Altered elementary calcium release events and enhanced calcium release by thymol in rat skeletal muscle

The effects of thymol on steps of excitation-contraction coupling were studied on fast-twitch muscles of rodents. Thymol was found to increase the depolarization-induced release of calcium from the sarcoplasmic reticulum, which could not be attributed to a decreased calcium-dependent inactivation of calcium release channels/ryanodine receptors or altered intramembrane charge movement, but rather to a more efficient coupling of depolarization to channel opening. Thymol increased ryanodine binding to heavy sarcoplasmic reticulum vesicles, with a half-activating concentration of 144 micro M and a Hill coefficient of 1.89, and the open probability of the isolated and reconstituted ryanodine receptors, from 0.09 +/- 0.03 to 0.22 +/- 0.04 at 30 micro M. At higher concentrations the drug induced long-lasting open events on a full conducting state. Elementary calcium release events imaged using laser scanning confocal microscopy in the line-scan mode were reduced in size, 0.92 +/- 0.01 vs. 0.70 +/- 0.01, but increased in duration, 56 +/- 1 vs. 79 +/- 1 ms, by 30 micro M thymol, with an increase in the relative proportion of lone embers. Higher concentrations favored long events, resembling embers in control, with duration often exceeding 500 ms. These findings provide direct experimental evidence that the opening of a single release channel will generate an ember, rather than a spark, in mammalian skeletal muscle.     

Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells

To examine the natureof inositol 1,4,5-trisphosphate (IP₃)-sensitive andryanodine (Ryn)-sensitive Ca²⁺ stores in isolated caninepulmonary arterial smooth cells (PASMC), agonist-induced changes inglobal intracellular Ca²⁺ concentration([Ca²⁺]_i) were measured using fura2-AM fluorescence. Properties of elementary local Ca²⁺release events were characterized using fluo 3-AM or fluo 4-AM, incombination with confocal laser scanning microscopy. In PASMC, depletion of sarcoplasmic reticulum Ca²⁺ stores with Ryn(300 µM) and caffeine (Caf; 10 mM) eliminated subsequent Caf-inducedintracellular Ca²⁺ transients but had little or no effecton the initial IP₃-mediated intracellular Ca²⁺transient induced by ANG II (1 µM). Cyclopiazonic acid (CPA; 10 µM) abolished IP₃-induced intracellularCa²⁺ transients but failed to attenuate the initialCaf-induced intracellular Ca²⁺ transient. These resultssuggest that in canine PASMC, IP₃-, and Ryn-sensitiveCa²⁺ stores are organized into spatially distinctcompartments while similar experiments in canine renal arterial smoothmuscle cells (RASMC) reveal that these Ca²⁺ stores arespatially conjoined. In PASMC, spontaneous local intracellular Ca²⁺ transients sensitive to modulation by Caf and Ryn weredetected, exhibiting spatial-temporal characteristics similar to thosepreviously described for "Ca²⁺ sparks" in cardiac andother types of smooth muscle cells. After depletion of Ryn-sensitiveCa²⁺ stores, ANG II (8 nM) induced slow, sustained[Ca²⁺]_i increases originating at sites nearthe cell surface, which were abolished by depleting IP₃stores. Discrete quantal-like events expected due to the coordinatedopening of IP₃ receptor clusters ("Ca²⁺puffs") were not observed. These data provide new information regarding the functional properties and organization of intracellular Ca²⁺ stores and elementary Ca²⁺ release eventsin isolated PASMC.

     

Increased sensitivity and clustering of elementary Ca2+ release events during oocyte maturation

The universal signal for egg activation at fertilization is a rise in cytoplasmic Ca(2+) with defined spatial and temporal kinetics. Mammalian and amphibian eggs acquire the ability to produce such Ca(2+) signals during a maturation period that precedes fertilization and encompasses resumption of meiosis and progression to metaphase II. In Xenopus, immature oocytes produce fast, saltatory Ca(2+) waves that can be oscillatory in nature in response to IP(3). In contrast, mature eggs produce a single continuous, sweeping Ca(2+) wave in response to IP(3) or sperm fusion. The mechanisms mediating the differentiation of Ca(2+) signaling during oocyte maturation are not well understood. Here, I characterized elementary Ca(2+) release events (Ca(2+) puffs) in oocytes and eggs and show that the sensitivity of IP(3)-dependent Ca(2+) release is greatly enhanced during oocyte maturation. Furthermore, Ca(2+) puffs in eggs have a larger spatial fingerprint, yet are short lived compared to oocyte puffs. Most interestingly, Ca(2+) puffs cluster during oocyte maturation resulting in a continuum of Ca(2+) release sites over space in eggs. These changes in the spatial distribution of elementary Ca(2+) release events during oocyte maturation explain the continuous nature and slower speed of the fertilization Ca(2+) wave.     

