Signal transduction in red blood cells of the lizards Ameiva ameiva and Tupinambis merianae (Squamata, Teiidae)

The fluorescent calcium probe, Fluo-3, AM was used to measure the intracellular calcium concentration in red blood cells (RBCs) of the teiid lizards Ameiva ameiva and Tupinambis merianae. The cytosolic [Ca2+] is maintained around 20 nM and the cells contain membrane-bound Ca2+ pools. One pool appears to be identifiable with the endoplasmic reticulum (ER) inasmuch as addition of the sarco-endoplasmic reticulum Ca2+ ATPase, SERCA, inhibitor thapsigargin induces an increase in cytosolic [Ca2+ both in the presence and in the absence of extracellular Ca2+. In addition to the ER, an acidic compartment appears to be involved in Ca2+ storage, as collapse of intracellular pHgradients by monensin, a Na+ -H+ exchanger, and nigericin, a K+ -H+ exchanger, induce the release of Ca2+ from internal pools. A vacuolar H+ pump, sensitive to NBD-Cl and bafilomycin appears to be necessary to load the acidic Ca2+ pools. Finally, the purinergic agonist ATP triggers a rapid and transient increase of [Ca2+]c in the cells from both lizard species, mostly by mobilization of the cation from internal stores.     

Role of proton gradients and vacuola H(+)-ATPases in the refilling of intracellular calcium stores in exocrine cells

Numerous hormones and neurotransmitters activate cells by increasing cytosolic calcium concentration ([Ca(2+)](i)), a key regulatory factor for many cellular processes. A pivotal feature of these Ca(2+) signals is the release of Ca(2+) from intracellular stores, which is followed by activation of extracellular calcium influx, allowing refilling of the stores by SERCA pumps associated with the endoplasmic reticulum. Although the mechanisms of calcium release and calcium influx have been extensively studied, the biology of the Ca(2+) stores is poorly understood. The presence of heterogeneous calcium pools in cells has been previously reported [1] [2] [3]. Although recent technical improvements have confirmed this heterogeneity [4], knowledge about the mechanisms underlying Ca(2+) transport within the stores is very scarce and rather speculative. A recent study in polarized exocrine cells [5] has revealed the existence of Ca(2+) tunneling from basolateral stores to luminal pools, where Ca(2+) is initially released upon cell activation. Here, we present evidence that, during stimulation, Ca(2+) transported into basolateral stores by SERCA pumps is conveyed toward the luminal pools driven by proton gradients generated by vacuolar H(+)-ATPases. This finding unveils a new aspect of the machinery of Ca(2+) stores.     

Modulation of intracellular free Ca2+ concentration by IP3-sensitive and IP3-insensitive nonmitochondrial Ca2+ pools

Intracellular Ca2+ pools play an important role in the adjustment of cytosolic free Ca2+ concentrations. This review summarizes the recent knowledge on receptor-mediated Ca2+ release and Ca2+ uptake mechanisms in Ca2+ stores of exocrine cells taking the exocrine pancreas and the parotid gland as an example. The intracellular mediator for agonist-induced Ca2+ release is inositol 1,4,5-trisphosphate (IP3) which acts by opening Ca2+ channels from the endoplasmic reticulum or a more specialized organelle called 'calciosome'. This Ca2+ release is the major event to increase cytosolic free Ca2+ concentrations of exocrine glands from a resting level of approximately 10(-7) mol/l to approximately 10(-6) mol/l. Subsequently also Ca2+ influx from the extracellular fluid into the cell is increased which involves the action of inositol 1,3,4,5-tetrakisphosphate (IP4). Intracellular nonmitochondrial Ca2+ reuptake occurs into IP3-sensitive (IsCaP) as well as into IP3-insensitive Ca2+ pools Ca2+ pools (IisCaP). While Ca2+ uptake into the IisCaP is mediated by a vanadate-sensitive Ca2+ pump, Ca2+ uptake into the IsCaP is mediated by a Ca2+/H+ exchanger at the expense of an H+ gradient which is established by a vacuolar type H+ pump present in the same Ca2+ pool. During stimulation both Ca2+ pools, IsCaP and IisCaP, are probably connected, the nature of which has not yet been clarified. It is suggested that GTP and/or IP4 control Ca2+ conveyance between intracellular Ca2+ pools by forming Ca2+-carrying junctions between membranes. Other models propose that Ca2+, which is released by IP3, induces Ca2+ release from another Ca2+ pool. Taking into account that H+ transport is present in IP3-sensitive Ca2+ pools the possibility of pH-regulated Ca2+ channels in the IisCaP, located in close neighbourhood to the IsCaP, is also considered.     

