The Role of Acidocalcisomes in Parasitic Protists1

ABSTRACT. Acidocalcisomes are acidic organelles with a high concentration of phosphorus present as pyrophosphate (PP_i) and polyphosphate (poly P) complexed with calcium and other cations. The acidocalcisome membrane contains a number of pumps (Ca²⁺‐ATPase, V‐H⁺‐ATPase, H⁺‐PPase), exchangers (Na⁺/H⁺, Ca²⁺/H⁺), and channels (aquaporins), while its matrix contains enzymes related to PP_i and poly P metabolism. Acidocalcisomes have been observed in pathogenic, as well as non‐pathogenic prokaryotes and eukaryotes, e.g. Chlamydomonas reinhardtii, and Dictyostelium discoideum. Some of the potential functions of the acidocalcisome are the storage of cations and phosphorus, the participation of phosphorus in PP_i and poly P metabolism, calcium homeostasis, maintenance of intracellular pH homeostasis, and osmoregulation. In addition, acidocalcisomes resemble lysosome‐related organelles (LRO) from mammalian cells in many of their properties. For example, we found that platelet dense granules, which are LROs, are very similar to acidocalcisomes. They share a similar size, acidic properties, and both contain PP_i, poly P, and calcium. Recent work that indicates that they also share the system for targeting of their membrane proteins through adaptor protein 3 reinforces this concept. The fact that acidocalcisomes interact with other organelles in parasitic protists, e.g. the contractile vacuole in Trypanosoma cruzi, and other vacuoles observed in Toxoplasma gondii, suggests that these cellular compartments may be associated with the endosomal/lysosomal pathway.     

Evolution of acidocalcisomes and their role in polyphosphate storage and osmoregulation in eukaryotic microbes

Acidocalcisomes are acidic electron-dense organelles, rich in polyphosphate (poly P) complexed with calcium and other cations. While its matrix contains enzymes related to poly P metabolism, the membrane of the acidocalcisomes has a number of pumps (Ca²⁺-ATPase, V-H⁺-ATPase, H⁺-PPase), exchangers (Na⁺/H⁺, Ca²⁺/H⁺), and at least one channel (aquaporin). Acidocalcisomes are present in both prokaryotes and eukaryotes and are an important storage of cations and phosphorus. They also play an important role in osmoregulation and interact with the contractile vacuole complex in a number of eukaryotic microbes. Acidocalcisomes resemble lysosome-related organelles (LRO) from mammalian cells in many of their properties. They share similar morphological characteristics, acidic properties, phosphorus contents and a system for targeting of their membrane proteins through adaptor complex-3 (AP-3). Storage of phosphate and cations may represent the ancestral physiological function of acidocalcisomes, with cation and pH homeostasis and osmoregulatory functions derived following the divergence of prokaryotes and eukaryotes.     

Calcium uptake and proton transport by acidocalcisomes of Toxoplasma gondii

Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to humans. They possess an acidic matrix that contains several cations bound to phosphates, which are mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. Calcium uptake occurs through a Ca(2+)/H(+) countertransporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. We report the purification and characterization of an acidocalcisome fraction from T. gondii tachyzoites after subcellular fractionation and further discontinuous iodixanol gradient purification. Proton and calcium transport activities in the fraction were characterized by fluorescence microscopy and spectrophotometric methods using acridine orange and arsenazo III, respectively. This work will facilitate the understanding of the function of acidocalcisomes in Apicomplexan parasites, as we can now isolate highly purified fractions that could be used for proteomic analysis to find proteins that may clarify the biogenesis of these organelles.     

