Activation of pre-synaptic M-type K+ channels inhibits [3H]d-aspartate release by reducing Ca2+ entry through P/Q-type voltage-gated Ca2+channels

In this study, the functional consequences of the pharmacological modulation of the M‐current (I_KM) on cytoplasmic Ca²⁺ intracellular Ca²⁺concentration ([Ca²⁺]_i) changes and excitatory neurotransmitter release triggered by various stimuli from isolated rat cortical synaptosomes have been investigated. K_v7.2 immunoreactivity was identified in pre‐synaptic elements in cortical slices and isolated glutamatergic cortical synaptosomes. In cerebrocortical synaptosomes exposed to 20 mM [K⁺]_e, the I_KM activator retigabine (RT, 10 μM) inhibited [³H]d ‐aspartate ([³H]d ‐Asp) release and caused membrane hyperpolarization; both these effects were prevented by the I_KM blocker XE‐991 (20 μM). The I_KM activators RT (0.1–30 μM), flupirtine (10 μM) and BMS‐204352 (10 μM) inhibited 20 mM [K⁺]_e‐induced synaptosomal [Ca²⁺]_i increases; XE‐991 (20 μM) abolished RT‐induced inhibition of depolarization‐triggered [Ca²⁺]_i transients. The P/Q‐type voltage‐sensitive Ca²⁺channel (VSCC) blocker ω‐agatoxin IVA prevented RT‐induced inhibition of depolarization‐induced [Ca²⁺]_i increase and [³H]d ‐Asp release, whereas the N‐type blocker ω‐conotoxin GVIA failed to do so. Finally, 10 μM RT did not modify the increase of [Ca²⁺]_i and the resulting enhancement of [³H]d ‐Asp release induced by [Ca²⁺]_i mobilization from intracellular stores, or by store‐operated Ca²⁺channel activation. Collectively, the present data reveal that the pharmacological activation of I_KM regulates depolarization‐induced [³H]d ‐Asp release from cerebrocortical synaptosomes by selectively controlling the changes of [Ca²⁺]_i occurring through P/Q‐type VSCCs.     

Chloride-induced release of actively loaded calcium from light and heavy sarcoplasmic reticulum vesicles

Summary Light and heavy sarcoplasmic reticulum vesicles (LSR, HSR) isolated from rabbit leg muscle have been used in a study of chloride-induced Ca²⁺ release. The biochemical and morphological data indicate that LSR is derived from the longitudinal reticulum and HSR is derived from the terminal cisternae of the sarcoplasmic reticulum. LSR and HSR were both able to accumulate Ca²⁺ in the presence of ATP to amounts greater than 100 nmol Ca²⁺/mg of protein in less than 1 min. LSR and HSR each had a biphasic time course of Ca²⁺ uptake. The initial uptake was followed by a rapid release, after approximately 1 min, of 30–40% of the accumulated Ca²⁺, which was then followed by a slower phase of Ca²⁺ accumulation. Ca²⁺ taken up by the SR vesicles could be released from both the LSR and HSR by changing the anion outside the vesicles from methanesulfonate to chloride. Due to the difference in permeability between methanesulfonate and chloride, this change should result in a decreased positivity inside the vesicles with respect to the exterior. It could also result in osmotic swelling of the vesicles. Changing the ionic medium from chloride to methanesulfonate caused no release of Ca²⁺. The amount of accumulated Ca²⁺ released in 6 sec by changing the anion outside the vesicles from methanesulfonate to chloride was 30–35 nmol/mg membrane protein for LSR and HSR, respectively. Osmotic buffering with 200mm sucrose caused a slight inhibition of chloride-induced Ca²⁺ release from HSR (17%15%) but it greatly reduced the release of Ca²⁺ from LSR (32%15%). The specificity of Ca²⁺ release was measured using SR vesicles which were passively loaded with 10mm ²²Na⁺. LSR released five times more²²Na⁺ than HSR under same conditions as chloride-induced Ca²⁺ release occurred. Na dantrolene (20 m) had no effect on the release of Ca²⁺ from LSR but it inhibited the chloride-induced Ca²⁺ release from HSR by more than 50%. Na dantrolene also increased the Ca²⁺ uptake in the HSR by 20% while not affecting LSR Ca²⁺ uptake. Our results indicate the presence of a chloride-induced, Na dantrolene inhibited, Ca²⁺ release from HSR, which is not due to osmotic swelling.     