Cytosolic acidification and intracellular zinc release in hippocampal neurons

In neurons exposed to glutamate, Ca2? influx triggers intracellular Zn2? release via an as yet unclear mechanism. As glutamate induces a Ca2?-dependent cytosolic acidification, the present work tested the relationships among intracellular Ca2? concentration ([Ca2?](i)), intracellular pH (pH(i) ), and [Zn2?](i). Cultured hippocampal neurons were exposed to glutamate and glycine (Glu/Gly), while [Zn2?](i), [Ca2?](i) and pH(i) were monitored using FluoZin-3, Fura2-FF, and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. Glu/Gly applications decreased pH(i) to 6.1 and induced intracellular Zn2? release in a Ca2?-dependent manner, as expected. The pH(i) drop reduced the affinity of FluoZin-3 and Fura-2-FF for Zn2?. The rate of Glu/Gly-induced [Zn2?](i) increase was not correlated with the rate of [Ca2?](i) increase. Instead, the extent of [Zn2?](i) elevations corresponded well to the rate of pH(i) drop. Namely, [Zn2?](i) increased more in more highly acidified neurons. Inhibiting the mechanisms responsible for the Ca2?-dependent pH(i) drop (plasmalemmal Ca2? pump and mitochondria) counteracted the Glu/Gly-induced intracellular Zn2? release. Alkaline pH (8.5) suppressed Glu/Gly-induced intracellular Zn2? release whereas acidic pH (6.0) enhanced it. A pH(i) drop to 6.0 (without any Ca2? influx or glutamate receptor activation) led to intracellular Zn2? release; the released Zn2? (free Zn2? plus Zn2?) bound to Fura-2FF and FluoZin-3) reached 1 μM.     

Imaging spatial and cellular characteristics of low temperature calcium signature after cold acclimation in Arabidopsis

Cooling-induced 'calcium signatures' were imaged in aequorin-expressing ARABIDOPSIS: plants after cold acclimation or growth at ambient temperature. In all tissues, signatures were altered after acclimation. Characterization of the components generating this response indicates that cold acclimation increases cold-induced vacuolar Ca(2+) release, but does not affect the influx of extracellular calcium.     

Investigation of carvedilol-evoked Ca²+ movement and death in human oral cancer cells

The effect of carvedilol on cytosolic free Ca2? concentrations ([Ca2?](i)) in OC2 human oral cancer cells is unknown. This study examined if carvedilol altered basal [Ca2?](i) levels in suspended OC2 cells by using fura-2 as a Ca2?-sensitive fluorescent probe. Carvedilol at concentrations between 10 and 40 μM increased [Ca2?](i) in a concentration-dependent fashion. The Ca2? signal was decreased by 50% by removing extracellular Ca2?. Carvedilol-induced Ca2? entry was not affected by the store-operated Ca2? channel blockers nifedipine, econazole, and SK&F96365, but was enhanced by activation or inhibition of protein kinase C. In Ca2?-free medium, incubation with the endoplasmic reticulum Ca2? pump inhibitor thapsigargin did not change carvedilol-induced [Ca2?](i) rise; conversely, incubation with carvedilol did not reduce thapsigargin-induced Ca2? release. Pretreatment with the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) inhibited carvedilol-induced [Ca2?](i) release. Inhibition of phospholipase C with U73122 did not alter carvedilol-induced [Ca2?](i) rise. Carvedilol at 5-50 μM 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 acetoxymethyl ester (BAPTA/AM). Annexin V/propidium iodide staining assay suggests that apoptosis played a role in the death. Collectively, in OC2 cells, carvedilol induced [Ca2?](i) rise by causing phospholipase C-independent Ca2? release from mitochondria and non-endoplasmic reticulum stores, and Ca2? influx via protein kinase C-regulated channels. Carvedilol (up to 50 μM) induced cell death in a Ca2?-independent manner that involved apoptosis.     

Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation.

