Subcellular analysis of Ca2+ homeostasis in primary cultures of skeletal muscle myotubes.

Specifically targeted aequorin chimeras were used for studying the dynamic changes of Ca2+ concentration in different subcellular compartments of differentiated skeletal muscle myotubes. For the cytosol, mitochondria, and nucleus, the previously described chimeric aequorins were utilized; for the sarcoplasmic reticulum (SR), a new chimera (srAEQ) was developed by fusing an aequorin mutant with low Ca2+ affinity to the resident protein calsequestrin. By using an appropriate transfection procedure, the expression of the recombinant proteins was restricted, within the culture, to the differentiated myotubes, and the correct sorting of the various chimeras was verified with immunocytochemical techniques. Single-cell analysis of cytosolic Ca2+ concentration ([Ca2+]c) with fura-2 showed that the myotubes responded, as predicted, to stimuli known to be characteristic of skeletal muscle fibers, i.e., KCl-induced depolarization, caffeine, and carbamylcholine. Using these stimuli in cultures transfected with the various aequorin chimeras, we show that: 1) the nucleoplasmic Ca2+ concentration ([Ca2+]n) closely mimics the [Ca2+]c, at rest and after stimulation, indicating a rapid equilibration of the two compartments also in this cell type; 2) on the contrary, mitochondria amplify 4-6-fold the [Ca2+]c increases; and 3) the lumenal concentration of Ca2+ within the SR ([Ca2+]sr) is much higher than in the other compartments (> 100 microM), too high to be accurately measured also with the aequorin mutant with low Ca2+ affinity. An indirect estimate of the resting value (approximately 1-2 mM) was obtained using Sr2+, a surrogate of Ca2+ which, because of the lower affinity of the photoprotein for this cation, elicits a lower rate of aequorin consumption. With Sr2+, the kinetics and amplitudes of the changes in [cation2+]sr evoked by the various stimuli could also be directly analyzed.     

A dynamic pool of calcium in catecholamine storage vesicles. Exploration in living cells by a novel vesicle-targeted chromogranin A-aequorin chimeric photoprotein

Chromaffin vesicles contain very high concentration of Ca2+ (approximately 20-40 mM total), compared with approximately 100 nM in the cytosol. Aequorin, a jellyfish photoprotein with Ca(2+)-dependent luminescence, measures [Ca2+] in specific subcellular compartments wherein proteins with organelle-specific trafficking domains are fused in-frame to aequorin. Because of the presence of vesicular trafficking domain within CgA we engineered sorting of an expressed human CgA-Aequorin fusion protein (hCgA-Aeq) into the vesicle compartment as confirmed by sucrose density gradients and confocal immunofluorescent co-localization studies. hCgA-Aeq and cytoplasmic aequorin (Cyto-Aeq) luminescence displayed linear functions of [Ca2+] in vitro, over >5 log10 orders of magnitude (r > 0.99), and down to at least 10(-7) M sensitivity. Calibrating the pH dependence of hCgA-Aeq luminescence allowed estimation of [Ca2+]ves at granule interior pH (approximately 5.5). In the cytoplasm, Cyto-Aeq accurately determined [Ca2+]cyto under both basal ([Ca2+]cyto = 130 +/- 35 nM) and exocytosis-stimulated conditions, confirmed by an independent reference technique (Indo-1 fluorescence). The hCgA-Aeq chimera determined vesicular free [Ca2+]ves = 1.4 +/- 0.3 microM under basal conditions indicating that >99% of granule total Ca2+ is in a "bound" state. The basal free [Ca2+]ves/[Ca2+]cyto ratio was thus approximately 10.8-fold, indicating active, dynamic Ca2+ uptake from cytosol into the granules. Stimulation of exocytotic secretion revealed prompt, dynamic increases in both [Ca2+](ves) and [Ca2+]cyto, and an exponential relation between the two (y = 0.99 x e(1.53x), r = 0.99), reflecting a persistent [Ca2+]ves/[Ca2+]cyto gradient, even during sharp increments of both values. Studies with inhibitors of Ca2+ translocation (Ca(2+)-ATPase), Na+/Ca(+)-exchange, Na+/H(+)-exchange, and vesicle acidification (H(+)-translocating ATPase), documented a role for these four ion transporter classes in accumulation of Ca2+ inside the vesicles.     

Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells.

Direct monitoring of the free Ca2+ concentration in the lumen of the endoplasmic reticulum (ER) is an important but still unsolved experimental problem. We have shown that a Ca(2+)-sensitive photoprotein, aequorin, can be addressed to defined subcellular compartments by adding the appropriate targeting sequences. By engineering a new aequorin chimera with reduced Ca2+ affinity, retained in the ER lumen via interaction of its N-terminus with the endogenous resident protein BiP, we show here that, after emptying the ER, Ca2+ is rapidly re-accumulated up to concentrations of > 100 microM, thus consuming most of the reporter photoprotein. An estimate of the steady-state Ca2+ concentration was obtained using Sr2+, a well-known Ca2+ surrogate which elicits a significantly slower rate of aequorin consumption. Under conditions in which the rate and extent of Sr2+ accumulation in the ER closely mimick those of Ca2+, the steady-state mean lumenal Sr2+ concentration ([Sr2+]er) was approximately 2 mM. Receptor stimulation causes, in a few seconds, a 3-fold decrease of the [Sr2+]er, whereas specific inhibition of the ER Ca2+ ATPase leads to an approximately 10-fold drop in a few minutes.     

Hypoosmotic Shock Induces Increases in Cytosolic Ca2+ in Tobacco Suspension-Culture Cells

Hypoosmotic shock treatment increased cytosolic Ca2+ ion concentration ([Ca2+]cyt) in tobacco (Nicotiana tabacum) suspension-culture cells. [Ca2+]cyt measurements were made by genetically transforming these cells to express apoaequorin and by reconstituting the Ca2+-dependent photoprotein, aequorin, in the cytosol by incubation with chemically synthesized coelenterazine. Measurement of Ca2+-dependent luminescence output thus allowed the direct monitoring of [Ca2+]cyt changes. When cells were added to a hypoosmotic medium, a biphasic increase in [Ca2+]cyt was observed; an immediate small elevation (phase 1) was observed first, followed by a rapid, large elevation (phase 2). Phase 1 [Ca2+]cyt was stimulated by the V-type ATPase inhibitor bafilomycin A1. Phase 2 was inhibited by the protein kinase inhibitor K-252a and required the continued presence of the hypoosmotic stimulus to maintain it. Although Ca2+ in the medium was needed to produce phase 2, it was not needed to render the cells competent to the hypoosmotic stimulus. If cells were subject to hypoosmotic shock in Ca2+- depleted medium, increases in luminescence could be induced up to 20 min after the shock by adding Ca2+ to the medium. These data suggest that hypoosmotic shock-induced [Ca2+]cyt elevation results from the activity of a Ca2+ channel in the plasma membrane or associated hypoosmotic sensing components that require Ca2+- independent phosphorylation and a continued stimulus to maintain full activity.     

Ca(2+)-activated anion channels and membrane depolarizations induced by blue light and cold in Arabidopsis seedlings.

The activation of an anion channel in the plasma membrane of Arabidopsis thaliana hypocotyls by blue light (BL) is believed to be a signal-transducing event leading to growth inhibition. Here we report that the open probability of this particular anion channel depends on cytoplasmic Ca2+ ([Ca2+]cyt) within the concentration range of 1 to 10 microM, raising the possibility that BL activates the anion channel by increasing [Ca2+]cyt. Arabidopsis seedlings cytoplasmically expressing aequorin were generated to test this possibility. Aequorin luminescence did not increase during or after BL, providing evidence that Ca2+ does not play a second-messenger role in the activation of anion channels. However, cold shock simultaneously triggered a large increase in [Ca2+]cyt and a 110-mV transient depolarization of the plasma membrane. A blocker of the anion channel, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, blocked 61% of the cold-induced depolarization without affecting the increase in [Ca2+]cyt. These data led us to propose that cold shock opens Ca2+ channels at the plasma membrane, allowing an inward, depolarizing Ca2+ current. The resulting large increase in [Ca2+]cyt activates the anion channel, which further depolarizes the membrane. Although an increase in [Ca2+]cyt may activate anion channels in response to cold, it appears that BL does so via a Ca(2+)-independent pathway.     

