Ca2+ signaling in the regulation of dendritic cell functions

Dendritic cells (DCs) are highly versatile antigen-presenting cells critically involved in both innate and adaptive immunity as well as maintenance of self-tolerance. DC function is governed by Ca(2+) signaling, which directs the DC responses to diverse antigens, including Toll-like receptor ligands, intact bacteria, and microbial toxins. Ca(2+)-sensitive DC functions include DC activation, maturation, migration, and formation of immunological synapses with T cells. Moreover, alterations of cytosolic Ca(2+) trigger immune suppression or switch off DC activity. Ca(2+) signals are generated by the orchestration of Ca(2+) transport processes across plasma, endoplasmic reticulum, and inner mitochondrial membrane. These processes include active pumping of Ca(2+), Ca(2+)/Na(+) antiport, and electrodiffusion through Ca(2+)-permeable channels or uniporters. Ca(2+) channels in the plasma membrane such as Ca(2+) release-activated Ca(2+) or L-type Ca(2+) channels are tightly regulated by the membrane potential which in turn depends on the activity of voltage-gated K(+) or Ca(2+)-activated nonselective cation channels. The rapidly growing knowledge on the function and regulation of these membrane transport proteins provides novel insight into pathophysiological mechanisms underlying dysfunction of the immune system and opens novel therapeutic opportunity to favorably influence the function of the immune system.     

Distinction between Ca(2+) pump and Ca(2+)/H(+) antiport activities in synaptic vesicles of sheep brain cortex

Synaptic vesicles, isolated from a sheep brain cortex, accumulate Ca(2+) in a manner that depends on the pH and pCa values. In the presence of 100 microM CaCl(2), most of the Ca(2+) taken up by the vesicles was vanadate-inhibited (86%) at pH 7.4, whereas at pH 8.5, part of the Ca(2+) accumulated (36%) was DeltapH-dependent (bafilomycin and CCCP inhibited) and part was insensitive to those drugs (31%). We also observed that both vanadate-sensitive and bafilomycin-sensitive Ca(2+) accumulations were completely released by the Ca(2+) ionophore, ionomycin, and that these processes work with high (K(0.5)=0.6 microM) and low (K(0.5)=217 microM) affinity for Ca(2+), respectively. The DeltapH-dependent Ca(2+) transport appears to be largely operative at Ca(2+) concentrations (>100 microM) which completely inhibited the vanadate-sensitive Ca(2+) uptake. These Ca(2+) effects on the Ca(2+) accumulation were well correlated with those observed on the vanadate-inhibited Ca(2+)-ATPase and bafilomycin-inhibited H(+)-ATPase, respectively. The Ca(2+)-ATPase activity reached a maximum at about 25 microM (pH 7.4) and sharply declined at higher Ca(2+) concentrations. In contrast, Ca(2+) had a significant stimulatory effect on the H(+)-ATPase between 250 and 500 microM Ca(2+) concentration. Furthermore, we found that DeltapH-sensitive Ca(2+) transport was associated with proton release from the vesicles. About 21% of the maximal proton gradient was dissipated by addition of 607.7 microM CaCl(2) to the reaction medium and, if CaCl(2) was present before the proton accumulation, lower pH gradients were reached. Both vanadate-inhibited and bafilomycin-inhibited systems transported Ca(2+) into the same vesicle pool of our preparation, suggesting that they belong to the same cellular compartment. These results indicate that synaptic vesicles of the sheep brain cortex contain two distinct mechanisms of Ca(2+) transport: a high Ca(2+) affinity, proton gradient-independent Ca(2+) pump that has an optimal activity at pH 7.4, and a low Ca(2+) affinity, proton gradient-dependent Ca(2+)/H(+) antiport that works maximally at pH 8.5.     

P-type proton ATPases are involved in intracellular calcium and proton uptake in the plant parasite Phytomonas francai

The use of digitonin to permeabilize the plasma membrane of promastigotes of Phytomonas francai allowed the identification of two non-mitochondrial Ca(2+) compartments; one sensitive to ionomycin and vanadate (neutral or alkaline), possibly the endoplasmic reticulum, and another sensitive to the combination of nigericin plus ionomycin (acidic), possibly the acidocalcisomes. A P-type (phospho-intermediate form) Ca(2+)-ATPase activity was found to be responsible for intracellular Ca(2+) transport in these cells, with no evidence of a mitochondrial Ca(2+) transport activity. ATP-driven acidification of internal compartments in cell lysates and cells mechanically permeabilized was assayed spectrophotometrically with acridine orange. This activity was inhibited by low concentrations of vanadate and digitonin, was insensitive to bafilomycin A(1), and stimulated by Na(+) ions. Taken together, our results indicate that P-type ATPases are involved in intracellular Ca(2+) and H(+) transport in promastigotes of P. francai.     

