The changes in Ca2+ sparks associated with measured modifications of intra-store Ca2+ concentration in skeletal muscle

In cardiac muscle and amphibian skeletal muscle, the intracellular Ca2+ release that signals contractile activation proceeds by discrete local packets, which result in Ca2+ sparks. The remarkably stereotyped duration of these release events requires a robustly timed termination mechanism. In cardiac muscle the mechanism of spark termination appears to crucially involve depletion of Ca2+ in the lumen of the sarcoplasmic reticulum (SR), but in skeletal muscle, the mechanism is unknown. We used SEER (shifted excitation and emission ratioing of fluorescence) of SR-trapped mag-indo-1 and confocal imaging of fluorescence of cytosolic rhod-2 to image Ca2+ sparks while reversibly changing and measuring [Ca2+] in the SR ([Ca2+]SR) of membrane-permeabilized frog skeletal muscle cells. Sparks were collected in cells immersed in a solution promoting production of events at moderate frequency. Just after permeabilization, event frequency was zero, and in 10 minutes it reached close to a steady value. Controlled interventions modified [Ca2+]SR reversibly between a low value (299 microM on average in 10 experiments) and a high value (433 microM, a 45% average increase). This change increased sparks frequency by 93%, spatial width by 7%, rise time by 10%, and peak amplitude by 38% (provided that it was calculated in absolute terms, rather than normalized by resting fluorescence). The changes in event frequency and amplitude were statistically significant. The "strength" of the effect of [Ca2+]SR on frequency, quantified by decomposition of variance, was <6%. While the average change in [Ca2+]SR was limited, it reached up to 200% in individual fibers, without causing massive Ca2+ release or an increase of >3.5-fold in event frequency. Taken together with existing evidence that depletion is modest during Ca2+ sparks or release elicited by an action potential, the mild effects of [Ca2+]SR reported here do not support a major role of depletion in either the termination of sparks or the strong inactivation that terminates Ca2+ release at the global level in frog skeletal muscle.     

FKBP12.6 overexpression decreases Ca2+ spark amplitude but enhances [Ca2+]i transient in rat cardiac myocytes

Ryanodine receptors/Ca2+-release channels (RyR2) from the sarcoplasmic reticulum (SR) provide the Ca2+ required for contraction at each cardiac twitch. RyR2 are regulated by a variety of proteins, including the immunophilin FK506 binding protein (FKBP12.6). FKBP12.6 seems to be important for coupled gating of RyR2 and its deficit and alteration may be involved in heart failure. The role of FKBP12.6 on Ca2+ release has not been analyzed directly, but rather it was inferred from the effects of immunophilins, such us FK506 and rapamycin, which, among other effects, dissociates FKBP12.6 from the RyR2. Here, we investigated directly the effects of FKBP12.6 on local (Ca2+ sparks) and global [intracellular Ca2+ concentration ([Ca2+]i) transients] Ca2+ release in single rat cardiac myocytes. The FKBP12.6 gene was transfected in single myocytes using the adenovirus technique with a reporter gene strategy based on green fluorescent protein (GFP) to check out the success of transfections. Control myocytes were transfected with only GFP (Ad-GFP). Rhod-2 was used as the Ca2+ indicator, and cells were viewed with a confocal microscope. We found that overexpression of FKBP12.6 decreases the occurrence, amplitude, duration, and width of spontaneous Ca2+ sparks. FK506 had diametrically opposed effects. However, overexpression of FKBP12.6 increased the [Ca2+]i transient amplitude and accelerated its decay in field-stimulated cells. The associated cell shortening was increased. SR Ca2+ load, estimated by rapid caffeine application, was increased. In conclusion, FKBP12.6 overexpression decreases spontaneous Ca2+ sparks but increases [Ca2+]i transients, in relation with enhanced SR Ca2+ load, therefore improving excitation-contraction coupling.     

