Uptake, retention, and efflux of Ca2+ by mitochondrial preparations from skeletal muscle

Functionally intact mitochondria, substantially free of contamination, were isolated from rabbit gastrocnemius muscle after protease digestion and their Ca2+-handling properties examined. When judged by their capacity to retain large Ca2+ loads and the magnitude of basal and Na+-stimulated Ca2+ effluxes, the most suitable isolation method was digestion of finely minced muscle in buffered isoosmotic KCl with low levels (0.4 mg/g) of trypsin or the bacterial protease nagarse, followed by differential centrifugation. Polytron disruption of skeletal muscle in both sucrose- and KCl-based media released mitochondria deficient in cytochrome c. Use of the divalent ion chelator EDTA rather than EGTA in the isolation medium sharply reduced Ca2+-dependent respiratory control and tolerance of the mitochondria to Ca2+ loads, probably by removing Mg2+ essential to membrane integrity. ADP-dependent respiratory control was not altered in mitochondria prepared in an EDTA-containing isolation medium. Purification of mitochondria on a Percoll density gradient did not improve their Ca2+-handling ability despite removal of minor contaminants. Mitochondria prepared by the protease method could accumulate micromole loads of Ca2+/mg while maintaining a low basal Ca2+ efflux. Addition of BSA to the assay medium slightly improved Ca2+ retention but was not essential either during isolation or assay. Ca2+-dependent state 3 respiration was maximal at pH 6.5-7.0 while respiratory control and Ca2+/O were optimal at pH 7.0-7.5. Neither Pi nor oxaloacetate induced Ca2+ release from loaded mitochondria when monitored for 30 min after ruthenium red addition. Na+-stimulated Ca2+ efflux had sigmoidal kinetics with a Hill coefficient of 3. Since skeletal muscle mitochondria can be isolated and assayed in simple media, functional deficiencies of mitochondria from diseased muscle are unlikely to be masked.     

Inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum during active Ca2+ transport

ATP-dependent Ca2+ uptake by subfractions of skeletal muscle sarcoplasmic reticulum (SR) was studied with the Ca2+ indicator dye, antipyrylazo III. Ca2+ uptake by heavy SR showed two phases, a slow uptake phase and a fast uptake phase. By contrast, Ca2+ uptake by light SR exhibited a monophasic time course. In both fractions a steady state of Ca2+ uptake was observed when the concentration of free Ca2+ outside the vesicles was reduced to less than 0.1 microM. In the steady state, the addition of 5 microM Ca2+ to the external medium triggered rapid Ca2+ release from heavy SR but not from light SR, indicating that the heavy fraction contains a Ca2+-induced Ca2+ release channel. During Ca2+ uptake, heavy SR showed a constant Ca2+-dependent ATPase activity (1 mumol/mg protein X min) which was about 150 times higher than the rate of Ca2+ uptake in the slow uptake phase. Ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, enhanced the rate of Ca2+ uptake during the slow phase without affecting Ca2+-dependent ATPase activity. Adenine nucleotides, activators of Ca2+ release, reduced the Ca2+ uptake rate. These results suggest that the rate of Ca2+ accumulation by heavy SR is not proportional to ATPase activity during the slow uptake phase due to the activation of the channel for Ca2+-induced Ca2+ release. In addition, they suggest that the release channel is inactivated during the fast Ca2+ uptake phase.     

Reduced Ca(2+)-induced Ca2+ release from skeletal muscle sarcoplasmic reticulum at low pH.

The purpose of this investigation was to determine the effects of reduced pH on Ca(2+)-induced Ca2+ release (CICR) from skeletal muscle sarcoplasmic reticulum (SR). Frog semitendinosus fiber bundles (1-3/bundle) were chemically skinned via saponin treatment (50 micrograms/mL, 20 min), which removes the sarcolemma and leaves the SR functional. The SR was first depleted of Ca2+ then loaded for 2 min at pCa (log free Ca2+ concentration) 6.6. CICR was then evoked by exposing the fibers to pCa 5-7 for 5-60 s. CICR was evoked both in the absence of ATP and Mg2+ and in the presence of beta, gamma-methyleneadenosine-5'-triphosphate (AMPPCP, a nonhydrolyzable form of ATP) and Mg2+. Ca2+ remaining in the SR was then assayed via caffeine (25 mM) contracture. In all cases, CICR evoked at pH 6.5 resulted in larger caffeine contractures than that evoked at 7.0, suggesting that more Ca2+ was released during CICR at the higher pH. Accordingly, rate constants for CICR were significantly greater at pH 7.0 than at pH 6.5. These results indicate that reduced pH depresses CICR from skeletal muscle SR.     