Cold shock elicits an immediate rise in cytosolic free calcium concentration ([Ca2+]cyt) in both chilling-resistant Arabidopsis and chilling-sensitive tobacco (Nicotiana plumbaginifolia). In Arabidopsis, lanthanum or EGTA caused a partial inhibition of both cold shock [Ca2+]cyt elevation and cold-dependent kin1 gene expression. This suggested that calcium influx plays a major role in the cold shock [Ca2+]cyt response and that an intracellular calcium source also might be involved. To investigate whether the vacuole (the major intracellular calcium store in plants) is involved, we targeted the calcium-dependent photoprotein aequorin to the cytosolic face of the vacuolar membrane. Cold shock calcium kinetics in this microdomain were consistent with a cold-induced vacuolar release of calcium. Treatment with neomycin or lithium, which interferes with phosphoinositide cycling, resulted in cold shock [Ca2+]cyt kinetics consistent with the involvement of inositol trisphosphate and inositide phosphate signaling in this response. We also investigated the effects of repeated and prolonged low temperature on cold shock [Ca2+]cyt. Differences were observed between the responses of Arabidopsis and N. plum-baginifolia to repeated cold stimulation. Acclimation of Arabidopsis by pretreatment with cold or hydrogen peroxide caused a modified calcium signature to subsequent cold shock. This suggests that acclimation involves modification of plant calcium signaling to provide a "cold memory."     

H+ uptake increases GTP-induced connection of inositol 1,4,5-trisphosphate- and caffeine-sensitive calcium pools in pancreatic microsomal vesicles.

Evidence suggests that GTP but not GTP gamma S activates Ca2+ movement between myo-inositol 1,4,5-trisphosphate (IP3)-sensitive and -insensitive Ca2+ pools (1). Measuring 45Ca2+ uptake into pancreatic microsomal vesicles we have determined the sizes of three different Ca2+ pools which release Ca2+ in response 1) to IP3, 2) to caffeine, and 3) to both IP3 and caffeine ("common" Ca2+ pool). In the presence of GTP the size of the IP3-sensitive Ca2+ pool is decreased whereas the "common" Ca2+ pool is increased as compared to control Ca2+ pool sizes in the presence of GTP gamma S. This effect of GTP is inhibited by bafilomycin B1, a specific inhibitor of vacuolar type H+ ATPases (2). We conclude that GTP induced connection between IP3- and caffeine-sensitive Ca2+ pools is triggered by intravesicular acidification and involves function of small GTP-binding proteins, known to mediate interorganelle transfer.     

Mobilization of vacuolar arginine in Neurospora crassa. Mechanism and role of glutamine

Nitrogen starvation has been shown to increase the cytosolic arginine concentration and to accelerate protein turnover in mycelia of Neurospora crassa. The cytosolic arginine is derived from a metabolically inactive vacuolar pool. Redistribution of arginine between cytosolic and vacuolar compartments is the result of mobilization of this metabolite in response to nitrogen starvation. Mobilization of arginine (and purines) also occurred in response to glutamine limitation, but arginine accumulated upon proline starvation. These observations indicate that mobilization is a consequence of glutamine limitation rather than a general response to amino acid starvation (or limitation). Analysis of the amino acid pools in mycelia subjected to starvation or limitation suggests that glutamine (or a metabolite derived from glutamine) provides a signal which determines the metabolic fate of vacuolar arginine. The results are consistent with the hypothesis that vacuolar compartmentation provides a readily available store of nitrogen-rich compounds to be utilized during differentiation or under conditions of nutritional stress.     

Effect of tumour-promoting phorbol ester on calcium homeostasis in human platelets.

The more interesting features of the effects or PMA on [Ca2+]i and ATP release were the following: 1. preincubation with PMA inhibited thrombin-evoked calcium transients; 2. PMA stimulated slightly the release of calcium and ATP whereas inhibited calcium and ATP pools sensitive to thrombin; 3. A23187 reversed the inhibitory effect of PMA; 4. subsaturating thrombin concentrations gave results similar to PMA on thrombin-induced calcium and ATP release but not on [Ca2+]i.     