Ca(2+) efflux in mitochondria from the yeast Endomyces magnusii.

Calcium release pathways in Ca(2+)-preloaded mitochondria from the yeast Endomyces magnusii were studied. In the presence of phosphate as a permeant anion, Ca(2+) was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis. Ca(2+) release was not affected by cyclosporin A, an inhibitor of the mitochondrial permeability transition. Aeration of the mitochondrial suspension inhibited the efflux of Ca(2+) and induced its re-uptake. With acetate as the permeant anion, a spontaneous net Ca(2+) efflux set in after uptake of approximately 150 nmol of Ca(2+)/mg of protein. The rate of this efflux was proportional to the Ca(2+) load and insensitive to aeration, protonophorous uncouplers, and Na(+) ions. Ca(2+) efflux was inhibited by La(3+), Mn(2+), Mg(2+), tetraphenylphosphonium, inorganic phosphate, and nigericin and stimulated by hypotonicity, spermine, and valinomycin in the presence of 4 mm KCl. Atractyloside and t-butyl hydroperoxide were without effect. Ca(2+) efflux was associated with contraction, but not with mitochondrial swelling. We conclude that the permeability transition pore is not involved in Ca(2+) efflux in preloaded E. magnusii mitochondria. The efflux occurs via an Na(+)-independent pathway, in many ways similar to the one in mammalian mitochondria.     

Mechanism of N-terminal autoinhibition in the Arabidopsis Ca(2+)/H(+) antiporter CAX1

Regulation of Ca(2+)/H(+) antiporters may be an important function in determining the duration and amplitude of cytosolic Ca(2+) oscillations. Previously the Arabidopsis Ca(2+)/H(+) transporter, CAX1 (cation exchanger 1), was identified by its ability to suppress yeast mutants defective in vacuolar Ca(2+) transport. Recently, a 36-amino acid N-terminal regulatory region on CAX1 has been identified that inhibits CAX1-mediated Ca(2+)/H(+) antiport. Here we show that a synthetic peptide designed against the CAX1 36 amino acids inhibited Ca(2+)/H(+) transport mediated by an N-terminal-truncated CAX1 but did not inhibit Ca(2+) transport by other Ca(2+)/H(+) antiporters. Ca(2+)/H(+) antiport activity measured from vacuolar-enriched membranes of Arabidopsis root was also inhibited by the CAX1 peptide. Through analyzing CAX chimeric constructs the region of interaction of the N-terminal regulatory region was mapped to include 7 amino acids (residues 56-62) within CAX1. The CAX1 N-terminal regulatory region was shown to physically interact with this 7-amino acid region by yeast two-hybrid analysis. Mutagenesis of amino acids within the N-terminal regulatory region implicated several residues as being essential for regulation. These findings describe a unique mode of antiporter autoinhibition and demonstrate the first detailed mechanisms for the regulation of a Ca(2+)/H(+) antiporter from any organism.     

Effects of 4,4'-diisothyocyanatostilbene-2,2'-disulfonic acid on Trypanosoma cruzi proliferation and Ca(2+) homeostasis

Cell viability requires the perfect functioning of the processes controlling ATP and Ca(2+) homeostasis. It is known that cell death caused by a variety of toxins or pathological conditions is associated with a disruption of ATP and Ca(2+) homeostasis. This study shows that 4,4'-diisothyocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibits Trypanosoma cruzi epimastigote cell growth. This thiol-reagent thiocyanate derivative was able to inhibit two ecto-enzymes present in this parasite. The ecto-ATPase and ecto-phosphatase activities were inhibited in a dose-dependent manner (K(i)=47.7 and 472.5 microM, respectively), but the 5'nucleotidase and 3'nucleotidase activities were not. DIDS uptake was approached by fluorescence microscopy. Pulse-chase experiments revealed the DIDS accumulation in compartments, presumably endocytic, in the posterior region of epimastigotes. In addition, we show that the T. cruzi mitochondria studied in permeabilized cells are able to accumulate and retain medium Ca(2+) in the absence of DIDS. However, in the presence of increasing concentrations of DIDS (50-200 microM), Ca(2+) transport was inhibited in a dose-dependent manner. DIDS also caused a disruption of the mitochondrial membrane potential, in the same concentration range, thus explaining its effect on Ca(2+) uptake. The presence of EGTA prevented the elimination of the mitochondrial membrane potential (DeltaPsi), supporting previous data suggesting that the binding of Ca(2+) to the mitochondrial membrane exposes buried thiols to react with DIDS. This thiocyanate derivative was also able to inhibit Ca(2+) uptake by the endoplasmic reticulum in a dose-dependent manner. Taken together, the data presented here provide further insights into the mechanisms underlying the antiproliferative actions of DIDS in T. cruzi.     