Muscle aging is associated with compromised Ca2+ spark signaling and segregated intracellular Ca2+ release

Reduced homeostatic capacity for intracellular Ca2+ ([Ca2+]i) movement may underlie the progression of sarcopenia and contractile dysfunction during muscle aging. We report two alterations to Ca2+ homeostasis in skeletal muscle that are associated with aging. Ca2+ sparks, which are the elemental units of Ca2+ release from sarcoplasmic reticulum, are silent under resting conditions in young muscle, yet activate in a dynamic manner upon deformation of membrane structures. The dynamic nature of Ca2+ sparks appears to be lost in aged skeletal muscle. Using repetitive voltage stimulation on isolated muscle preparations, we identify a segregated [Ca2+]i reserve that uncouples from the normal excitation-contraction process in aged skeletal muscle. Similar phenotypes are observed in adolescent muscle null for a synaptophysin-family protein named mitsugumin-29 (MG29) that is involved in maintenance of muscle membrane ultrastructure and Ca2+ signaling. This finding, coupled with decreased expression of MG29 in aged skeletal muscle, suggests that MG29 expression is important in maintaining skeletal muscle Ca2+ homeostasis during aging.     

Redox regulation of calcium release in skeletal and cardiac muscle

In skeletal and cardiac muscle cells, specific isoforms of the Ryanodine receptor channels mediate Ca2+ release from the sarcoplasmic reticulum. These channels are highly susceptible to redox modifications, which regulate channel activity. In this work, we studied the effects of Ca2+ (endogenous agonist) and Mg2+ (endogenous inhibitor) on the kinetics of Ca2+ release from sarcoplasmic reticulum vesicles isolated from skeletal or cardiac mammalian muscle. Native skeletal vesicles exhibited maximal stimulation of release kinetics by 10-20 microM [Ca2+], whereas in native cardiac vesicles, maximal stimulation of release required only 1 microM [Ca2+]. In 10 microM [Ca2+], free [Mg2+] < 0.1 mM produced marked inhibition of release from skeletal vesicles but free [Mg2+] < or = 0.8 mM did not affect release from cardiac vesicles. Incubation of skeletal or cardiac vesicles with the oxidant thimerosal increased their susceptibility to stimulation by Ca2+ and decreased the inhibitory effect of Mg2+ in skeletal vesicles. Sulfhydryl-reducing agents fully reversed the effects of thimerosal. The endogenous redox species, glutathione disulfide and S-nitrosoglutathione, also stimulated release from skeletal sarcoplasmic reticulum vesicles. In 10 microM [Ca2+], 35S-nitrosoglutathione labeled a protein fraction enriched in release channels through S-glutathiolation. Free [Mg2+] 1 mM or decreasing free [Ca2+] to the nM range prevented this reaction. Possible physiological and pathological consequences of redox modification of release channels on Ca2+ signaling in heart and muscle cells are discussed.     

Role of mitochondria in calcium regulation of spontaneously contracting cardiac muscle cells.

Mitochondrial involvement in the regulation of cytosolic calcium concentration ([Ca2+]i) in cardiac myocytes has been largely discounted by many authors. However, recent evidence, including the results of this study, has forced a reappraisal of this role. [Ca2+]i and Ca2+ in the mitochondria ([Ca2+]m) were measured in this study with specific fluorescent probes, fluo-3 and di-hydro-rhod-2, respectively; mitochondrial membrane potential (DeltaPsim) was monitored with JC-1. Addition of uncouplers or inhibitors of the mitochondrial respiratory chain was found to cause a twofold decrease in the rate of removal of Ca2+ from the cytosol after a spontaneously generated Ca2+ wave. These agents also caused a progressive elevation of [Ca2+]i, an increase in the number of hotspots of Ca2+ release (Ca2+ sparks), and depression of mitochondrial potential. The Ca2+-indicative fluorophore dihydro-rhod-2 has a net positive charge that contributes to selective accumulation by mitochondria, as supported by its co-localization with other mitochondrial-specific probes (MitoTracker Green). Treatment of dihydro-rhod-2-loaded cells with NaCN resulted in rapid formation of "black holes" in the otherwise uniformly banded pattern. These are likely to represent individual or small groups of mitochondria that have depressed mitochondrial potential, or have lost accumulated rhod-2 and/or Ca2+; all of these eventualities are possible upon onset of the mitochondrial permeability transition. Release of Ca2+ from the sarcoplasmic reticulum and the resultant spontaneous contractility of cardiac muscle are proposed to be triggered by the induction of the mitochondrial permeability transition and the subsequent loss of [Ca2+]m.     

