We demonstrated that l-glutamate is transported by GVs with an apparent affinity and a biphasic chloride dependency comparable to that reported for SVs (Naito and Ueda, 1985; Maycox et al

We demonstrated that l-glutamate is transported by GVs with an apparent affinity and a biphasic chloride dependency comparable to that reported for SVs (Naito and Ueda, 1985; Maycox et al., 1988). before being processed for gel electrophoresis and immunoblotting with standard methods or in appropriate buffer for additional analysis. Isolations with murine IgG or at 21C were used as a control. Immunoisolation of SVs was performed Fluorocurarine chloride following the same protocol, with the following modifications. The rat cerebral cortex of a male Wistar rat (2 months) was homogenized with a glass/Teflon homogenizer (10 strokes at 2500 rpm) in homogenization buffer immediately after decapitation. The homogenate was centrifuged for 25 min at 35,000 = 0 and corrected for baseline shifts induced by volume changes associated with the additions. Uptake of radiolabeled amino acids. Amino acid uptake inside immunoisolated vesicles was analyzed by a filtration assay with radiolabeled amino acids as explained previously (Burger et al., 1991). One-hundred-microliter aliquots of Sb2-immunoisolated beads resuspended in assay buffer (0.32 m sucrose, 4 mm KCl, 4 mm MgSO4, and 10 mm HEPES-KOH, pH 7.4) were incubated with 400 m d-serine or 40 m l-glutamate containing 4 Ci d-[3H]serine or 2 Ci l-[3H]glutamate (PerkinElmer Life and Analytical Sciences). Uptake activities were assayed by quick filtration Rabbit Polyclonal to PRKAG2 on glass fiber filters (GF/C) using a cell harvester system (Connectorate) and by scintillation counting. Determination of amino acid vesicular content. Immunoisolation was performed at 0C to prevent vesicular content leakage (Burger et al., 1991). Starting material and immunoisolates were treated with 5% (w/v) trichloroacetic acid to extract free amino acids as explained previously (Hashimoto et al., 1992). Amino acids were determined by a laboratory-assembled capillary electrophoresis with laser-induced fluorescence (CECLIF) system (Scanlan et al., 2010) using pre-column derivatization with 10 mm naphthalene-2,3-dicarboxaldehyde and 20 mm KCN for 20 min in the dark. Chiral separation and quantitation were achieved by using a separation buffer consisting of 10 mm -cyclodextrin, 30 mm SDS, and 75 mm borate buffer at pH 10.5. In addition, both a CECLIF separation with non-chiral separation conditions (50 mm borate buffer at pH 8.42) and capillary electrophoresis-mass spectrometry (CECMS) were used to verify analyte identifications and quantitation. The laboratory-assembled CECMS instrument and separation conditions were as explained previously (Lapainis et al., 2009; Nemes et al., 2011). Amino acid contents were corrected for nonspecific adsorption on beads determined by isolating vesicles at 21C Fluorocurarine chloride and normalized to Sb2 quantified by immunoblotting. Immunostainings. Astrocytes cultured on glass coverslips were immunostained with pairs of antibodies as explained previously (Martineau et al., 2008). Immunostainings of cerebral cortex with rabbit polyclonal anti-d-serine antibody was performed as explained previously (Puyal et al., 2006) on 30 m coronal brain sections of 2-month-old male rats. Images were acquired using a confocal microscope (Leica TCS SP2), and colocalization was quantified using NIH ImageJ software as explained previously (Martineau et al., 2008). Statistical analyses. Uptake and acidification kinetics were analyzed using Prism4 software (GraphPad Software). Results are reported as mean SEM from three to six impartial experiments. Statistical significance was defined at 0.05 using the Student’s test. Results Glial Sb2-made up of vesicles are similar to SVs Although d-serine was originally shown to be present in brain astrocytes (Schell et al., 1995; Panatier et al., 2006; Williams et al., 2006), recent investigations have revealed that both d-serine and its synthesizing enzyme SR are also present in neurons (Kartvelishvily et al., 2006; Miya et al., 2008). Therefore, to clarify the respective contribution of astrocytes and neurons in d-serine disposition in the mature rat cerebral cortex, we performed immunostainings for d-serine. Double immunostainings for d-serine with the astroglial marker glutamine synthase revealed that d-serine is mainly present in the soma and processes of Fluorocurarine chloride astroglia (Fig. 1(Harris and Sultan, 1995; Bezzi et al., 2004; Bergersen et al., 2012). Control beads coupled to non-immune IgG were devoid of bound membranes, suggesting that nonspecific contamination by other membranes is usually negligible. We next resolved the protein composition of immunoisolated GVs by immunoblotting (Fig. 2for numerous marker proteins specific for SVs (for comparison with GVs. Vesicular d-serine uptake is dependent on chloride concentration and is Fluorocurarine chloride specific to astroglia The presence of d-serine in GVs suggests that GVs possess a specific transporter for this gliotransmitter. Properties of vesicular transporters have been mainly analyzed in purified SV fractions. All of them are driven by a proton electrochemical potential (H+) generated by a V-ATPase (Edwards, 2007). We therefore examined.

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