A quantitative model for linking Na+/Ca2+ exchanger to SERCA during refilling of the sarcoplasmic reticulum to sustain [Ca2+] oscillations in vascular smooth muscle

We have developed a quantitative model for the creation of cytoplasmic Ca²⁺ gradients near the inner surface of the plasma membrane (PM). In particular we simulated the refilling of the sarcoplasmic reticulum (SR) via PM–SR junctions during asynchronous [Ca²⁺]_i oscillations in smooth muscle cells of the rabbit inferior vena cava. We have combined confocal microscopy data on the [Ca²⁺]_i oscillations, force transduction data from cell contraction studies and electron microscopic images to build a basis for computational simulations that model the transport of calcium ions from Na⁺/Ca²⁺ exchangers (NCX) on the PM to sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) pumps on the SR as a three-dimensional random walk through the PM–SR junctional cytoplasmic spaces. Electron microscopic ultrastructural images of the smooth muscle cells were elaborated with software algorithms to produce a very clear and dimensionally accurate picture of the PM–SR junctions. From this study, we conclude that it is plausible and possible for enough Ca²⁺ to pass through the PM–SR junctions to replete the SR during the regenerative Ca²⁺ release, which underlies agonist induced asynchronous Ca²⁺ oscillations in vascular smooth muscle.     

Quercetin stimulation of calcium release from rabbit skeletal muscle sarcoplasmic reticulum

To elucidate the mechnism by which quercetin enhances the rate of tension development in skinned muscle fibers, effects on calcium release from longitudinal tubule-derived SR (LSR) after phosphate-supported calcium uptake were examined. In all studies, 100 μM quercetin (which inhibits initial calcium uptake velocity 85%) was added at or shortly after the time calcium content reached a maximum at various extravesicular Ca²⁺ concentrations (Ca_o). At moderate Ca_o (0.2–1.0 μM). where spontaneous calcium release rate depended on Ca_o, quercetin caused a marked stimulation of calcium release. This was accompanied by a 60% reduction in calcium influx and a 30-fold increase in calcium efflux. Thus, the previously reported quercetin-induced increase in the rate of tension development by skinned muscle fibers may result, at least in part, from sensitization of Ca²⁺-triggered calcium release to lower Ca_o.     

The concentration of free Ca(2+) in the sarcoplasmic reticulum of frog cut twitch skeletal muscle fibers estimated with tetramethylmurexide

One aim of this article was to determine the resting concentration of free Ca²⁺ in the sarcoplasmic reticulum (SR) of frog cut skeletal muscle fibers ([Ca²⁺]_SR,R) using the calcium absorbance indicator dye tetramethylmurexide (TMX). Another was to determine the ratio of [Ca²⁺]_SR,R to TMX's apparent dissociation constant for Ca²⁺ (K_app) in order to establish the capability of monitoring [Ca²⁺]_SR(t) during SR Ca²⁺ release – a signal needed to determine the Ca²⁺ permeability of the SR. To reveal the properties of TMX in the SR, the surface membrane was rapidly permeabilized with saponin to rapidly dissipate myoplasmic TMX. Results indicated that the concentration of Ca-free TMX in the SR was 2.8-fold greater than that in the myoplasm apparently due to binding of TMX to sites in the SR. Taking into account that such binding might influence K_app as well as a dependence of K_app on TMX concentration, the results indicate an average [Ca²⁺]_SR,R ranging from 0.43 to 1.70 mM. The ratio [Ca²⁺]_SR,R/K_app averaged 0.256, a relatively low value which should not depend on factors influencing K_app. As a result, the time course of [Ca²⁺]_SR(t) in response to electrical stimulation is well determined by, and approximately linearly related to, the active TMX absorbance signal.     