A probability density approach to modeling local control of calcium-induced calcium release in cardiac myocytes

We present a probability density approach to modeling localized Ca2+ influx via L-type Ca2+ channels and Ca2+-induced Ca2+ release mediated by clusters of ryanodine receptors during excitation-contraction coupling in cardiac myocytes. Coupled advection-reaction equations are derived relating the time-dependent probability density of subsarcolemmal subspace and junctional sarcoplasmic reticulum [Ca2+] conditioned on "Ca2+ release unit" state. When these equations are solved numerically using a high-resolution finite difference scheme and the resulting probability densities are coupled to ordinary differential equations for the bulk myoplasmic and sarcoplasmic reticulum [Ca2+], a realistic but minimal model of cardiac excitation-contraction coupling is produced. Modeling Ca2+ release unit activity using this probability density approach avoids the computationally demanding task of resolving spatial aspects of global Ca2+ signaling, while accurately representing heterogeneous local Ca2+ signals in a population of diadic subspaces and junctional sarcoplasmic reticulum depletion domains. The probability density approach is validated for a physiologically realistic number of Ca2+ release units and benchmarked for computational efficiency by comparison to traditional Monte Carlo simulations. In simulated voltage-clamp protocols, both the probability density and Monte Carlo approaches to modeling local control of excitation-contraction coupling produce high-gain Ca2+ release that is graded with changes in membrane potential, a phenomenon not exhibited by so-called "common pool" models. However, a probability density calculation can be significantly faster than the corresponding Monte Carlo simulation, especially when cellular parameters are such that diadic subspace [Ca2+] is in quasistatic equilibrium with junctional sarcoplasmic reticulum [Ca2+] and, consequently, univariate rather than multivariate probability densities may be employed.     

Nitric oxide affects sarcoplasmic calcium release in skeletal myotubes.

In the present study, we used real-time confocal microscopy to examine the effects of two nitric oxide (NO) donors on acetylcholine (ACh; 10 microM)- and caffeine (10 mM)-induced intracellular calcium concentration ([Ca2+]i) responses in C2C12 mouse skeletal myotubes. We hypothesized that NO reduces [Ca2+]i in activated skeletal myotubes through oxidation of thiols associated with the sarcoplasmic reticulum Ca2+-release channel. Exposure to diethylamine NONOate (DEA-NO) reversibly increased resting [Ca2+]i level and resulted in a dose-dependent reduction in the amplitude of ACh-induced [Ca2+]i responses (25 +/- 7% reduction with 10 microM DEA-NO and 78 +/- 14% reduction with 100 microM DEA-NO). These effects of DEA-NO were partly reversible after subsequent exposure to dithiothreitol (10 mM). Preexposure to DEA-NO (1, 10, and 50 microM) also reduced the amplitude of the caffeine-induced [Ca2+]i response. Similar data were obtained by using the chemically distinct NO donor S-nitroso-N-acetyl-penicillamine (100 microM). These results indicate that NO reduces sarcoplasmic reticulum Ca2+ release in skeletal myotubes, probably by a modification of hyperreactive thiols present on the ryanodine receptor channel.     

Temperature dependence of Ca2+ wave properties in cardiomyocytes: implications for the mechanism of autocatalytic Ca2+ release in wave propagation.

Digital imaging microscopy of fluo-3 fluorescence was used to study the velocity and shape of intracellular Ca2+ waves in isolated rat cardiomyocytes as a function of temperature. Decreasing the temperature from 37 to 17 degrees C reduced the longitudinal wave velocity by a factor of 1.8 and remarkably slowed the decay of [Ca2+]i in the trailing flank of a wave. Using image analysis, rise times, and half-maximum decay times of local Ca2+ transients, which characterize the processes of local Ca2+ release and removal, were determined as a function of temperature. Apparent activation energies for wave front propagation, local Ca2+ release, and local Ca2+ removal were derived from Arrhenius plots and amounted to -23, -28, and -46 kJ/mol, respectively. The high activation energy of Ca2+ removal, which arises from the activity of the sarcoplasmic reticulum (SR) Ca2+ ATPase, relative to those of longitudinal wave propagation and local Ca2+ release excludes the hypothetical mechanism of regenerative "spontaneous Ca2+ release," in which Ca2+ that has been taken up from the approaching wavefront triggers Ca2+ release at a luminal site of the SR. It is consistent, however, with the hypothesis that Ca2+ wave propagation is based on Ca(2+)-induced Ca2+ release where Ca2+ triggers release on the cytosolic face of the SR.     