Proton-pyrophosphatase and polyphosphate in acidocalcisome-like vesicles from oocytes and eggs of Periplaneta americana

Acidocalcisomes are acidic organelles containing large amounts of polyphosphate (poly P), a number of cations, and a variety of cation pumps in their limiting membrane. The vacuolar proton-pyrophosphatase (V-H⁺-PPase), a unique electrogenic proton-pump that couples pyrophosphate (PPi) hydrolysis to the active transport of protons across membranes, is commonly present in membranes of acidocalcisomes. In the course of insect oogenesis, a large amount of yolk protein is incorporated by the oocytes and stored in organelles called yolk granules (YGs). During embryogenesis, the content of these granules is degraded by acid hydrolases. These enzymes are activated by the acidification of the YG by a mechanism that is mediated by proton-pumps present in their membranes. In this work, we describe an H⁺-PPase activity in membrane fractions of oocytes and eggs of the domestic cockroach Periplaneta americana. The enzyme activity was optimum at pH around 7.0, and was dependent on Mg²⁺ and inhibited by NaF, as well as by IDP and Ca²⁺. Immunolocalization of the yolk preparation using antibodies against a conserved sequence of V-H⁺-PPases showed labeling of small vesicles, which also showed the presence of high concentrations of phosphorus, calcium and other elements, as revealed by electron probe X-ray microanalysis. In addition, poly P content was detected in ovaries and eggs and localized inside the yolk granules and the small vesicles. Altogether, our results provide evidence that numerous small vesicles of the eggs of P. americana present acidocalcisome-like characteristics. In addition, the possible role of these organelles during embryogenesis of this insect is discussed.     

Proteomic Analysis of the Acidocalcisome,an Organelle Conserved from Bacteria to Human Cells

Acidocalcisomes are acidic organelles present in a diverse range of organisms from bacteria to human cells. In this study acidocalcisomes were purified from the model organism Trypanosoma brucei, and their protein composition was determined by mass spectrometry. The results, along with those that we previously reported, show that acidocalcisomes are rich in pumps and transporters, involved in phosphate and cation homeostasis, and calcium signaling. We validated the acidocalcisome localization of seven new, putative, acidocalcisome proteins (phosphate transporter, vacuolar H⁺-ATPase subunits a and d, vacuolar iron transporter, zinc transporter, polyamine transporter, and acid phosphatase), confirmed the presence of six previously characterized acidocalcisome proteins, and validated the localization of five novel proteins to different subcellular compartments by expressing them fused to epitope tags in their endogenous loci or by immunofluorescence microscopy with specific antibodies. Knockdown of several newly identified acidocalcisome proteins by RNA interference (RNAi) revealed that they are essential for the survival of the parasites. These results provide a comprehensive insight into the unique composition of acidocalcisomes of T. brucei, an important eukaryotic pathogen, and direct evidence that acidocalcisomes are especially adapted for the accumulation of polyphosphate.     

Acidocalcisomes - conserved from bacteria to man

Recent work has shown that acidocalcisomes, which are electron-dense acidic organelles rich in calcium and polyphosphate, are the only organelles that have been conserved during evolution from prokaryotes to eukaryotes. Acidocalcisomes were first described in trypanosomatids and have been characterized in most detail in these species. Acidocalcisomes have been linked with several functions, including storage of cations and phosphorus, polyphosphate metabolism, calcium homeostasis, maintenance of intracellular pH homeostasis and osmoregulation. Here, we review acidocalcisome ultrastructure, composition and function in different trypanosomatids and other organisms.     

Effects of W-7, W-5, Verapamil and Diltiazem on vacuolar proton transport. Comparison of vacuolar H+-ATPase and H+-PPase from roots of Zea mays

