Bax affects intracellular Ca2+ stores and induces Ca2+ wave propagation

In the present study, we evaluated proapoptotic protein Bax on mitochondria and Ca2+ homeostasis in primary cultured astrocytes. We found that recombinant Bax (rBax, 10 and 100 ng/ml) induces a loss in mitochondrial membrane potential (Delta Psi m). This effect might be related to the inhibition of respiratory rates and a partial release of cytochrome c, which may change mitochondrial morphology. The loss of Delta Psi m and a selective permeabilization of mitochondrial membranes contribute to the release of Ca2+ from the mitochondria. This was inhibited by cyclosporin A (5 microM) and Ruthenium Red (1 microg/ml), indicating the involvement of mitochondrial Ca2+ transport mechanisms. Bax-induced mitochondrial Ca2+ release evokes Ca2+ waves and wave propagation between cells. Our results show that Bax induces mitochondrial alteration that affects Ca2+ homeostasis and signaling. These changes show that Ca2+ signals might be correlated with the proapoptotic activities of Bax.     

Ca2+ channels and intracellular Ca2+ stores in neuronal and neuroendocrine cells

Changes in [Ca2+]i are essential in modulating a variety of cellular functions. In no other cell type does the regulation of [Ca2+]i reach the level of sophistication observed in cells of neuronal origin. Because of its physicochemical characteristics, the fluorescent Ca2+ indicator Fura-2 has become extremely popular among neuroscientists. The use of this probe, however, has generated a number of problems, in particular, extracytosolic trapping and leakage from intact cells. In the first part of this contribution we briefly discuss the practical application of Fura-2 to the study of [Ca2+]i in primary cultures of neurons and astrocytes. In the second part, we review some recent data (mainly from our laboratories) obtained in neurons and neuroendocrine cells, concerning the regulation of different types of Ca2+ channels and the role and mechanism of intracellular Ca2+ mobilization. The experimental evidence supporting the existence of a previously unrecognised organelle, the calciosome, that we hypothesize represents the functional equivalent in non-muscle cells of sarcoplasmic reticulum, will also briefly be discussed.     

Functionally separate intracellular Ca2+ stores in smooth muscle

In smooth muscle, release via the inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)R) and ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR) controls oscillatory and steady-state cytosolic Ca(2+) concentrations ([Ca(2+)](c)). The interplay between the two receptors, itself determined by their organization on the SR, establishes the time course and spatial arrangement of the Ca(2+) signal. Whether or not the receptors are co-localized or distanced from each other on the same store or whether they exist on separate stores will significantly affect the Ca(2+) signal produced by the SR. To date these matters remain unresolved. The functional arrangement of the RyR and Ins(1,4,5)P(3)R on the SR has now been examined in isolated single voltage-clamped colonic myocytes. Depletion of the ryanodine-sensitive store, by repeated application of caffeine, in the presence of ryanodine, abolished the response to Ins(1,4,5)P(3), suggesting that Ins(1,4,5)P(3)R and RyR share a common Ca(2+) store. Ca(2+) release from the Ins(1,4,5)P(3)R did not activate Ca(2+)-induced Ca(2+) release at the RyR. Depletion of the Ins(1,4,5)P(3)-sensitive store, by the removal of external Ca(2+), on the other hand, caused only a small decrease ( approximately 26%) in caffeine-evoked Ca(2+) transients, suggesting that not all RyR exist on the common store shared with Ins(1,4,5)P(3)R. Dependence of the stores on external Ca(2+) for replenishment also differed; removal of external Ca(2+) depleted the Ins(1,4,5)P(3)-sensitive store but caused only a slight reduction in caffeine-evoked transients mediated at RyR. Different mechanisms are presumably responsible for the refilling of each store. Refilling of both Ins(1,4,5)P(3)-sensitive and caffeine-sensitive Ca(2+) stores was inhibited by each of the SR Ca(2+) ATPase inhibitors thapsigargin and cyclopiazonic acid. These results may be explained by the existence of two functionally distinct Ca(2+) stores; the first expressing only RyR and refilled from [Ca(2+)](c), the second expressing both Ins(1,4,5)P(3)R and RyR and dependent upon external Ca(2+) for refilling.     

Interaction between injected Ca2+ and intracellular Ca2+ stores in Xenopus oocytes.