Predicting Local SR Ca Dynamics during Ca Wave Propagation in Ventricular Myocytes

Of the many ongoing controversies regarding the workings of the sarcoplasmic reticulum (SR) in cardiac myocytes, two unresolved and interconnected topics are 1), mechanisms of calcium (Ca²⁺) wave propagation, and 2), speed of Ca²⁺ diffusion within the SR. Ca²⁺ waves are initiated when a spontaneous local SR Ca²⁺ release event triggers additional release from neighboring clusters of SR release channels (ryanodine receptors (RyRs)). A lack of consensus regarding the effective Ca²⁺ diffusion constant in the SR (D_Ca,SR) severely complicates our understanding of whether dynamic local changes in SR [Ca²⁺] can influence wave propagation. To address this problem, we have implemented a computational model of cytosolic and SR [Ca²⁺] during Ca²⁺ waves. Simulations have investigated how dynamic local changes in SR [Ca²⁺] are influenced by 1), D_Ca,SR; 2), the distance between RyR clusters; 3), partial inhibition or stimulation of SR Ca²⁺ pumps; 4), SR Ca²⁺ pump dependence on cytosolic [Ca²⁺]; and 5), the rate of transfer between network and junctional SR. Of these factors, D_Ca,SR is the primary determinant of how release from one RyR cluster alters SR [Ca²⁺] in nearby regions. Specifically, our results show that local increases in SR [Ca²⁺] ahead of the wave can potentially facilitate Ca²⁺ wave propagation, but only if SR diffusion is relatively slow. These simulations help to delineate what changes in [Ca²⁺] are possible during SR Ca²⁺release, and they broaden our understanding of the regulatory role played by dynamic changes in [Ca²⁺]_SR.     

RyR2 Modulates a Ca2+-Activated K+ Current in Mouse Cardiac Myocytes

In cardiomyocytes, Ca²⁺ entry through voltage-dependent Ca²⁺ channels (VDCCs) binds to and activates RyR2 channels, resulting in subsequent Ca²⁺ release from the sarcoplasmic reticulum (SR) and cardiac contraction. Previous research has documented the molecular coupling of small-conductance Ca²⁺-activated K⁺ channels (SK channels) to VDCCs in mouse cardiac muscle. Little is known regarding the role of RyRs-sensitive Ca²⁺ release in the SK channels in cardiac muscle. In this study, using whole-cell patch clamp techniques, we observed that a Ca²⁺-activated K+ current (I_K,Ca) recorded from isolated adult C57B/L mouse atrial myocytes was significantly decreased by ryanodine, an inhibitor of ryanodine receptor type 2 (RyR2), or by the co-application of ryanodine and thapsigargin, an inhibitor of the sarcoplasmic reticulum calcium ATPase (SERCA) (p<0.05, p<0.01, respectively). The activation of RyR2 by caffeine increased the I_K,Ca in the cardiac cells (p<0.05, p<0.01, respectively). We further analyzed the effect of RyR2 knockdown on I_K,Ca and Ca²⁺ in isolated adult mouse cardiomyocytes using a whole-cell patch clamp technique and confocal imaging. RyR2 knockdown in mouse atrial cells transduced with lentivirus-mediated small hairpin interference RNA (shRNA) exhibited a significant decrease in I_K,Ca (p<0.05) and [Ca²⁺]i fluorescence intensity (p<0.01). An immunoprecipitated complex of SK2 and RyR2 was identified in native cardiac tissue by co-immunoprecipitation assays. Our findings indicate that RyR2-mediated Ca²⁺ release is responsible for the activation and modulation of SK channels in cardiac myocytes.     

Modeling CICR in rat ventricular myocytes: voltage clamp studies

Background  

The past thirty-five years have seen an intense search for the molecular mechanisms underlying calcium-induced calcium-release (CICR) in cardiac myocytes, with voltage clamp (VC) studies being the leading tool employed. Several VC protocols including lowering of extracellular calcium to affect Ca ²⁺ loading of the sarcoplasmic reticulum (SR), and administration of blockers caffeine and thapsigargin have been utilized to probe the phenomena surrounding SR Ca ²⁺ release. Here, we develop a deterministic mathematical model of a rat ventricular myocyte under VC conditions, to better understand mechanisms underlying the response of an isolated cell to calcium perturbation. Motivation for the study was to pinpoint key control variables influencing CICR and examine the role of CICR in the context of a physiological control system regulating cytosolic Ca ²⁺ concentration ([Ca ²⁺] _myo ).     

