Elsevier

Neuropharmacology

Volume 37, Issue 1, January 1998, Pages 83-91
Neuropharmacology

Gabapentin inhibits calcium currents in isolated rat brain neurons

https://doi.org/10.1016/S0028-3908(97)00189-5Get rights and content

Abstract

Gabapentin (1(aminomethyl) cyclohexane acetic acid; GBP) is a recently developed anticonvulsant, for which the mechanism of action remains quite elusive. Besides its possible interaction with glutamate synthesis and/or GABA release, in cerebral membranes gabapentin has been shown to bind directly to the α2δ subunit of the calcium channel. Therefore, we have tested the possibility that gabapentin affects high threshold calcium currents in central neurons. Calcium currents were recorded in whole-cell patch-clamp mode in neurons isolated from neocortex, striatum and external globus pallidus of the adult rat brain. A large inhibition of calcium currents by gabapentin was observed in pyramidal neocortical cells (up to 34%). Significantly, the gabapentin-mediated inhibition of calcium currents saturated at particularly low concentrations (around 10 μM), at least in neocortical neurons (IC50 about 4 μM). A less significant inhibition was seen in medium spiny neurons isolated from striatum (−12.4%) and in large globus pallidus cells (−10.4%). In all these areas, however, the GBP-induced block was fast and largely voltage-independent. Dihydropyridines (nimodipine, nifedipine) prevented the gabapentin response. ω-conotoxin GVIA and ω-conotoxin MVIIC, known to interfere with the currents driven by α1b and α1a calcium channels, did not prevent but partially reduced the response. These findings imply that voltage-gated calcium channels, predominately the L-type channel, are a direct target of gabapentin and may support its use in different clinical conditions, in which intracellular calcium accumulation plays a central role in neuronal excitability and the development of cellular damage.

Introduction

Several novel antiepileptic agents, such as lamotrigine, riluzole, topiramate and gabapentin, have been introduced successfully in the last few years (Macdonald and Kelly, 1994, Meldrum, 1996Macdonald and Greenfield 1997). Aside from their effectiveness as anticonvulsants, these drugs are currently being investigated as promising neuroprotectants (Stefani et al., 1997a). Gabapentin (1 (aminomethyl) cyclohexane acetic acid; GBP), in particular, is used as add-on therapy for partial and secondary generalized seizures (Leiderman, 1994, Beydoun et al., 1995) but it is also utilized in a rather broad range of diseases, including neurodegenerative pathologies, restless legs syndrome, chronic neuralgia and others (Welty et al., 1995, Cochran, 1996, Gurney et al., 1996, Kanthasamy et al., 1996, Mellick and Mellick, 1996, Patel and Naritoku, 1996, Segal and Rordorf, 1996). Yet, its precise mechanism of action is far from being established. When tested on different enzymes of the metabolic pathways of glutamate and GABA in vivo, GBP was demonstrated to inhibit potently the branched-chain amino acid aminotransferase (BCAA-T) (Goldlust et al., 1995), thus reducing the endogenous synthesis of glutamate. In addition, in patients taking GBP, GABA levels were shown to increase (and in a dose-dependent fashion, Petroff et al., 1996), supporting a potential GBP-mediated facilitation of inhibitory transmission (Kocsis and Honmou, 1994, Honmou et al., 1995). GBP was also capable, after prolonged preincubation, to limit the sodium-driven repetitive firing in mammalian cell cultures (Wamil and McLean, 1994). From pig cerebral cortex, however, a GBP-binding protein has recently been isolated, of which the N-terminal sequence matched with the α2δ subunit of voltage-gated calcium (Ca2+) channels (VGCC) (Gee et al., 1996). The brain α2δ subunit, an alternatively spliced form of the skeletal muscle subunit (Witcher et al., 1993, Isom et al., 1994), derives from a single gene (Ellis et al., 1988) and through functional coexpression with the different α1 subunits which form the Ca2+ channel pores (Mikami et al., 1989, Williams et al., 1992, Brust et al., 1993), contributes effectively to the stimulation of Ca2+ current amplitude (Catterall, 1995, Gurnett et al., 1996). These findings might imply that VGCC are also one of the critical targets at which GBP exerts it actions. At present, this possibility has not been sufficiently addressed.

The efficacy of several AEDs may depend, at least in part, on their ability to reduce calcium (Ca2+) conductances (Stefani et al., 1997a). The findings obtained by our group in both isolated neurons and brain slice preparations has provided strong evidence of the inhibition of VGCC by lamotrigine (LTG), riluzole, oxcarbazepine (OCBZ) and felbamate (FBM) (Stefani et al., 1995, Calabresi et al., 1995, Calabresi et al., 1996, Siniscalchi et al., 1996, Stefani et al., 1996a, Stefani et al., 1996b). Since the accumulation of intracellular Ca2+ is a crucial step in the spread of epileptic discharges as well as in the sequence of events leading to cell damage and death (Siesjo and Bengtsson, 1989, Tymianski and tator, 1996), we have suggested that the AED-mediated reduction of voltage-dependent Ca2+ fluxes underlies their potential usefulness as neuroprotective agents (Stefani et al., 1997a, Stefani et al., 1997b). In particular, the reduction of the Ca2+ fluxes through channels which are known to govern transmitter release at axon terminals (N and P/Q type channels) (Takahashi and Momiyama, 1993) caused LTG and OCBZ to have a powerful modulatory effect on glutamate-mediated synaptic potentials (Calabresi et al., 1995, Calabresi et al., 1996). The inhibition of predominantly L-type channels (mainly distributed in the somatodendritic regions) by FBM should be reflected as an impact on cellular integrative properties and excitability (Stefani et al., 1996b, Stefani et al., 1997a). The physiological impact of drugs interfering with regulatory subunits of the Ca2+ channels, such as the α2δ subunit, is less predictable. Using molecular investigations, however, it has been clearly documented how procedures which manipulate the α2δ subunit (such as N-glycosylation or cleavage into disulfide-linked α2 and δ subunits; Gurnett et al., 1996) may reduce dramatically the stimulated Ca2+ current, even in the absence of consistent changes in the voltage-dependence of its activation.