Upon two repetitive deep injections of Ca2+ into Xenopus oocyte (200-300 microns under the membrane), the amplitude of the transient Cl- current induced by the second injection is several-fold higher than that of the first one. This 'potentiation' persists even at 60-90 min intervals between injections. However, in oocytes permeabilized to Ca2+ by the ionophore A23187 in a Ca2(+)-free solution, the potentiation completely disappears after 30 min. It is proposed that the injected Ca2+ is largely taken up by the stores, whereas following the second injection, a higher proportion of Ca2+ reaches the membrane, since the stores are already loaded. In ionophore-treated oocytes, the stores lose the accumulated Ca2+ over several minutes and are then ready to take up Ca2+ again, hindering its arrival at the membrane.     

Mechanosensitive Ca2+ release from intracellular stores in Nitella flexilis

We found previously that the cytoplasmic drop isolated from internodal cells of Nitella flexilis releases Ca2+ in response to hypotonic treatment and named the phenomenon hydration-induced Ca2+ release (HICR). The HICR is assumed to be a result of activation of Ca2+ permeable channels in the membrane of Ca2+ stores in a stretch-activated manner. To prove this idea, mechanical stimulus was applied to the drop by means of shooting isotonic/hypnotic medium or silicon oil into the drop, or compressing the drop. All these mechanical stimuli induced a rapid increase in the Ca2+ concentration of the drop. The chloroplast fraction isolated from the cytoplasmic drop released Ca2+ on compression, while the chloroplast-free cytoplasm did not. In Chara corallina, the cytoplasmic drop, which shows a very weak HICR, also responded weakly to the mechanical stimulus, but the chloroplast fraction was inert. When chloroplasts from Chara were added to the chloroplast-free cytoplasm of N. flexilis, the cytoplasm recovered the mechanoresponse. Starch grains were as effective as chloroplasts. The data indicate that Ca2+ permeable channels in the membrane of Ca2+ stores in N. flexilis are really mechano-sensitive.     

Ca2+ influx-dependent refilling of intracellular Ca2+ stores determines the frequency of Ca2+ oscillations in fertilized mouse eggs

On mammalian fertilization, long-lasting Ca²⁺ oscillations are induced in the egg by the fusing spermatozoon. While each transient Ca²⁺ increase in Ca²⁺ concentration ([Ca²⁺]) in the cytosol is due to Ca²⁺ release from the endoplasmic reticulum (ER), Ca²⁺ influx from outside is required for Ca²⁺ oscillations to persist. In this study, we investigated how Ca²⁺ influx is interrelated to the cycle of Ca²⁺ release and uptake by the intracellular Ca²⁺ stores during Ca²⁺ oscillations in fertilized mouse eggs. In addition to monitoring cytosolic [Ca²⁺] with fura-2, the influx rate was evaluated using Mn²⁺ quenching technique, and the change in [Ca²⁺] in the ER lumen was visualized with a targeted fluorescent probe. We found that the influx was stimulated after each transient Ca²⁺ release and then diminished gradually to the basal level, and demonstrated that the ER Ca²⁺ stores once depleted by Ca²⁺ release were gradually refilled until the next Ca²⁺ transient to be initiated. Experiments altering extracellular [Ca²⁺] in the middle of Ca²⁺ oscillations revealed the dependence of both the refilling rate and the oscillation frequency on the rate of Ca²⁺ influx, indicating the crucial role of Ca²⁺ influx in determining the intervals of Ca²⁺ transients. As for the influx pathway supporting Ca²⁺ oscillations to persist, STIM1/Orai1-mediated store-operated Ca²⁺ entry (SOCE) may not significantly contribute, since neither known SOCE blockers nor the expression of protein fragments that interfere the interaction between STIM1 and Orai1 inhibited the oscillation frequency or the influx rate.     