Increased calcium release from sarcoplasmic reticulum stimulated by inositol trisphosphate in spontaneously hypertensive rat heart cells

It is known that inositol (1, 4, 5)-trisphosphate (IP₃) stimulates Ca²⁺ release from sarcoplasmic reticulum (SR) in several tissues, but in cardiac myocytes this phenomenon has not been confirmed. The purpose of the present study was to confirm the effect of (1, 4, 5)-IP₃ on Ca²⁺ release from SR in cardiac myocytes. The effect of IP₃ on Ca²⁺ release from SR in hypertrophic cardiac cells was also determined.We examined the effects of IP₃ on Ca²⁺ release from cardiac myocyte SR by the bigital-image method in a single cell. We also determined the effect of IP₃ on calcium release from isolated SR. SR was prepared from spontaneous hypertensive rat hearts and Wistar kyoto rat hearts. The SR was prelabeled with⁴⁵Ca²+, and then incubated with the indicated concentrations of IP³ for 1 min at 37°C. In cardiac myocytes treated with saponin, Ca²⁺ release stimulated by 10 M (1, 4, 5)-IP₃ was detected by fura-2. In⁴⁵Ca²⁺ prelabeled SR, the maximal Ca²⁺ release was achieved at 10 M IP₃ incubated for 1 min. The release of Ca²⁺ was higher in Sr of SHR than in the SR of WKY. IP₃ stimulates Ca²⁺ release from cardiac SR, and this release is greater in SHR than in WKY. However, it is uncertain whether this phenomenon plays a role in cardiac hypertrophy.     

Mitochondria in cardiomyocyte Ca signaling

Ca²⁺ signaling is of vital importance to cardiac cell function and plays an important role in heart failure. It is based on sarcolemmal, sarcoplasmic reticulum and mitochondrial Ca²⁺ cycling. While the first two are well characterized, the latter remains unclear, controversial and technically challenging.In mammalian cardiac myocytes, Ca²⁺ influx through L-type calcium channels in the sarcolemmal membrane triggers Ca²⁺ release from the nearby junctional sarcoplasmic reticulum to produce Ca²⁺ sparks. When this triggering is synchronized by the cardiac action potential, a global [Ca²⁺]_i transient arises from coordinated Ca²⁺ release events. The ends of intermyofibrillar mitochondria are located within 20 nm of the junctional sarcoplasmic reticulum and thereby experience a high local [Ca²⁺] during the Ca²⁺ release process. Both local and global Ca²⁺ signals may thus influence calcium signaling in mitochondria and, reciprocally, mitochondria may contribute to the local control of calcium signaling. In addition to the intermyofibrillar mitochondria, morphologically distinct mitochondria are also located in the perinuclear and subsarcolemmal regions of the cardiomyocyte and thus experience a different local [Ca²⁺].Here we review the literature in regard to several issues of broad interest: (1) the ultrastructural basis for mitochondrion – sarcoplasmic reticulum cross-signaling; (2) mechanisms of sarcoplasmic reticulum signaling; (3) mitochondrial calcium signaling; and (4) the possible interplay of calcium signaling between the sarcoplasmic reticulum and adjacent mitochondria.Finally, this review discusses experimental findings and mathematical models of cardiac calcium signaling between the sarcoplasmic reticulum and mitochondria, identifies weaknesses in these models, and suggests strategies and approaches for future investigations.     

Mitochondrial Ca2+-Handling in Fast Skeletal Muscle Fibers from Wild Type and Calsequestrin-Null Mice