Spread of Ca2+ in the sarcomere during fast and slow activation of mammalian cardiac myocytes

A multi-compartment model was used to estimate Ca2+ gradients in a sarcomere of a cardiac myocyte. The mathematical model assumed Ca2+ release from the sarcoplasmic reticulum as a driving function, and calculated Ca2+ binding to myoplasmic buffers, Ca2+ uptake by the sarcoplasmic reticulum, and diffusion of Ca2+ (and the buffers). During the fast Ca2+ transient similar to those observed during a twitch, the model predicted a large Ca2+ gradient in the sarcomere. A trajectory of the instantaneous relation between spatially averaged concentrations of Ca2+ and the Ca2+-troponin complex showed a counterclockwise loop, indicating non-equilibrium Ca2+ binding to troponin. During slow changes in [Ca2+] with time to peaks of approximately 500 ms or longer, the gradient of [Ca2+] was largely dissipated and the apparent equilibrium of the Ca2+-troponin binding reaction was suggested with little hysteresis of the trajectory. We conclude that a steady-state relation between [Ca2+] and mechanical activity can be achieved uniformly in the sarcomere by slowing the rate of Ca2+ release from the sarcoplasmic reticulum.     

Calcium sparks in mouse ventricular myocytes at physiological temperature

In cardiac muscle, Ca2+ is released from the sarcoplasmic reticulum (SR) in units called Ca2+ sparks. Ca2+ spark characteristics have been studied almost entirely at room temperature. This study compares characteristics of spontaneous sparks detected with fluo 3 in resting mouse ventricular myocytes at 22 and 37 degrees C. The incidence and frequency of Ca2+ sparks decreased dramatically at 37 degrees C compared with 22 degrees C. Also, spark amplitudes and times to peak were significantly reduced at 37 degrees C. In contrast, spatial width and decay times were unchanged. During field stimulation, peak spatially averaged transients were similar at 22 and 37 degrees C, and experiments with fura 2 demonstrated that diastolic and systolic Ca2+ concentrations were unchanged. However, SR Ca2+ content decreased significantly at 37 degrees C. Restoration of SR Ca2+ by superfusion with 5 mM Ca2+ increased spark frequency but did not reverse the effects of temperature on spark parameters. Thus effects of temperature on spark frequency may reflect changes in SR stores, whereas changes in spark amplitude and rise time may reflect known effects of temperature on ryanodine receptor function.     

Ca2+ signaling in human fetoplacental vasculature: effect of CGRP on umbilical vein smooth muscle cytosolic Ca2+ concentration