ATP Release, Adenosine Formation, and Modulation of Dynorphin and Glutamic Acid Release by Adenosine Analogues in Rat Hippocampal Mossy Fiber Synaptosomes

Using a hippocampal subcellular fraction enriched in mossy fiber synaptosomes, evidence was obtained indicating that adenosine derived from a presynaptic pool of ATP may modulate the release of prodynorphin-derived peptides. and glutamic acid from mossy fiber terminals. Synaptosomal ATP was released in a Ca2+-dependent manner by K+-induced depolarization. The rapid hydrolysis of extracellular [14C]ATP in the presence of intact mossy fiber synaptosomes resulted in the production of [14C]adenosine. Micromolar concentrations of a stable adenosine analogue, 2-chloroadenosine, inhibited the K+-stimulated release of both dynorphin B and dynorphin A(1-8). 2-Chloroadenosine failed to suppress the evoked release of glutamic acid, measured in these same superfusates, unless the mossy fiber synaptosomes were pretreated with D-aspartic acid to deplete the cytosolic, Ca2+-independent, pool of this acidic amino acid. In synaptosomes pretreated in this manner, release of the remaining Ca2+-dependent pool of glutamic acid was significantly inhibited by NiCl2, 2-chloroadenosine, 5'-N-ethylcarboxamidoadenosine, cyclohexyladenosine, and R(-)-N6(2-phenylisopropyl)adenosine, but not by ATP. 2-Chloroadenosine-induced inhibition was reversed when the external CaCl2 concentration was raised from 1.8 mM to 6 mM. 8-Phenyltheophylline, an adenosine receptor antagonist, effectively blocked the inhibitory effects of 2-chloroadenosine on mossy fiber synaptosomes and significantly enhanced the K+-evoked release of both glutamic acid and dynorphin A(1-8) when added alone to the superfusion medium. These results support the proposition that depolarized hippocampal mossy fiber synaptosomes release endogenous ATP and are capable of forming adenosine from extracellular ATP, and that endogenous adenosine may act at a presynaptic site to inhibit the further release of glutamic acid and the prodynorphin-derived peptides.     

Changes induced in the permeability barrier of the yeast plasma membrane by cupric ion.

A specific effect of Cu2+ eliciting selective changes in the permeability of intact Saccharomyces cerevisiae cells is described. When 100 microM CuCl2 was added to a cell suspension in a buffer of low ionic strength, the permeability barrier of the plasma membranes of the cells was lost within 2 min at 25 degrees C. The release of amino acids was partial, and the composition of the amino acids released was different from that of those retained in the cells. Mostly glutamate was released, but arginine was mainly retained in the cells. Cellular K+ was released rapidly after CuCl2 addition, but 30% of the total K+ was retained in the cells. These and other observations suggested that Cu2+ caused selective lesions of the permeability barrier of the plasma membrane but did not affect the permeability of the vacuolar membrane. These selective changes were not induced by the other divalent cations tested. A novel and simple method for differential extraction of vacuolar and cytosolic amino acid pools by Cu2+ treatment was established. When Ca2+ was added to Cu2+-treated cells, a large amount of Ca2+ was sequestered into vacuoles, with formation of an inclusion of a Ca2+-polyphosphate complex in the vacuoles. Cu2+-treated cells also showed enhanced uptake of basic amino acids and S-adenosylmethionine. The transport of these substrates showed saturable kinetics with low affinities, reflecting the vacuolar transport process in situ. With Cu2+ treatment, selective leakage of K+ from the cytosolic compartment appears to create a large concentration gradient of K+ across the vacuolar membrane and generates an inside-negative membrane potential, which may provide a driving force of uptake of positively charged substances into vacuoles. Cu2+ treatment provides a useful in situ method for investigating the mechanisms of differential solute pool formation and specific transport phenomena across the vacuolar membrane.     

Calcium pools mobilized by calcium or inositol 1,4,5-trisphosphate are differentially localized in rat heart and brain.

Calcium-induced calcium release (CICR) pools have been demonstrated in brain and heart microsomes biochemically and autoradiographically by the sensitivity of 45Ca2+ accumulation to Mg2+, ATP, ruthenium red, caffeine, and tetracaine. The CICR pool colocalizes with [3H]ryanodine binding sites, supporting the notion that [3H]ryanodine labels CICR pools. Sites of CICR pools in the brain contrast with those of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools with reciprocal localizations between the two Ca2+ pools in several structures. Thus, in the hippocampus CA-1 is enriched in IP3-sensitive Ca2+ pools, whereas CICR pools are highest in CA-3 and the dentate gyrus. The corpus striatum and cerebellum are enriched in IP3 pools, whereas the medial septum and olfactory bulb have high CICR densities. In cardiac tissue, CICR is localized to atrial and ventricular muscle, whereas IP3 pools are concentrated in coronary vessels and cardiac conduction fibers. The reciprocal enrichment of IP3 and CICR Ca2+ pools implies differential regulation of Ca2+ hemostasis in these tissues.     

