Amperometric measurement of glutamate release modulation by gabapentin and pregabalin in rat neocortical slices: role of voltage-sensitive Ca2+ α2δ-1 subunit

Abstract

Gabapentin (GBP; Neurontin) and pregabalin (PGB; Lyrica, S-(+)-3-isobutylgaba) are used clinically to treat several disorders associated with excessive or inappropriate excitability, including epilepsy; pain from diabetic neuropathy, postherpetic neuralgia, and fibromyalgia; and generalized anxiety disorder. The molecular basis for these drugs' therapeutic effects are believed to involve the interaction with the auxiliary α(2)δ subunit of voltage-sensitive Ca(2+) channel (VSCC) translating into a modulation of pathological neurotransmitter release. Glutamate as the primary excitatory neurotransmitter in the mammalian central nervous system contributes, under conditions of excessive glutamate release, to neurological and psychiatric disorders. This study used enzyme-based microelectrode arrays to directly measure extracellular glutamate release in rat neocortical slices and determine the modulation of this release by GBP and PGB. Both drugs attenuated K(+)-evoked glutamate release without affecting basal glutamate levels. PGB (0.1-100 μM) exhibited concentration-dependent inhibition of K(+)-evoked glutamate release with an IC(50) value of 5.3 μM. R-(-)-3-Isobutylgaba, the enantiomer of PGB, did not significantly reduce K(+)-evoked glutamate release. The decrease of K(+)-evoked glutamate release by PGB was blocked by the l-amino acid l-isoleucine, a potential endogenous ligand of the α(2)δ subunit. In neocortical slices from transgenic mice having a point mutation (i.e., R217A) of the α(2)δ-1 (subtype) subunit of VSCC, PGB did not affect K(+)-evoked glutamate release yet inhibited this release in wild-type mice. The results show that GBP and PGB attenuated stimulus-evoked glutamate release in rodent neocortical slices and that the α(2)δ-1 subunit of VSCC appears to mediate this effect.

Fig. 1.

Effect of GBP (100 μM) on K⁺-evoked glutamate release from rat neocortical slices. A, glutamate release evoked by repeated pressure-ejection delivery of 70 mM K⁺ solution (arrowheads on abscissa) in the absence (first three traces) and presence of GBP (last three traces). B, amplitude of K⁺-evoked glutamate release (as derived from A) was decreased by GBP. Values given are X ± S.E. (n = 6). The paired t statistic gave t(5) = 2.930 (p = 0.0326). A significant difference from the control value is indicated by an asterisk (*, p ≤ 0.05).

Fig. 2.

Effect of PGB (100 μM) on K⁺-evoked glutamate release in rat neocortical slices. A, detection of glutamate release by MEAs after repeat superfusion with 70 mM K⁺ (arrowheads; S₁, S₂) for 50 s. B, PGB (closed bar), present 15 min before S₂, attenuated glutamate release.

Fig. 3.

Effects of PGB (0.1–100 μM), R-(−)-3-isobutylgaba (100 μM), and l-isoleucine (100 μM) to inhibit K⁺-evoked glutamate release in rat neocortical slices. A, concentration–effect relationship of PGB and inactivity of R-(−)-3-isobutylgaba, l-isoleucine, and PGB (100 μM) and l-isoleucine (100 μM) combination after repeat superfusion with 70 mM K⁺ (S₁, S₂) for 50 s. Substances were present 15 min before S₂. Values given are X ± S.E. (n = 7). Analysis of variance of S₂/S₁ values for control and PGB concentrations gave F(4,30) = 5.17 (p = 0.003). A significant difference from the control value is indicated by an asterisk (*, p ≤ 0.05 and ***, p ≤ 0.001). The S₂/S₁ ratios obtained for the other substances, including the PGB and l-isoleucine combination, were not significantly different from the control value. B, the transformed data from A depict inhibition (%) by PGB relative to the mean control S_2/S₁ ratio of 0.97 normalized to 1.0; the corresponding IC₅₀ value was 5.3 μM.

Fig. 4.

Effects of PGB (100 μM) on K⁺-evoked glutamate release in neocortical slices from wild-type mice and transgenic mice having a point mutation (i.e., R217A) of the VSCC α₂δ-1 subunit. Glutamate release was assessed with MEAs after repeated superfusion with 70 mM K⁺ (S₁, S₂) for 50 s. Slices from wild-type mice were treated as control (A) and exposed to PGB (15 min before S₂) (B). Meanwhile, slices from transgenic mice (R217A) were treated as control (C) and exposed to PGB (15 min before S₂) (D). Open bars, 70 mK K⁺; closed bars, PGB.

Fig. 5.

The point mutation R217A prevents PGB from attenuating K⁺-evoked glutamate release in slices. The S₂/S₁ ratio derived from repeated K⁺ superfusion of slices was decreased by PGB. A main effect of PGB treatment was identified with a two-way analysis of variance [F(1,41) = 5.57 (p = 0.023)], and a post hoc Bonferroni test revealed a significant effect (p < 0.05) of PGB only in slices from wild-type animals. A significant difference from the control value is indicated by an asterisk (*, p ≤ 0.05). Values given are X ± S.E.