Polycystin-2 accelerates Ca2+ release from intracellular stores in Caenorhabditis elegans

Polycystin-2, a member of the TRP family of calcium channels, is encoded by the human PKD2 gene. Mutations in that gene can lead to swelling of nephrons into the fluid-filled cysts of polycystic kidney disease. In addition to expression in tubular epithelial cells, human polycystin-2 is found in muscle and neuronal cells, but its cell biological function has been unclear. A homologue in Caenorhabditis elegans is necessary for male mating behavior. We compared the behavior, calcium signaling mechanisms, and electrophysiology of wild-type and pkd-2 knockout C. elegans. In addition to characterizing PKD-2-mediated aggregation and mating behaviors, we found that polycystin-2 is an intracellular Ca(2+) release channel that is required for the normal pattern of Ca(2+) responses involving IP(3) and ryanodine receptor-mediated Ca(2+) release from intracellular stores. Activity of polycystin-2 creates brief cytosolic Ca(2+) transients with increased amplitude and decreased duration. Polycystin-2, along with the IP(3) and ryanodine receptors, acts as a major calcium-release channel in the endoplasmic reticulum in cells where rapid calcium signaling is required, and polycystin-2 activity is essential in those excitable cells for rapid responses to stimuli.     

The control of Ca2+ homeostasis: role of intracellular rapidly exchanging Ca2+ stores.

The role of rapidly exchanging intracellular Ca²⁺ stores in the control of Ca²⁺ homeostasis is reviewed. The following issues are discussed: the reasons why such stores exist in eukaryotic cells; the differences between the terminal cisternae of the skeletal muscle sarcoplasmic reticulum, which have direct, physical connection with the T tubules of the plasmalemma, and the Ca²⁺ stores located in the depth of the cytoplasm, which are stimulated by second messengers; the cytological nature (subcompartments of the ER) of the rapidly exchanging Ca²⁺ stores and their functional significance. The conclusions introduce recent developments in which intracellular Ca²⁺ stores have been investigated also by molecular biology techniques.     

Interferon-gamma elicits a G-protein-dependent Ca2+ signal in human neutrophils after depletion of intracellular Ca2+ stores

Interferon-gamma (IFN-gamma) has multiple effects on Ca2+ signalling in polymorphonuclear neutrophils (PMNs), including evoked cytosolic Ca2+ transients, increased capacitative calcium influx and increased sequestration of Ca2+ in intracellular stores. The present study was conducted to elucidate the mechanism behind the Ca2+ transients. As observed before, the IFN-gamma-evoked Ca2+ signals were apparent when extracellular Ca2+ was removed. A new finding was that the proportion of responding cells and the extent of calcium release increased with increasing time in EGTA buffer. As assessed by N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated Ca2+ release, the intracellular stores were depleted during this incubation period, and the extent of depletion correlated well with the appearance of IFN-gamma-induced Ca2+ signals. This store dependence of the IFN-gamma-induced Ca2+ signals was confirmed by the appearance of IFN-gamma-evoked Ca2+ signals in the presence of extracellular Ca2+ after store depletion by thapsigargin. The appearance of IFN-gamma-mediated Ca2+-signals in the presence of EGTA indicates that IFN-gamma stimulates Ca2+ release from intracellular stores. This was confirmed by the inability of the calcium transportation blocker La3+ to abolish the IFN-gamma response and the total abrogation of the response by the phospholipase C inhibitor U73122. Although these latter results imply a role for inositol 1,4,5-trisphosphate(IP3) in IFN-gamma signalling, comparison of IFN-gamma-evoked responses with fMLP responses revealed clear differences that suggest different signal-transduction pathways. However, responses to fMLP and IFN-gamma were both depressed by pertussis toxin, and the IFN-gamma responses were, in addition, inhibited by the tyrosine kinase inhibitor genistein. Further evidence of the involvement of tyrosine kinase was a slight stimulatory effect of the protein tyrosine phosphatase inhibitor sodium orthovanadate. The PI-3K activity was of minor importance. In conclusion, we present evidence of a novel signal-transduction mechanism for IFN-gamma in PMNs, dependent on tyrosine kinase activity, a pertussis toxin-sensitive G protein and phospholipase C activity.     

Early developmental changes in intracellular Ca2+ stores in rat brain.

