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.

Intact skeletal muscle fibres from adult mammals exhibit neither spontaneous nor stimulated Ca 2+ sparks. Mechanical or chemical skinning procedures have been reported to unmask sparks. The present study investigates the mechanisms that determine the development of Ca 2+ spark activity in permeabilized fibres dissected from muscles with different metabolic capacity. Spontaneous Ca 2+ sparks were detected with fluo-3 and single photon confocal microscopy; mitochondrial redox potential was evaluated from mitochondrial NADH signals recorded with two-photon confocal microscopy, and Ca 2+ load of the sarcoplasmic reticulum (SR) was estimated from the amplitude of caffeine-induced Ca 2+ transients recorded with fura-2 and digital photometry. In three fibre types studied, there was a time lag between permeabilization and spark development. Under all experimental conditions, the delay was the longest in slow-twitch oxidative fibres, intermediate in fast-twitch glycolytic-oxidative fibres, and the shortest in fast-twitch glycolytic cells. The temporal evolution of Ca 2+ spark frequencies was bell-shaped, and the maximal spark frequency was reached slowly in mitochondria-rich oxidative cells but quickly in mitochondria-poor glycolytic fibres. The development of spontaneous Ca 2+ sparks did not correlate with the SR Ca 2+ content of the fibre, but did correlate with the redox potential of their mitochondria. Treatment of fibres with scavengers of reactive oxygen species (ROS), such as superoxide dismutase (SOD) and catalase, dramatically and reversibly reduced the spark frequency and also delayed their appearance. In contrast, incubation of fibres with 50 µM H 2 O 2 sped up the development of Ca 2+ sparks and increased their frequency. These results indicate that the appearance of Ca 2+ sparks in permeabilized skeletal muscle cells depends on the fibre's oxidative strength and that misbalance between mitochondrial ROS production and the fibre's ability to fight oxidative stress is likely to be responsible for unmasking Ca 2+ sparks in skinned preparations. They also suggest that under physiological and pathophysiological conditions the appearance of Ca 2+ sparks may be, at least in part, limited by the fine-tuned equilibrium between mitochondrial ROS production and cellular ROS scavenging mechanisms. Skeletal muscle depends on ATP supply to meet its energy demands. There are three major sources of ATP in muscle: creatine phosphate, anaerobic glycolysis and oxidative phosphorylation. The relative contribution of each ATP source varies among muscle fibre types. Type I (slow-twitch, oxidative) and type IIa (fast-twitch, glycolytic-oxidative) fibres are rich in mitochondria. They rely for their ATP production on oxidative phosphorylation. In contrast, type IIb (fast-twitch, glycolytic) fibres, are mitochondria-poor and have a E. V. Isaeva and V. M. Shkryl contributed equally to this work. very effective glycolytic ATP synthesis. Thus, muscle mitochondrial content is a reflection of the relative importance of mitochondria to th...

The role of mitochondrial Ca 2؉ transport in regulating intracellular Ca 2؉ signaling and mitochondrial enzymes involved in energy metabolism is widely recognized in many tissues. However, the ability of skeletal muscle mitochondria to sequester Ca 2؉ released from the sarcoplasmic reticulum (SR) during the muscle contractionrelaxation cycle is still disputed. To assess the functional cross-talk of Ca 2؉ between SR and mitochondria, we examined the mutual relationship connecting cytosolic and mitochondrial Ca 2؉ dynamics in permeabilized skeletal muscle fibers. Cytosolic and mitochondrial Ca 2؉ transients were recorded with digital photometry and confocal microscopy using fura-2 and mag-rhod-2, respectively. In the presence of 0.5 mM slow Ca Mitochondria are one of the major subcellular structures in mammalian skeletal muscle. The key role of these organelles in muscle physiology was always considered to be the energy production via generation of ATP. In addition to this well documented function, more evidence has recently accumulated regarding the importance of mitochondrial Ca 2ϩ