High- and low-calcium-dependent mechanisms of mitochondrial calcium signalling

The Ca(2+) coupling between endoplasmic reticulum (ER) and mitochondria is central to multiple cell survival and cell death mechanisms. Cytoplasmic [Ca(2+)] ([Ca(2+)](c)) spikes and oscillations produced by ER Ca(2+) release are effectively delivered to the mitochondria. Propagation of [Ca(2+)](c) signals to the mitochondria requires the passage of Ca(2+) across three membranes, namely the ER membrane, the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM). Strategic positioning of the mitochondria by cytoskeletal transport and interorganellar tethers provides a means to promote the local transfer of Ca(2+) between the ER membrane and OMM. In this setting, even >100 microM [Ca(2+)] may be attained to activate the low affinity mitochondrial Ca(2+) uptake. However, a mitochondrial [Ca(2+)] rise has also been documented during submicromolar [Ca(2+)](c) elevations. Evidence has been emerging that Ca(2+) exerts allosteric control on the Ca(2+) transport sites at each membrane, providing mechanisms that may facilitate the Ca(2+) delivery to the mitochondria. Here we discuss the fundamental mechanisms of ER and mitochondrial Ca(2+) transport, particularly the control of their activity by Ca(2+) and evaluate both high- and low-[Ca(2+)]-activated mitochondrial calcium signals in the context of cell physiology.     

Evidence of differential pH regulation of the Arabidopsis vacuolar Ca2+/H+ antiporters CAX1 and CAX2.

The Arabidopsis Ca(2+)/H(+) antiporters cation exchanger (CAX) 1 and 2 utilise an electrochemical gradient to transport Ca(2+) into the vacuole to help mediate Ca(2+) homeostasis. Previous whole plant studies indicate that activity of Ca(2+)/H(+) antiporters is regulated by pH. However, the pH regulation of individual Ca(2+)/H(+) antiporters has not been examined. To determine whether CAX1 and CAX2 activity is affected by pH, Ca(2+)/H(+) antiport activity was measured in vacuolar membrane vesicles isolated from yeast heterologously expressing either transporter. Ca(2+) transport by CAX1 and CAX2 was regulated by cytosolic pH and each transporter had a distinct cytosolic pH profile. Screening of CAX1/CAX2 chimeras identified an amino acid domain within CAX2 that altered the pH-dependent Ca(2+) transport profile so that it was almost identical to the pH profile of CAX1. Results from mutagenesis of a specific His residue within this domain suggests a role for this residue in pH regulation.     

P(5A)-type ATPase Cta4p is essential for Ca2+ transport in the endoplasmic reticulum of Schizosaccharomyces pombe

This study establishes the role of P(5A)-type Cta4 ATPase in Ca(2+) sequestration in the endoplasmic reticulum by detecting an ATP-dependent, vanadate-sensitive and FCCP insensitive (45)Ca(2+)-transport in fission yeast membranes isolated by cellular fractionation. Specifically, the Ca(2+)-ATPase transport activity was decreased in ER membranes isolated from cells lacking a cta4(+) gene. Furthermore, a disruption of cta4(+) resulted in 6-fold increase of intracellular Ca(2+) levels, sensitivity towards accumulation of misfolded proteins in ER and ER stress, stimulation of the calcineurin phosphatase activity and vacuolar Ca(2+) pumping. These data provide compelling biochemical evidence for a P(5A)-type Cta4 ATPase as an essential component of Ca(2+) transport system and signaling network which regulate, in conjunction with calcineurin, the ER functionality in fission yeast.     

