The muscle relaxant thiocolchicoside is an antagonist of GABA_A receptor function in the central nervous system

Abstract

Thiocolchicoside (TCC) is used clinically for its muscle relaxant, anti-inflammatory, and analgesic properties, and it has been shown to interact with γ-aminobutyric acid (GABA) type A receptors (GABA_ARs) and strychnine-sensitive glycine receptors in the rat central nervous system. In contrast to a proposed agonistic action at these two types of inhibitory receptors, pharmacological evidence has shown that, under certain conditions, TCC manifests convulsant activity in animals and humans. We now show that the phasic and tonic GABA_AR-mediated currents recorded from Purkinje cells and granule neurons, respectively, in parasagittal cerebellar slices from adult male rats were inhibited by TCC in a concentration-dependent manner. The median inhibitory concentrations of TCC for these effects were ∼0.15 and ∼0.9 μM, respectively. TCC did not potentiate GABA_BR-mediated currents in hippocampal slices, suggesting that its muscle relaxant action is not mediated by GABA_BRs. Intraperitoneal injection of TCC in rats either alone or in combination with negative modulators of GABAergic transmission revealed convulsant and proconvulsant actions of this drug. Our data, consistent with clinical observations of the epileptogenic effect of this compound, suggest that TCC is a potent competitive antagonist of GABA_AR function.

Introduction

Thiocolchicoside (TCC) (Fig. 1A) is a semisynthetic sulfur derivative of colchicoside, a naturally occurring glucoside present in the plant Gloriosa superba. TCC has been used clinically for more than 35 years as a muscle-relaxant, anti-inflammatory, and analgesic drug (Janbroers, 1987), but its molecular targets and mechanisms of action are still under investigation. This compound has been shown to inhibit the binding of [³H]GABA (γ-aminobutyric acid) or [³H]strychnine to rat cerebrocortical or spinal cord membranes, respectively, in vitro as well as to corresponding autoradiographic sections in vivo (Balduini et al., 1999, Balduini et al., 2001, Cimino et al., 1996). [³H]TCC was also displaced in a concentration-dependent manner by GABA or by several agonists or antagonists of the type A receptor for GABA (GABA_AR). Moreover, TCC was shown to interact preferentially with a subpopulation of GABA_ARs with low-affinity binding sites for GABA (Balduini et al., 2001).

Although its precise mechanisms of action remain unknown, TCC has been thought to act as a GABA_AR agonist that induces depression of the central nervous system and, in turn, myorelaxation (Biziere et al., 1981). However, under specific experimental or clinical conditions, TCC has been shown to induce epileptic seizures (De Riu et al., 2001, Sechi et al., 2003), challenging the notion that this drug acts as an agonist at GABA_ARs and raising the possibility that it may directly or indirectly inhibit GABAergic transmission. Moreover, TCC possesses a molecular structure similar to that of colchicine, a plant alkaloid that binds to tubulin and induces the depolymerization of microtubules (Osborn and Weber, 1976), disrupts axonal transport (Karlsson and Sjostrand, 1969), and inhibits mitosis (Wilson and Friedkin, 1966). Colchicine induces epileptic seizures in rodents (Dasheiff and Ramirez, 1985), and its intracranial administration triggers generalized convulsions and death (Wisniewski and Terry, 1967). In addition to its well-characterized effects on microtubules, colchicine acts directly as a competitive antagonist of GABA_AR function (Weiner et al., 1998), possibly explaining, at least in part, its epileptogenic action.

In view of these contrasting observations with TCC, we have now investigated the effects of this drug on the function of native GABA_ARs in cerebellar slices with the patch-clamp technique. Purkinje neurons and cerebellar granule cells of adult rats manifest a relatively uniform expression of GABA_AR isoforms. In particular, Purkinje neurons express mostly GABA_ARs that contain the α1, β2 or β3, and γ2 subunits; these receptors exhibit a relatively low affinity for GABA and are localized in the synaptic cleft (Pirker et al., 2000, Wisden et al., 1996). In contrast, granule cells, in their extrasynaptic regions, express GABA_ARs that mostly comprise the α6, β2 or β3, and δ subunits, exhibit a higher affinity for GABA, and mediate the tonic component of inhibitory transmission. In addition to these extrasynaptic receptors, granule cells express synaptic GABA_ARs that contain α1 or α6, β2 or β3, and γ2 subunits; these low-affinity receptors bind GABA that is released from Golgi cells and mediate fast-rising spontaneous inhibitory postsynaptic currents (sIPSCs) (Brickley et al., 1996, Brickley et al., 2001, Hamann et al., 2002, Rossi and Hamann, 1998, Rossi et al., 2003, Tia et al., 1996, Wall and Usowicz, 1997). We now show that TCC is a potent competitive antagonist of GABA_AR function. This compound shows a greater potency at the synaptic GABA_ARs of Purkinje cells than at the extrasynaptic GABA_ARs of granule cells. Consistent with our electrophysiological observations, in vivo experiments revealed that TCC acts as a convulsant and proconvulsant drug. Our results are thus in accord with the previous demonstration of focal and secondarily generalized convulsive epileptic activity of TCC in rats and humans (De Riu et al., 2001, Sechi et al., 2003).