Ca2+-induced Ca2+ release from fragmented sarcoplasmic reticulum: Ca2+-dependent passive Ca2+ efflux

Characterization of the putative Ca2+-gated Ca2+ channel of sarcoplasmic reticulum, which is thought to mediate Ca2+-induced Ca2+ release, was carried out in order to elucidate the mechanism of Ca2+-induced Ca2+ release. Heavy and light fractions of fragmented sarcoplasmic reticulum isolated from rabbit skeletal muscle were loaded passively with Ca2+, and then passive Ca2+ efflux was measured under various conditions. The fast phase of the Ca2+ efflux depended on the extravesicular free Ca2+ concentration and was assigned to the Ca2+ efflux through the Ca2+-gated Ca2+ channel. Vesicles with the Ca2+-gated Ca2+ channels comprised about 85% of the heavy fraction and about 40% of the light fraction. The amount of Ca2+ loaded in FSR was found to be much larger than that estimated on the basis of vesicle inner volume and the equilibration of intravesicular with extravesicular Ca2+, indicating Ca2+ binding inside FSR. Taking this fact into account, the Ca2+ efflux curve was quantitatively analyzed and the dependence of the Ca2+ efflux rate constant on the extravesicular free Ca2+ concentration was determined. The Ca2+ efflux was maximal, with the rate constant of 0.75 s-1, when the extravesicular free Ca2+ was at 3 microM. Caffeine increased the affinity for Ca2+ of Ca2+-binding sites for opening the channel with only a slight change in the maximum rate of Ca2+ efflux. Mg2+ inhibited the Ca2+ binding to the sites for opening the channel while procaine seemed to inhibit the Ca2+ efflux by blocking the ionophore moiety of the channel.     

Effect of halothane on Ca2+-induced Ca2+ release from sarcoplasmic reticulum vesicles isolated from rat skeletal muscle

Halothane induces the release of Ca2+ from a subpopulation of sarcoplasmic reticulum vesicles that are derived from the terminal cisternae of rat skeletal muscle. Halothane-induced Ca2+ release appears to be an enhancement of Ca2+-induced Ca2+ release. The low-density sarcoplasmic reticulum vesicles which are believed to be derived from nonjunctional sarcoplasmic reticulum lack the capability of both Ca2+-induced and halothane-induced Ca2+ release. Ca2+ release from terminal cisternae vesicles induced by halothane is inhibited by Ruthenium red and Mg2+, and require ATP (or an ATP analogue), KCl (or similar salt) and extravesicular Ca2+. Ca2+-induced Ca2+ release has similar characteristics.     

Activation of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum by caffeine and related compounds

The caffeine-sensitive Ca2+ release pathway in skeletal muscle was identified and characterized by studying the release of 45Ca2+ from heavy sarcoplasmic reticulum (SR) vesicles and by incorporating the vesicles or the purified Ca2+ release channel protein complex into planar lipid bilayers. First-order rate constants for 45Ca2+ efflux of 1 s-1 were obtained in the presence of 1-10 microM free Ca2+ or 2 X 10(-9) M free Ca2+ plus 20 mM caffeine. Caffeine- and Ca2+-induced 45Ca2+ release were potentiated by ATP and Mg.ATP, and were both inhibited by Mg2+. Dimethylxanthines were similarly (3,9-dimethylxanthine) or more (1,7-, 1,3-, and 3,7-dimethylxanthine) effective than caffeine in increasing the 45Ca2+ efflux rate. 1,9-Dimethylxanthine and 1,3-dimethyluracil (which lacks the imidazole ring) did not appreciably stimulate 45Ca2+ efflux. Recordings of calcium ion currents through single channels showed that the Ca2+- and ATP-gated SR Ca2+ release channel is activated by addition of caffeine to the cis (cytoplasmic) and not the trans (lumenal) side of the channel in the bilayer. The single channel measurements further revealed that caffeine activated Ca2+ release by increasing the number and duration of open channel events without a change of unit conductance (107 pS in 50 mM Ca2+ trans). These results suggest that caffeine exerts its Ca2+ releasing effects in muscle by activating the high-conductance, ligand-gated Ca2+ release channel of sarcoplasmic reticulum.     