Effects of ryanoids on spontaneous and depolarization-evoked calcium release events in frog muscle

The effects of ryanoids on calcium sparks and transients were studied in voltage-clamped cut frog muscle fibers with a laser scanning confocal microscope. For each ryanoid employed, several sequential effects were observed, including: a), transient increases in spontaneous spark frequency; b), conversions of sparks to long-lasting steady glows; and c), occasional interruptions of the glows. The ratio of the amplitude of the glow induced by a ryanoid to that of the precursory spark followed the order: ryanodol > ryanodine > C(10)-O(eq)-glycyl-ryanodine > C(10)-O(eq)-beta-alanyl-ryanodol. This sequence of glow amplitudes parallels that of the subconductances induced by these ryanoids in single-channel studies, suggesting that the glows reflect Ca(2+) fluxes through semiopen calcium release channels. Ryanoids also abolished depolarization-evoked sparks elicited with small pulses, and transformed the calcium release during depolarization to a uniform nonsparking fluorescence signal. The ratio of this signal, averaged spatially, to that of the control followed the order: ryanodol < ryanodine < C(10)-O(eq)-glycyl-ryanodine < C(10)-O(eq)-beta-alanyl-ryanodol, implying an inverse relationship with the amplitudes of ryanoid-induced glows. The observation that depolarization-evoked calcium release can occur after ryanoid suppression of calcium sparks suggests the possibility of a new strategic approach for treating skeletal muscle diseases resulting from leaky calcium release channels.     

Modeling of quantal neurotransmitter release kinetics in the presence of fixed and mobile calcium buffers

The local calcium concentration in the active zone of secretion determines the number and kinetics of neurotransmitter quanta released after the arrival of a nerve action potential in chemical synapses. The small size of mammalian neuromuscular junctions does not allow direct measurement of the correlation between calcium influx, the state of endogenous calcium buffers determining the local concentration of calcium and the time course of quanta exocytosis. In this work, we used computer modeling of quanta release kinetics with various levels of calcium influx and in the presence of endogenous calcium buffers with varying mobilities. The results of this modeling revealed the desynchronization of quanta release under low calcium influx in the presence of an endogenous fixed calcium buffer, with a diffusion coefficient much smaller than that of free Ca²⁺, and synchronization occurred upon adding a mobile buffer. This corresponds to changes in secretion time course parameters found experimentally (Samigullin et al., Physiol Res 54:129–132, 2005; Bukharaeva et al., J Neurochem 100:939–949, 2007).     

Modeling study of the effects of membrane surface charge on calcium microdomains and neurotransmitter release

Synchronous neurotransmitter release is mediated by the opening of voltage-gated Ca²⁺ channels and the build-up of submembrane Ca²⁺ microdomains. Previous models of Ca²⁺ microdomains have neglected possible electrostatic interactions between Ca²⁺ ions and negative surface charges on the inner leaflet of the plasma membrane. To address the effects of these interactions, we built a computational model of ion electrodiffusion described by the Nernst-Planck and Poisson equations. We found that inclusion of a negative surface charge significantly alters the spatial characteristics of Ca²⁺ microdomains. Specifically, close to the membrane, Ca²⁺ ions accumulate, as expected from the strong electrostatic attraction exerted on positively charged Ca²⁺ ions. Farther away from the membrane, increasing the surface charge density results in a reduction of the Ca²⁺ concentration because of the preferential spread of Ca²⁺ ions along lateral directions. The model also predicts that the negative surface charge will decrease the spatial gradient of the Ca²⁺ microdomain in the lateral direction, resulting in increased overlap of microdomains originating from different Ca²⁺ channels. Finally, we found that surface charge increases the probability of vesicle release if the Ca²⁺ sensor is located within the electrical double layer, whereas this probability is decreased if the Ca²⁺ sensor lies at greater distances from the membrane. Our data suggest that membrane surface charges exert a significant influence on the profile of Ca²⁺ microdomains, and should be taken into account in models of neurotransmitter release.     

