/SN1 and SNAT1, was inhibited after 100 and 500 microM Mn treatment for 24 h. Our results demonstrate that induction of oxidative stress, associated mitochondrial dysfunction, and alterations in GLN/glutamate cycling in astrocytes represent key mechanisms by which Mn exerts its neurotoxicity. (+info)
Segmental and complementary expression of L-serine biosynthetic enzyme 3-phosphoglycerate dehydrogenase and neutral amino acid transporter ASCT1 in the mouse kidney.
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3-Phosphoglycerate dehydrogenase (Phgdh) is the initial step enzyme in the phosphorylated pathway of L-serine biosynthesis. We have previously revealed in the brain that Phgdh is preferentially expressed in glial cells, but not in neurons, and that glia-borne L-serine exerts strong neurotrophic actions to neuronal survive, differentiation, and development. To investigate whether such an L-serine-meditated intercellular relationship is constructed in peripheral organs and tissues, we examined the kidney, which is one of the organs with the highest expression of Phgdh mRNA in the body. We found that Phgdh was distributed highly in the renal papilla and inner layer of the outer zone and moderately in the cortex, whereas it was almost negative in the outer layer of the outer zone. This heterogeneous distribution was due to selective expression in distinct tubular segments, i.e., the Bowman's capsule, proximal tubule, and thin limbs of the Henle's loop. Interestingly, neutral amino acid transporter ASCT1, which preferentially transports alanine, serine, cysteine, and threonine, was selectively expressed in Phgdh-negative tubular segments, i.e., the distal tubule and collecting duct. Therefore, either Phgdh or ASCT1 is provided to each segment of renal tubules, suggesting that metabolic interplay mediated by L-serine biosynthesis and supply may exist in the kidney too. (+info)
The human endogenous retrovirus envelope glycoprotein, syncytin-1, regulates neuroinflammation and its receptor expression in multiple sclerosis: a role for endoplasmic reticulum chaperones in astrocytes.
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Retroviral envelopes are pathogenic glycoproteins which cause neuroinflammation, neurodegeneration, and endoplasmic reticulum stress responses. The human endogenous retrovirus (HERV-W) envelope protein, Syncytin-1, is highly expressed in CNS glia of individuals with multiple sclerosis (MS). In this study, we investigated the mechanisms by which Syncytin-1 mediated neuroimmune activation and oligodendrocytes damage. In brain tissue from individuals with MS, ASCT1, a receptor for Syncytin-1 and a neutral amino acid transporter, was selectively suppressed in astrocytes (p < 0.05). Syncytin-1 induced the expression of the endoplasmic reticulum stress sensor, old astrocyte specifically induced substance (OASIS), in cultured astrocytes, similar to findings in MS brains. Overexpression of OASIS in astrocytes increased inducible NO synthase expression but concurrently down-regulated ASCT1 (p < 0.01). Treatment of astrocytes with a NO donor enhanced expression of early growth response 1, with an ensuing reduction in ASCT1 expression (p < 0.05). Small-interfering RNA molecules targeting Syncytin-1 selectively down-regulated its expression, preventing the suppression of ASCT1 and the release of oligodendrocyte cytotoxins by astrocytes. A Syncytin-1-transgenic mouse expressing Syncytin-1 under the glial fibrillary acidic protein promoter demonstrated neuroinflammation, ASCT1 suppression, and diminished levels of myelin proteins in the corpus callosum, consistent with observations in CNS tissues from MS patients together with neurobehavioral abnormalities compared with wild-type littermates (p < 0.05). Thus, Syncytin-1 initiated an OASIS-mediated suppression of ASCT1 in astrocytes through the induction of inducible NO synthase with ensuing oligodendrocyte injury. These studies provide new insights into the role of HERV-mediated neuroinflammation and its contribution to an autoimmune disease. (+info)
Differential regulation of homocysteine transport in vascular endothelial and smooth muscle cells.
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OBJECTIVE: We previously reported that homocysteine (Hcy) inhibits endothelial cell (EC) growth and promotes vascular smooth muscle cell (VSMC) proliferation. This study characterized and directly compared Hcy transport in cultured human aortic ECs (HAECs) and smooth muscle cells (HASMCs). METHODS AND RESULTS: Hcy (10 micromol/L) was transported into both cell types in a time-dependent fashion but was approximately 4-fold greater in HASMCs, and is nonstereoenantiomer specific. Hcy transport in HAECs had a Michaelis-Menten constant (Km) of 39 micromol/L and a maximal transport velocity (Vmax) of 873 pmol/mg protein/min. In contrast, Hcy transport in HASMCs had a lower affinity (Km = 106 micromol/L) but a higher transport capacity (Vmax = 4192 pmol/mg protein/min). Competition studies revealed that the small neutral amino acids tyrosine, cysteine, glycine, serine, alanine, methionine, and leucine inhibited Hcy uptake in both cell types, but the inhibition was greater for tyrosine, serine, glycine, and alanine in HAECs. Sodium-depletion reduced Hcy transport to 16% in HAECs and 56% in HASMCs. Increases in pH from 6.5 to 8.2 or lysosomal inhibitors blocked Hcy uptake only in HAECs. In addition, Hcy shares carrier systems with cysteine, in a preferable order of alanine-serine-cysteine (ASC) > aspartate and glutamate (X(AG)) = large branched-chain neutral amino acids (L) transporter systems in HAECs and ASC > L > X(AG) in HASMCs. The sodium-dependent system ASC plays a predominant role for Hcy transport in vascular cells. CONCLUSIONS: Transport system ASC predominantly mediates Hcy transport in EC and is lysosomal dependent. (+info)
Mapping of glutathione and its precursor amino acids reveals a role for GLYT2 in glycine uptake in the lens core.