Effects of thyroid hormone on Ca2+ efflux and Ca2+ transport capacity in rat skeletal muscle

The present study was undertaken to investigate the effects of 3,5,3'-triiodothyronine (T3) treatment on passive Ca2+ efflux, Ca2(+)-dependent Mg2(+)-ATPase (Ca2(+)-ATPase) concentration and active Ca2+ transport in isolated rat skeletal muscle. In addition, the question was examined whether changes in Ca2+ efflux at rest and during electrical stimulation in the hyperthyroid state were accompanied by parallel changes in 3-O-methylglucose efflux. The resting Ca2+ efflux from rat soleus muscle was increased by 25% after 8 days of treatment with T3 (20 micrograms/100 g body weight). This was associated with a 78% increase in the basal efflux of 3-O-methylglucose. Electrical stimulation resulted in a rapid stimulation of Ca2+ efflux and 3-O-methylglucose efflux in the two groups of rats, and the levels obtained were significantly higher in the T3-treated group. The stimulating effect of the alkaloid veratridine on Ca2+ efflux was 60% larger in 8-day hyperthyroid rats. Within 24 h after the start of T3 treatment, a significant (21%) increase in Ca2(+)-ATPase concentration was detected. Significant increases in active Ca2+ uptake and passive Ca2+ efflux were not observed until after 2 and 3 days of T3 treatment, respectively. It is concluded that T3 stimulates the synthesis of Ca2+ ATPase and augments the intracellular Ca2+ pools (sarcoplasmic reticulum and mitochondria). The latter results in enhancement of the passive Ca2+ leak, which in turn, may lead to activation of substrate transport systems. The suggested increase in intracellular Ca2+ cycling after T3 treatment may, at least partly, explain the T3-induced stimulation of energy metabolism.     

Sarcoplasmic reticulum lumenal Ca2+ has access to cytosolic activation and inactivation sites of skeletal muscle Ca2+ release channel.

The effects of sarcoplasmic reticulum lumenal (trans) Ca2+ on cytosolic (cis) ATP-activated rabbit skeletal muscle Ca2+ release channels (ryanodine receptors) were examined using the planar lipid bilayer method. Single channels were recorded in symmetric 0.25 M KCl media with K+ as the major current carrier. With nanomolar [Ca2+] in both bilayer chambers, the addition of 2 mM cytosolic ATP greatly increased the number of short channel openings. As lumenal [Ca2+] was increased from < 0.1 microM to approximately 250 microM, increasing channel activities and events with long open time constants were seen at negative holding potentials. Channel activity remained low at positive holding potentials. Further increase in lumenal [Ca2+] to 1, 5, and 10 mM resulted in a decrease in channel activities at negative holding potentials and increased activities at positive holding potentials. A voltage-dependent activation by 50 microM lumenal Ca2+ was also observed when the channel was minimally activated by < 1 microM cytosolic Ca2+ in the absence of ATP. With microM cytosolic Ca2+ in the presence or absence of 2 mM ATP, single-channel activities showed no or only a weak voltage dependence. Other divalent cations (Mg2+, Ba2+) could not replace lumenal Ca2+. On the contrary, cytosolic ATP-activated channel activities were decreased as lumenal Ca2+ fluxes were reduced by the addition of 1-5 mM BaCl2 or MgCl2 to the lumenal side, which contained 50 microM Ca2+. An increase in [KCl] from 0.25 M to 1 M also reduced single-channel activities. Addition of the "fast" Ca2+ buffer 1,2-bis(2-aminophenoxy)ethanetetraacetic acid (BAPTA) to the cls chamber increased cytosolic ATP-, lumenal Ca(2+)-activated channel activities to a nearly maximum level. These results suggested that lumenal Ca2+ flowing through the skeletal muscle Ca2+ release channel may regulate channel activity by having access to cytosolic Ca2+ activation and Ca2+ inactivation sites that are located in "BAPTA-inaccessible" and "BAPTA-accessible" spaces, respectively.     