Mitochondrial calcium handling and its relation with calcium released from sarcoplasmic reticulum (SR) in muscle tissue are subject of lively debate. In this study we aimed to clarify how the SR determines mitochondrial calcium handling using dCASQ-null mice which lack both isoforms of the major Ca²⁺-binding protein inside SR, calsequestrin. Mitochondrial free Ca²⁺-concentration ([Ca²⁺]_mito) was determined by means of a genetically targeted ratiometric FRET-based probe. Electron microscopy revealed a highly significant increase in intermyofibrillar mitochondria (+55%) and augmented coupling (+12%) between Ca²⁺ release units of the SR and mitochondria in dCASQ-null vs. WT fibers. Significant differences in the baseline [Ca²⁺]_mito were observed between quiescent WT and dCASQ-null fibers, but not in the resting cytosolic Ca²⁺ concentration. The rise in [Ca²⁺]_mito during electrical stimulation occurred in 20−30 ms, while the decline during and after stimulation was governed by 4 rate constants of approximately 40, 1.6, 0.2 and 0.03 s⁻¹. Accordingly, frequency-dependent increase in [Ca²⁺]_mito occurred during sustained contractions. In dCASQ-null fibers the increases in [Ca²⁺]_mito were less pronounced than in WT fibers and even lower when extracellular calcium was removed. The amplitude and duration of [Ca²⁺]_mito transients were increased by inhibition of mitochondrial Na⁺/Ca²⁺ exchanger (mNCX). These results provide direct evidence for fast Ca²⁺ accumulation inside the mitochondria, involvement of the mNCX in mitochondrial Ca²⁺-handling and a dependence of mitochondrial Ca²⁺-handling on intracellular (SR) and external Ca²⁺ stores in fast skeletal muscle fibers. dCASQ-null mice represent a model for malignant hyperthermia. The differences in structure and in mitochondrial function observed relative to WT may represent compensatory mechanisms for the disease-related reduction of calcium storage capacity of the SR and/or SR Ca²⁺-leakage.     

Cellular mechanisms of paired electrical stimulation in ferret ventricular myocardium: relationship between myocardial force and stimulus interval change

Summary The subcellular mechanisms of twitch-force potentiation with paired electrical stimulation was studied in ferret ventricular myocardium using the bioluminescent calcium indicator aequorin. It is demonstrated for the first time that interpolation of an extrasystole in a train of conditioned twitches results in a beat-to-beat change in [Ca²⁺]_i and force. Steady-state twitch force and Ca _i ²⁺ were increased with paired stimulation. Increased [Ca²⁺]₀ in the setting of paired stimulation resulted in an increase in the amplitude of the postextrasystole and associated Ca²⁺ transient. Verapamil, a Ca²⁺ channel antagonist, had the opposite effect of increased [Ca²⁺]₀. Postextrasystole potentiation was still present, but diminished in amplitude. These results indicate that postextrasystole potentiation is in part due to a verapamil-depletable store (Ca²⁺). Postextrasystole potentiation is therefore predominantly dependent on sarcoplasmic reticulum (SR) Ca²⁺ loading. Ryanodine, an alkaloid which induces Ca²⁺ leakage from the SR, abolished postextrasystole potentiation; however, in the presence of ryanodine the extrasystole was potentiated. Caffeine, a phosphodiesterase inhibitor which induces SR Ca²⁺ release and impairs uptake, also abolished postextrasystole potentiation. As with ryanodine there was resultant potentiation of the extrasystole. In the case of caffeine the calcium transient consisted of a second slow component associated with extrasystole twitch potentiation. The results are consistent with sarcolemmal Ca²⁺ influx playing a role in potentiation of the extrasystole in the presence of an impaired SR. These data indicate that transsarcolemmal Ca²⁺ influx in the presence of impaired intracellular Ca²⁺ buffering can directly activate the myofilaments in agreement with reports on human myocardium.Abbreviations C conditioned stimulus - ESI extrasystolic interval - L_max active tension - PES postextrasystole - PESI postextrasystolic interval - SR sarcoplasmic reticulum - T test stimulus     