The vacuolar membrane of plant cells is characterized by two proton pumps: the vacuolar H⁺-ATPase (V-ATPase; EC 3.6.1.3) and the vacuolar H⁺-PPase (V-PPase; EC 3.6.1.1). Recently, Du Pont and Morrissey reported that Ca²⁺ stimulates hydrolytic activity of purified V-ATPase (Arch. Biochim. Biophys., 1992. 294: 341–346). Since this effect may be due to degradation during purification further investigation of Ca²⁺ regulation of native V-ATPase was done. However, native tonoplast membranes contain a Ca²⁺/H⁺ antiport activity, which interferes with effects of calcium ions on proton transport activity of vacuolar ATPase. Therefore, the effects of anti-calmodulin drugs (W-7, W-5, calmidazolium), and calcium channel antagonists (Verapamil, Diltiazem) on proton transport activities of the vacuolar-type H⁺-ATPase and H⁺-PPase in tonoplast enriched membrane vesicle preparations from roots of Zea mays L. were studied. The concentrations for half maximal inhibition of vacuolar H⁺-ATPase (H⁺-PPase) were: 71 (191) μM W-7, 470 (> 800) μM W-5, 26 (24) μM calmidazolium (= compound R 24571). 398 (700) μM Verapamil, and 500 (1 330) μM Diltiazem. Estimation of Hill coefficients (n_H) for the inhibition by Verapamil showed a further difference between the two vacuolar proton pumps (H⁺-ATPase, n_H= 2.02; H⁺-PPase, n_n= 0.96). The data indicate that the vacuolar H⁺-ATPase itself is affected by these chemicals. It is suggested that some biological activities of W-7, W-5, Verapamil, and Diltiazem are due to their effects on proton translocation by the vacuolar-type H⁺-ATPase.     

Characterization of the tonoplast proton pumps in Cucumis sativus L. root cells

Large-scale preparation of highly purified tonoplast from cucumber (Cucumis sativus L.) roots was obtained after centrifugation of microsome pellet (10,000 – 80,000 g) on discontinuous sucrose density gradient (20, 28, 32 and 42 %). Lack of PEP carboxylase (cytosol marker) and cytochrome c oxidase (mitochondrial marker) together with a slight activity of VO₄-ATPase (plasma membrane marker) and NADH-cytochrome c reductase (ER marker) in tonoplast preparation confirmed its high purity. Using latency of nitrate-inhibited ATPase and H⁺ pumping as criteria it was established that the majority of tonoplast vesicles were sealed and oriented right(cytoplasmic)-side-out. Strong acidification of the interior of vesicles observed at the presence of both, ATP and PP_i, confirmed that obtained tonoplast contains two classes of proton pumps: V-ATPase and H⁺PP_iase. To examine and characterise of proton-transport systems in tonoplast, the effect of various inhibitors on H⁺ pumping and hydrolytic activities of ATPase and PP_iase were measured. ATP-dependent activities (H⁺ flux and ATP hydrolysis) were specifically decreased by nitrate and bafilomycin A₁, whereas the PP_iase activities were reduced in the presence of fluoride and Na⁺ ions. Both enzymes showed a similar sensitivity to DCCD and DES. The results of experiments with KCl and NaCl suggested that the vacuolar ATPase was stimulated by Cl⁻, whereas the vacuolar Pp_iase requires K⁺ ions for its activity.     

Calcium‐ and polyphosphate‐containing acidocalcisomes in chicken egg yolk

Background information. Poly P (inorganic polyphosphate) is a polymer formed by P_i residues linked by high‐energy phosphoanhydride bonds. The presence of poly P in bacteria, fungi, algae and protists has been widely recognized, but the distribution of poly P in more complex eukaryotes has been poorly studied. Poly P accumulates, together with calcium, in acidic vesicles or acidocalcisomes in a number of organisms and possesses a diverse array of functions, including roles in stress response, blood clotting, inflammation, calcification, cell proliferation and apoptosis. Results. We report here that a considerable amount of phosphorus in the yolk of chicken eggs is in the form of poly P. DAPI (4′,6‐diamidino‐2‐phenylindole) staining showed that poly P is localized mainly in electron‐dense vesicles located inside larger vacuoles (compound organelles) that are randomly distributed in the yolk. These internal vesicles were shown to contain calcium, potassium, sodium, magnesium, phosphorus, chlorine, iron and zinc, as detected by X‐ray microanalysis and elemental mapping. These vesicles stain with the acidophilic dye Acridine Orange. The presence of poly P in organellar fractions of the egg yolk was evident in agarose gels stained with Toluidine Blue and DAPI. Of the total phosphate (P_i) of yolk organelles, 16% is present in the form of poly P. Total poly P content was not altered during the first 4 days of embryogenesis, but poly P chain length decreased after 1 day of development. Conclusions. The results of the present study identify a novel organelle in chicken egg yolk comprising acidic vesicles with a morphology, physiology and composition similar to those of acidocalcisomes, within larger acidic vacuoles. The elemental composition of these acidocalcisomes is proportionally similar to the elemental composition of the yolk, suggesting that most of these elements are located in these organelles, which might be an important storage compartment in eggs.     

