Summary: Purpose:We investigated the effect of the new antiepileptic drug (AED) levetiracetam (LEV) on different types of high-voltage-activated (HVA) Ca 2+ channels in freshly isolated CA1 hippocampal neurons of rats.Methods: Patch-clamp recordings of HVA Ca 2+ channel activity were obtained from isolated hippocampal CA1 neurons. LEV was applied by gravity flow from a pipette placed near the cell, and solution changes were made by electromicrovalves. Ca 2+ channel blockers were used for separation of the channel subtypes.Results: The currents were measured in controls and after application of 1-200 M LEV. LEV irreversibly inhibited the HVA calcium current by ∼18% on the average. With a prepulse stimulation protocol, which can eliminate direct inhibition of Ca 2+ channels by G proteins, we found that G proteins were not involved in the pathways underlying the LEV inhibitory effect. This suggested that the inhibitory effect arises from a direct action of LEV on the channel molecule. The blocking mechanism of LEV was not related to changes in steady-state activation or inactivation of Ca 2+ channels. LEV also did not influence the rundown of the HVA Ca 2+ current during experimental protocols lasting ∼10 min. Finally, LEV at the highest concentration used (200 M) did not influence the activity of L-, P-or Q-type Ca 2+ channels in CA1 neurons, while selectively influencing the activity of N-type calcium channels. The maximal effect on these channels separated from other channel types was ∼37%.Conclusions: Our results provide evidence that LEV selectively inhibits N-type Ca 2+ channels of CA1 pyramidal hippocampal neurons. These data suggest the existence of a subtype of N-type channels sensitive to LEV, which might be involved in the molecular basis of its antiepileptic action. Key Words: Levetiracetam-Antiepileptic drugs-Calcium channels-Hippocampal neurons-Epilepsy.Levetiracetam (LEV) is a new antiepileptic drug (AED) with a unique pharmacologic profile, exerting potent seizure suppression in kindling models of epilepsy (1-3). It substantially inhibits neuronal hypersynchronization in hippocampal slices induced by application of high potassium-low calcium perfusion solutions, without any intrinsic effects on normal electrophysiologic responses. Therefore it is of obvious importance to evaluate possible cellular mechanisms of the antiepileptic action of LEV that might be related to its specific interaction with molecular structures responsible for the generation of electrical activity in brain neurons.Previous investigations have failed to find any modulatory activity of levetiracetam on voltage-gated Na + and low-voltage-activated Ca 2+ currents in rat neocortical neurons (4,5). Therefore special attention was devoted to high-voltage-activated (HVA) Ca 2+ currents, which also can be responsible for changes in the firing pattern of corresponding neurons. Recently it was shown that LEV can inhibit HVA calcium channels in pyramidal hippocampal neurons (6,7). Therefore it was of special interest to evaluate whether L...

Shkryl VM, Maxwell JT, Domeier TL, Blatter LA. Refractoriness of sarcoplasmic reticulum Ca 2ϩ release determines Ca 2ϩ alternans in atrial myocytes.

Muscular dystrophies are among the most severe inherited muscle diseases. The genetic defect is a mutation in the gene for dystrophin, a cytoskeletal protein which protects muscle cells from mechanical damage. Mechanical stress, applied as osmotic shock, elicits an abnormal surge of Ca(2+) spark-like events in skeletal muscle fibers from dystrophin deficient (mdx) mice. Previous studies suggested a link between changes in the intracellular redox environment and appearance of Ca(2+) sparks in normal mammalian skeletal muscle. Here, we tested whether the exaggerated Ca(2+) responses in mdx fibers are related to oxidative stress. Localized intracellular and mitochondrial Ca(2+) transients, as well as ROS production, were assessed with confocal microscopy. The rate of basal cellular but not mitochondrial ROS generation was significantly higher in mdx cells. This difference was abolished by pre-incubation of mdx fibers with an inhibitor of NAD(P)H oxidase. In addition, immunoblotting showed a significantly stronger expression of NAD(P)H oxidase in mdx muscle, suggesting a major contribution of this enzyme to oxidative stress in mdx fibers. Osmotic shock produced an abnormal and persistent Ca(2+) spark activity, which was suppressed by ROS-reducing agents and by inhibitors of NAD(P)H oxidase. These Ca(2+) signals resulted in mitochondrial Ca(2+) accumulation in mdx fibers and an additional boost in cellular and mitochondrial ROS production. Taken together, our results indicate that the excessive ROS production and the simultaneous activation of abnormal Ca(2+) signals amplify each other, finally culminating in a vicious cycle of damaging events, which may contribute to the abnormal stress sensitivity in dystrophic skeletal muscle.

Ca2+ sparks, localized elevations in cytosolic [Ca 2+ ], are rarely detected in intact adult mammalian skeletal muscle under physiological conditions. However, they have been observed in permeabilized cells and in intact fibres subjected to stresses, such as osmotic shock and strenuous exercise. Our previous studies indicated that an excess in cellular reactive oxygen species (ROS) generation over the ROS scavenging capabilities could be one of the up-stream causes of Ca 2+ spark appearance in permeabilized muscle fibres. Here we tested whether the cytosolic ROS balance is compromised in intact skeletal muscle fibres that underwent osmotic shock and whether this misbalance contributes to unmasking Ca 2+ sparks. Spontaneous Ca 2+ sparks and the rate of ROS generation were assessed with single photon confocal microscopy and fluorescent indicators fluo-4, CM-H 2 DCFDA and MitoSOX Red. Osmotic shock produced spontaneous Ca 2+ sparks and a concomitant significant increase in ROS production. Preincubation of muscle cells with ROS scavengers (e.g. MnTBAP, Mn-cpx 3, TIRON) nearly eliminated Ca 2+ sparks. In addition, inhibitors of NAD(P)H oxidase (DPI and apocynin) significantly reduced ROS production and suppressed the appearance of Ca 2+ sparks. Taken together, the data suggest that ROS contribute to the abnormal Ca 2+ spark activity in mammalian skeletal muscle subjected to osmotic stress and also indicate that NAD(P)H oxidase is a possible source of ROS. We propose that ROS-dependent Ca 2+ sparks are an important component of adaptive/maladaptive muscle responses under various pathological conditions such as eccentric stretch, osmotic changes during ischaemia and reperfusion, and some muscle diseases.