Imaging calcium dynamics in living plants using semi-synthetic recombinant aequorins

The genetic transformation of the higher plant Nicotiana plumbaginifolia to express the protein apoaequorin has recently been used as a method to measure cytosolic free calcium ([Ca2+]i) changes within intact living plants (Knight, M. R., A. K. Campbell, S. M. Smith, and A. J. Trewavas. 1991. Nature (Lond.). 352:524-526; Knight, M. R., S. M. Smith, and A. J. Trewavas. 1992. Proc. Natl. Acad. Sci. USA. 89:4967-4971). After treatment with the luminophore coelenterazine the calcium-activated photoprotein aequorin is formed within the cytosol of the cells of the transformed plants. Aequorin emits blue light in a dose-dependent manner upon binding free calcium (Ca2+). Thus the quantification of light emission from coelenterazine-treated transgenic plant cells provides a direct measurement of [Ca2+]i. In this paper, by using a highly sensitive photon-counting camera connected to a light microscope, we have for the first time imaged changes in [Ca2+]i in response to cold-shock, touch and wounding in different tissues of transgenic Nicotiana plants. Using this approach we have been able to observe tissue-specific [Ca2+]i responses. We also demonstrate how this method can be tailored by the use of different coelenterazine analogues which endow the resultant aequorin (termed semi-synthetic recombinant aeqorin) with different properties. By using h-coelenterazine, which renders the recombinant aequorin reporter more sensitive to Ca2+, we have been able to image relatively small changes in [Ca2+]i in response to touch and wounding: changes not detectable when standard coelenterazine is used. Reconstitution of recombinant aequorin with another coelenterazine analogue (e-coelenterazine) produces a semi-synthetic recombinant aequorin with a bimodal spectrum of luminescence emission. The ratio of luminescence at two wavelengths (421 and 477 nm) provides a simpler method for quantification of [Ca2+]i in vivo than was previously available. This approach has the benefit that no information is needed on the amount of expression, reconstitution or consumption of aequorin which is normally required for calibration with aequorin.     

Measurement of ionized calcium in blood platelets with the photoprotein aequorin. Comparison with Quin 2

The Ca2+-sensitive photoprotein aequorin (Mr = 20,000) was introduced into human blood platelets by incubation with 10 mM EGTA and 5 mM ATP. Platelet cytoplasmic and granule contents were retained during the loading procedure, and platelet morphology, aggregation, and secretion in response to agonists were normal after aequorin loading. Luminescence indicated an apparent resting cytoplasmic ionized calcium concentration [( Cai2+]) of 2-4 microM in media containing 1 mM Ca2+ and of 0.8-2 microM in 2-4 mM EGTA. The Ca2+ ionophore A23187 and the enzyme thrombin produced dose-related luminescent signals in both Ca2+-containing and EGTA-containing media. Peak [Cai2+] after A23187 or thrombin stimulation of aequorin-loaded platelets was 2-10 microM, while peak [Cai2+] determined using Quin 2 as the [Cai2+] indicator was at least 1 log unit lower. In platelets loaded with both aequorin and Quin 2, the aequorin signal was delayed but not reduced in amplitude. Aequorin loading of Quin 2-loaded cells had no effect on the Quin 2 signal. Ca2+ buffering by Quin 2 (intracellular concentration greater than 1 mM) is also supported by a reciprocal relationship between [Quin 2] and peak [Cai2+] stimulated by A23187 in the presence of EGTA. Parallel experiments with Quin 2 and aequorin may identify inhomogeneous [Cai2+] in platelets and give a more complete picture of platelet Ca2+ homeostasis than either indicator alone.     

The deposition of suberin lamellae determines the magnitude of cytosolic Ca2+ elevations in root endodermal cells subjected to cooling

