Localization And Expression Of K_ATP Channels In Chronic Epileptic Rat Brain, And The Effects On The GABA Transporters By Pretreatment With Diazoxide, A K_ATP Channel Opener

ObjectivesIt is generally hypothesized that the increased cerebral excitability associated with epilepsy/seizure is caused by a disturbance in the balance between excitation and inhibition. However, how epilepsy develops remains elusive. Most of previous studies were focused on the epilepsy relevant cascades happened as a consequence of its action on the brain, such as glutamic acid, NO and peptide etc., but the real situation is that the ion channels may play a key role in the development of eplepsy as a crucial seizure gate. ATP-sensitive K+ (KATP) channels are, expressed pre- and post-synaptically in many brain regions. KATP channels couple the intracellular metabolic state to electrical activity at the plasma membrane. Recent studies have provided evidence to support a role for KATP channels in the control of neuronal excitability and seizure propagation. However, little is known about its subunit gene/protein expression and loalization in chronic epilepsy, its role in the seizure kindling, and its effects on the GABA transportation during epileptic discharge. Therefore, in the present study, changes in Kir6.1, Kir6.2, SUR1, SUR2A/B, VGAT, GAT-1 and GAT-3 gene expression, and in Kir6.2, SUR1 and VGAT protein expression in hippocampus of picrotoxin (PTX)-induced kindling rats were investigated, and also the changes of these genes and proteins after re-kindling and the effectiveness of diazoxide (DIZ) a KATP channel opener on their expression were studied to identify the role of KATP channels in epilepsy.MethodsPart 1 Changes in the gene and protein expression of KATP channel subunits inthe hippocampus of rats subjected to PTX-induced kindlingChemical kindling was induced in rats by a long-term administration (i.p.) of PTX (0.0225 mg per 10 g body weight, one times a day for 20 days). Control animals underwent saline injections in the same volume. Rats were observed for 45 min aftereach administration of PTX or vehicle, and seizures were recorded according to the following scale devised by Racine; stage 1, facial clonus; stage 2, nodding; stage 3, forelimb clonus; stage 4, forelimb clonus with rearing; stage 5, rearing, jumping and falling. Animals were scored after having had three consecutive seizures at each stage. The stage 4 and 5 animals were employed in this study. The effects of such kindling on the abundance of transcripts encoding subunits of the KATP channels in the brain were measured by reverse transcription polymerase chain reaction (RT-PCR) assay. Fourteen days after the last drug administration of the long-term protocol, kindled rats were re-injected with PTX or vehicle and examined at 0 h, 12 h, 1 day or 3 days after the re-treatment.Part 2 Localization of the KATP K+ channel subunits, Kir6.2 and SUR1, inPTX-induced kindling rat brainFourteen days after the last PTX administration of the long-term protocol, kindled rats were re-injected with PTX or vehicle and examined at 0 h, 12 h, 1 day or 3 days after the re-treatment. Kir6.2 and SUR1 were localized in adult rat brain by in situ hybridization histochemistry.Part 3 Evidence for an involvement of vesicular GABA transporter, GABAtransporters GAT-1 and GAT-3 in the anticonvulsant action of Diazoxide, a KATP channel opener on PTX-induced convulsions in ratsRats with chronic seizures were induced by a long-term administration with PTX. 'Fourteen days after the last drug administration of the long-term protocol, rats were treated (i.p.) with DIZ (1.9 or 3.8 mg/kg) or equivalent volumes of saline one hour prior to the re-injection with PTX or equal volumes of saline. RT-PCR and Western blotting were used to measure the quantity of VGAT, GAT-1 and GAT-3 mRNAs or VGAT protein in hippocampus.Results PartiThe amounts of Kir6.1, Kir6.2, SUR1 and SUR2B subunit mRNAs were decreased in hippocampus and cerebral cortex of kindled rats at 0 h after the re-treatment with vehicle, compared with those observed in control rats, demonstrating a significant difference in SUR2B expression from cortical samples (P< 0...