Inhibition of Na+-dependent Ca2+ efflux from heart mitochondria by amiloride analogues

The Na+-induced release of accumulated Ca2+ from heart mitochondria is inhibited by amiloride, benzamil and several other amiloride analogues. These drugs do not affect uptake or release of Ca2+ mediated by the ruthenium red-sensitive uniporter and their effects, like those of diltiazem and other Ca2+-antagonists, appear to be localized principally at the Na+/Ca2+ antiporter of the mitochondrion. Benzamil inhibits Na+/Ca2+ antiport non-competitively with respect to [Na+] with a Ki of 167 microM. In the presence of 1.5 mM Pi the Ki for benzamil inhibition of this reaction is decreased to 87 microM.     

Comparison of sarcoplasmic reticulum Ca2+-adenosine triphosphatase from vitamin E-deficient dystrophic rabbit skeletal muscle with iron-ascorbate-treated and untreated enzyme

Sarcoplasmic reticulum Ca2+-ATPase from rabbit skeletal muscle has an Arrhenius curve of enzyme activity with a discontinuity at about 20 degrees C. Preparations treated with FeSO4 and ascorbic acid and from a vitamin E-deficient dystrophic rabbit have 22% of the normal activity and a linear Arrhenius curve (Promkhatkaew, D., Komaratat, P., & Wilairat, P. (1985) Biochem. Int. 10, 937-943). All three preparations were cross-linked to the same extent by dimethyl suberimidate and copper-phenanthroline reagent at temperatures above and below the temperature of the Arrhenius discontinuity. Both iron-ascorbate-treated Ca2+-ATPase and that from a vitamin E-deficient animal had 50% of the normal sulfhydryl content, but the disulfide and free amino contents were unaltered. These observations suggest that loss of sulfhydryl groups through lipid peroxidation, both in vivo and in vitro, resulted in reduction of Ca2+-ATPase activity and loss of the break in the Arrhenius plot. Changes in Ca2+-ATPase polypeptide aggregational state could not account for the discontinuity in the Arrhenius curve as revealed by the similar extent of cross-linking of the three enzyme preparations at temperatures above and below the temperature of the Arrhenius discontinuity.     

Crystallization of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum. Inhibition by myotoxin a

Decavanadate produces extensive ordered arrays of Ca2+-ATPase molecules on sarcoplasmic reticulum (SR) vesicle surfaces [(1984) J. Bioenerg. Biomembranes 16, 491-505] and the basic unit of these crystalline structures seems to be a dimer of Ca2+-ATPase [(1983) J. Ultrastruct. Res. 24, 454-464; (1984) J. Mol. Biol. 174, 193-204]. Myotoxin a, isolated from the venom of the prairie rattlesnake Crotalus viridis viridis, is a muscle-degenerating polypeptide and its primary site of interaction is the SR membrane, where it uncouples CA2+-translocation from CA2+-dependent ATP hydrolysis [(1986) Arch. Biochem. Biophys. 246, 90-97]. The effect of myotoxin a on decavanadate-induced two-dimensional Ca2+-ATPase crystals of SR membranes has been investigated. The toxin inhibits the formation of two-dimensional SR-membrane crystals and disrupts previously formed crystals in a time- and concentration-dependent manner, which parallels the uncoupling of ATP hydrolysis from Ca2+ translocation. Two-dimensional crystalline arrays of the SR membrane have a typical diffraction pattern which, after myotoxin a treatment, displays a progressive loss of order. Decavanadate is an uncompetitive inhibitor of the Ca2+-ATPase enzyme-myotoxin a complex. The present results suggest that a Ca2+-ATPase dimer is required for coupling Ca2+ translocation to Ca2+-dependent ATP hydrolysis.     