A transient increase in cytosolic Ca2+ concentration ([Ca2+]cyt) is thought to be a prerequisite for an appropriate physiological response to both chilling and salt stress. The [Ca2+]cyt is raised by Ca2+ influx to the cytosol from the apoplast and/or intracellular stores. It has been speculated that different signals mobilise Ca2+ from different stores, but little is known about the origin(s) of the Ca2+ entering the cytosol in response to specific environmental challenges. We have utilised the developmentally regulated suberisation of endodermal cells, which is thought to prevent Ca2+ influx from the apoplast, to ascertain whether Ca2+ influx is required to increase [Ca2+]cyt in response to chilling or salt stress. Perturbations in [Ca2+]cyt were studied in transgenic Arabidopsis thaliana, expressing aequorin fused to a modified yellow fluorescent protein solely in root endodermal cells, during slow cooling of plants from 20 to 0.5 degrees C over 5 min and in response to an acute salt stress (0.333 m NaCl). Only in endodermal cells in the apical 4 mm of the Arabidopsis root did [Ca2+]cyt increase significantly during cooling, and the magnitude of the [Ca2+]cyt elevation elicited by cooling was inversely related to the extent of suberisation of the endodermal cell layer. No [Ca2+]cyt elevations were elicited by cooling in suberised endodermal cells. This is consistent with the hypothesis that suberin lamellae isolate the endodermal cell protoplast from the apoplast and, thereby, prevent Ca2+ influx. By contrast, acute salt stress increased [Ca2+]cyt in endodermal cells throughout the root. These results suggest that [Ca2+]cyt elevations, upon slow cooling, depend absolutely on Ca2+ influx across the plasma membrane, but [Ca2+]cyt elevations in response to acute salt stress do not. They also suggest that Ca2+ release from intracellular stores contributes significantly to increasing [Ca2+]cyt upon acute salt stress.     

Beat-to-beat oscillations of mitochondrial [Ca2+] in cardiac cells

The Ca2+-sensitive photoprotein aequorin and the new green fluorescent protein-based fluorescent Ca2+ indicators 'ratiometric-pericam' were selectively expressed in the mitochondria, cytosol and/or nucleus of spontaneously beating ventricular myocytes from neonatal rats. This combined strategy reveals that mitochondrial [Ca2+] oscillates rapidly and in synchrony with cytosolic and nuclear [Ca2+]. The Ca2+ oscillations were reduced in frequency and/or amplitude by verapamil and carbachol and were enhanced by isoproterenol and elevation of extracellular [Ca2+]. An increased frequency and/or amplitude of cytosolic Ca2+ spikes was rapidly mirrored by similar changes in mitochondrial Ca2+ spikes and more slowly by elevations of the interspike Ca2+ levels. The present data unequivocally demonstrate that in cardiac cells mitochondrial [Ca2+] oscillates synchronously with cytosolic [Ca2+] and that mitochondrial Ca2+ handling rapidly adapts to inotropic or chronotropic inputs.     

Aequorin detects increased cytoplasmic calcium in platelets stimulated with phorbol ester or diacylglycerol

A biochemical pathway to platelet activation involving protein kinase C has been deemed "Ca2+-independent", because the intracellular fluorophore quin2 indicates no rise in cytoplasmic [Ca2+] in platelets stimulated by certain agonists. However, unlike quin2, the Ca2+-sensitive photoprotein aequorin demonstrates a rise in [Ca2+] when platelet aggregation is induced by phorbol ester or diacylglycerol. Aequorin and quin2 appear to report different aspects of Ca2+ homeostasis, and the absence of a quin2 signal may not be sufficient to establish that a metabolic pathway is "Ca2+-independent".     

Phorbol-ester-induced alterations of free calcium ion transients in single rat hepatocytes.

The effect of the phorbol esters phorbol 12-myristate 13-acetate (TPA) and phorbol 12,13-dibutyrate (PDB) on changes in free Ca2+ concentration ([Ca2+]i) in single rat hepatocytes, microinjected with the photoprotein aequorin, were investigated. [Arg8]vasopressin and phenylephrine induced a series of repetitive [Ca2+]i transients. Phorbol esters inhibited the alpha 1-adrenoceptor-induced response; sub-nanomolar concentrations decreased the transient frequency, and higher concentrations abolished the transients. The inhibitory effect of PDB was readily reversible. Phorbol esters were less effective in decreasing the frequency of [Arg8]-vasopressin-induced transients, and the inhibition could be overcome by high [Arg8]vasopressin concentrations.     

