Article Figures & Data
Figures
- Figure 1.
Inhibition of glutamate uptake by S1P contrasts with S1P receptor modulator effects. Primary astrocytes were incubated with labeled glutamate, S1P, or respective agonists for 1 h at 37°C. The glutamate uptake assay was performed as described. A, S1P inhibited glutamate uptake by astrocytes in a dose-dependent manner, while the S1P1-specific agonist, RP001, did not. B, The relative expression of S1P1-5 (normalized to β-actin) in wild-type primary astrocytes derived from cerebral cortices of P0 pups. C, The S1P1,5 modulator, BAF312, did not induce glutamate uptake inhibition at the indicated doses. D, The S1P1,3,4,5 modulator, FTY720-P, induced partial inhibition. A, C, D, Data are from two independent experiments and curve fitting was performed by nonlinear regression. E, Incubation with FTY720-P for 3.5 d, followed by stimulation with S1P, induced glutamate uptake inhibition similar to no FTY720-P pretreatment controls. Data are representative of three independent experiments. Error bars indicate (mean ± SD). Statistics are calculated by two-way ANOVA performing Sidak’s multiple comparisons test (***p < 0.0005, **p < 0.005; ns, not significant).
- Figure 2.
Glutamate uptake inhibition is not eliminated in S1P3-null and astrocyte-specific (As)-S1P1/S1P3-double-null astrocytes. Primary astrocytes from As-S1P1, S1P3, and As-S1P1/S1P3 receptor-null mice were incubated with labeled glutamate and S1P for 1 h at 37°C. A, Glutamate uptake in As-S1P1−/− astrocytes exhibited a slight left shift (decrease in glutamate uptake) compared with littermate controls. S1P3−/− (B) and As-S1P1/S1P3−/− (C) astrocytes exhibited control levels of glutamate uptake. Data in each panel are from three independent experiments. Curve fitting was performed by nonlinear regression.
- Figure 3.
Pharmacological inhibition of S1P2, but not S1P1 or S1P3, eliminates astrocytic inhibition of glutamate uptake by S1P. Primary astrocytes were preincubated (A) with or without 10 μM VPC23019, an antagonist for S1P1 and S1P3, for 45 min, (B) 10 μM JTE 013, an S1P2 antagonist, for 30 min, (C) 200 ng/mL PTX overnight, and (D) 10 μM Y27632 for 30 min at 37°C followed by incubation with or without 20 nM S1P for 1 h. Glutamate uptake assays were performed as described. Data in each panel are representative of two independent experiments (n = 3 or 4). Error bars represent mean ± SD; ns, not significant; **p < 0.005, ***p < 0.001, two-way ANOVA with Sidak’s multiple comparisons test.
- Figure 4.
Genetic removal of S1P2 or Gα12 downstream signaling reduces astrocytic glutamate uptake inhibition by S1P. Primary astrocytes from S1P2-null or Gα12-hemizygous-null mice were incubated with labeled glutamate and S1P for 1 h at 37°C. Glutamate uptake in S1P2−/− (A) and Gα12+/− (B) astrocytes exhibited a right shift (increase in glutamate uptake) when compared with controls. Data in each panel are from three independent experiments. Curve fitting was performed by nonlinear regression.
- Figure 5.
S1P increases astrocyte mitochondrial oxygen consumption rate (OCR) that is S1P2 dependent. A, Schematic of Seahorse Mito Stress analysis. B, Primary astrocytes were exposed to four conditions: vehicle, 100 μM glutamate, 1 μM S1P, or a combination of glutamate and S1P for 1 h at 37°C. After washing, Seahorse assay medium containing 1 mM pyruvate and 10 mM glucose was added and the OCR measured with an Agilent Seahorse XF24 Cell Mito Stress kit. OCR was measured three times per injection and was read in triplicate. C, S1P increased endogenous OCR when compared with vehicle *p < 0.05 unpaired t test. Non-mitochondrial respiratory rate (NMMR = OCR after rotenone/antimycin A addition; D), basal OCR (= endogenous OCR – NMRR; E), maximal OCR (= OCR after FCCP addition – NMRR; F), proton leak (= OCR after oligomycin injection – NMRR; G), and spare respiratory capacity (= maximal OCR – basal OCR; H) is represented under different conditions as indicated, n = 5 with OCR measured in triplicate at each step. Data are representative of two independent experiments with similar results. D–H, Ordinary one-way ANOVA with Tukey’s multiple comparisons test (*p < 0.05, **p < 0.005, ***p ≤ 0.0005, ****p < 0.0001). I, Wild-type and S1P2 null primary astrocytes were incubated with Seahorse assay medium containing 1 mM pyruvate and 10 mM glucose followed by treatment with 10 nM S1P for 1 h. OCR was then measured using the Agilent Seahorse XF24 analyzer. % OCR in the presence of S1P was calculated with respect to vehicle as indicated, ****p < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test. ns, not significant.
Tables
Figure Method performed 1A,C,D Non-linear regression 1E Two-way ANOVA/Sidak’s multiple comparisons test 2A–C Non-linear regression 3A–D Two-way ANOVA/Sidak’s multiple comparisons test 4A,B Non-linear regression 5C Unpaired t test 5D–H Ordinary one-way ANOVA/Tukey’s multiple
comparisons test5I Two-way ANOVA/Tukey’s multiple comparisons test
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