Acidocalcisomes in Apicomplexan parasites

Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to man. They posses an acidic matrix that contains several cations bound to phosphates, mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. The calcium uptake occurs through a Ca2+/H+ counter transporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. In this paper, we review the structural, biochemical and physiological aspects of acidocalcisomes in Apicomplexan parasites and discuss their functional roles in the maintenance of intracellular ion homeostasis.     

Ablation of a small transmembrane protein of Trypanosoma brucei (TbVTC1) involved in the synthesis of polyphosphate alters acidocalcisome biogenesis and function, and leads to a cytokinesis defect

Inorganic poly P (polyphosphate) is an abundant component of acidocalcisomes of Trypanosoma brucei. In the present study we report the presence of a protein homologous with the yeast Vtc1p (vacuolar transporter chaperone 1) in T. brucei that is essential for poly P synthesis, acidocalcisome biogenesis and cytokinesis. Localization studies in a cell line expressing a TbVTC1 fused to GFP (green fluorescent protein) revealed its co-localization with the V-H+-PPase (vacuolar H+-pyrophosphatase), a marker for acidocalcisomes. Western blot analysis of acidocalcisome fractions and immunogold electron microscopy using polyclonal antibodies against a fragment of TbVTC1 confirmed the acidocalcisome localization. Ablation of TbVTC1 expression by RNA interference caused an abnormal morphology of acidocalcisomes, indicating that their biogenesis was disturbed, with a decreased pyrophosphate-driven H+ uptake and Ca2+ content, a significant decrease in the amount of poly P and a deficient response to hyposmotic stress. Ablation of TbVTC1 expression for longer periods produced marked gross morphological alterations compatible with a defect in cytokinesis, followed by cell death. Overexpression of the TbVTC1 gene caused mild alterations in growth rate, but had no perceptible effect on acidocalcisome morphology. We propose that the PP(i)-driven H+ pumping deficiency induced by ablation of TbVTC1 leads to alterations in the protonmotive force of acidocalcisomes, which results in deficient fusion or budding of the organelles, decreased H+ and Ca2+ content, and decreased synthesis of poly P. A decrease in the poly P content would lead to osmotic sensitivity and defects in cytokinesis.     

ATP synthesis catalyzed by a V-ATPase: an alternative pathway for energy conservation operating in plant vacuoles?

The electrochemical H⁺ gradient generated in tonoplast vesicles isolated from maize seeds was found to be able to drive the reversal of the catalytic cycle of both vacuolar H⁺-pumps (Façanha and de Meis, 1998). Here we describe the reversibility of the vacuolar V-type H⁺-ATPase (V-ATPase) even in the absence of the H⁺ gradient in a water-Me₂SO co-solvent mixture, resulting in net synthesis of [γ-³²P]ATP from [³²P]P_i and ADP. The water-Me₂SO (5 to 20 %) media promoted inhibition of both PP_i hydrolysis and synthesis reactions whereas it slightly affected the ATP hydrolysis and clearly stimulated the ATP synthesis, which was unaffected by uncoupling agents (FCCP, Triton X-100 or NH₄⁺). This effect of Me₂SO on the ATP⇔³²P exchange reaction seems to be related to a decrease of the apparent K_m of the V-ATPase for P_i. The results are in accordance to the concept that the energetics of ATP synthesis catalysis depends on the solvation energies interacting in the enzyme microenvironment. A possible physiological significance of this phenomenon for the metabolism of desiccation-tolerant plant cells is discussed.Key words: bind energy, proton pumps, proton gradient, DMSO, corn seeds, V₁V₀-ATPase, membrane bound H⁺-pyrophosphatase     