Excitation-contraction coupling in cardiac Purkinje fibers. Effects of cardiotonic steroids on the intracellular [Ca2+] transient, membrane potential, and contraction

The [Ca2+]-activated photoprotein aequorin was used to measure [Ca2+] in canine cardiac Purkinje fibers during the positive inotropic and toxic effects of ouabain, strophanthidin, and acetylstrophanthidin. The positive inotropic effect of these substances was associated with increases in the two components of the aequorin signal, L1 and L2. On the average, strophanthidin at 10(-7) M produced steady, reversible increases in L1, L2, and peak twitch tension of 20, 91, and 240%, respectively. This corresponds to increases in the upper-limit spatial average [Ca2+] from 1.9 X 10(-6) M to 2.1 X 10(-6) M at L1 and from 1.4 X 10(-6) M to 1.8 X 10(-6) M at L2. Elevation of diastolic luminescence above the control level was not detected. At higher concentrations (5 X 10(-7) M), strophanthidin produced aftercontractions, diastolic depolarization, and transient depolarizations, all of which were associated with temporally similar changes in [Ca2+]. During these events, diastolic [Ca2+] rose from the normal level of approximately 3 X 10(-7) M up to 1-2 X 10(-6) M. The negative inotropic effect of 5 X 10(-7) M strophanthidin was not associated with a corresponding decrease in the [Ca2+] transient but was associated with a change in the relationship between [Ca2+] and tension. Assuming the Na+-lag mechanism of cardiotonic steroid action, we conclude the following: at low concentrations of drug, increased Ca2+ uptake by the sarcoplasmic reticulum prevents a detectable rise in cytoplasmic [Ca2+] during diastole, but this increased Ca2+ uptake results in increased release of Ca2+ during the action potential. At higher drug concentrations, observable [Ca2+] changes during diastole activate tension and membrane conductance changes.     

Enzyme-substrate interactions in the hydrolysis of peptides by cathepsins B and H from rat liver.

Free Ca2+ in the cytosol ([Ca2+]i) of individual rat ventricle cells injected with aequorin was measured under anoxia. In glucose-free medium myocytes spontaneously shortened after about 60 min, although [Ca2+]i was still at or near resting levels. However, within minutes a net inward movement of Ca2+ across the sarcolemma developed and [Ca2+]i began to rise. Provided oxygen was readmitted before [Ca2+]i exceeded 2-3 microM, cells were able to restore [Ca2+]i to resting levels through caffeine-sensitive sequestration of Ca2+ in the sarcoplasmic reticulum. We suggest that Ca2+-independent shortening of anoxic cardiomyocytes reflects onset of rigor which triggers loss of [Ca2+]i homoeostasis.     

Simultaneous measurement of Ca2+ in muscle with Ca electrodes and aequorin. Diffusible cytoplasmic constituent reduces Ca(2+)-independent luminescence of aequorin

Estimates of cytoplasmic Ca2+ concentration ([Ca2+]i) were made essentially simultaneously in the same intact frog skeletal muscle fibers with aequorin and with Ca-selective microelectrodes. In healthy fibers under truly resting conditions [Ca2+]i was too low to be measured reliably with either technique. The calibration curves for both indicators were essentially flat in this range of [Ca2+], and the aequorin light signal was uniformly below the level to be expected in the total absence of Ca2+. When [Ca2+]i had been raised to a stable level below the threshold for contracture by increasing [K+]o to 12.5 mM, [Ca2+]i was 38 nM according to aequorin and 59 nM according to the Ca-selective microelectrodes. These values are not significantly different. Our estimates of [Ca2+]i are lower than most others obtained with microelectrodes, probably because the presence of aequorin in the cells allowed us to detect damaging microelectrode impalements that otherwise we would have had no reason to reject. The observation that the light emission from aequorin-injected fibers in normal Ringer solution was below the level expected from the Ca(2+)-independent luminescence of aequorin in vitro was investigated further, with the conclusion that the myoplasm contains a diffusible macromolecule (between 10 and 30 kD) that interacts with aequorin to reduce light emission in the absence of Ca2+.     