Characteristics of phosphate-induced Ca2+ efflux from the SR in mechanically skinned rat skeletal muscle fibers

The effects of P_i onsarcoplasmic reticulum (SR) Ca²⁺ regulation were studied inmechanically skinned rat skeletal muscle fibers. Brief application ofcaffeine was used to assess the SR Ca²⁺ content, andchanges in concentration of Ca²⁺([Ca²⁺]) within the cytosol were detected withfura 2 fluorescence. Introduction of P_i (1-40 mM)induced a concentration-dependent Ca²⁺ efflux from the SR.In solutions lacking creatine phosphate (CP), the amplitude of theP_i-induced Ca²⁺ transient approximatelydoubled. A similar potentiation of P_i-induced Ca²⁺ release occurred after inhibition of creatine kinase(CK) with 2,4-dinitrofluorobenzene. In the presence of ruthenium red or ryanodine, caffeine-induced Ca²⁺ release was almostabolished, whereas P_i-induced Ca²⁺ release wasunaffected. However, introduction of the SR Ca²⁺ ATPaseinhibitor cyclopiazonic acid effectively abolishedP_i-induced Ca²⁺ release. These data suggestthat P_i induces Ca²⁺ release from the SR byreversal of the SR Ca²⁺ pump but not via the SRCa²⁺ channel under these conditions. If this occurs inintact skeletal muscle during fatigue, activation of a Ca²⁺efflux pathway by P_i may contribute to the reporteddecrease in net Ca²⁺ uptake and increase in resting[Ca²⁺].

     

The effects of Mg2+ on the Ca2+-binding properties and Ca2+-induced tyrosine-fluorescence changes of calmodulin isolated from rabbit skeletal muscle

Calmodulin from phosphorylase kinase (the delta subunit) was obtained as a homogeneous protein in a spectroscopically pure form, and its interaction with Ca2+ and Mg2+ was studied. 1. Determination of the binding of Ca2+ to calmodulin in a buffer of low ionic strength (0.001 M) show that it contained six binding sites for this divalent cation. 2. Employment of a buffer of high ionic strength (0.18 M) allowed two Ca2+/Mg2+-binding sites (KdCa2+ = 4.0 microM), which showed Ca2+ - Mg2+ competition (KdMg2+ = 0.75 mM), to be distinguished from two Ca2+-specific binding sites (KdCa2+ = 40 microM). The remaining two Ca2+-binding sites are not observed under these conditions and are probably Mg2+-specific binding sites. Thus, the binding sites on calmodulin are remarkably similar to those of the homologous Ca2+-binding protein, troponin C [Potter and Gergely (1975) J. Biol. Chem. 250, 4628, 4633]. 3. The conformational states of calmodulin are defined by Ca2+, Mg2+ and salt concentrations, which can be differentiated by their Ca2+ affinity and their relative tyrosine fluorescence intensity. In a buffer of high ionic strength, Mg2+ induces a conformation which enhances the apparent affinity for Ca2+. Addition of Ca2+ leads to an enhancement of the tyrosine fluorescence intensity, which remains enhanced even upon removal of Ca2+ by chelation with EGTA. Only additional chelation of Mg2+ with EDTA reduces the tyrosine fluorescence intensity. 4. Comparison of the Ca2+-binding parameters of phosphorylase kinase, which were previously determined under identical experimental conditions [Kilimann and Heilmeyer (1977) Eur. J. Biochem. 73, 191-197], with those reported here on calmodulin isolated from this enzyme, allows the conclusion that Ca2+ binding to the holoenzyme occurs by binding to the delta subunit exclusively. 5. Ca2+ binding and Ca2+ activation of phosphorylase kinase are compared and discussed in relation to the Ca2+ and Mg2+-induced conformation changes of calmodulin.     

Increase in cytosolic Ca2+ induced by elevation of extracellular Ca2+ in skeletal myogenic cells

Cytoplasmic Ca²⁺concentration ([Ca²⁺]_i) variation is akey event in myoblast differentiation, but the mechanism by which itoccurs is still debated. Here we show that increases of extracellular Ca²⁺ concentration ([Ca²⁺]_o)produced membrane hyperpolarization and a concentration-dependent increase of [Ca²⁺]_i due to Ca²⁺influx across the plasma membrane. Responses were not related toinositol phosphate turnover and Ca²⁺-sensing receptor.[Ca²⁺]_o-induced[Ca²⁺]_i increase was inhibited byCa²⁺ channel inhibitors and appeared to be modulated byseveral kinase activities. [Ca²⁺]_i increasewas potentiated by depletion of intracellular Ca²⁺ storesand depressed by inactivation of the Na⁺/Ca²⁺exchanger. The response to arginine vasopressin (AVP), which inducesinositol 1,4,5-trisphosphate-dependent[Ca²⁺]_i increase in L6-C5 cells, was notmodified by high [Ca²⁺]_o. On the contrary,AVP potentiated the [Ca²⁺]_i increase in thepresence of elevated [Ca²⁺]_o. Other clones ofthe L6 line as well as the rhabdomyosarcoma RD cell line and thesatellite cell-derived C2-C12 line expressed similar responses to high[Ca²⁺]_o, and the amplitude of the responseswas correlated with the myogenic potential of the cells.