Developmental changes in intracellular Ca2+ stores in brain was studied by examining: (1) IP3- and cADPR-induced increase in [Ca2+]i in synaptosomes; (2) Ca(2+)-ATPase activity and ATP-dependent 45Ca2+ uptake into Ca2+ store in ER microsomes; (3) TG-induced inhibition of Ca(2+)-ATPase activity and ATP-dependent 45Ca2+ uptake into Ca2+ store in ER microsomes; and (4) gene expression of Ca(2+)-ATPase pump in neurons obtained from brains of the new-born and the 3-week-old rats. IP3 (EC50 310 +/- 8 nM, 200% maximum increase in [Ca2+]i) and cADPR (EC50 25 +/- 3 nM, greater than 170% maximum increase in [Ca2+]i) both were potent agonist of Ca2+ release from internal stores in synaptosomes obtained from the 3-week-old rats. However, IP3 (EC50 250 +/- 10 nM, 175 maximum increase in [Ca2+]i) was a potent, but cADPR (EC50 300 +/- 20 nM, 75% maximum increase) was a poor agonist of Ca2+ release from intracellular stores in synaptosomes obtained from the new-born rats. [3H]IP3, [32P]cADPR and [3H]Ry binding in the new-born samples were significantly less than that in the 3-week-old samples. [3H]Ry binding to its receptor was more sensitive to cADPR in microsomes from the 3-week-old rats than those from the new-born rats. Microsomes from the new-born rats exhibited TG-sensitive (IC50 30 +/- 4 nM) and TG-insensitive forms of Ca(2+)-ATPase, while microsomes from the 3-week-old rats exhibited only the TG-sensitive form of Ca(2+)-ATPase (5 +/- 1 nM IC50). Microsomes from the 3-week-old rats were more sensitive to TG but less sensitive to IP3, while microsomes from the new-born rats were more sensitive to IP3 but less sensitive to TG. The lower TG sensitivity of the new-born Ca2+ store may be because they poorly express a 45 amino acid C-terminal tail of Ca(2+)-ATPase that contains the TG regulatory sites. This site is adequately expressed in the older brain. This suggests that: (1) the new-born brain contains fully operational IP3 pathway but poorly developed cADPR pathway, while the older brain contains both IP3 and cADPR pathways; and (2) a developmental switch occurs in the new-born Ca(2+)-ATPase as a function of maturity.     

Cytotoxic hyperthermia and Ca2+ homeostasis: the effect of heat on Ca2+ uptake by nonmitochondrial intracellular Ca2+ stores

The effect of cytotoxic hyperthermia on Ca2+ transport by intracellular, nonmitochondrial Ca2+ stores of the human colon cancer cell line, HT-29, was studied using cells permeabilized with saponin. Saponin treatment permitted equilibration of the cytosol with a defined extracellular medium consisting of an intracellular-like ionic composition, ATP and an ATP-regenerating system, and Ca2+/EGTA buffers to adjust the free [Ca2+]. Under the conditions employed, ATP-dependent Ca2+ uptake in saponin-permeabilized cells was demonstrated to be exclusively due to nonmitochondrial Ca2+ stores, e.g., endoplasmic reticulum or calciosomes. Heat treatment for 120 min at 44.5 degrees C sufficient to kill 80% of the cells inhibited ATP-dependent Ca2+ uptake by 50% in terms of rate and total Ca2+ accumulated. With cells made thermotolerant by either arsenite or heat treatment 24 h prior to challenge heating, ATP-dependent Ca2+ uptake was resistant to a second equivalent heat dose. Efflux of Ca2+ from saponin-permeabilized cells when measured at 37 degrees C was unaffected by a prior heat treatment (44.5 degrees C for 120 min).     

Extracellular pH modulates the Ca2+ current activated by depletion of intracellular Ca2+ stores in human macrophages

