Michael Madeja

Reduction of voltage-operated potassium currents by levetiracetam : a novel antiepileptic mechanism of action?

Levetiracetam (ucb L059; Keppra) is a novel antiepileptic drug. Its effects on action potential generation and voltage-operated potassium currents were studied in acutely isolated hippocampal CA1 neurones from rat and guinea pig, using the patch-clamp technique in the whole-cell configuration. (i) Levetiracetam reduced repetitive action potential generation and affected the single action potential. Levetiracetam, 100 microM, decreased the total number of action potentials and reduced the total depolarisation area of repetitive action potentials by 21%. Furthermore, levetiracetam increased the duration of the first action potential slightly, prolonged that of the second action potential by 13% and decreased the slope of rise by 23%. (ii) Levetiracetam decreased the voltage-operated potassium current. Without effect on sodium and A-type potassium currents, levetiracetam, 100 microM, reduced the delayed rectifier current by 26%. The concentration of half-maximal block was 47 microM for guinea pig and 6 microM for rat neurones. Thus, the reduction of repetitive action potential generation by levetiracetam can be attributed, unexpectedly, to a moderate reduction of the delayed rectifier potassium current, as supported by a simulation of action potential generation. This suggests that a reduction of potassium currents may contribute to the antiepileptic effect(s) of levetiracetam.

A concentration-clamp system allowing two-electrode voltage-clamp investigations in oocytes of Xenopus laevis

A system for rapid solution exchange (concentration-clamp) was developed for Xenopus laevis oocytes. The exchange time (t90) was 10 ms. The system allows two-electrode voltage-clamp investigations.

Follicular Tissues Reduce Drug Effects on Ion Channels in Oocytes of Xenopus laevis

The influence of follicular tissues on drug effects on ion channels in Xenopus oocytes was tested by investigating the pharmacological properties of a cloned potassium channel in oocytes with and without follicular tissues. The data show that the efficacy of blocking agents (ranging from metal ions to peptides) is drastically reduced by the follicular tissues (reductions by as much as 90% and increases of the IC50 values up to 30–fold). Furthermore, the time course of the blocking effect was slowed down by the tissues (increases of the t50 values up to 40–fold). The described impairment could be mitigated, but not abolished by partial removal of the follicular tissues (so‐called defolliculation, leaving only the vitelline envelope and part of the follicle cells on the oocyte surface). The results indicate that the follicular tissues can induce significant errors in pharmacological measurements on membrane proteins in Xenopus oocytes.

Effect of levetiracetam on epileptiform discharges in human neocortical slices.

Summary:  Purpose: The anticonvulsant effects of the novel antiepileptic drug (AED) levetiracetam (LEV) were tested in neocortical slice preparations from 23 patients who underwent surgery for the treatment of refractory epilepsy.

Improvement and testing of a concentration-clamp system for oocytes of Xenopus laevis

A system for rapid solution exchange on Xenopus laevis oocytes in the two-electrode voltage-clamp mode (Madeja et al., 1991) was improved for investigations on oocytes with removed follicular tissues. The speed of application and the time for potassium ions to reach the oocyte membrane (t50) was found to be about 40 ms in oocytes without follicular tissues and 360 ms for oocytes with follicular tissues. This rate of solution exchange is fast enough to measure the fast desensitizing component of the AMPA current response.

Effects of 2-phenoxyethanol on N-methyl-d-aspartate (NMDA) receptor-mediated ion currents

Abstract The actions were examined of 17 frequently used glycol ether compounds on the glutamate receptor-mediated ion currents. The receptors were expressed in Xenopus oocytes by injection of rat brain mRNA. Most of the 17 glycol ethers exerted no effects on the glutamate subreceptors activated by kainate and N-methyl-d-aspartate (NMDA), whereas 2-phenoxyethanol (ethylene glycol monophenyl ether) caused a considerable reduction of NMDA-induced membrane currents in a reversible and concentration-dependent manner. The threshold concentration of the ethylene glycol monophenyl ether effect was <10 μmol/l. The concentration for a 50% inhibition (IC50) was ∼360 μmol/l. The results indicate a neurotoxic potential for 2-phenoxyethanol.