PPiase-Activated ATP-Dependent H+ Transport at the Tonoplast of Mesophyll Cells of the CAM Plant Kalanchoë daigremontiana*

The co-ordinated action of the two proton-transporting enzymes at the tonoplast of the CAM plants. daigremontiana, viz. the ATPase and the PP_iase, was studied by measuring fluorescent dye quenching. The initial rates of ATP and PP_i-dependent H⁺ transport into tonoplast vesicles were additive, i.e. the sum of the rates obtained with each substrate alone was in the range obtained with both substrates added together at the same time. Conversely, the activities of the two H⁺ pumps were non-additive in establishing the steady-state level, indicating that the final steady state was under thermodynamic control of a maximal attainable proton gradient. The initial rates of ATP-dependent H⁺ transport were stimulated enormously if ATP was added a few minutes after pre-energization of the vesicles with PP_i. This stimulation was observed only when the PP_iase was active. A similar effect was not found for PP_i-dependent H⁺ transport after pre-energization with ATP. Hence, a PP_iase-activated ATP-dependent H⁺ transport can be distinguished from the basic ATP- and the basic PP_i-dependent H⁺ transport. In parallel a PP_i-dependent stimulation of ATP hydrolysis in the absence of ionophores was measured, which can only be attributed to the activity of the PP_iase. PP_iase-activated ATP-dependent H⁺ transport depends on the presence of permeant anions. It shows properties of both H⁺ transport activities, i.e. the chloride and malate stimulation and the DCCD inhibition of the ATP-dependent H⁺ transport activity, the nitrate stimulation and the KF inhibition of the PP_i-dependent H⁺ transport activity. Only MgPP_i and MgATP were effective as the respective substrates. The PP_iase-activated ATP-dependent H⁺ transport had a half life of about 5–9 minutes. It is concluded that the PP_iase may play an important role in kinetic regulation of the ATPase, and implications for CAM metabolism are discussed.     

The polyphosphate bodies of Chlamydomonas reinhardtii possess a proton-pumping pyrophosphatase and are similar to acidocalcisomes

Acidocalcisomes are acidic calcium storage compartments described initially in trypanosomatid and apicomplexan parasites. In this work, we describe organelles with properties similar to acidocalcisomes in the green alga Chlamydomonas reinhardtii. Nigericin and NH(4)Cl released (45)Ca(2+) from preloaded permeabilized cells, suggesting the incorporation of a significant amount of this cation into an acidic compartment. X-ray microanalysis of the electron-dense vacuoles or polyphosphate bodies of C. reinhardtii showed large amounts of phosphorus, magnesium, calcium, and zinc. Immunofluorescence microscopy, using antisera raised against a peptide sequence of the vacuolar type proton pyrophosphatase (H(+)-PPase) of Arabidopsis thaliana which is conserved in the C. reinhardtii enzyme, indicated localization in the plasma membrane, in intracellular vacuoles, and the contractile vacuole where it colocalized with the vacuolar proton ATPase (V-H(+)-ATPase). Purification of the electron-dense vacuoles using iodixanol density gradients indicated a preferential localization of the H(+)-PPase and the V-H(+)-ATPase activities in addition to high concentrations of PP(i) and short and long chain polyphosphate, but lack of markers for mitochondria and chloroplasts. In isolated electron-dense vacuoles, PP(i)-driven proton translocation was stimulated by potassium ions and inhibited by the PP(i) analog aminomethylenediphosphonate. Potassium fluoride, imidodiphosphate, N,N'-dicyclohexylcarbodiimide, and N-ethylmaleimide also inhibited PP(i) hydrolysis in the isolated organelles in a dose-dependent manner. These results indicate that the electron-dense vacuoles of C. reinhardtii are very similar to acidocalcisomes with regard to their chemical composition and the presence of proton pumps. Polyphosphate was also localized to the contractile vacuole by 4',6-diamidino-2-phenylindole staining, suggesting, with the immunochemical data, a link between these organelles and the acidocalcisomes.     