     

Fusion of proteoliposomes and cells. ATP-dependent Ca2+ uptake into erythrocytes catalyzed by Ca2+-ATPase from skeletal muscle

Purified Ca2+-ATPase from rabbit skeletal muscle has been incorporated into intact erythrocyte membranes by a two-step procedure. The isolated protein was reconstituted into proteoliposomes composed of phosphatidylethanolamine, phosphatidylcholine, and cardiolipin (50:20:30%, respectively). The resulting proteoliposomes were fused with erythrocytes in presence of La3+, Ca2+, or Mg2+. Subsequently, 45Ca uptake into the cells could be demonstrated. It was dependent on externally added ATP, inhibited by N-ethylmaleimide and p-hydroxymercuribenzoate, and enhanced by inactivation of the endogenous Ca2+-ATPase which catalyzes Ca2+ extrusion from the cells. The insertion of the protein did not induce cell lysis, but the cells did become more fragile. Functional insertion of isolated membrane proteins into cell membranes allows a new approach to research of plasma membranes.     

Tributyltin increases cytosolic free Ca2+ concentration in thymocytes by mobilizing intracellular Ca2+, activating a Ca2+ entry pathway, and inhibiting Ca2+ efflux.

The immunotoxic environmental pollutant tri-n-butyltin (TBT) kills thymocytes by apoptosis through a mechanism that requires an increase in intracellular Ca2+ concentration. The addition of TBT (EC50 = 2 microM) to fura-2-loaded rat thymocytes resulted in a rapid and sustained increase in the cytosolic free Ca2+ concentration ([Ca2+]i) to greater than 1 microM. In nominally Ca(2+)-free medium, TBT slightly but consistently increased thymocyte [Ca2+]i by about 0.11 microM. The subsequent restoration of CaCl2 to the medium resulted in a sustained overshoot in [Ca2+]i; similarly, the addition of MnCl2 produced a rapid decrease in the intracellular fura-2 fluorescence in thymocytes exposed to TBT. The rates of Ca2+ and Mn2+ entry stimulated by TBT were essentially identical to the rates stimulated by 2,5-di-(tert.-butyl)-1,4-benzohydroquinone (tBuBHQ), which has previously been shown to empty the agonist-sensitive endoplasmic reticular Ca2+ store and to stimulate subsequent Ca2+ influx by a capacitative mechanism. The addition of excess [ethylenebis(oxyethylenenitrilo)]tetraacetic acid to thymocytes produced a rapid return to basal [Ca2+]i after tBuBHQ treatment but a similar rapid return to basal [Ca2+]i was not observed after TBT treatment. In addition, TBT produced a marked inhibition of both Ca2+ efflux from the cells and the plasma membrane Ca(2+)-ATPase activity. Also, TBT treatment resulted in a rapid decrease in thymocyte ATP level. Taken together, our results show that TBT increases [Ca2+]i in thymocytes by the combination of intracellular Ca2+ mobilization, stimulation of Ca2+ entry, and inhibition of the Ca2+ efflux process. Furthermore, the ability of TBT to apparently mobilize the tBuBHQ-sensitive intracellular Ca2+ store followed by Ca2+ and Mn2+ entry suggests that the TBT-induced [Ca2+]i increase involves a capacitative type of Ca2+ entry.     