Methylglyoxal-induced stomatal closure accompanied by peroxidase-mediated ROS production in Arabidopsis

Methylglyoxal (MG) is an oxygenated short aldehyde and a glycolytic intermediate that accumulates in plants under environmental stresses. Being a reactive α-oxoaldehyde, MG may act as a signaling molecule in plants during stresses. We investigated whether MG induces stomatal closure, reactive oxygen species (ROS) production, and cytosolic free calcium concentration ([Ca2?](cyt)) to clarify roles of MG in Arabidopsis guard cells. MG induced production of ROS and [Ca2?](cyt) oscillations, leading to stomatal closure. The MG-induced stomatal closure and ROS production were completely inhibited by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), but were not affected by an NAD(P)H oxidase mutation, atrbohD atrbohF. Furthermore, the MG-elicited [Ca2?](cyt) oscillations were significantly suppressed by SHAM but not by the atrbohD atrbohF mutation. Neither endogenous abscisic acid nor endogenous methyl jasmonate was involved in MG-induced stomatal closure. These results suggest that intrinsic metabolite MG can induce stomatal closure in Arabidopsis accompanied by extracellular ROS production mediated by SHAM-sensitive peroxidases, intracellular ROS accumulation, and [Ca2?](cyt) oscillations.     

Effect of pH on the calcium metabolism of isolated rat kidney cells

Summary The effects of metabolic and respiratory acidosis and alkalosis on cellular calcium metabolism were studied in rat kidney cells dispersed with collagenase. In both types of acidosis, the intracellular pH, total cell calcium, and the cell relative radioactivity after 60 min of labeling are significantly depressed. Kinetic analysis of⁴⁵Ca desaturation curves shows that acidosis decreases all three cellular calcium pools and depresses calcium fluxes between the superficial and cytosolic pools and between the cytosolic and mitochondrial pools. In alkalosis the intracelluar pH, the total cell calcium, and the cell relative radioactivity are significantly increased. Kinetic studies show that in alkalosis, only the mitochondrial pool is consistently increased. Calcium exchange between the mitochondrial and cytosolic pool is increased in metabolic alkalosis only. These results suggest that hydrogen ion is an important modulator of calcium metabolism, and that the intracellular pH rather than extracellular pH is the critical factor in determining the calcium status of cells during altered acid-base conditions.     

CA(2+) mobilization in the human submandibular duct cell line A253

Ca(2+)mobilization induced by ATP, isoproterenol and the Ca(2+)-ATPase inhibitor thapsigargin in the human submandibular duct cell line A253 was investigated using the Ca(2+)-sensitive fluorescent indicator fura-2. ATP and isoproterenol increased cytosolic free Ca(2+)([Ca(2+)](i)) and subsequent exposure to thapsigargin after ATP or isoproterenol stimulation caused a further increase in [Ca(2+)](i). However, ATP and isoproterenol were not able to elicit a further increase in [Ca(2+)](i)after exposure of the cells to thapsigargin. Relatively few cells reacted to isoproterenol stimulation, but nearly all cells reacted to isoproterenol if ATP was added together with, or prior to isoproterenol stimulation. Moreover, the effect of ATP was potentiated by prior or simultaneous addition of isoproterenol. Furthermore, ATP decreased [Ca(2+)](i)in the presence of thapsigargin probably due to agonist-induced export of intracellular calcium. The results may suggest the existence of three thapsigargin sensitive pools; one opened by ATP acting through P(2)-purinergic receptors and IP(3), one opened by isoproterenol acting through beta2-adrenergic receptors, and a third pool not sensitive to ATP or isoproterenol.     

Signal transduction in red bloodcells of the lizards Ameiva ameiva and Tupinambis merianae (Squamata, Teiidae)

The fluorescent calcium probe, Fluo-3, AM was used to measure the intracellular calcium concentration in red blood cells (RBCs) of the teiid lizards Ameiva ameiva and Tupinambis merianae. The cytosolic [Ca²⁺] is maintained around 20nM and the cells contain membrane-bound Ca²⁺pools. One pool appears to be identifiable with the endoplasmic reticulum (ER) inasmuch as addition of the sarco-endoplasmic reticulum Ca²⁺ATPase, SERCA, inhibitor thapsigargin induces an increase in cytosolic [Ca²⁺both in the presence and in the absence of extracellular Ca²⁺. In addition to the ER, an acidic compartment appears to be involved in Ca²⁺storage, as collapse of intracellular pHgradients by monensin, a Na⁺–H⁺exchanger, and nigericin, a K⁺–H⁺exchanger, induce the release of Ca²⁺from internal pools. A vacuolar H⁺pump, sensitive to NBD-Cl and bafilomycin appears to be necessary to load the acidic Ca²⁺pools. Finally, the purinergic agonist ATP triggers a rapid and transient increase of [Ca²⁺]_cin the cells from both lizard species, mostly by mobilization of the cation from internal stores.     