Calcium overload and cardiac function

The changes in cardiac function caused by calcium overload are reviewed. Intracellular Ca²⁺ may increase in different structures [e.g. sarcoplasmic reticulum (SR), cytoplasm and mitochondria] to an excessive level which induces electrical and mechanical abnormalities in cardiac tissues. The electrical manifestations of Ca²⁺ overload include arrhythmias caused by oscillatory (V_os) and non-oscillatory (V_ex) potentials. The mechanical manifestations include a decrease in force of contraction, contracture and aftercontractions. The underlying mechanisms involve a role of Na⁺ in electrical abnormalities as a charge carrier in the Na⁺-Ca²⁺ exchange and a role of Ca²⁺ in mechanical toxicity. Ca²⁺ overload may be induced by an increase in [Na⁺]_i through the inhibition of the Na⁺-K⁺ pump (e.g. toxic concentrations of digitalis) or by an increase in Ca²⁺ load (e.g. catecholamines). The Ca²⁺ overload is enhanced by fast rates. Purkinje fibers are more susceptible to Ca²⁺ overload than myocardial fibers, possibly because of their greater Na⁺ load. If the SR is predominantly Ca²⁺ overloaded, V_os and fast discharge are induced through an oscillatory release of Ca²⁺ in diastole from the SR; if the cytoplasm is Ca²⁺ overloaded, the non-oscillatory V_ex tail is induced at negative potentials. The decrease in contractile force by Ca²⁺ overload appears to be associated with a decrease in high energy phosphates, since it is enhanced by metabolic inhibitors and reduced by metabolic substrates. The ionic currents I_os and I_ex underlie V_os and V_ex, respectively, both being due to an electrogenic extrusion of Ca²⁺ through the Na⁺-Ca²⁺ exchange. I_os is an oscillatory current due to an oscillatory release of Ca²⁺ in early diastole from the Ca²⁺-overloaded SR, and I_ex is a non-oscillatory current due to the extrusion of Ca²⁺ from the Ca²⁺-overloaded cytoplasm. I_os and I_ex can be present singly or simultaneously. An increase in [Ca²⁺]_i appears to be involved in the short- and long-term compensatory mechanisms that tend to maintain cardiac output in physiological and pathological conditions. Eventually, [Ca²⁺]_i may increase to overload levels and contribute to cardiac failure. Experimental evidence suggests that clinical concentrations of digitalis increase force in Ca²⁺-overloaded cardiac cells by decreasing the inhibition of the Na⁺-K⁺ pump by Ca²⁺, thereby leading to a reduction in Ca²⁺ overload and to an increase in force of contraction.     

Ca disorder caused by rapid electrical field stimulation can be modulated by CaMKIIδ expression in primary rat atrial myocytes

Abnormalities in intracellular Ca²⁺ handing are believed to contribute to arrhythmogenesis during atrial fibrillation (AF). Ca²⁺/calmodulin-dependent protein kinaseII δ (CaMKIIδ) overexpression was detected in atrial myocytes from patients and animal models with persistent AF. In the present study, we found that rapid electrical field stimulation applied to primary atrial myocytes altered the CaMKIIδ activity, not expression level, resulting in Ca²⁺ disorder. By lentivirus mediated delivery of CaMKIIδ gene or siRNA into atrial myocytes, cells with different CaMKIIδ expression were generated. Changes of CaMKIIδ expression altered the sarcoplasmic reticulum (SR) Ca²⁺ release and L-type Ca²⁺ channels current (I_Ca) in both steady and electrical stimulating state. These results revealed the important role of CaMKIIδ in Ca²⁺ disorder caused by electrical field stimulation. It also provided a potential method to improve Ca²⁺ disorder in AF by modulating CaMKIIδ expression level.     

Thermogenic activity of Ca-ATPase from skeletal muscle heavy sarcoplasmic reticulum: The role of ryanodine Ca channel

The sarcoplasmic reticulum Ca²⁺ ATPase 1 (SERCA 1) is able to handle the energy derived from ATP hydrolysis in such a way as to determine the parcel of energy that is used for Ca²⁺ transport and the fraction that is converted into heat. In this work we measured the heat production by SERCA 1 in the two sarcoplasmic reticulum (SR) fractions: the light fraction (LSR), which is enriched in SERCA and the heavy fraction (HSR), which contains both the SERCA and the ryanodine Ca²⁺ channel. We verified that although HSR cleaved ATP at faster rate than LSR, the amount of heat released during ATP hydrolysis by HSR was smaller than that measured by LSR. Consequently, the amount of heat released per mol of ATP cleaved (ΔH^cal) by HSR was lower compared to LSR. In HSR, the addition of 5 mM Mg²⁺ or ruthenium red, conditions that close the ryanodine Ca²⁺ channel, promoted a decrease in the ATPase activity, but the amount of heat released during ATP hydrolysis remained practically the same. In this condition, the ΔH^cal values of ATP hydrolysis increased significantly. Neither Mg²⁺ nor ruthenium red had effect on LSR. Thus, we conclude that heat production by SERCA 1 depends on the region of SR in which the enzyme is inserted and that in HSR, the ΔH^cal of ATP hydrolysis by SERCA 1 depends on whether the ryanodine Ca²⁺ channel is opened or closed.     

