The inhibitory effect of trimethylamine on the anticonvulsant activities of quinine in the pentylenetetrazole model in rats

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Abstract

Quinine specifically blocks connexin 36 (Cx36), one of the proteins that form gap junction channels. Quinine suppressed ictal epileptiform activity in in vitro and in vivo studies without decreasing neuronal excitability. In this study, we considered the possible mechanism of anticonvulsant effects of quinine (1, 250, 500, 1000 and 2000 μM, i.c.v.) in the pentylenetetrazole (PTZ) model of seizure. Thus, we used trimethylamine (TMA) (0.05 μM, 5 μM, 50 μM), a gap junction channel opener, to examine whether it could reverse the effects of quinine in rats. Intracerebroventricular (i.c.v.) injection of quinine affected generalized tonic–clonic seizure (GTCS) induced by PTZ by increments in seizure onset and reducing seizure duration. Additionally, pretreatment with different doses of TMA (i.c.v.) attenuated the anticonvulsant effects of quinine on the latency and duration of GTCS. It can be concluded that quinine possesses anticonvulsant effects via modulation of gap junction channels, which could contribute to the control of GTCS.

Introduction

Epilepsy is one of the most common serious neurological conditions, with an annual incidence of 50/100,000 per year (Poole et al., 2000, Ropper and Brown, 2005). Abnormal synchronization of neuronal discharges is of recognized critical importance in seizures; however, the mechanisms underlying this pathological synchrony remain uncertain. In this context, there is growing interest in electrotonic communication via gap junctions and speculation based largely on studies in vitro and on ex vivo brain tissue that gap junctions may be important in the generation and propagation of seizures (Carlen et al., 2000, Kohling et al., 2001, Proulx et al., 2006). The pathogenesis of abnormal neuronal synchrony underlying seizures, formerly thought to be based mainly on chemical synaptic transmission, now includes a role for gap junctional communication. This concept has been strengthened by evidence from several in vitro models, in which pharmacological manipulations of gap junctional communication predictably affect the generation of seizures, with blockers diminishing seizures, and enhancers increasing seizures (Carlen et al., 2000, Kohling et al., 2001). Thus, it seems that gap junctions may represent a novel therapeutic target for the future. Gap junction channels of vertebrates are formed by a family of proteins known as connexins (Cx) that are expressed in an overlapping pattern of tissue distribution. Quinine, an anti-malarial drug, specifically blocks Cx36 and with lesser potency Cx50 in mammalian cells (Srinivas et al., 2001). Cx36 is exclusively expressed in neurons, is the principal connexin in adult neurons, and has been linked to other genetic markers of juvenile myoclonic epilepsy (JME) (Belluardo et al., 2000, Mas et al., 2004). In contrast, Cx50 is not expressed in the mammalian brain (Nagy et al., 2004). Quinine was reported to suppress ictal epileptiform activity in vitro without decreasing neuronal excitability (Bikson et al., 2002). Recently, it was shown that quinine suppressed epileptiform activity by decreasing the amplitude and frequency of epileptiform spikes and by attenuating the epileptiform behavior in rats (Bostanci and Bagirici, 2007). Moreover, it inhibited cortical spreading depression (SD) on rat neocortical slices in vitro (Margineanu and Klitgaard, 2006). On the other hand, Wambebe and colleagues have shown that quinine has anticonvulsant properties and that a dopaminergic mechanism may be involved in the protective influence of quinine against pentylenetetrazole (PTZ)-induced seizures in mice (Wambebe et al., 1990).

Thus, in this study, we considered the possible mechanism of the anticonvulsant effects of quinine in the PTZ model of generalized tonic–clonic seizure (GTCS); we hypothesized that if gap junction channels are important in seizure generation and/or propagation, quinine will reduce the frequency or severity of seizures and this might suggest novel treatment strategies for seizure in humans. Furthermore, trimethylamine (TMA), a gap junction channel opener, could reverse the effects of quinine. It was reported that TMA opens gap junctions as a result of intracellular alkalinization (Spray et al., 1981, Lee et al., 1996, Willoughby et al., 2001). Previous works have shown that gap junction openers exacerbate seizure activity (Gajda et al., 2003, Gajda et al., 2006, Sayyah et al., 2007). To date, the role of TMA as a gap junction channel opener on the anticonvulsant activities of quinine in the different models of epilepsy in vivo and in vitro has not been studied. Thus, we examined TMA in this study.

Section snippets

Animals

Male Wistar rats (250–300 g) were obtained from the Razi Institute (Karaj, Iran) and housed in groups of four per cage under standard laboratory conditions. They were kept at constant room temperature (21 ± 2 °C) under a normal 12 L:12D regime with free access to food and water. All animal experiments were carried out in accordance with the European Communities Council directive of 24 November 1986 (86/609/EEC) in such a way as to minimize the number of animals and their suffering.

Chemicals

Quinine

Results

All rats in experimental groups were positively verified with histological examination. All the animals that were treated with vehicle 30 min before PTZ (90 mg/kg, i.p.) underwent GTCS (Fig. 1, Fig. 2). Microinjection of quinine (500–1000 μM) 30 min before injection with PTZ significantly prolonged the latency to GTCS compared to vehicle [F(5, 50) = 3.7, P < 0.05, P < 0.01, respectively] (Fig. 1). However, quinine at a dose of 2000 μM did not significantly increase the latency to GTCS compared to

Discussion

Our results confirm that quinine has anticonvulsant activities in the PTZ model. The inhibitory effect of quinine affected the latency and duration of GTCS. However, the effects of quinine decreased at the highest dose we tested, 2000 μM. Similar to our results, it was shown that quinine at low concentrations (20–40 μM) in 4-aminopyridine in vivo epilepsy model, suppressed the expression of seizures, but at higher concentrations (> 80 μM) its effect was rather proconvulsive (Gajda et al., 2005).

Conclusions

In brief, the present study provides evidence for the anticonvulsant activity of quinine in the GTCS of PTZ model. As a result, we suggest that gap junctions represent an appropriate target for the development of drugs aimed at decreasing epileptiform synchronization and preventing epileptogenesis. Structure-activity studies of quinine will perhaps lead to the synthesis of quinine based derivatives that will be effective in the treatment of seizure disorders.

Acknowledgement

This study was supported by a grant from the research council of Qazvin University of Medical Sciences, Qazvin, Iran.

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