Mini-dystrophin expression down-regulates IP3-mediated calcium release events in resting dystrophin-deficient muscle cells

We present here evidence for the enhancement, at rest, of an inositol 1,4,5-trisphosphate (IP3)-mediated calcium signaling pathway in myotubes from dystrophin-deficient cell lines (SolC1(-)) as compared to a cell line from the same origin but transfected with mini-dystrophin (SolD(+)). With confocal microscopy, the number of sites discharging calcium (release site density [RSD]) was quantified and found more elevated in SolC1(-) than in SolD(+) myotubes. Variations of membrane potential had no significant effect on this difference, and higher resting [Ca2+]i in SolC1(-) (Marchand, E., B. Constantin, H. Balghi, M.C. Claudepierre, A. Cantereau, C. Magaud, A. Mouzou, G. Raymond, S. Braun, and C. Cognard. 2004. Exp. Cell Res. 297:363-379) cannot explain alone higher RSD. The exposure with SR Ca(2+) channel inhibitors (ryanodine and 2-APB) and phospholipase C inhibitor (U73122) significantly reduced RSD in both cell types but with a stronger effect in dystrophin-deficient SolC1(-) myotubes. Immunocytochemistry allowed us to localize ryanodine receptors (RyRs) as well as IP3 receptors (IP3Rs), IP3R-1 and IP3R-2 isoforms, indicating the presence of both RyRs-dependent and IP3-dependent release systems in both cells. We previously reported evidence for the enhancement, through a Gi protein, of the IP3-mediated calcium signaling pathway in SolC1(-) as compared to SolD(+) myotubes during a high K(+) stimulation (Balghi, H., S. Sebille, B. Constantin, S. Patri, V. Thoreau, L. Mondin, E. Mok, A. Kitzis, G. Raymond, and C. Cognard. 2006. J. Gen. Physiol. 127:171-182). Here we show that, at rest, these regulation mechanisms are also involved in the modulation of calcium release activities. The enhancement of resting release activity may participate in the calcium overload observed in dystrophin-deficient myotubes, and our findings support the hypothesis of the regulatory role of mini-dystrophin on intracellular signaling.     

Elevated InsP3R expression underlies enhanced calcium fluxes and spontaneous extra-systolic calcium release events in hypertrophic cardiac myocytes

Cardiac hypertrophy is associated with profound remodelling of Ca²⁺ signalling pathways. During the early, compensated stages of hypertrophy, Ca²⁺ fluxes may be enhanced to facilitate greater contraction, whereas as the hypertrophic heart decompensates, Ca²⁺ homeostatic mechanisms are dysregulated leading to decreased contractility, arrhythmia and death. Although ryanodine receptor Ca²⁺ release channels (RyR) on the sarcoplasmic reticulum (SR) intracellular Ca²⁺ store are primarily responsible for the Ca²⁺ flux that induces myocyte contraction, a role for Ca²⁺ release via the inositol 1,4,5-trisphosphate receptor (InsP₃R) in cardiac physiology has also emerged. Specifically, InsP₃-induced Ca²⁺ signals generated following myocyte stimulation with an InsP₃-generating agonist (e.g. endothelin, ET-1), lead to modulation of Ca²⁺ signals associated with excitation-contraction coupling (ECC) and the induction of spontaneous Ca²⁺ release events that cause cellular arrhythmia. Using myocytes from spontaneously hypertensive rats (SHR), we recently reported that expression of the type 2 InsP₃R (InsP₃R2) is significantly increased during hypertrophy. Notably, this increased expression was restricted to the junctional SR in close proximity to RyRs. There, enhanced Ca²⁺ release via InsP₃Rs serves to sensitise neighbouring RyRs causing an augmentation of Ca²⁺ fluxes during ECC as well as an increase in non-triggered Ca²⁺ release events. Although the sensitization of RyRs may be a beneficial consequence of elevated InsP₃R expression during hypertrophy, the spontaneous Ca²⁺ release events are potentially of pathological significance giving rise to cardiac arrhythmia. InsP₃R2 expression was also increased in hypertrophic hearts from patients with ischemic dilated cardiomyopathy and aortically-banded mice demonstrating that increased InsP₃R expression may be a general phenomenon that underlies Ca²⁺ changes during hypertrophy.     