Mechanisms of calcium signaling in smooth muscle cells explored with fluorescence confocal imaging

A rise in the intracellular concentration of ionized calcium ([Ca²⁺]_i) is a primary signal for contraction in all types of muscles. Recent progress in the development of imaging techniques, with special accent on fluorescence confocal microscopy, and new achievements in the synthesis of organelle- and ion-specific fluorochromes provide an experimental basis for studying the relationship between the structural organization of living smooth muscle cells (SMCs) and features of calcium signaling at the subcellular level. Applying fluorescent confocal imaging, patch-clamp recording, immunostaining, and flash photolysis techniques to freshly isolated SMCs, we have demonstrated that: (i) Ca²⁺ sparks are mediated by spontaneous clustered opening of ryanodine receptors (RyRs) and occur at the highest rate at preferred sites (frequent discharge sites, FDSs), the number of which depends on SMC type; (ii) FDSs are associated with sub-plasmalemmal sarcoplasmic reticulum (SR) elements, but not with polarized mitochondria; (iii) Ca²⁺ spark frequency increases with membrane depolarization in voltage-clamped SMCs or following neurotransmitter application to SMCs, in which the membrane potential was not controlled, leading to spark summation and resulting in a cell-wide increase in [Ca²⁺]_i and myocyte contraction; (iv) cross-talk between RyRs and inositol trisphosphate receptors (IP₃Rs) is an important determinant of the [Ca²⁺]_i dynamics and recruits neighboring Ca²⁺-release sites to generate [Ca²⁺]_i waves; (v) [Ca²⁺]_i waves induced by depolarization of the plasma membrane or by noradrenaline or caffeine, but not by carbachol (CCh), originate at FDSs; (vi) Ca²⁺-dependent K⁺ and Cl^- channels sense the local changes in [Ca²⁺]_i during a Ca²⁺ spark and thereby may couple changes in [Ca²⁺]_i within a microdomain to changes in the membrane potential, thus affecting the cell excitability; (vii) the muscarinic cation current (mI _cat) does not mirror changes in [Ca²⁺]_i, thus reflecting the complexity of [Ca²⁺]_i — muscarinic cationic channel coupling; (viii) RyR-mediated Ca²⁺ release, either spontaneous or caffeine-induced, does not augment mI _cat; (ix) intracellular flash release of Ca²⁺ is less effective in augmentation of mI _cat than flash release of IP₃, suggesting that IP₃ may sensitize muscarinic cationic channels to Ca²⁺; (x) intracellular flash release of IP₃ fails to augment mI _cat in SMCs, in which [Ca²⁺]_i was strongly buffered, suggesting that IP₃ exerts no direct effect on muscarinic cationic channel gating, and that these channels sense an increase in [Ca²⁺]_i rather than depletion of the IP₃-dependent Ca²⁺ store; and (xi) predominant expression of IP₃R type 1 in the peripheral SR provides a structural basis for a tight functional coupling between IP₃R-mediated Ca²⁺ release and muscarinic cationic channel opening.Neirofiziologiya/Neurophysiology, Vol. 36, Nos. 5/6, pp. 455–465, September–December, 2004.This revised version was published online in April 2005 with a corrected cover date and copyright year.     

Relationship between Extra- and Intracellular Calcium in Distal Segments of the Renal Tubule. Role of the Ca2+ Receptor RaKCaR

The effect of extracellular calcium ([Ca²⁺] _e ) on cytosolic calcium ([Ca²⁺] _i ) was investigated in thick ascending limbs and collecting ducts from the rat kidney, using the fluorescent dye fura-2. In cortical collecting ducts, basolateral but not apical changes in [Ca²⁺] _e were associated with parallel changes in [Ca²⁺] _i . Basal [Ca²⁺] _i was hardly modified by nifedipine and verapamil but was decreased by 60% by basolateral La³⁺. Increasing peritubular [Ca²⁺] _e triggered Ca²⁺ release from intracellular stores. This effect was not reproduced by agonists of the renal Ca²⁺-receptor RaKCaR, e.g., Ba²⁺, Mg²⁺, Gd³⁺, and neomycin, but was reproduced by Ni²⁺. Ni²⁺-induced mobilization of intracellular Ca²⁺ was larger in the inner medullary collecting duct, a segment which poorly responds to increasing [Ca²⁺] _e . In the cortical thick ascending limb, removing basolateral Ca²⁺ hardly altered [Ca²⁺] _i but increasing [Ca²⁺] _e or adding Ba²⁺, Mg²⁺, Gd³⁺ and neomycin released intracellular calcium. These data demonstrate that (1) basolateral influx of calcium occurs in cortical collecting ducts, under basal conditions; (2) this influx occurs through nonvoltage gated channels, permeable to Ba²⁺, insensitive to verapamil and nifedipine, and blocked by La³⁺; (3) increasing [Ca²⁺] _e stimulates the influx and triggers intracellular calcium release, independently of the phospholipase C-coupled receptor RaKCaR; (4) RaKCaR is functionally expressed in thick ascending limbs; (5) another membrane receptor, sensitive to Ni²⁺ but not to Ca²⁺ is present in the collecting duct. Received: 12 July 1996/Revised: 28 October 1996     