Intracellular Ca²⁺ (Ca_i) signaling following the binding of surface receptors activates a Ca²⁺ permeable plasma membrane conductance which has been shown to be associated with store depletion in a number of cell types. We examined the activation of this conductance in human monocyte-derived macrophages (HMDMs) using whole-cell voltage-clamp techniques coupled with fura-2 microfluorimetry and characterized the importance of external pH (pH_o) as a modulator of current amplitude. Current activation was observed following experimental maneuvers designed to deplete intracellular Ca²⁺-stores including: (i) dialysis of the cell with 100 m inositol 1,4,5-triphosphate (IP₃), (ii) intracellular dialysis with high concentrations of the Ca²⁺ buffers EGTA and BAPTA, or (iii) exposure of the cell to the Ca²⁺-ATPase inhibitor thapsigargin (1 m). Currents associated with store depletion were inwardly rectifying with kinetics, inactivation, and selectivity that appeared similar irrespective of the mode of activation. Currents were Ca²⁺ selective with a selectivity sequence of Ca²⁺ > Sr²⁺ Mg²⁺ = Mn²⁺ = Ni²⁺. The Ca²⁺ influx current was modulated by changes in pH_o; modulation was not produced as a consequence of changes in internal pH (pH_i). External acidification led to a reversible reduction in current amplitude with a pKa at pH 8.2. Changes in pH_o alone failed to induce current activation. These observations are consistent with a scheme by which changes in pH_o, as would be encountered by macrophages at sites of inflammation, could change the time course and magnitude of the Ca_i transient associated with receptor activation by regulating the influx of Ca²⁺ ions.The authors wish to gratefully acknowledge the expert technical assistance of Weiwen Xie without whom the study could not have been completed. This work was supported by National Institutes of Health GM36823.     

Uptake of Ca2+ and refilling of intracellular Ca2+ stores in Ehrlich-ascites-tumour cells and in rat thymocytes.

We have studied the uptake of Ca2+ and its redistribution between the cytoplasm and the intracellular stores in Ehrlich-ascites-tumour cells and rat thymocytes previously depleted of Ca2+ by incubation in Ca2(+)-free medium. Measurements included changes of the cytoplasmic Ca2+ concentration ([Ca2+]i), uptake of 45Ca2+ and uptake of Mn2+, a Ca2+ surrogate for Ca2+ channels. Refilling of the Ca2+ stores in thymocytes was very fast (half-filling time: 4 s at 37 degrees C) and very sensitive to temperature (10 times slower at 20 degrees C). It was always preceded by increase of [Ca2+]i. In the Ehrlich cell, both refilling and increase of [Ca2+]i were about one order of magnitude slower. The increase of [Ca2+]i and the refilling of the intracellular stores were both almost completely blocked by Ni2+ in thymocytes, but only partially in the Ehrlich cell. The rates of 45Ca2+ and Mn2+ uptake varied consistently with temperature and the kind of cell. These results suggest that the intracellular stores are refilled by Ca2+ taken up from the cytoplasm. We also find that filling of the Ca2+ stores decreases by about 90% the rate of Mn2+ uptake in thymocytes. This is direct evidence of modulation of the plasma-membrane Ca2+ entry by the degree of filling of the intracellular stores. This modulation occurs in the absence of agonists, suggesting some kind of signalling between the intracellular stores and the Ca2+ entry pathways of the plasma membrane.     

Signaling between intracellular Ca2+ stores and depletion-activated Ca2+ channels generates [Ca2+]i oscillations in T lymphocytes

Stimulation through the antigen receptor (TCR) of T lymphocytes triggers cytosolic calcium ([Ca2+]i) oscillations that are critically dependent on Ca2+ entry across the plasma membrane. We have investigated the roles of Ca2+ influx and depletion of intracellular Ca2+ stores in the oscillation mechanism, using single-cell Ca2+ imaging techniques and agents that deplete the stores. Thapsigargin (TG; 5-25 nM), cyclopiazonic acid (CPA; 5-20 microM), and tert- butylhydroquinone (tBHQ; 80-200 microM), inhibitors of endoplasmic reticulum Ca(2+)-ATPases, as well as the Ca2+ ionophore ionomycin (5-40 nM), elicit [Ca2+]i oscillations in human T cells. The oscillation frequency is approximately 5 mHz (for ATPase inhibitors) to approximately 10 mHz (for ionomycin) at 22-24 degrees C. The [Ca2+]i oscillations resemble those evoked by TCR ligation in terms of their shape, amplitude, and an absolute dependence on Ca2+ influx. Ca(2+)- ATPase inhibitors and ionomycin induce oscillations only within a narrow range of drug concentrations that are expected to cause partial depletion of intracellular stores. Ca(2+)-induced Ca2+ release does not appear to be significantly involved, as rapid removal of extracellular Ca2+ elicits the same rate of [Ca2+]i decline during the rising and falling phases of the oscillation cycle. Both transmembrane Ca2+ influx and the content of ionomycin-releasable Ca2+ pools fluctuate in oscillating cells. From these data, we propose a model in which [Ca2+]i oscillations in T cells result from the interaction between intracellular Ca2+ stores and depletion-activated Ca2+ channels in the plasma membrane.     