Extracellular NAD induces a rise in [Ca]i in activated human monocytes via engagement of P2Y1 and P2Y11 receptors

Extracellular nicotinamide adenine dinucleotide (NAD⁺) is known to increase the intracellular calcium concentration [Ca²⁺]_i in different cell types and by various mechanisms. Here we show that NAD⁺ triggers a transient rise in [Ca²⁺]_i in human monocytes activated with lipopolysaccharide (LPS), which is caused by a release of Ca²⁺ from IP₃-responsive intracellular stores and an influx of extracellular Ca²⁺. By the use of P2 receptor-selective agonists and antagonists we demonstrate that P2 receptors play a role in the NAD⁺-induced calcium response in activated monocytes. Of the two subclasses of P2 receptors (P2X and P2Y) the P2Y receptors were considered the most likely candidates, since they share calcium signaling properties with NAD⁺. The identification of P2Y₁ and P2Y₁₁ as receptor subtypes responsible for the NAD⁺-triggered increase in [Ca²⁺]_i was supported by several lines of evidence. First, specific P2Y₁ and P2Y₁₁ receptor antagonists inhibited the NAD⁺-induced increase in [Ca²⁺]_i. Second, NAD⁺ was shown to potently induce calcium signals in cells transfected with either subtype, whereas untransfected cells were unresponsive. Third, NAD⁺ caused an increase in [cAMP]_i, prevented by the P2Y₁₁ receptor-specific antagonist NF157.     

Release of mouse eggs from metaphase arrest by protein synthesis inhibition in the absence of a calcium signal or microtubule assembly

Mouse egg activation, which includes release from meiotic metaphase II arrest, results from fertilization-induced increase in intracellular calcium concentration ([Ca²⁺]_i). However, during egg activation caused by exposure to the protein synthesis inhibitor, cycloheximide, [Ca²⁺]_i did not change. Although eggs fertilized in the presence of microtubule inhibitors remain arrested at metaphase, eggs treated for 32 hr with cycloheximide and the microtubule inhibitor, colcemid, formed nuclei. In untreated eggs aged in culture for 24 hr, the microtubule spindles became deformed. These eggs formed nuclei after exposure to cycloheximide, but not the calcium ionophore A23187. Our results indicate that eggs in which protein synthesis is inhibited are released from metaphase without an increase in [Ca²⁺]_i, and despite disruption of the Spindle. © 1995 Wiley-Liss, Inc.     

The extracellular calcium‐sensing receptor promotes porcine egg activation via calcium/calmodulin‐dependent protein kinase II

Extracellular calcium is required for intracellular Ca²⁺ oscillations needed for egg activation, but the regulatory mechanism is still poorly understood. The present study was designed to demonstrate the function of calcium‐sensing receptor (CASR), which could recognize extracellular calcium as first messenger, during porcine egg activation. CASR expression was markedly upregulated following egg activation. Functionally, the addition of CASR agonist NPS R‐568 significantly enhanced pronuclear formation rate, while supplementation of CASR antagonist NPS2390 compromised egg activation. There was no change in NPS R‐568 group compared with control group when the egg activation was performed without extracellular calcium addition. The addition of NPS2390 precluded the activation‐dependent [Ca²⁺]_i rise. When egg activation was conducted in intracellular Ca²⁺ chelator BAPTA‐AM and NPS R‐568 containing medium, CASR function was abolished. Meanwhile, CASR activation increased the level of the [Ca²⁺]_i effector p‐CAMKII, and the presence of KN‐93, an inhibitor of CAMKII, significantly reduced the CASR‐mediated increasement of pronuclear formation rate. Furthermore, the increase of CASR expression following activation was reversed by inhibiting CAMKII activity, supporting a positive feedback loop between CAMKII and CASR. Altogether, these findings provide a new pathway of egg activation about CASR, as the extracellular Ca²⁺ effector, promotes egg activation via its downstream effector and upstream regulator CAMKII.     