Excitation-contraction coupling in heart muscle

We have investigated the links between electrical excitation and contraction in mammalian heart muscle. Using isolated single cells from adult rat ventricle, a whole-cell voltage-clamp technique and quantitative fluorescence microscopy, we have measured simultaneously calcium current (I_ca) and [Ca²⁺]_i (with fura-2). We find that the voltage-dependence of Ica and the [Ca ²⁺]_i-transient and the dependence of [Ca²⁺]_i-transient on depolarization-duration cannot both be readily explained by a simple calcium-induced Ca-release (CICR) mechanism. Additionally, we find that when [Ca²⁺]_i and [Na⁺]_i are at their diastolic levels, activation of the Na-Ca exchange mechanism by depolarization does not measurably trigger the release of Ca²⁺i. Finally, measuring I_ca in adult and neonatal rat heart cells and using the alkaloid ryanodine, we have carried out complementary experiments. These experiments show that there may be an action of ryanodine on I_ca that is independent of [Ca²⁺]_i and independent of a direct action of the alkaloid on the calcium channel itself. Along with experiments of others showing that ryanodine binds to the sarcoplasmic reticulum calcium-release channel/spanning protein complex, our data suggests a model to explain our findings. The model links the calcium channels responsible for Ica to the sarcoplasmic reticulum by means of one or more of the spanning protein(s). Information from the calcium channel can be communitated to the sarcoplasmic reticulum by this route and, presumably, information can move in the opposite direction from the sarcoplasmic reticulum to the calcium channel.     

Calcium-sensing receptors regulate cardiomyocyte Ca2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation

Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca²⁺ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP₃Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca²⁺ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP₃Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP₃Rs were located in the SR. [Ca²⁺]i, [Ca²⁺]_m and [Ca²⁺]_SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca²⁺]_SR, increased [Ca²⁺]_i and [Ca²⁺]_m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca²⁺ release from the SR into the mitochondria through IP₃Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.     

Calcium efflux from sarcoplasmic reticulum microsomes due to oxidation and sulfhydryl-binding agents

Calcium permeability of sarcoplasmic reticulum (SR) microsomes was measured after aging or after exposure to peroxydisulfate or to sulfhydryl-binding agents. Under conditions where the Ca²⁺-ATPase was active, the maximum net release of Ca²⁺ was not significantly different between control and oxidized SR. However, when calcium uptake was prevented by EGTA or apyrase, the Ca²⁺ permeability of oxidized microsomes was 2 to 3 times greater than control of low (10⁻⁹, 10⁻⁷ M) but not high (10⁻⁶ M) levels of external calcium. The observation that vesicles preincubated with 5 mM dithiothreitol loaded up to 3 times as much calcium and had a slightly lower calcium permeability coefficient than control vesicles suggested that suflhydryl oxidation might modulate calcium flux. This hypothesis was tested by exposing to sulfydryl-binding agents:silver, arsenite, and p-chloromercuri-phenylsulfonic acid. Sulfhydryl-binding agents initiated a rapid release of calcium from microsomes, and release was halted by dithiothreitol. Inhibition of calcium transport could not entirely account for the apparent increase in permeability because the calcium permeability of SR treated with sulfhydryl-binding agents was 5 times greater than that of SR exposed to Ca²⁺-ATPase inhibitors. These results suggest that oxidation may increase the calcium permeability of SR. by allowing calcium loss through a channel that can be gated by sulfhydryl oxidation.