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PURPOSE: To correlate the distribution of glutathione (GSH) and its precursor amino acids (cysteine, glycine, and glutamate) with the expression of their respective amino acid transporters in the rat lens. METHODS: Whole rat lenses were fixed, cryoprotected, and cryosectioned in either an equatorial or axial orientation. Sections were double labeled with cystine, glycine, glutamate, GSH, GLYT1, or GLYT2 antibodies, and the membrane marker wheat germ agglutinin (WGA). Sections were imaged by confocal laser scanning microscopy. Cystine, glycine, glutamate, and GSH labeling were quantified by using image-analysis software and intensity profiles plotted as a function of distance from the lens periphery. Western blot analysis was used to verify regional differences in amino acid transporter expression. RESULTS: Cystine and glycine labeling in equatorial sections was most intense in the outer cortex, was diminished in the inner cortex, but was increased again in the core relative to the inner cortex. Glutamate and GSH labeling was most intense in the outer cortex and was diminished in the inner cortex to a minimum that was sustained throughout the core. The distribution of cystine and glutamate levels correlated well with the expression patterns observed previously for the cystine/glutamate exchanger (Xc-) and the glutamate transporter (EAAT4/5), respectively. Although high levels of glycine labeling in the outer cortex correlated well with the expression of the glycine transporter GLYT1, the absence of GLYT1 in the core, despite an increase of glycine in this region, suggests an alternative glycine uptake system such as GLYT2 exists in the core. Equatorial sections labeled with GLYT2 antibodies, showed that labeling in the outer cortex was predominantly cytoplasmic, but progressively became more membranous with distance into the lens. In the inner cortex and core, GLYT2 labeling was localized around the entire membrane of fiber cells. Western blot analysis confirmed GLYT2 to be expressed in the outer cortex, inner cortex, and core of the lens. Axial sections labeled for glycine revealed a track of high-intensity glycine labeling that extended from the anterior pole through to the core that was associated with the sutures. CONCLUSIONS: The mapping of GSH and its precursor amino acids has shown that an alternative glycine uptake pathway exists in mature fiber cells. Although GLYT1 and -2 are likely to mediate glycine uptake in cortical fiber cells, GLYT2 alone appears responsible for the accumulation of glycine in the center of the lens. Enhancing the delivery of glycine to the core via the sutures may represent a pathway to protect the lens against the protein modifications associated with age-related nuclear cataract. (+info)
Acute administration of cefepime lowers L-carnitine concentrations in early lactation stage rat milk.
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Our study investigated the potential for important in vivo drug-nutrient transport interactions at the lactating mammary gland using the L-carnitine transporter substrates, cefepime and L-carnitine, as proof-of-concept. On d 4 (n = 6/treatment) and d 10 (n = 6/treatment) of lactation, rats were administered cefepime (250 mg/h) or saline by continuous i.v. infusion (4 h). Serum and milk L-carnitine and cefepime concentrations were quantified by HPLC-UV. In whole mammary gland, organic cation/carnitine transporter (OCTN)1, OCTN2, OCTN3, amino acid transporter B(0,+) (ATB(0,+)), and L-carnitine transporter 2 expression were determined by quantitative RT-PCR and by western blot and immunohistochemistry when possible. Cefepime caused a 56% decrease in milk L-carnitine concentrations on lactation d 4 (P = 0.0048) but did not affect milk L-carnitine at lactation d 10 or serum L-carnitine concentrations at either time. The mean L-carnitine and cefepime milk:serum ratios (M/S) decreased from 9.1 +/- 0.4 to 4.9 +/- 0.6 (P < 0.0001) and 0.89 +/- 0.3 to 0.12 +/- 0.02 (P = 0.0473), respectively, between d 4 and d 10 of lactation. In both groups, OCTN2 (P < 0.0001), OCTN3 (P = 0.0039), and ATB(0,+) (P = 0.004) mRNA expression and OCTN2 protein (P < 0.0001) were higher in mammary glands at d 4 of lactation compared with d 10. Immunohistochemistry revealed OCTN1 and OCTN2 localization in the mammary alveolar epithelium and OCTN3 expression in the interstitial space and blood vessel endothelium. In conclusion, cefepime significantly decreased milk L-carnitine concentrations only at d 4 of lactation. Relative to d 10, enhanced expression of OCTN2 and ATB(0,+) in mammary glands at d 4 of lactation and higher M/S (L-carnitine and cefepime) suggests cefepime competes with L-carnitine for L-carnitine transporters expressed in the lactating mammary gland to adversely affect L-carnitine milk concentrations and these effects depend upon lactation stage. (+info)
Association study of polymorphisms in the neutral amino acid transporter genes SLC1A4, SLC1A5 and the glycine transporter genes SLC6A5, SLC6A9 with schizophrenia.
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