CGRP is a potent vasodilator with increased levels in fetoplacental circulation during late pregnancy. We have recently demonstrated that acute CGRP exposure to fetoplacental vessels in vitro induced vascular relaxation, but the signaling pathway of CGRP in fetoplacental vasculature remains unclear. We hypothesized that CGRP relaxes fetoplacental vasculature via regulating smooth muscle cytosolic Ca2+ concentrations. In the present study, by using human umbilical vein smooth muscle (HUVS) cells (HUVS-112D), we examined CGRP receptors, cAMP generation, and changes in cellular Ca2+ concentrations on CGRP treatment. These cells express mRNA for CGRP receptor components, calcitonin receptor-like receptor, and receptor activity-modifying protein-1. Direct saturation binding for 125I-labeled CGRP to HUVS cells and Scatchard analysis indicate specificity of the receptors for CGRP [dissociation constant (K(D)) = 67 nM, maximum binding capcity (Bmax) = 2.7 pmol/million cells]. Exposure of HUVS cells to CGRP leads to a dose-dependent increase in intracellular cAMP accumulation, and this increase is prevented by CGRP antagonist CGRP(8-37). Using fura-2-loaded HUVS cells, we monitored the effects of CGRP on intracellular Ca2+ concentration ([Ca2+]i). In the presence of extracellular Ca2+, bradykinin (10(-6) M), a fetoplacental vasoconstrictor, increases HUVS cells [Ca2+]i concentration. CGRP (10(-8) M) abolishes bradykinin-induced [Ca2+]i elevation. When the cells were pretreated with glibenclamide, an ATP-sensitive potassium channel blocker, the CGRP actions on bradykinin-induced Ca2+ influx were profoundly inhibited. In the absence of extracellular Ca2+, CGRP (10(-8) M) attenuated the increase of [Ca2+]i induced by a sarcoplasmic reticulum Ca2+ pump ATPase inhibitor thapsigargin (10(-5) M). Furthermore, Rp-cAMPS, a cAMP-dependent protein kinase A inhibitor, blocks CGRP actions on thapsigargin-induced Ca2+ release from sarcoplasmic reticulum. Our results suggested that CGRP relaxes human fetoplacental vessels by not only inhibiting the influx of extracellular Ca2+ but also attenuating the release of intracellular Ca2+ from the sarcoplasmic reticulum, and these actions might be attributed to CGRP-induced intracellular cAMP accumulation.     

Effect of the antidepressant sertraline on Ca2+fluxes in Madin-Darby canine renal tubular cells

The effect of the antidepressant sertraline on cytosolic-free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells is unclear. This study explored whether sertraline changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Sertraline at concentrations between 1and 100 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+ implicating Ca2+ entry and release both contributed to the [Ca2+]i rise. Sertraline induced Mn2+ influx, leading to quench of fura-2 fluorescence, suggesting Ca2+ influx. This Ca2+ influx was inhibited by suppression of phospholiapase A2 but not by store-operated Ca2+ channel blockers and protein kinase C/A modulators. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitors nearly abolished sertraline-induced Ca2+ release. Conversely, pretreatment with sertraline partly reduced inhibitor-induced [Ca2+]i rise, suggesting that sertraline released Ca2+ from endoplasmic reticulum. Inhibition of phospholipase C did not much alter sertraline-induced [Ca2+]i rise. Collectively, in MDCK cells, sertraline induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-sensitive Ca2+ channels.     

Expression of active p21-activated kinase-1 induces Ca2+ flux modification with altered regulatory protein phosphorylation in cardiac myocytes

p21-Activated kinase-1 (Pak1) is a serine-threonine kinase that associates with and activates protein phosphatase 2A in adult ventricular myocytes and, thereby, induces increased Ca2+ sensitivity of skinned-fiber tension development mediated by dephosphorylation of myofilament proteins (Ke Y, Wang L, Pyle WG, de Tombe PP, Solaro RJ. Circ Res 94: 194-200, 2004). We test the hypothesis that activation of Pak1 also moderates cardiac contractility through regulation of intracellular Ca2+ fluxes. We found no difference in field-stimulated intracellular Ca2+ concentration ([Ca2+]i) transient amplitude and extent of cell shortening between myocytes expressing constitutively active Pak1 (CA-Pak1) and controls expressing LacZ; however, time to peak shortening was significantly faster and rate of [Ca2+]i decay and time of relengthening were slower. Neither caffeine-releasable sarcoplasmic reticulum (SR) Ca2+ content nor fractional release was different in CA-Pak1 myocytes compared with controls. Isoproterenol application revealed a significantly blunted increase in [Ca2+]i transient amplitude, as well as a slowed rate of [Ca2+]i decay, increased SR Ca2+ content, and increased cell shortening, in CA-Pak1 myocytes. We found no significant change in phospholamban phosphorylation at Ser16 or Thr17 in CA-Pak1 myocytes. Analysis of cardiac troponin I revealed a significant reduction in phosphorylated species that are primarily attributable to Ser(23/24) in CA-Pak1 myocytes. Nonstimulated, spontaneous SR Ca2+ release sparks were significantly smaller in amplitude in CA-Pak1 than LacZ myocytes. Propagation of spontaneous Ca2+ waves resulting from SR Ca2+ overload was significantly slower in CA-Pak1 myocytes. Our data indicate that CA-Pak1 expression has significant effects on ventricular myocyte contractility through altered myofilament Ca2+ sensitivity and modification of the [Ca2+]i transient.     