Modeling and analysis of calcium signaling events leading to long-term depression in cerebellar Purkinje cells

Modeling and simulation of the calcium signaling events that precede long-term depression of synaptic activity in cerebellar Purkinje cells are performed using the Virtual Cell biological modeling framework. It is found that the unusually high density and low sensitivity of inositol-1,4,5-trisphosphate receptors (IP3R) are critical to the ability of the cell to generate and localize a calcium spike in a single dendritic spine. The results also demonstrate the model's capability to simulate the supralinear calcium spike observed experimentally during coincident activation of the parallel and climbing fibers. The sensitivity of the calcium spikes to certain biological and geometrical effects is investigated as well as the mechanisms that underlie the cell's ability to generate the supralinear spike. The sensitivity of calcium release rates from the IP3R to calcium concentrations, as well as IP3 concentrations, allows the calcium spike to form. The diffusion barrier caused by the small radius of the spine neck is shown to be important, as a threshold radius is observed above which a spike cannot be formed. Additionally, the calcium buffer capacity and diffusion rates from the spine are found to be important parameters in shaping the calcium spike.     

Single synaptic events evoke NMDA receptor-mediated release of calcium from internal stores in hippocampal dendritic spines

We have used confocal microscopy to monitor synaptically evoked Ca2+ transients in the dendritic spines of hippocampal pyramidal cells. Individual spines respond to single afferent stimuli (<0.1 Hz) with Ca2+ transients or failures, reflecting the probability of transmitter release at the activated synapse. Both AMPA and NMDA glutamate receptor antagonists block the synaptically evoked Ca2+ transients; the block by AMPA antagonists is relieved by low Mg2+. The Ca2+ transients are mainly due to the release of calcium from internal stores, since they are abolished by antagonists of calcium-induced calcium release (CICR); CICR antagonists, however, do not depress spine Ca2+ transients generated by backpropagating action potentials. These results have implications for synaptic plasticity, since they show that synaptic stimulation can activate NMDA receptors, evoking substantial Ca2+ release from the internal stores in spines without inducing long-term potentiation (LTP) or depression (LTD).     

Double crossing over: elementary events and the sequence of their occurrence

Analysis of the crossing over increment in the structurally normal chromosome of Drosophila caused by a rearrangement in nonhomologous chromosome (interchromosomal effect on crossing over, IEC) was carried out based on the author's personal and literature data. The IEC in the left arm of chromosome 2 caused by inversions in chromosomes X and 3, as well as the IEC in X chromosome caused by inversions in chromosomes 2 and 3, were examined. The IEC-induced increment of crossing over results from the increase of the number of double exchanges under the constant or reduced number of single exchanges. Tetrad analysis showed that the given alternation of the crossing over processes could occur only in the case of conversion of the tetrads with single exchanges into the tetrads with double exchanges. In other words, the events leading to the formation of double exchanges occur consecutively. The borders of the IEC-induced double exchanges can be seen all over the chromosome body. However, the IEC-induced increase of chromosome recombination length occurs only in the proximal region (in rare cases, in proximal and distal regions) of the chromosome arm. This means that a double exchange is formed when the first event with predominant location in the middle of the arm is supplemented with the second event predominantly localized at the arm T end, most frequently in the proximal region. The pattern of the IEC-induced double exchange formation can be satisfactorily described in terms of the contact model of the crossing over. According to the model, an elementary crossing-over event is the local contact between the homologues. Neither single exchange nor a double-stranded DNA break can serve as an elementary event in the process of any multiple exchange formation.     