Calcium entry in squid axons during voltage clamp pulses

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with sodium ion sensitive, current and voltage electrodes. The axons were usually bathed in a solution of varying Ca2+ concentration ([Ca2+]o) containing 150mM each of Na+, K+ and an inert cation such as Li+, Tris or N-methylglucamine and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic Ca2+ level, [Ca2+]i. The effect of membrane voltage on [Ca2+]i was found to depend on the concentration of internal Na+ ([Na+]i). Voltage clamp hyperpolarizing pulses were found to cause a reduction of [Ca2+]i. For depolarizing pulses a relationship between [Ca2+]i gain and [Na+]i indicates that Ca2+ entry is sigmoid with a half maximal response at 22 mM Na+. This Ca2+ entry is a steep function of [Na+]i suggesting that 4 Na+ ions are required to promote the influx of 1 Ca2+. There was little change in Ca2+ entry with depolarizing pulses when [Ca2+]o is varied from 1 to 10mM, while at 50mM [Ca2+]o calcium entry clearly increases suggesting an alternate pathway from that of Na+/Ca2+ exchange. This entry of Ca2+ at high [Ca2+]o, however, was not blocked by Cs+o. The results obtained lend further support to the notion that Na+/Ca2+ exchange in squid giant axon is sensitive to membrane voltage no matter whether this is applied as a constant change in membrane potential or as an intermittent one.     

Evaluation, using targeted aequorins, of the roles of the endoplasmic reticulum and its (Ca2++Mg2+)ATP-ases in the activation of store-operated Ca2+ channels in liver cells

The process by which store-operated Ca2+ channels (SOCs) deliver Ca2+ to the endoplasmic reticulum (ER) and the role of (Ca2++Mg2+)ATP-ases of the ER in the activation of SOCs in H4-IIE liver cells were investigated using cell lines stably transfected with apo-aequorin targeted to the cytoplasmic space or the ER. In order to measure the concentration of Ca2+ in the ER ([Ca2+]er), cells were pre-treated with 2,5-di-tert-butylhydroquinone (DBHQ) to deplete Ca2+ in the ER before reconstitution of holo-aequorin. The addition of extracellular Ca2+ (Cao2+) to Ca2+-depleted cells induced refilling of the ER, which was complete within 5 min. This was associated with a sharp transient increase in the cytoplasmic Ca2+ concentration ([Ca2+]cyt) of about 15 s duration (a Cao2+-induced [Ca2+]cyt spike) after which [Ca2+]cyt remained elevated slightly above the basal value for a period of about 2 min (low [Ca2+]cyt plateau). The Cao2+-induced [Ca2+]cyt spike was inhibited by Gd3+, not affected by tetrakis-(2-pyridymethyl) ethylenediamine (TPEN), and broadened by ionomycin and the intracellular Ca2+ chelators BAPTA and EGTA. Refilling of the ER was inhibited by caffeine. Neither thapsigargin nor DBHQ caused a detectable inhibition or change in shape of the Cao2+-induced [Ca2+]cyt spike or the low [Ca2+]cyt plateau whereas each inhibited the inflow of Ca2+ to the ER by about 80%. Experiments conducted with carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) indicated that thapsigargin did not alter the amount of Ca2+ accumulated in mitochondria. The changes in [Ca2+]cyt reported by aequorin were compared with those reported by fura-2. It is concluded that (i) there are significant quantitative differences between the manner in which aequorin and fura-2 sense changes in [Ca2+]cyt and (ii) thapsigargin and DBHQ inhibit the uptake of Ca2+ to the bulk of the ER but this is not associated with inhibition of the activation of SOCs. The possible involvement of a small sub-region of the ER (or another intracellular Ca2+ store), which contains thapsigargin-insensitive (Ca2++Mg2+)ATP-ases, in the activation of SOCs is briefly discussed.     

BAY-K-8644, a calcium channel agonist, induces a rise in cytoplasmic free calcium and iodide discharge in thyroid cells

BAY-K-8644, a calcium channel agonist, induces a rise in cytoplasmic free calcium and iodide discharge in cultured porcine thyroid cells. The cytoplasmic free calcium concentration, [Ca2+]i, was measured using aequorin, a calcium-sensitive photoprotein. BAY-K-8644, a dihydropyridine derivative, acts as a Ca channel agonist and induces a rise in [Ca2+]i and iodide discharge; 0.5 nM BAY-K-8644 is a minimal dose to effect a rise in [Ca2+]i and iodide discharge and 50 nM BAY-K-8644 produces the maximal effect. The data indicate that BAY-K-8644-induced iodide discharge is mediated by a rise in [Ca2+]i.     