Expression of Arabidopsis CAX1 in tobacco: altered calcium homeostasis and increased stress sensitivity

Calcium (Ca(2)+) efflux from the cytosol modulates Ca(2+) concentrations in the cytosol, loads Ca(2+) into intracellular compartments, and supplies Ca(2+) to organelles to support biochemical functions. The Ca(2+)/H(+) antiporter CAX1 (for CALCIUM EXCHANGER 1) of Arabidopsis is thought to be a key mediator of these processes. To clarify the regulation of CAX1, we examined CAX1 RNA expression in response to various stimuli. CAX1 was highly expressed in response to exogenous Ca(2+). Transgenic tobacco plants expressing CAX1 displayed symptoms of Ca(2+) deficiencies, including hypersensitivity to ion imbalances, such as increased magnesium and potassium concentrations, and to cold shock, but increasing the Ca(2+) in the media abrogated these sensitivities. Tobacco plants expressing CAX1 also demonstrated increased Ca(2+) accumulation and altered activity of the tonoplast-enriched Ca(2+)/H(+) antiporter. These results emphasize that regulated expression of Ca(2+)/H(+) antiport activity is critical for normal growth and adaptation to certain stresses.     

pH and monovalent cations regulate cytosolic free Ca(2+) in E. coli

The results here show for the first time that pH and monovalent cations can regulate cytosolic free Ca(2+) in E. coli through Ca(2+) influx and efflux, monitored using aequorin. At pH 7.5 the resting cytosolic free Ca(2+) was 0.2-0.5 microM. In the presence of external Ca(2+) (1 mM) at alkaline pH this rose to 4 microM, being reduced to 0.9 microM at acid pH. Removal of external Ca(2+) caused an immediate decrease in cytosolic free Ca(2+) at 50-100 nM s(-1). Efflux rates were the same at pH 5.5, 7.5 and 9.5. Thus, ChaA, a putative Ca(2+)/H(+)exchanger, appeared not to be a major Ca(2+)-efflux pathway. In the absence of added Na(+), but with 1 mM external Ca(2+), cytosolic free Ca(2+) rose to approximately 10 microM. The addition of Na(+)(half maximum 60 mM) largely blocked this increase and immediately stimulated Ca(2+) efflux. However, this effect was not specific, since K(+) also stimulated efflux. In contrast, an increase in osmotic pressure by addition of sucrose did not significantly stimulate Ca(2+) efflux. The results were consistent with H(+) and monovalent cations competing with Ca(2+) for a non-selective ion influx channel. Ca(2+) entry and efflux in chaA and yrbG knockouts were not significantly different from wild type, confirming that neither ChaA nor YrbG appear to play a major role in regulating cytosolic Ca(2+) in Escherichia coli. The number of Ca(2+) ions calculated to move per cell per second ranged from <1 to 100, depending on conditions. Yet a single eukaryote Ca(2+) channel, conductance 100 pS, should conduct >6 million ions per second. This raises fundamental questions about the nature and regulation of Ca(2+) transport in bacteria, and other small living systems such as mitochondria, requiring a new mathematical approach to describe such ion movements. The results have important significance in the adaptation of E. coli to different ionic environments such as the gut, fresh water and in sea water near sewage effluents.     

Regulation of intracellular calcium homeostasis in Trypanosoma cruzi. Effects of calmidazolium and trifluoperazine

Trypanosoma cruzi epimastigotes maintained an intracellular free calcium concentration of about 0.15 microM, as measured with the fluorescent indicator Fura-2. The maintenance of low [Ca2+]i is energy-dependent since it is disrupted by KCN and FCCP. When the cells were permeabilized with digitonin, the steady-state free Ca2+ concentration in the absence of ATP was about 0.7 microM. The additional presence of ATP resulted in a steady-state level close to 0.1-0.2 microM which compares favorably with the concentration detected in intact cells. Intracellular Ca2+ uptake at high levels of free Ca2+ (greater than 1 microM) was due to energy-dependent mitochondrial uptake as indicated by its FCCP-sensitivity. However, as the free Ca2+ concentration was lowered from 1 microM, essentially all uptake was due to the ATP-dependent Ca2+ sequestration by the endoplasmic reticulum as indicated by its stimulation by ATP, and its inhibition by sodium vanadate. High concentrations of the calmodulin antagonist trifluoperazine, inhibited both the Ca2+ uptake by the endoplasmic reticulum and by the mitochondria, while calmidazolium released Ca2+ from both compartments. In addition, trifluoperazine and calmidazolium inhibited respiration and collapsed the mitochondrial membrane potential of T. cruzi, thus indicating non-specific effects unrelated to calmodulin.     