Characterization of hypoxia-induced [Ca2+]i rise in rabbit pulmonary arterial smooth muscle cells

We have investigated the effects of hypoxia on the intracellular Ca2+ concentration ([Ca2+]i) in rabbit pulmonary (PASMCs) and coronary arterial smooth muscle cells with fura-2. Perfusion of a glucose-free and hypoxic (PO2<50 mmHg) external solution increased [Ca2+]i in cultured as well as freshly isolated PASMCs. However it had no effect on [Ca2+]i in freshly isolated coronary arterial myocytes. In the absence of extracellular Ca2+, hypoxic stimulation elicited a transient [Ca2+]i increase in cultured PASMCs which was abolished by the simultaneous application of cyclopiazonic acid and ryanodine, suggesting the involvement of sarcoplasmic reticulum (SR) Ca2+ store. Pretreatment with the mitochondrial protonophore, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) enhanced the [Ca2+]i rise in response to hypoxia. A short application of caffeine gave a transient [Ca2+]i rise which was prolonged by CCCP. Decay of the caffeine-induced [Ca2+]i transients was significantly slowed by treatment of CCCP or rotenone. After full development of the hypoxia-induced [Ca2+]i rise, nifedipine did not decrease [Ca2+]i. These data suggest that the [Ca2+]i increase in response to hypoxia may be ascribed to both Ca2+ release from the SR and the subsequent activation of nifedipine-insensitive capacitative Ca2+ entry. Mitochondria appear to modulate hypoxia induced Ca2+ release from the SR.     

Modulation of agonist-induced Ca2+ release by SR Ca2+ load: direct SR and cytosolic Ca2+ measurements in rat uterine myocytes

Release of Ca2+ from sarcoplasmic reticulum (SR) is one of the most important mechanisms of smooth muscle stimulation by a variety of physiologically active substances. Agonist-induced Ca2+ release is considered to be dependent on the Ca2+ content of the SR, although the mechanism underlying this dependence is unclear. In the present study, the effect of SR Ca2+ load on the amplitude of [Ca2+]i transients elicited by application of the purinergic agonist ATP was examined in uterine smooth muscle cells isolated from pregnant rats. Measurement of intraluminal Ca2+ level ([Ca2+]L) using a low affinity Ca indicator, mag-fluo-4, revealed that incubation of cells in a high-Ca2+ (10 mM) extracellular solution leads to a substantial increase in [Ca2+]L (SR overload). However, despite increased SR Ca2+ content this did not potentiate ATP-induced [Ca2+]i transients. Repetitive applications of ATP in the absence of extracellular Ca2+, as well as prolonged incubation in Ca2+-free solution without agonist, depleted the [Ca2+]L (SR overload). In contrast to overload, partial depletion of the SR substantially reduced the amplitude of Ca2+ release. ATP-induced [Ca2+]i transients were completely abolished when SR Ca2+ content was decreased below 80% of its normal value indicating a steep dependence of the IP3-mediated Ca2+ release on the Ca2+ load of the store. Our results suggest that in uterine smooth muscle cells decrease in the SR Ca2+ load below its normal resting level substantially reduces the IP3-mediated Ca2+ release, while Ca2+ overload of the SR has no impact on such release.     