For many AEDs, the mechanisms responsible for anticonvulsant activity may be a combination of effects at different receptors or channels (Macdonald and Greenfield, 1997). Phenytoin is known to decrease Ca2+ currents, but only at supratherapeutic concentrations, well above those required to inhibit action potential discharge (McLean and Macdonald, 1983); whereas low μM concentrations of LTG have been shown to decrease both sodium and Ca2+-dependent events (Stefani et al., 1996a, Stefani et al., 1997b). For FBM, the block of the inactivated sodium channel and the reduction of NMDA-mediated transmission occur in the 50–300 μM range (Pisani et al., 1996); yet, the saturating dose for the inhibition of L-type Ca2+ currents by FBM is close to 500 nM (Stefani et al., 1996b). These examples emphasize the opportunity to characterize the different pharmacological aspects of these AED’s which have been recently introduced.

The aim of our study was to assess the putative effect of GBP on high-voltage-activated (HVA) Ca2+ currents in rat central neurons. Since GBP is used in several disease states, presumed to involve both cortical and subcortical regions, GBP-mediated responses were evaluated in different structures, namely cortical, striatal and pallidal neurons.

Section snippets

Methods

Neocortical, striatal and pallidal neurons were dissociated from 60 male Wistar rats aged 1–2 months. Briefly, as previously reported (Stefani et al., 1996b, Stefani et al., 1997b) either neostriatum or the surrounding neocortex or external GP was dissected under stereomicroscope from coronal slices 350–400 μm thick. Slices were incubated in a Hepes-buffered Hank's balanced salt solution (HBSS), bubbled with 100% O2 and warmed at 35°C. From 30 to 60 min later, one slice (two microslices for GP)

Results

In this study, we have investigated the GBP effect on VGCC by: (i) characterizing the GBP-mediated inhibition of HVA Ca2+ currents isolated in neurons obtained from cortex; (ii) comparing the modulation produced by GBP in the neocortex with the inhibitory response caused in the striatum and GP; (iii) identifying, by utilizing selective channel blockers, the Ca2+ channel types targeted by GBP.

As previously described (Sayer et al., 1993, Lorenzon and Foehring, 1995, Stefani et al., 1996a, Stefani

Discussion

Biochemical studies have proposed that GBP binds to the α2δ subunit of VGCC (Taylor et al., 1993, Gee et al., 1996). Recently, a renewed interest has focused on the roles played by the auxiliary α2δ subunit (Isom et al., 1994, Catterall, 1995); in particular, it was shown unequivocally that its coexpression (and coassembly with the α1 subunit) is required for the physiological activation of Ca2+ currents (Gurnett et al., 1996). Therefore, it is not surprising per se that GBP, as shown, indeed

Acknowledgements

This work was supported by CNR Grants to AS, GB and by Ministero della Sanità (‘Progetto Finalizzato’) to FS.

References (51)

  • A.W Wamil et al.

    Limitation by gabapentin of high frequency action potential firing by mouse central neurons in cell culture

    Epilepsy Res.

    (1994)
  • A Beydoun et al.

    Gabapentin: pharmacokinetics, efficacy and safety

    Clin. Neuropharmacol.

    (1995)
  • C.M Buetefisch et al.

    Choreoathetotic movements: a possible side effect of gabapentin

    Neurology

    (1996)
  • P Calabresi et al.

    Action of GP 47779, the active metabolite of oxcarbazepine, on the corticostriatal system. I. Modulation of corticostriatal synaptic transmission

    Epilepsia

    (1995)
  • A Calabresi et al.

    A field potential analysis on the effects of lamotrigine, GP 47779 and felbamate in neocortical slices

    Neurology

    (1996)
  • W.A Catterall

    Structure and function of voltage-gated ion channels

    Annu. Rev. Biochem.

    (1995)
  • S Choi et al.

    Metabotropic glutamate receptor modulation of voltage-gated Ca2+ channels involves multiple receptor subtypes in cortical neurons

    J. Neurosci.

    (1996)
  • J.W Cochran

    Restless legs syndrome

    J. Am. Med. Assoc.

    (1996)
  • S.B Ellis et al.

    Sequence and expression of mRNAs encoding the alfa 1 and alfa 2 subunit of a DHP-sensitive calcium channel

    Science

    (1988)
  • A.C Gurnett et al.

    Dual function of the voltage-dependent Ca2+ channel α2δ subunit in current stimulation and subunit interaction

    Neuron

    (1996)
  • M.E Gurney et al.

    Benefit of vitamin E, Riluzole and Gabapentin in a transgenic model of familail amyotrophic lateral sclerosis

    Ann. Neurol.

    (1996)
  • J.G Larkin et al.

    Nifedipine for epilepsy? A double-blind, placebo-controlled study

    Epilepsia

    (1992)
  • D.B Leiderman

    Gabapentin as add-on therapy for refractory partial epilepsy: results of five placebo-controlled trials

    Epilepsia

    (1994)
  • N.M Lorenzon et al.

    Characterization of pharmacologically identified voltage-gated calcium channels in acutely isolated rat neocortical neurons. I Adult neurons

    J. Neurophysiol.

    (1995)
  • R.L Macdonald et al.

    Mechanisms of action of currently prescribed and newly developed antiepileptic drugs

    Epilepsia

    (1994)
  • Cited by (0)

    View full text