Elsevier

Neuropharmacology

Volume 42, Issue 2, February 2002, Pages 229-236
Neuropharmacology

Inhibition of neuronal Ca2+ influx by gabapentin and pregabalin in the human neocortex

https://doi.org/10.1016/S0028-3908(01)00172-1Get rights and content

Abstract

Gabapentin and pregabalin (S-(+)-3-isobutylgaba) produced concentration-dependent inhibitions of the K+-induced [Ca2+]i increase in fura-2-loaded human neocortical synaptosomes (IC50=17 μM for both compounds; respective maximal inhibitions of 37 and 35%). The weaker enantiomer of pregabalin, R-(−)-3-isobutylgaba, was inactive. These findings were consistent with the potency of these drugs to inhibit [3H]-gabapentin binding to human neocortical membranes. The inhibitory effect of gabapentin on the K+-induced [Ca2+]i increase was prevented by the P/Q-type voltage-gated Ca2+ channel blocker ω-agatoxin IVA. The α2δ-1, α2δ-2, and α2δ-3 subunits of voltage-gated Ca2+ channels, presumed sites of gabapentin and pregabalin action, were detected with immunoblots of human neocortical synaptosomes. The K+-evoked release of [3H]-noradrenaline from human neocortical slices was inhibited by gabapentin (maximal inhibition of 31%); this effect was prevented by the AMPA receptor antagonist NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydro[f]quinoxaline-7-sulphonamide). Gabapentin and pregabalin may bind to the Ca2+ channel α2δ subunit to selectively attenuate depolarization-induced Ca2+ influx of presynaptic P/Q-type Ca2+ channels; this results in decreased glutamate/aspartate release from excitatory amino acid nerve terminals leading to a reduced activation of AMPA heteroreceptors on noradrenergic nerve terminals.

Introduction

Gabapentin (GBP) and pregabalin (PGB; S-(+)-3-isobutylgaba) are efficacious drugs in the treatment of epilepsy, neuropathic pain, and anxiety states (Rosenberg et al., 1997, Chadwick et al., 1998 Abou-Khalil et al., 1999, Pande et al., 1999, Pande et al., 2000, Iacobellis et al., 2000). This broad spectrum of clinical utility has generated considerable interest; at the same time, significant preclinical efforts have been directed at unraveling the complicated mechanism(s) of action of these drugs (Taylor et al., 1998).

Both GBP and PGB are considered to bind to the α2δ subunit of voltage-gated Ca2+ channels (VGCC) with nanomolar affinity (Suman-Chauhan et al., 1993, Gee et al., 1996, Bryans and Wustrow, 1999, Gong et al., 2001). One functional consequence of this interaction, occurring at micromolar concentrations of these drugs, is the inhibition of Ca2+ influx through presynaptic P/Q-type VGCCs. For example, GBP decreased K+-induced [Ca2+]i increase in rat neocortical synaptosomes [IC50=14 μM (Fink et al., 2000); IC50=9.7 μM (Meder and Dooley, 2000a)] and monkey neocortical synaptosomes [IC50=4.8 μM (Meder and Dooley, 2000b)]. Other researchers have demonstrated a similar decrease of Ca2+ influx with GBP in rat neocortical and hippocampal synaptosomes (van Hooft et al., 2000); a high concentration (i.e. 300 μM) of this compound did not, however, alter VGCC currents recorded from acutely dissociated rat neocortical and hippocampal pyramidal cells. As a presumed result of presynaptic VGCC modulation, GBP and PGB inhibited K+-evoked [3H]-noradrenaline ([3H]-NA) release from rat neocortical slices [GBP IC50=8.9 μM, PGB IC50=11.8 μM (Dooley et al., 2000a)]. These various studies suggest that the binding affinity of GBP and PGB to the α2δ subunit is 10–100 times higher than the potency of these drugs to produce inhibitory effects on Ca2+ influx and neurotransmitter release.

In the present study, we have extended our previous results on the effects of GBP on Ca2+ influx in rat neocortical synaptosomes (Fink et al., 2000, Meder and Dooley, 2000a) to include effects of this drug, PGB, and R-(−)-3-isobutylgaba (the enantiomer of PGB) on K+-induced [Ca2+]i increase in human neocortical synaptosomes. In addition, we evaluated the effects of GBP, PGB, and R-(−)-3-isobutylgaba to inhibit [3H]-GBP binding to human neocortical membranes, the presence of α2δ subunit isoforms in human neocortical synaptosomes using immunoblots, and the role of excitatory amino acid release in the inhibitory effects of GBP on K+-evoked [3H]-NA release from human neocortical slices.

Section snippets

Subjects

Neocortical tissue was obtained from 41 patients (25 male, 16 female; age range of 3–59 yr, mean age of 31.7±2.3 yr) undergoing neurosurgery for drug resistant epilepsy as described previously (Meder et al., 1999). Patients had been diagnosed with mesial temporal lobe sclerosis based on MR imaging and Ammon's horn sclerosis based on histology. The neocortical specimens which were macroscopically, and in MRI, considered normal had to be removed to enable access for selective

Immunoblotting of α2δ VGCC subunits in neocortical synaptosome lysates

Immunoreactivity of α2δ-1, α2δ-2 and α2δ-3 subunit isoforms was detected with immunoblots, indicating a constitutive expression of these α2δ subunits in the human neocortex (Fig. 1).

Effect of GBP on basal [Ca2+]i and K+-induced [Ca2+]i increase in neocortical synaptosomes

The basal [Ca2+]i in human neocortical synaptosomes, under control conditions as measured after 360 s, was 174±6 nM (n=41). Basal [Ca2+]i was not affected by the presence of GBP or the 3-isobutylgaba enantiomers at the concentrations investigated in this study (results not shown). GBP caused a

Discussion

One aspect of this study was to evaluate the effects of GBP and PGB on depolarization-induced [Ca2+]i increase in human neocortical synaptosomes. These synaptosomes predominantly represent glutamatergic and GABAergic axon terminals. The neocortical tissue used in the preparation of synaptosomes was from patients undergoing neurosurgery, Although the tissue was histologically normal, it is possible that the patients' disease state, anticonvulsant drugs, surgical manipulations, and/or anesthetics

Acknowledgements

We thank Ms Hannelore Burisch for skilled technical assistance, and Ms Hua Gong (Pfizer) for providing GKS 02 and GKS 07 cells. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 400) and Pfizer.

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