Figure 3.

Glutamate Interferes with Pi Uptake by VGLUTs

(A) (Left) The Pi content of VGLUT-expressing COS7 cell lines was determined as in Figure 1. The cells were incubated in Pi-containing uptake buffer (10 mM) in the presence of increasing glutamate concentrations. (Right) In parallel experiments, glutamate content was measured after starving the cells in glutamate-free KREBS 4.7 mM [K⁺] and then incubating them in uptake buffer ± 10 mM [Glu] ± 10 mM [Pi]. Note that all cell lines showed 2.5- to 4-fold higher glutamate content when incubated in 10 mM glutamate compared to cells receiving no glutamate (control). Glutamate uptake was not inhibited by 10 mM [Pi] (right Glu + Pi). In addition, it was not significantly changed by the expression of VGLUTs (Figure S3). The line colors given to the COS7-VGLUT cell lines (left panel) also apply to the bars (right panel).

(B) Pi uptake by synaptosomes prepared from adult rats (left) or from E18 mice of the VGLUT2 strains (right) after incubation with 10 mM [Pi] in the presence of increasing concentrations of glutamate. Inhibition saturated around 10 mM glutamate. In E18 synaptosomes, the extent of inhibition of Pi uptake by 10 mM glutamate (bars) correlated with VGLUT expression.

Data are shown as % of control (samples incubated with 10 mM [Pi] uptake buffer without glutamate, A, left and B, or glutamate and Pi-free buffer, A, right). Dotted lines in the left panels represent the condition Pi + RB and show the effect of complete VGLUT inhibition. Values represent the mean ± SEM from at least 3 independent experiments/animals (A and B). Significance toward: (1) no glutamate added; (2) Pi; (9) MOCK; and (10) glutamate.