Ca2+ -induced Ca2+ release through localized Ca2+ uncaging in smooth muscle

Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) occurs in smooth muscle as spontaneous SR Ca(2+) release or Ca(2+) sparks and, in some spiking tissues, as Ca(2+) release that is triggered by the activation of sarcolemmal Ca(2+) channels. Both processes display spatial localization in that release occurs at a higher frequency at specific subcellular regions. We have used two-photon flash photolysis (TPFP) of caged Ca(2+) (DMNP-EDTA) in Fluo-4-loaded urinary bladder smooth muscle cells to determine the extent to which spatially localized increases in Ca(2+) activate SR release and to further understand the molecular and biophysical processes underlying CICR. TPFP resulted in localized Ca(2+) release in the form of Ca(2+) sparks and Ca(2+) waves that were distinguishable from increases in Ca(2+) associated with Ca(2+) uncaging, unequivocally demonstrating that Ca(2+) release occurs subsequent to a localized rise in [Ca(2+)](i). TPFP-triggered Ca(2+) release was not constrained to a few discharge regions but could be activated at all areas of the cell, with release usually occurring at or within several microns of the site of photolysis. As expected, the process of CICR was dominated by ryanodine receptor (RYR) activity, as ryanodine abolished individual Ca(2+) sparks and evoked release with different threshold and kinetics in FKBP12.6-null cells. However, TPFP CICR was not completely inhibited by ryanodine; Ca(2+) release with distinct kinetic features occurred with a higher TPFP threshold in the presence of ryanodine. This high threshold release was blocked by xestospongin C, and the pharmacological sensitivity and kinetics were consistent with CICR release at high local [Ca(2+)](i) through inositol trisphosphate (InsP(3)) receptors (InsP(3)Rs). We conclude that CICR activated by localized Ca(2+) release bears essential similarities to those observed by the activation of I(Ca) (i.e., major dependence on the type 2 RYR), that the release is not spatially constrained to a few specific subcellular regions, and that Ca(2+) release through InsP(3)R can occur at high local [Ca(2+)](i).     

Rapid filtration studies of Ca2+-induced Ca2+ release from skeletal sarcoplasmic reticulum. Role of monovalent ions

We have developed a rapid filtration technique for the measurement of Ca2+ release from isolated sarcoplasmic reticulum vesicles. Using this technique, we have studied the Ca2+-induced Ca2+ release of sarcoplasmic reticulum vesicles from rabbit skeletal muscle passively loaded with 5 mM Ca2+. The effect of known effectors (adenine nucleotides and caffeine) and inhibitors (Mg2+ and ruthenium red) of this release were investigated. In a medium composed of 100 mM KCl buffered at pH 6.8 with 20 mM K/3-(N-morpholino)propanesulfonic acid the Ca2+ release rate was maximal (500 nmol of Ca2+ released.(mg of protein)-1.s-1) at 1 micron external Ca2+ and 5 mM ATP. We also observed a rapid Ca2+ release induced by micromolar Ag+ in the presence of ATP (at 1 nM Ca2+). The Ag+-induced Ca2+ release was totally inhibited by 5 micron ruthenium red. We have also investigated the effect of monovalent ions on the Ca2+ release elicited by Ca2+ or Ag+. We show that the Ca2+ release rate: 1) was dependent upon the presence of K+ or Na+ in the release medium and 2) was influenced by a K+ gradient created across the sarcoplasmic reticulum membrane. These results directly support the idea of the involvement of an influx of K+ (through K+ channels) during the Ca2+ release and allow to reconsider a possible influence of the membrane potential of the sarcoplasmic reticulum on the Ca2+ release.     

Ca2+ sparks in skeletal muscle

The study of Ca²⁺ sparks has led to extensive new information regarding the gating of the Ca²⁺ release channels underlying these events in skeletal, cardiac and smooth muscle cells, as well as the possible roles of these local Ca²⁺ release events in muscle function. Here we review basic procedures for studying Ca²⁺sparks in skeletal muscle, primarily from frog, as well as the basic results concerning the properties of these events, their pattern and frequency of occurrence during fiber depolarization and the mechanisms underlying their termination. Finally, we also consider the contribution of different ryanodine receptor (RyR) isoforms to Ca²⁺ sparks and the number of RyR Ca²⁺ release channels that may contribute to the generation of a Ca²⁺ spark. Over the decade since their discovery, Ca²⁺ sparks have provided a wealth of information concerning the function of Ca²⁺ release channels within their intracellular environment.