Monazomycin-induced single channels. I. Characterization of the elementary conductance events

Monazomycin (a positively charged, polyene-like antibiotic) induces voltage-dependent conductance changes in lipid bilayer membranes when added to one of the bathing solutions. These conductance changes have generally been attributed to the existence of channels spanning the membrane. In this article we characterize the behavior of the individual conductance events observed when adding small amounts of monazomycin to one side of a lipid bilayer. We find that there are several apparent channel types with one or sometimes two amplitudes predominating. We find further that these fairly similar amplitudes represent two different states of the same fundamental channel entity, presumed to be the monazomycin channel. The current-voltage characteristics of these channels are weakly hyperbolic functions of applied potential. The average lifetimes are essentially voltage independent (between 50 and 400 mV). The average channel intervals, on the other hand, can be strongly voltage dependent, and we can show that the time-averaged conductance of a membrane is proportional to the average channel frequency.     

Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release

Calcium-induced calcium release (CICR) has been observed in cardiac myocytes as elementary calcium release events (calcium sparks) associated with the opening of L-type Ca(2+) channels. In heart cells, a tight coupling between the gating of single L-type Ca(2+) channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca(2+) channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca(2+) sparks and propagated Ca(2+) waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca(2+) channels. L-type Ca(2+) channels can open without triggering Ca(2+) sparks and triggered Ca(2+) sparks are often observed after channel closure. CICR is a function of the net flux of Ca(2+) ions into the cytosol, rather than the single channel amplitude of L-type Ca(2+) channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca(2+) channels are loosely coupled to RYR through an increase in global [Ca(2+)] due to an increase in the effective distance between L-type Ca(2+) channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release.     

Model-based analysis of elementary Ca<Superscript>2+</Superscript> release events in skinned mammalian skeletal muscle fibres

Using high temporally and spatially resolved confocal laser scanning microscopy, we have recently demonstrated the existence of elementary Ca(2+) release events (ECRE) in chemically and mechanically skinned fibres from adult mammalian skeletal muscle. Here, we present a first approach to the analysis of mammalian ECRE with a spatio-temporal mathematical model of Ca(2+) ion distribution in skinned muscle fibre preparations. The differential equations for the main processes, including sarcoplasmic reticulum Ca(2+) handling, are solved in a 2-D cylindrical geometry by the method of explicit finite differences. By calculating the various spatio-temporal ion concentrations as well as the theoretical fluorescence signals for confocal microscopy, corrected for the point spread function, the model output can be directly correlated with the experimental data. Thus, the basic features of mammalian ECRE were successfully reproduced with our model. In particular, under our model assumptions a considerable depletion of luminal free calcium is predicted even for short spark-like ECRE. For a full understanding of the molecular and sub-cellular events responsible for EC coupling it is vitally important to combine the experimental and modelling approaches to elucidate the contribution of mammalian ECRE to the global Ca(2+) release and its alteration under various physiological and also pathophysiological conditions.     

Luminal calcium regulation of calcium release from sarcoplasmic reticulum

This article discusses how changes in luminal calcium concentration affect calcium release rates from triad-enriched sarcoplasmic reticulum vesicles, as well as single channel opening probability of the ryanodine receptor/calcium release channels incorporated in bilayers. The possible participation of calsequestrin, or of other luminal proteins of sarcoplasmic reticulum in this regulation is addressed. A comparison with the regulation by luminal calcium of calcium release mediated by the inositol 1,4,5-trisphosphate receptor/calcium channel is presented as well.     

Two elementary models for the regulation of skeletal muscle contraction by calcium

It is shown by use of an extremely simple explicit two-state model that two basic ideas may be sufficient to understand at least qualitatively the sensitive activation of isometric muscle contraction by Ca2+. (a) Ca2+ binds much more strongly on troponin if myosin is already attached to actin. The steady state analogue of this is that the single rate constant (in the two-state model) for myosin attachment plus Pi release is much larger if Ca2+ is bound to troponin. (b) End-to-end tropomyosin interactions are responsible for positive cooperativity. Although these ideas seem to be sufficient, this of course does not mean that they are necessary. These same ingredients were used in two previous, more elaborate models for the cooperative equilibrium binding of myosin subfragment-1 on actin-tropomyosin-troponin, with and without Ca2+, and for a study of the steady state ATPase activity of the same system. Essentially as an appendix, the above-mentioned simple treatment is extended to a somewhat more realistic and complicated model of isometric contraction.