Key points• The signal for skeletal muscle contraction is a rapid increase in cytosolic Ca 2+ concentration, which requires the coordinated opening of ryanodine receptor (RyR) channels in the sarcoplasmic reticulum.• Channel opening is controlled by voltage-sensing dihydropyridine receptors (DHPRs) of plasma membrane and T tubules. Whether or not their signal is amplified by Ca 2+ -induced Ca 2+ release (CICR) is controversial.• We used two-photon lysis of an advanced Ca 2+ cage to produce local Ca 2+ concentration transients that were large, fast, reproducible and quantifiable, while monitoring the cellular response with a dual confocal laser scanner.• Single frog muscle cells in physiological solutions responded to transients greater than 0.28 μM with propagated CICR waves.• Mouse cells did not respond to stimuli up to 8 μM, unless channel opening drugs were present.• We conclude that CICR contributes to physiological Ca 2+ release in frog but not mouse muscle.• Mice and presumably other mammals do have a capability for CICR that is normally inhibited.It could be manifested under special circumstances, including diseases.Abstract The contribution of Ca 2+ -induced Ca 2+ release (CICR) to trigger muscle contraction is controversial. It was studied on isolated muscle fibres using synthetic localized increases in Ca 2+ concentration, SLICs, generated by two-photon photorelease from nitrodibenzofuran (NDBF)-EGTA just outside the permeabilized plasma membrane. SLICs provided a way to increase cytosolic [Ca 2+ ] rapidly and reversibly, up to 8 μM, levels similar to those reached during physiological activity. They improve over previous paradigms in rate of rise, locality and reproducibility. Use of NDBF-EGTA allowed for the separate modification of resting [Ca 2+ ], trigger [Ca 2+ ] and resting [Mg 2+ ]. In frog muscle, SLICs elicited propagated responses that had the characteristics of CICR. The threshold [Ca 2+ ] for triggering a response was 0.5 μM or less. As this value is much lower than concentrations prevailing near channels during normal activity, the result supports participation of CICR in the physiological control of contraction in amphibian muscle. As SLICs were applied outside cells, the primary stimulus was Ca 2+ , rather than the radiation or subproducts of photorelease. Therefore the responses qualify as 'classic' CICR. By contrast, mouse muscle fibres did not respond unless channel-opening drugs were present at substantial concentrations, an observation contrary to the physiological involvement of CICR in mammalian excitation-contraction coupling. In mouse muscle, the propagating wave had a substantially lower release flux, which together with a much higher threshold justified the absence of response when drugs were not present. The differences in flux and threshold may be ascribed to the absence of ryanodine receptor 3 (RyR3) isoforms in adult mammalian muscle.

Parameters (amplitude, width, kinetics) of Ca2+ sparks imaged confocally are affected by errors when the spark source is not in focus. To identify sparks that were in focus, we used fast scanning (LSM 5 LIVE; Carl Zeiss) combined with fast piezoelectric focusing to acquire x–y images in three planes at 1-µm separation (x-y-z-t mode). In 3,000 x–y scans in each of 34 membrane-permeabilized cat atrial cardiomyocytes, 6,906 sparks were detected. 767 sparks were in focus. They had greater amplitude, but their spatial width and rise time were similar compared with all sparks recorded. Their distribution of amplitudes had a mode at ΔF/F0 = 0.7. The Ca2+ release current underlying in-focus sparks was 11 pA, requiring 20 to 30 open channels, a number at the high end of earlier estimates. Spark frequency was greater than in earlier imaging studies of permeabilized ventricular cells, suggesting a greater susceptibility to excitation, which could have functional relevance for atrial cells. Ca2+ release flux peaked earlier than the time of peak fluorescence and then decayed, consistent with significant sarcoplasmic reticulum (SR) depletion. The evolution of fluorescence and release flux were strikingly similar for in-focus sparks of different rise time (T). Spark termination involves both depletion of Ca2+ in the SR and channel closure, which may be synchronized by depletion. The observation of similar flux in sparks of different T requires either that channel closure and other termination processes be independent of the determinants of flux (including [Ca2+]SR) or that different channel clusters respond to [Ca2+]SR with different sensitivity.