Kinetic studies of calcium release from sarcoplasmic reticulum in vitro

Release of Ca2+ from a heavy fraction of rabbit skeletal muscle sarcoplasmic reticulum was triggered by several different methods: (a) increasing extravesicular [Ca2+] [( CaO2+] from about 0.1 microM to 10 microM), (b), adding caffeine, (c) adding quercetin, and (d) substituting a solution containing equimolar choline+ for K+-containing solution (depolarization-induced Ca2+ release). The maximal rate of Ca2+ release triggered by caffeine or quercetin in the presence of 12.5 microM [CaO2+] (21-25 nmol of Ca2+/mg/s) is similar to that of the depolarization-induced Ca2+ release (19 nmol of Ca2+/mg/s), as determined by stopped flow spectrometry of changes in [CaO2+] with arsenazo III. The release is transient and all of the released Ca2+ is reaccumulated. The rates of Ca2+ release triggered by caffeine, quercetin, or membrane depolarization sharply decrease at high [CaO2+], suggesting a negative feedback effect of the released Ca2+. Inhibition of the release pathway allows the sarcoplasmic reticulum to reaccumulate Ca2+. The rate of Ca2+ release triggered by caffeine or quercetin, but not that triggered by membrane depolarization, is also reduced upon decreasing [CaO2+] to the submicromolar range. Passive efflux of intravesicular Ca2+ in solutions containing lower [CaO2+] in the absence of Mg.ATP is attenuated at about the same time (congruent to 1 min) regardless of the amounts of Ca2+ released, indicating that the opened Ca2+ channels close spontaneously. These results suggest that kinetically identical channels are responsible for Ca2+ release independent of the methods of triggering and this in vitro release is consistent with the physiological mechanism both in terms of the rapidity and the reversibility of Ca2+ release.     

Paroxetine-induced Ca2+ movement and death in OC2 human oral cancer cells

The effect of the antidepressant paroxetine on cytosolic free Ca2+ concentrations ([Ca2+]i) in OC2 human oral cancer cells is unclear. This study explored whether paroxetine changed basal [Ca2+]i levels in suspended OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Paroxetine at concentrations between 100-1,000 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 50% by removing extracellular Ca2+. Paroxetine-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, and protein kinase C modulators. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished paroxetine-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not alter paroxetine-induced [Ca2+]i rise. Paroxetine at 10-50 microM induced cell death in a concentration-dependent manner. The death was not reversed when cytosolic Ca2+ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Propidium iodide staining suggests that apoptosis plays a role in the death. Collectively, in OC2 cells, paroxetine induced [Ca2+]i rise by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels in a manner regulated by protein kinase C and phospholipase A2. Paroxetine (up to 50 microM) induced cell death in a Ca2+-independent manner.     

Calcium-induced calcium release from purified cardiac sarcoplasmic reticulum vesicles. General characteristics

Isolated canine cardiac sarcoplasmic reticulum exhibits Ca2+-induced Ca2+ release from both actively and passively loaded vesicles. The rate and extent of Ca2+ release depend on the extravesicular ionized Ca2+ concentration ( [Ca2+]o) at the onset of release. Maximal release following ATP-dependent, phosphate-facilitated Ca2+ loading (up to 360 nmol of Ca2+/mg of protein/min at 37 degrees C) occurs at 1.5-2 microM [Ca2+]o, with reduced release at both lower and higher Ca2+ concentrations (half-maximal Ca2+ release at approximately 0.8 and 5.5 microM [Ca2+]o). Only a portion of the accumulated Ca2+ is released and the release is followed by reuptake of Ca2+. A similar Ca2+ dependence is obtained in the absence of ATP and Pi by measuring unidirectional Ca2+ efflux from passively loaded vesicles (maximal Ca2+ efflux at 1 microM [Ca2+]o; half-maximal Ca2+-dependent efflux at approximately 0.15 and 13 microM [Ca2+]o). Although the Ca2+ release rates observed in this study are several orders of magnitude lower than the rate of Ca2+ release which occurs in muscle cells in vivo, this Ca2+ release phenomenon may be related to the Ca2+-induced Ca2+ release which has been described for skinned cardiac cells ( Fabiato , A. (1983) Am. J. Physiol. 245, C1-C14). Ca2+ release occurs in the presence of an ATP-regenerating system and is not accompanied by a reduction in ATP hydrolysis. Also, since unidirectional Ca2+ efflux (as high as 860 nmol of Ca2+/mg of protein/min at 37 degrees C) exceeds net Ca2+ release under similar conditions, Ca2+ influx proceeds during the period of net Ca2+ release. Therefore, Ca2+ release does not involve reversal or cessation of inward Ca2+ pumping. Other data indicate that Ca2+ release is not mediated through the Ca2+ pump protein, but occurs through a separate Ca2+-dependent efflux pathway, possibly a channel.     