Calcium and mitochondria

The literature suggests that the physiological functions for which mitochondria sequester Ca(2+) are (1). to stimulate and control the rate of oxidative phosphorylation, (2). to induce the mitochondrial permeability transition (MPT) and perhaps apoptotic cell death, and (3). to modify the shape of cytosolic Ca(2+) pulses or transients. There is strong evidence that intramitochondrial Ca(2+) controls both the rate of ATP production by oxidative phosphorylation and induction of the MPT. Since the results of these processes are so divergent, the signals inducing them must not be ambiguous. Furthermore, as pointed out by Balaban [J. Mol. Cell. Cardiol. 34 (2002 ) 11259-11271], for any repetitive physiological process dependent on intramitochondrial free Ca(2+) concentration ([Ca(2+)](m)), a kind of intramitochondrial homeostasis must exist so that Ca(2+) influx during the pulse is matched by Ca(2+) efflux during the period between pulses to avoid either Ca(2+) buildup or depletion. In addition, mitochondrial Ca(2+) transport modifies both spatial and temporal aspects of cytosolic Ca(2+) signaling. Here, we look at the amounts of Ca(2+) necessary to mediate the functions of mitochondrial Ca(2+) transport and at the mechanisms of transport themselves in order to set up a hypothesis about how the mechanisms carry out their roles. The emphasis here is on isolated mitochondria and on general mitochondrial properties in order to focus on how mitochondria alone may function to fulfill their physiological roles even though the interactions of mitochondria with other organelles, particularly with endoplasmic and sarcoplasmic reticulum [Sci. STKE re1 (2004) 1-9], may also influence this story.     

Mitochondria buffer NCX-mediated Ca2+-entry and limit its diffusion into vascular smooth muscle cells

The reverse-mode of the Na(+)/Ca(2+)-exchanger (NCX) mediates Ca(2+)-entry in agonist-stimulated vascular smooth muscle (VSM) and plays a central role in salt-sensitive hypertension. We investigated buffering of Ca(2+)-entry by peripheral mitochondria upon NCX reversal in rat aortic smooth muscle cells (RASMC). [Ca(2+)] was measured in mitochondria ([Ca(2+)](MT)) and the sub-plasmalemmal space ([Ca(2+)](subPM)) with targeted aequorins and in the bulk cytosol ([Ca(2+)](i)) with fura-2. Substitution of extracellular Na(+) by N-methyl-d-glucamine transiently increased [Ca(2+)](MT) ( approximately 2microM) and [Ca(2+)](subPM) ( approximately 1.3microM), which then decreased to sustained plateaus. In contrast, Na(+)-substitution caused a delayed and tonic increase in [Ca(2+)](i) (<100nM). Inhibition of Ca(2+)-uptake by the sarcoplasmic reticulum (SR) (30microM cyclopiazonic acid) or mitochondria (2microM FCCP or 2microM ruthenium red) enhanced the elevation of [Ca(2+)](subPM). These treatments also abolished the delay in the [Ca(2+)](i) response to 0Na(+) and increased its amplitude. Extracellular ATP (1mM) caused a peak and plateau in [Ca(2+)](i), and only the plateau was inhibited by KB-R7943 (10microM), a selective blocker of reverse-mode NCX. Evidence for ATP-mediated NCX-reversal was also found in changes in [Na(+)](i). Mitochondria normally exhibited a transient elevation of [Ca(2+)] in response to ATP, but inhibiting the mitochondrial NCX with CGP-37157 (10microM) unmasked an agonist-induced increase in mitochondrial Ca(2+)-flux. This flux was blocked by KB-R7943. In summary, mitochondria and the sarcoplasmic reticulum co-operate to buffer changes in [Ca(2+)](i) due to agonist-induced NCX reversal.     