Sequential mechanism of calcium binding and translocation in sarcoplasmic reticulum adenosine triphosphatase

Cooperative calcium binding (apparent Kd = 1.04 X 10(-6) M) to the ATPase of sarcoplasmic reticulum vesicles occurs with a maximal stoichiometry of 2 mols of divalent cation/mol of enzyme in the absence of ATP. The bound calcium is distributed into two pools which undergo fast or slow isotopic exchange, respectively. The two pools retain a 1:1 molar ratio under various conditions and are both located within a protein crevice, as suggested by their cooperative interaction and exchange kinetics. Following enzyme phosphorylation by ATP, both pools of bound calcium are "internalized" (cannot be displaced by quench reagents). If following 45Ca2+ binding, isotopic dilution is obtained in the medium by adding 40Ca2+ with ATP, internalization of both pools of bound 45Ca2+ (2 mol/mol of phosphoenzyme) is still observed within the first enzyme cycle. When the cycle is reversed by addition of excess ADP soon after ATP, only half of the internalized 45Ca2+ is released from the enzyme into the medium outside the vesicles, while the other half remains with the vesicles. If half of the bound 45Ca2+ is exchanged (fast exchange) with 40Ca2+ previous to the addition of ATP, none of the remaining 45Ca2+ is released outside the vesicles upon reversal of the enzyme cycle. Therefore, the pool of bound calcium which undergoes slower exchange with the outside medium, is the first to be released inside the vesicles upon enzyme phosphorylation. A sequential mechanism of calcium binding and translocation is proposed, that accounts for binding cooperativity and exchange kinetics, presteady state transients following addition of ATP, and the Ca2+ concentration dependence of ATPase activity in steady state.     

Differential involvement of vacuolar H(+)-ATPase in the refilling of thapsigargin- and agonist-mobilized Ca(2+) stores

Our objective was to evaluate the role of vacuolar H(+)-ATPase and proton gradients in the refilling of Ca(2+) stores in fura-2-loaded pancreatic acinar cells. Once depleted with a high level of ACh, the Ca(2+) stores were replenished with a Ca(2+)-containing solution. The degree of refilling was estimated with a second release in response to either ACh (ACh-releasable store) or thapsigargin (thapsigargin-releasable store), a specific inhibitor of the endoplasmic reticulum Ca(2+) pumps. Both the protonophore nigericin and folimycin, a specific inhibitor of the vacuolar H(+)-ATPase, reduced reuptake into the ACh-mobilized stores but not into the thapsigargin-releasable pools. These treatments effectively dissipated the subcellular pH gradients (revealed by confocal observation of the distribution of a marker for acidic compartments), and did not impair the [Ca(2+)](i) response to ACh in control cells. Our results indicate that thapsigargin and ACh release heterogeneous Ca(2+) stores which are differently operated by vacuolar proton ATPase.     

Red blood cells of the lizards Ameiva ameiva (Squamata, Teiidae) display multiple mechanisms to control cytosolic calcium

We have previously reported that lizard red blood cells control their cytosolic calcium concentration by sequestering calcium ions in pools, which could be discharged by thapsigargin, by the Na+/H+ ionophore, monensin, by the K+/H+ ionophore, nigericin and by the proton pump inhibitor, bafilomycin A1 [1]. We have now demonstrated, with the aid of confocal microscopy, the presence in these cells of organelles, which accumulate the dye acridine orange and are thus by inference the sites of proton pools. We have found, moreover, that monensin, nigericin and bafilomycin all act to discharge these pools. We further show that calcium release ensues when the calcium ionophore, ionomycin, is added after thapsigargin and monensin; this implies the existence of a third pool, besides the acidic pool and the Endoplasmic Reticulum (ER), which participates in calcium homeostasis. The ER calcium pool can de discharged by the addition of the second messenger, IP3, and we present evidence, based on confocal microscopy, that the IP3 receptors are located in or close to the nucleus.