Role of sarcoplasmic reticulum and mitochondria in Ca2+ removal in airway myocytes

The aim of this study was to use both a theoretical and experimental approach to determine the influence of the sarco-endoplasmic Ca2+-ATPase (SERCA) activity and mitochondria Ca2+ uptake on Ca2+ homeostasis in airway myocytes. Experimental studies were performed on myocytes freshly isolated from rat trachea. [Ca2+]i was measured by microspectrofluorimetry using indo-1. Stimulation by caffeine for 30 s induced a concentration-graded response characterized by a transient peak followed by a progressive decay to a plateau phase. The decay phase was accelerated for 1-s stimulation, indicating ryanodine receptor closure. In Na2+-Ca2+-free medium containing 0.5 mM La3+, the [Ca2+]i response pattern was not modified, indicating no involvement of transplasmalemmal Ca2+ fluxes. The mathematical model describing the mechanism of Ca2+ handling upon RyR stimulation predicts that after Ca2+ release from the sarcoplasmic reticulum, the Ca2+ is first sequestrated by cytosolic proteins and mitochondria, and pumped back into the sarcoplasmic reticulum after a time delay. Experimentally, we showed that the [Ca2+]i decay after Ca2+ increase was not altered by the SERCA inhibitor cyclopiazonic acid, but was slightly but significantly modified by the mitochondria uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone. The experimental and theoretical results indicate that, although Ca2+ pumping back by SERCA is active, it is not primarily involved in [Ca2+]i decrease that is due, in part, to mitochondrial Ca2+ uptake.     

Enhanced Ca2+-induced calcium release by isolated sarcoplasmic reticulum vesicles from malignant hyperthermia susceptible pig muscle

To further define the possible involvement of sarcoplasmic reticulum calcium accumulation and release in the skeletal muscle disorder malignant hyperthermia (MH), we have examined various properties of sarcoplasmic reticulum fractions isolated from normal and MH-susceptible pig muscle. A sarcoplasmic reticulum preparation enriched in vesicles derived from the terminal cisternae, was further fractionated on discontinuous sucrose density gradients (Meissner, G. (1984) J. Biol. Chem. 259, 2365-2374). The resultant MH-susceptible and normal sarcoplasmic reticulum fractions, designated F0-F4, did not differ in yield, cholesterol and phospholipid content, or nitrendipine binding capacity. Calcium accumulation (0.27 mumol Ca/mg per min at 22 degrees C), Ca2+-ATPase activity (0.98 mumol Pi/mg per min at 22 degrees C), and calsequestrin content were also similar for MH-susceptible and normal sarcoplasmic reticulum fraction F3. To examine sarcoplasmic reticulum calcium release, fraction F3 vesicles were passively loaded with 45Ca (approx. 40 nmol Ca/mg), and rapidly diluted into a medium of defined Ca2+ concentration. Upon dilution into 1 microM Ca2+, the extent of Ca2+-dependent calcium release measured after 5 s was significantly greater for MH-susceptible than for normal sarcoplasmic reticulum, 65.9 +/- 2.8% vs. 47.7 +/- 3.9% of the loaded calcium, respectively. The C1/2 for Ca2+ stimulation of this calcium release (5 s value) from MH-susceptible sarcoplasmic reticulum also appeared to be shifted towards a higher Ca2+-sensitivity when compared to normal sarcoplasmic reticulum. Dantrolene had no effect on calcium release from fraction F3, however, halothane (0.1-0.5 mM) increased the extent of calcium release (5 s) similarly in both MH-susceptible and normal sarcoplasmic reticulum. Furthermore, Mg2+ was less effective at inhibiting, while ATP and caffeine were more effective in stimulating, this Ca2+-dependent release of calcium from MH-susceptible, when compared to normal sarcoplasmic reticulum. Our results demonstrate that while sarcoplasmic reticulum calcium-accumulation appears unaffected in MH, aspect(s) of the sarcoplasmic reticulum Ca2+-induced calcium release mechanism are altered. Although the role of the Ca2+-induced calcium release mechanism of sarcoplasmic reticulum in situ is not yet clear, our results suggest that an abnormality in the regulation of sarcoplasmic reticulum calcium release may play an important role in the MH syndrome.