Gabapentin inhibits calcium currents in isolated rat brain neurons
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.
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