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Exp Eye Res
2007 Jan 01;841:191-9. doi: 10.1016/j.exer.2006.09.015.
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Functional and molecular analysis of D-serine transport in retinal Müller cells.
Dun Y
,
Mysona B
,
Itagaki S
,
Martin-Studdard A
,
Ganapathy V
,
Smith SB
.
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D-serine, an endogenous co-agonist of NMDA receptors in vertebrate retina, may modulate glutamate sensitivity of retinal neurons. This study determined at the functional and molecular level the transport process responsible for D-serine in retinal Müller cells. RT-PCR and immunoblotting showed that serine racemase (SR), the synthesizing enzyme for D-serine, is expressed in the rMC-1 Müller cell line and primary cultures of mouse Müller cells (1 degrees MCs). The relative contributions of different amino acid transport systems to d-serine uptake were determined based on differential substrate specificities and ion dependencies. D-serine uptake was obligatorily dependent on Na+, eliminating Na+-independent transporters (asc-1 and system L) for D-serine in Müller cells. The Na+:substrate stoichiometry for the transport process was 1:1. D-serine transport was inhibited by alanine, serine, cysteine, glutamine, and asparagine, but not anionic amino acids or cationic amino acids, suggesting that D-serine transport in Müller cells occurs via ASCT2 rather than ASCT1 or ATB0,+. The expression of mRNAs specific for ASCT1, ASCT2, and ATB0,+ was analyzed by RT-PCR confirming the expression of ASCT2 (and ASCT1) mRNA, but not ATB0,+, in Müller cells. Immunoblotting detected ASCT2 in neural retina and in 1 degrees MCs; immunohistochemistry confirmed these data in retinal sections and in cultures of 1 degrees MCs. The efflux of D-serine via ASCT2 by ASCT2 substrates was demonstrable using the Xenopus laevis oocyte heterologous expression system. These data provide the first molecular evidence for SR and ASCT2 expression in a Müller cell line and in 1 degrees MCs and suggest that D-serine, synthesized in Müller cells by SR, is effluxed via ASCT2 to regulate NMDA receptors in adjacent neurons.
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Fig. 1. Expression of serine racemase (SR) mRNA and protein in retinal Müller cells. (A) Total RNA (1 μg) isolated from rMC-1 cells was reverse-transcribed into cDNA and subjected to PCR using rat-specific SR primers (415 bp) (right lane). Molecular size markers were loaded in the left lane (M). The RT-PCR products were analyzed by 1% agarose gel electrophoresis. (B) Western blot analysis of SR (Mr ∼ 38 kD) and β-actin (Mr ∼ 45 kD) with proteins extracted from the rat retinal Müller cell line (rMC-1) and primary mouse Müller cells (1°MC).
Fig. 2. Ion dependence of d-serine uptake in retinal Muller cells. (A) Time course and ion dependence of d-serine uptake in rMC-1 cells. Uptake of [3H] d-serine (0.1 μM) in rMC-1 cells incubated at various times in NaCl-, Na-gluconate- and NMDG-Cl-containing uptake buffers. (B) Na+-activation kinetics of d-serine uptake in rMC-1 cells. Uptake of [3H] d-serine (0.1 μM) was determined following a 30 min incubation in uptake medium containing increasing concentrations of Na+ (0–140 mM). The concentration of Cl− was kept constant at 140 mM (inset, Hill plot). (C) Ion dependence of d-serine uptake in primary Müller cells; uptake of [3H] d-serine (0.1 μM) was determined following a 30 min incubation in NaCl-, Na-gluconate- and NMDG-Cl-containing uptake medium. Values are means ± SE for three determinations from two independent experiments. (*Significantly different from NaCl value, p < 0.05.)
Fig. 3. Substrate specificity of the transport system responsible for d-serine uptake in Müller cells. Uptake of 0.1 μM [3H] d-serine was measured in the absence or presence of various unlabeled amino acids (5 mM) in (A) rMC-1 cells and (B) 1°MCs. Uptake was measured for 30 min in the presence of NaCl. Data are presented as the percentage of control uptake (100%) measured in the absence of competing amino acids. Values are means ± SE for three determinations from two independent experiments. (*Significantly different from control value, p < 0.05.)
Fig. 4. Relative affinities of d-serine and l-serine for the Na+-dependent d-serine uptake process in Müller cells. The Na+-dependent uptake of [3H] d-serine (0.1 μM) was measured for 30 min in the presence of increasing concentrations of unlabeled d-serine (○) and l-serine (●) in rMC-1 cells (A) and 1°MCs (B). Data are presented as the percentage of control uptake (100%) measured in the absence of competing amino acids.
Fig. 5. RT-PCR analysis of the expression of ASCT1, ASCT2 and ATB0,+ in rMC-1 and 1°MCs. (A) Total RNA (1 μg) isolated from rMC-1 cells was reverse-transcribed into cDNA and subjected to PCR using rat-specific ASCT1 and ASCT2 primers yielding products with the expected band sizes of 399 and 478 bp, respectively. (B) Total RNA (1 μg) isolated from 1°MCs reverse-transcribed into cDNA and subjected to PCR using mouse-specific ASCT1 and ASCT2 primers yielded products with the expected sizes 536 and 506, respectively. (C) Total RNA (1 μg) was isolated from mouse lung (positive control), rMC-1 and 1°MCs, reverse-transcribed into cDNA, and subjected to PCR using primers specific for mouse or rat ATB0,+. The mouse lung yielded a product with the expected band size of 617 bp; however, neither rMC-1 nor 1°MCs yielded any PCR product for ATB0,+. In each panel, the left lane represents DNA markers and the band sizes of 500 and 650 bp are labeled.
Fig. 6. Immunodetection of ASCT2 in retina and primary mouse Müller cells (1°MCs). (A) Immunoblotting of lung, brain, kidney and neural retina showing detection of ASCT2 (Mr ∼ 36–45 kD). (B) Immunoblotting of 1°MCs to detect ASCT2. (C) Immunohistochemical detection of ASCT2 in intact retina. Cyrosections were prepared from mouse retina and subjected to immunohistochemistry as described to detect ASCT2 followed by the Alexa fluor 488 conjugated secondary antibody (green staining); DAPI was used to label nuclei (blue). Calibration bar = 50 μm. (D) ASCT2 was detected in 1°MCs, and as shown in the inset, the labeling in these primary cells was largely cytoplasmic.
Fig. 7. [3H]-d-serine efflux in hASCT2-expressing oocytes. (A) Oocytes injected with water (H2O) or hASCT2 cRNA were incubated with [3H] d-serine (5 μM) for 1 h in an Na+-containing medium. At the end of the incubation, oocytes were washed and the radioactivity associated with the oocytes was determined to calculate d-serine uptake. (B) Oocytes injected with hASCT2 cRNA were preloaded with [3H] d-serine by incubation for 1 h in an Na+-containing medium with 5 μM [3H] d-serine. Oocytes were then washed and used for efflux measurements. Efflux of radiolabel was measured for 30 min by incubating the oocytes in a medium containing either no amino acid or 2.5 mM unlabeled l-alanine (Ala), l-glutamine (Gln) or l-glutamate (Glu) in the presence of NaCl. Data are expressed as mean ± SE. (*Significantly different from control value p < 0.05.)
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