H+-Coupled sugar transporter, an initiator of sugar-induced Ca2+-signaling in plant cells

Using Ca2+-dependent photoprotein aequorin-transformed tobacco BY-2 cell suspensions, the sugar-induced increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) was investigated by measuring the luminescence intensity. When 0.5 M sucrose or some other sugars were fed to the cells, strong and transient luminescence was observed. Salts or sugar analogues didn't show this effect. In addition, the intensity of sucrose-induced aequorin luminescence was gradually enhanced when cells were exposed to sugar-starvation. This was observed with the concurrent expression of the sucrose/H+ co-transporter, NtSUT1A. The [Ca2+]cyt increase may initiate Ca2+-signaling leading to the expression of genes related to biosynthesis of storage carbohydrates in a sink organ. The sugar-signaling may play an important role in the conversion on nutritional stage of plant tissue, source organ to sink organ.     

Secretagogues modulate the calcium concentration in the endoplasmic reticulum of insulin-secreting cells. Studies in aequorin-expressing intact and permeabilized ins-1 cells

The precise regulation of the Ca2+ concentration in the endoplasmic reticulum ([Ca2+]er) is important for protein processing and signal transduction. In the pancreatic beta-cell, dysregulation of [Ca2+]er may cause impaired insulin secretion. The Ca2+-sensitive photoprotein aequorin mutated to lower its Ca2+ affinity was stably expressed in the endoplasmic reticulum (ER) of rat insulinoma INS-1 cells. The steady state [Ca2+]er was 267 +/- 9 microM. Both the Ca2+-ATPase inhibitor cyclopiazonic acid and 4-chloro-m-cresol, an activator of ryanodine receptors, caused an almost complete emptying of ER Ca2+. The inositol 1,4,5-trisphosphate generating agonists, carbachol, and ATP, reduced [Ca2+]er by 20-25%. Insulin secretagogues that raise cytosolic [Ca2+] by membrane depolarization increased [Ca2+]er in the potency order K+ > glucose > leucine, paralleling their actions in the cytosolic compartment. Glucose, which augmented [Ca2+]er by about 25%, potentiated the Ca2+-mobilizing effect of carbachol, explaining the corresponding observation in cytosolic [Ca2+]. The filling of ER Ca2+ by glucose is not directly mediated by ATP production as shown by the continuous monitoring of cytosolic ATP in luciferase expressing cells. Both glucose and K+ increase [Ca2+]er, but only the former generated whereas the latter consumed ATP. Nonetheless, drastic lowering of cellular ATP with a mitochondrial uncoupler resulted in a marked decrease in [Ca2+]er, emphasizing the requirement for mitochondrially derived ATP above a critical threshold concentration. Using alpha-toxin permeabilized cells in the presence of ATP, glucose 6-phosphate did not change [Ca2+]er, invalidating the hypothesis that glucose acts through this metabolite. Therefore, insulin secretagogues that primarily stimulate Ca2+ influx, elevate [Ca2+]er to ensure beta-cell homeostasis.     

Mitochondrial localization as a determinant of capacitative Ca2+ entry in HeLa cells

Whether different subsets of mitochondria play distinct roles in shaping intracellular Ca2+ signals is presently unresolved. Here, we determine the role of mitochondria located beneath the plasma membrane in controlling (a) Ca2+ release from the endoplasmic reticulum (ER) and (b) capacitative Ca2+ entry. By over-expression of the dynactin subunit dynamitin, and consequent inhibition of the fission factor, dynamin-related protein (Drp-1), mitochondria were relocalised from the plasma membrane towards the nuclear periphery in HeLa cells. The impact of these changes on free calcium concentration in the cytosol ([Ca2+]c), mitochondria ([Ca2+]m) and ER ([Ca2+]ER) was then monitored with specifically-targeted aequorins. Whilst dynamitin over-expression increased the number of close contacts between the ER and mitochondria by >2.5-fold, assessed using organelle-targeted GFP variants, histamine-induced changes in organellar [Ca2+] were unaffected. By contrast, Ca2+ influx elicited significantly smaller increases in [Ca2+]c and [Ca2+]m in dynamitin-expressing than in control cells. These data suggest that the strategic localisation of a subset of mitochondria beneath the plasma membrane is required for normal Ca2+ influx, but that the transfer of Ca2+ ions between the ER and mitochondria is relatively insensitive to gross changes in the spatial relationship between these two organelles.