Sensitivity of native and cloned hippocampal delayed-rectifier potassium channels to verapamil

The effects of the phenylalkylamine verapamil on native and cloned hippocampal voltage-operated potassium channels were investigated. Native channels were studied in acutely isolated CA1 neurons from the guinea pig with the whole-cell patch-clamp technique. Cloned channels were expressed in oocytes of Xenopus laevis and studied with the two-electrode voltage-clamp technique. Native potassium channels: Verapamil suppressed the potassium currents in micro- and submicromolar concentrations. The current suppression increased during the voltage step. The IC50 value of verapamil was 3 micromol/l and the Hill coefficient was 0.5 indicating a mixed population of potassium channels with distinct verapamil sensitivity. Cloned potassium channels: The hippocampal potassium channels Kv1.1, Kv1.2, Kv1.3, Kv2.1, Kv3.1 and Kv3.2 were affected by verapamil in micromolar concentrations. The effect increased with depolarization time, was voltage-dependent, reached 90% of the maximum within around 40 s after start of verapamil application, recovered slowly after wash-out and did not reach control values even after wash-out times of six minutes. The IC50 values differed markedly and were 35 micromol/l for the Kv1.1 channel, 98 micromol/l for the Kv1.2 channel, 12 micromol/l for the Kv1.3 channel, 226 micromol/l for the Kv2.1 channel, 6 micromol/l for the Kv3.1 channel and 11 micromol/l for the Kv3.2 channel.

Divergent Effect of Acute Ventricular Dilatation on the Electrophysiologic Characteristics of d,I‐Sotalol and Flecainide in the Isolated Rebbit heart

Ventricular Dilatation and d,l‐Sotalol/Flecainide in Isolated Rabbit Heart. Introduction: The interaction between acute ventricular dilatation (AVD) as one aspect of ventricular dysfunction and Class I and III antiarrhythmic drugs is uncertain. We therefore investigated the effects of AVD on the electrophysiologic properties of d,l‐sotalol and flecainide.

Blockade of glutamatergic and GABAergic receptor channels by trimethyltin chloride

1 Organotin compounds such as trimethyltin chloride (TMT) are among the most toxic of the organometallics. As their main target for toxicity is the central nervous system, the aim of the present study was to investigate the effects of TMT on receptor channels involved in various processes of synaptic transmission. 2 The Xenopus oocyte expression system was chosen for direct assessment of TMT effects on voltage‐operated potassium channels and glutamatergic and GABAergic receptors, and hippocampal slices from rat brain for analyzing TMT effects on identified synaptic sites. 3 TMT was found to be ineffective, at 100 μmol l−1, against several potassium‐ and sodium‐operated ion channel functions as well as the metabotropic glutamate receptor. 4 The functions of the ionotropic glutamate and the GABAA receptor channels were inhibited by TMT in micromolar concentrations. Thus, at a maximum concentration of 100 μmol l−1, around 20–30% of the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid and GABAA receptor‐mediated ion currents and 35% of the N‐methyl‐D‐aspartate receptor‐mediated ion currents were blocked. 5 In the hippocampal slice model, the inhibitory effects of TMT were much stronger than expected from the results on the ion channels. Bath application of TMT significantly reduced the amplitudes of evoked excitatory postsynaptic field potentials in a concentration‐dependent and nonreversible manner. 6  Induction of long‐term potentiation, recorded from the CA1 dendritic region, was inhibited by TMT and failed completely at a concentration of 10 μmol l−1. 7 In general, TMT affects the excitatory and inhibitory synaptic processes in a receptor specific manner and is able to disturb the activity within a neuronal network.

Tunicamycin-induced inhibition of functional expression of glutamate receptors in Xenopus oocytes

The effects of tunicamycin, a specific inhibitor of N-linked glycosylation, on functional expression of glutamate receptor subtypes were investigated in RNA-injected oocytes. In the presence of tunicamycin the expression of ligand-operated receptors sensitive to kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and quisqualate were completely blocked. The inhibitory effect was reversible after removal of tunicamycin from the culture medium.