Riluzole and gabapentinoids activate glutamate transporters to facilitate glutamate-induced glutamate release from cultured astrocytes

Abstract

We have recently demonstrated that the glutamate transporter activator riluzole paradoxically enhanced glutamate-induced glutamate release from cultured astrocytes. We further showed that both riluzole and the α(2)δ subunit ligand gabapentin activated descending inhibition in rats by increasing glutamate receptor signaling in the locus coeruleus and hypothesized that these drugs share common mechanisms to enhance glutamate release from astrocytes. In the present study, we examined the effects of riluzole and gabapentin on glutamate uptake and release and glutamate-induced Ca(2+) responses in primary cultures of astrocytes. Riluzole and gabapentin facilitated glutamate-induced glutamate release from astrocytes and significantly increased glutamate uptake, the latter being completely blocked by the non-selective glutamate transporter blocker DL-threo-β-benzyloxyaspartic acid (DL-TBOA). Riluzole and gabapentin also enhanced the glutamate-induced increase in intracellular Ca(2+) concentrations. Some α(2)δ subunit ligands, pregabalin and L-isoleucine, enhanced the glutamate-induced Ca(2+) response, whereas another, 3-exo-aminobicyclo[2.2.1]heptane-2-exo-carboxylic acid (ABHCA), did not. The enhancement of glutamate-induced intracellular Ca(2+) response by riluzole and gabapentin was blocked by the DL-TBOA and an inhibitor of Na(+)/Ca(2+) exchange, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiurea (KB-R7943). Gabapentin's enhancement of Ca(2+) increase was specific to glutamate stimulation, as it was not mimicked with stimulation by ATP. These results suggest that riluzole and gabapentin enhance Na(+)-glutamate co-transport through glutamate transporters, induce subsequent Ca(2+) influx via the reverse mode of Na(+)/Ca(2+) exchange, and thereby facilitate Ca(2+)-dependent glutamate release by glutamate in astrocytes. The present study also demonstrates a novel target of gabapentinoid action in astrocytes other than α(2)δ subunits in neurons.

Fig. 1

[³H]-glutamate uptake in astrocytes is presented as a total radioactivivity (cpm) per well. Cells were pretreated with buffer (control), gabapentin (GBP, 1–100 μM) or riluzole (RIL, 1 μM) in the presence or absence of TBOA (50 μM) for 5 min and glutamate uptake was performed for 1 min in the presence of test drugs. In non-pretreatment group, GBP (100 μM) was co-applied with glutamate without the pretreatment. Number in each column represents sample size. *P<0.01 vs. control.

Fig. 2

Effects of gabapentin, pregabalin and riluzole on glutamate-induced glutamate release from astrocytes. Cells were treated with buffer or glutamate (10 μM) for 5 min in the presence of buffer alone, gabapentin (GBP, 1–100 μM), pregabalin (PGB, 10–100 μM), or riluzole (RIL, 0.1–1 μM). [³H]-glutamate release is presented as a percentage of total radioactivivity. Number in each column represents sample size. *P<0.01 vs. buffer alone. #P<0.01 vs. glutamate alone.

Fig. 3

(A) A representative intracellular Ca²⁺ response in an astrocyte stimulated with glutamate, presented as change in Fura-2 fluorescence ratio. (B) Concentration response of glutamate (10–400 μM)-induced Fura-2 response. Each box represents the 25^th, 50^th, and 75^th percentiles of Fura-2 fluorescence ratio response. Number under each box represents sample size.

Fig. 4

(A) Representative intracellular Ca²⁺ responses in astrocytes stimulated with glutamate (Glu) after perfused with gabapentin (GBP) or riluzole (RIL), presented as change in Fura-2 fluorescence ratio. (B) Effects of RIL and GBP on basal Fura-2 fluorescence ratio. Basal values were determined from the average ratio for 1 min before and after starting vehicle, RIL, or GBP perfusion. Number in each column represents sample size. (C) Effects of RIL (0.01–1 μM) and GBP (1–100 μM) on glutamate-induced intracellular Fura-2 response. Each box represents the 25^th, 50^th, and 75^th percentiles of Fura-2 fluorescence ratio response. Number under each box represents sample size. *P<0.05 vs. control (10 μM glutamate alone).

Fig. 5

Effects of pregabalin (PGB, 10–100 μM), L-isoleucine (L-ILe, 10–100 μM), and ABHCA (100 μM) on glutamate-induced intracellular Fura-2 response. Each box represents the 25^th, 50^th, and 75^th percentiles of Fura-2 fluorescence ratio response. Number under each box represents sample size. *P<0.05 vs. control (10 μM glutamate alone).

Fig. 6

(A) A representative intracellular Ca²⁺ concentration response in an astrocyte stimulated with glutamate (10 μM) after perfused with DL-TBOA (50 μM), presented as change in Fura-2 fluorescence ratio. (B) Effects of TBOA (5–50 μM) on gabapentin (GBP, 10 and 100 μM) and riluzole (Ril, 0.1 and 1 μM)-induced enhancement of glutamate-induced intracellular Fura-2 fluorescence ratio response. Each box represents the 25^th, 50^th, and 75^th percentiles of Fura-2 fluorescence ratio response in astrocytes. Number under each box represents sample size. *P<0.05 vs. without TBOA.

Fig. 7

(A) A representative intracellular Ca²⁺ concentration response in an astrocyte stimulated with glutamate (10 μM) after perfusion with KB-R7943 (30 μM), presented as change in Fura-2 fluorescence ratio. (B) Effects of KB-R7943 (30 μM) on gabapentin (GBP, 100 μM) and riluzole (Ril, 0.1–1 μM)-induced enhancement of glutamate-induced intracellular Fura-2 fluorescence ratio response. Each box represents the 25^th, 50^th, and 75^th percentiles of Fura-2 fluorescence ratio response. Number under each box represents sample size. *P<0.05 vs. without KB-R7943.