In planta regulation of the Arabidopsis Ca(2+)/H(+) antiporter CAX1

Vacuolar localized Ca(2+)/H(+) exchangers such as Arabidopsis thaliana cation exchanger 1 (CAX1) play important roles in Ca(2+) homeostasis. When expressed in yeast, CAX1 is regulated via an N-terminal autoinhibitory domain. In yeast expression assays, a 36 amino acid N-terminal truncation of CAX1, termed sCAX1, and variants with specific mutations in this N-terminus, show CAX1-mediated Ca(2+)/H(+) antiport activity. Furthermore, transgenic plants expressing sCAX1 display increased Ca(2+) accumulation and heightened activity of vacuolar Ca(2+)/H(+) antiport. Here the properties of N-terminal CAX1 variants in plants and yeast expression systems are compared and contrasted to determine if autoinhibition of CAX1 is occurring in planta. Initially, using ionome analysis, it has been demonstrated that only yeast cells expressing activated CAX1 transporters have altered total calcium content and fluctuations in zinc and nickel. Tobacco plants expressing activated CAX1 variants displayed hypersensitivity to ion imbalances, increased calcium accumulation, heightened concentrations of other mineral nutrients such as potassium, magnesium and manganese, and increased activity of tonoplast-enriched Ca(2+)/H(+) transport. Despite high in planta gene expression, CAX1 and N-terminal variants of CAX1 which were not active in yeast, displayed none of the aforementioned phenotypes. Although several plant transporters appear to contain N-terminal autoinhibitory domains, this work is the first to document clearly N-terminal-dependent regulation of a Ca(2+) transporter in transgenic plants. Engineering the autoinhibitory domain thus provides a strategy to enhance transport function to affect agronomic traits.     

Ca(2+) sequestering in the early-branching amitochondriate protozoan Tritrichomonas foetus: an important role of the Golgi complex and its Ca(2+)-ATPase

Total membrane vesicles isolated from Tritrichomonas foetus showed an ATP-dependent Ca(2+) uptake, which was not sensitive to 10 microM protonophore FCCP but was blocked by orthovanadate, the inhibitor of P-type ATPases (I(50)=130 microM), and by the Ca(2+)/H(+) exchanger, A-23187. The Ca(2+) uptake was prevented also by thapsigargin, an inhibitor of the SERCA Ca(2+)-ATPases. The sensitivity of the Ca(2+) uptake by the protozoan membrane vesicles to thapsigargin was similar to that of Ca(2+)-ATPase from rabbit muscle sarcoplasmic reticulum. Fractionation of the total membrane vesicles in sucrose density gradient revealed a considerable peak of Ca(2+) transport activity that co-migrated with the Golgi marker guanosine diphosphatase (GDPase). Electron microscopy confirmed that membrane fractions of the peak were enriched with the Golgi membranes. The Golgi Ca(2+)-ATPase contributed to the Ca(2+) uptake by all membrane vesicles 80-85%. We conclude that: (i) the Golgi and/or Golgi-like vesicles form the main Ca(2+) store compartment in T. foetus; (ii) Ca(2+) ATPase is responsible for the Ca(2+) sequestering in this protozoan, while Ca(2+)/H(+) antiporter is not involved in the process; (iii) the Golgi pump of this ancient eukaryotic microorganism appears to be similar to the enzymes of the SERCA family by its sensitivity to thapsigargin.     

Mitochondrial modulation of calcium signaling at the initiation of development

Fertilization triggers cytosolic Ca(2+) oscillations that activate mammalian eggs and initiate development. Extensive evidence demonstrates that Ca(2+) is released from endoplasmic reticulum stores; however, less is known about how the increased Ca(2+) is restored to its resting level, forming the Ca(2+) oscillations. We investigated whether mitochondria also play a role in activation-associated Ca(2+) signaling. Mitochondrial dysfunction induced by the mitochondrial uncoupler FCCP or antimycin A disrupted cytosolic Ca(2+) oscillations, resulting in sustained increase in cytosolic Ca(2+), followed by apoptotic cell death. This suggests that functional mitochondria may participate in sequestering the released Ca(2+), contributing to cytosolic Ca(2+) oscillations and preventing cell death. By centrifugation, mouse eggs were stratified and separated into fractions containing both endoplasmic reticulum and mitochondria and fractions containing endoplasmic reticulum with no mitochondria. The former showed Ca(2+) oscillations by activation, whereas the latter exhibited sustained elevation in cytosolic Ca(2+) but no Ca(2+) oscillations, suggesting that mitochondria take up released cytosolic Ca(2+). Further, using Rhod-2 for detection of mitochondrial Ca(2+), we found that mitochondria exhibited Ca(2+) oscillations, the frequency of which was not different from that of cytosolic Ca(2+) oscillations, indicating that mitochondria are involved in Ca(2+) signaling during egg activation. Therefore, we propose that mitochondria play a crucial role in Ca(2+) signaling that mediates egg activation and development, and apoptotic cell death.     