Nanomolar concentrations of Cd2+ inhibit Ca2+ transport systems in plasma membranes and intracellular Ca2+ stores in intestinal epithelium

The interactions of Cd2+ with active Ca2+ transport systems in rat intestinal epithelial cells have been investigated. ATP-driven Ca2+ transport in basolateral plasma membrane vesicles was inhibited by Cd2+ with an I50 value of 1.6 nM free Cd2+ at 1 microM free Ca2+, using EGTA and HEEDTA to buffer Ca2+ and Cd2+ concentrations, respectively. The inhibition was competitive in nature since the Km value of Ca2+ increased with increasing Cd2+ concentrations while the Vmax remained constant. Cd2+ had similar effects on ATP-dependent Ca2+ uptake by permeabilized enterocytes, indicating that non-mitochondrial and mitochondrial Ca2+ stores are also inhibited by nanomolar concentrations of Cd2+. We conclude that ATP-driven Ca2+ transport systems are the most sensitive elements so far reported in Cd2+ intoxication.     

Early effects of metabolic inhibition on intracellular Ca2+ in toad pacemaker cells: involvement of Ca2+ stores

The early effects of metabolic inhibition on intracellular Ca(2+) concentration ([Ca(2+)](i)), Ca(2+) current, and sarcoplasmic reticulum (SR) Ca(2+) content were studied in single pacemaker cells from the sinus venosus of the cane toad. The amplitude of the spontaneous elevations of systolic [Ca(2+)](i) (Ca(2+) transients) was reduced after 5-min exposure to 2 mM NaCN from 338 +/- 30 to 189 +/- 37 nM (P < 0.005, n = 9), and the spontaneous firing rate was reduced from 27 +/- 2 to 12 +/- 4 beats/min (P < 0.002, n = 9). It has been proposed that CN(-) acts by inhibition of cytochrome P-450, resulting in a reduction of cAMP and Ca(2+) current. To test this proposal, we used clotrimazole, a cytochrome P-450 inhibitor, which also decreased the Ca(2+) transients and firing rate. CN(-) caused an insignificant fall of Ca(2+) current (23 +/- 11%) but a substantial reduction of SR Ca(2+) content (by 65 +/- 5%), whereas clotrimazole produced a larger reduction of Ca(2+) current and did not affect the SR Ca(2+) content. Thus the main effect of CN(-) does not seem to be through inhibition of cytochrome P-450. In conclusion, CN(-) appears to reduce Ca(2+) release from the SR mainly by reducing SR Ca(2+) content. A likely cause of the decreased SR content is reduced Ca(2+) uptake by the SR pump.     

Ca2+ dependence of Ca2+ release from intracellular stores of intact and permeabilized Ehrlich ascites tumour cells

Effects of Ca2+ ions on the mobilization of Ca2+ from intracellular stores of intact and permeabilized (15 microM digitonin) Ehrlich ascites tumour cells (EATC) have been compared. For permeabilized cells, the dependences of the initial rate and amplitude of Ca2+ mobilization evoked by the addition of 100 nM inositol 1,4,5-trisphosphate (IP3) on preexisting [Ca2+] were bell-shaped within a [Ca2+] range 10(-7)-10(-6) M with the maxima at [Ca2+] = 166 nM. In intact cells, different concentrations of free cytosolic Ca2+ ([Ca2+]i) were produced using low (up to 0.005%) concentrations of digitonin which selectively increased the permeability of the plasma membrane. Stimulation of cells by exogenous ATP at [Ca2+]i = 10(-8)-10(-6) M resulted in Ca2+ mobilization the rate and amplitude of which were maximal at 102-115 nM Ca2+. The experimental Ca2+ dependences were fit by a model which includes channel opening upon Ca2+ binding and transition to the inactive states upon Ca2+ binding to the closed and open channel forms. Three inactivation types (including two particular cases) demonstrate a slight priority of inhibitory binding of Ca2+ only to the open channel, but predict markedly different parameter values. We conclude that an increase in [Ca2+] can stimulate IP3-induced mobilization, but in intact EATC, deviations of [Ca2+]i from the resting level (about 100 nM) attenuate responses to the agonist stimulation.