Biochemical approaches to the study of cytosolic calcium regulation in nerve endings

The nerve ending cytosol is bounded by the plasma membrane, the mitochondrial inner membrane and the endoplasmic reticulum membrane, transport across each of which is capable, in theory, of regulating the cytosolic free Ca2+ concentration. By parallel monitoring of mitochondrial and plasma membrane potentials, ATP levels, Na+ gradients and intrasynaptosomal Ca2+ distribution in preparations of isolated synaptosomes, we conclude the following: (a) mitochondria in situ represent a major Ca2+ pool, regulating the upper steady-state limit of the cytosolic free Ca2+ concentration by sequestering Ca2+ reversibly; (b) this limit is responsive to the cytosolic Na+ concentration, but is below the concentration required for significant exocytosis; (c) plasma membrane Ca2+ transport can be resolved into a constant slow influx, a voltage-dependent and verapamil-sensitive influx and an ATP-dependent efflux, while Ca2+ efflux driven by the sodium electrochemical potential cannot be detected; (d) Ca2+ regulation by intrasynaptosomal endoplasmic reticulum appears to be of minor significance in the present preparation.     

Neuronal-specific endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration in mixed primary hippocampal culture homogenates

Endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration is crucial for maintenance of neuronal Ca(2+) homeostasis. The use of cell culture in conjunction with modern Ca(2+) imaging techniques has been invaluable in elucidating these mechanisms. While imaging protocols evaluate endoplasmic reticulum Ca(2+) loads, measurement of Mg(2+)/Ca(2+) ATPase activity is indirect, comparing cytosolic Ca(2+) levels in the presence or absence of the Mg(2+)/Ca(2+) ATPase inhibitor thapsigargin. Direct measurement of Mg(2+)/Ca(2+) ATPase by isolation of microsomes is impossible due to the minuscule amounts of protein yielded from cultures used for imaging. In the current study, endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration was measured in mixed homogenates of neurons and glia from primary hippocampal cultures. It was demonstrated that Ca(2+) uptake was mediated by the endoplasmic reticulum Mg(2+)/Ca(2+) ATPase due to its dependence on ATP and Mg(2+), enhancement by oxalate, and inhibition by thapsigargin. It was also shown that neuronal Ca(2+) uptake, mediated by the type 2 sarco(endo)plasmic reticulum Ca(2+) ATPase isoform, could be distinguished from glial Ca(2+) uptake in homogenates composed of neurons and glia. Finally, it was revealed that Ca(2+) uptake was sensitive to incubation on ice, extremely labile in the absence of protease inhibitors, and significantly more stable under storage conditions at -80 degrees C.     

Mitochondria recycle Ca(2+) to the endoplasmic reticulum and prevent the depletion of neighboring endoplasmic reticulum regions

To study Ca(2+) fluxes between mitochondria and the endoplasmic reticulum (ER), we used "cameleon" indicators targeted to the cytosol, the ER lumen, and the mitochondrial matrix. High affinity mitochondrial probes saturated in approximately 20% of mitochondria during histamine stimulation of HeLa cells, whereas a low affinity probe reported averaged peak values of 106 +/- 5 microm, indicating that Ca(2+) transients reach high levels in a fraction of mitochondria. In concurrent ER measurements, [Ca(2+)](ER) averaged 371 +/- 21 microm at rest and decreased to 133 +/- 14 microm and 59 +/- 5 microm upon stimulation with histamine and thapsigargin, respectively, indicating that substantial ER refilling occur during agonist stimulation. A larger ER depletion was observed when mitochondrial Ca(2+) uptake was prevented by oligomycin and rotenone or when Ca(2+) efflux from mitochondria was blocked by CGP 37157, indicating that some of the Ca(2+) taken up by mitochondria is re-used for ER refilling. Accordingly, ER regions close to mitochondria released less Ca(2+) than ER regions lacking mitochondria. The ER heterogeneity was abolished by thapsigargin, oligomycin/rotenone, or CGP 37157, indicating that mitochondrial Ca(2+) uptake locally modulate ER refilling. These observations indicate that some mitochondria are very close to the sites of Ca(2+) release and recycle a substantial portion of the captured Ca(2+) back to vicinal ER domains. The distance between the two organelles thus determines both the amplitude of mitochondrial Ca(2+) signals and the filling state of neighboring ER regions.