The differentiation inducer,dimethyl sulfoxide,transiently increases the intracellular calcium ion concentration in various cell types

Dimethyl sulfoxide (DMSO) initiates a coordinated differentiation program in various cell types but the mechanism(s) by which DMSO does this is not understood. In this study, the effect of DMSO on intracellular calcium ion concentration ([Ca²⁺]_i) was determined in primary cultures of chicken ovarian granulosa cells from the two largest preovulatory follicles of laying hens, and in three cell lines: undifferentiated P19 embryonal carcinoma cells, 3T3-L1 fibroblasts, and Friend murine erythroleukemia (MEL) cells. [Ca²⁺]_i was measured in cells loaded with the Ca²⁺ -specific fluoroprobe Fura-2. There was an immediate (i.e., within 5 sec), transient, two to sixfold increase in [Ca²⁺]_i after exposing all cell types to 1% DMSO. DMSO was effective between 0.2 and 1%. The prompt DMSO-induced [Ca²⁺]_i spike in all of the cell types was not prevented by incubating the cells in Ca²⁺ -free medium containing 2 mM EGTA or by pretreating them with the Ca²⁺-channel blockers methoxyverapamil (D600; 100 μM), nifedipine (20 μM), or cobalt (5 mM). However, when granulosa cells, 3T3-L1 cells, or MEL cells were pretreated with lanthanum (La³⁺; 1 mM), which blocks both Ca²⁺ channels and membrane Ca²⁺ pumps, there was a sustained increase in [Ca²⁺]_i in response to 1% DMSO. By contrast, pretreating P19 cells with La³⁺ (1 mM) did not prolong the DMSO-triggered [Ca²⁺]_i transient. In all cases, the DMSO-induced [Ca²⁺]_i surge was unaffected by pretreating the cells with the inhibitors of inositol phospholipid hydrolysis, neomycin (1.5 mM) or U-73, 122 (2.5 μM). These results suggest that DMSO almost instantaneously triggers the release of Ca²⁺ from intracellular stores through a common mechanism in cells in primary cultures and in cells of a variety of established lines, but, this release is not mediated through phosphoinositide breakdown. This large, DMSO-induced Ca²⁺ spike may play a role in the induction of cell differentiation by DMSO. © 1993 Wiley-Liss, Inc.     

Dual Effect of Phosphate Transport on Mitochondrial Ca2+ Dynamics

The large inner membrane electrochemical driving force and restricted volume of the matrix confer unique constraints on mitochondrial ion transport. Cation uptake along with anion and water movement induces swelling if not compensated by other processes. For mitochondrial Ca²⁺ uptake, these include activation of countertransporters (Na⁺/Ca²⁺ exchanger and Na⁺/H⁺ exchanger) coupled to the proton gradient, ultimately maintained by the proton pumps of the respiratory chain, and Ca²⁺ binding to matrix buffers. Inorganic phosphate (P_i) is known to affect both the Ca²⁺ uptake rate and the buffering reaction, but the role of anion transport in determining mitochondrial Ca²⁺ dynamics is poorly understood. Here we simultaneously monitor extra- and intra-mitochondrial Ca²⁺ and mitochondrial membrane potential (ΔΨ_m) to examine the effects of anion transport on mitochondrial Ca²⁺ flux and buffering in P_i-depleted guinea pig cardiac mitochondria. Mitochondrial Ca²⁺ uptake proceeded slowly in the absence of P_i but matrix free Ca²⁺ ([Ca²⁺]_mito) still rose to ∼50 μm. P_i (0.001–1 mm) accelerated Ca²⁺ uptake but decreased [Ca²⁺]_mito by almost 50% while restoring ΔΨ_m. P_i-dependent effects on Ca²⁺ were blocked by inhibiting the phosphate carrier. Mitochondrial Ca²⁺ uptake rate was also increased by vanadate (V_i), acetate, ATP, or a non-hydrolyzable ATP analog (AMP-PNP), with differential effects on matrix Ca²⁺ buffering and ΔΨ_m recovery. Interestingly, ATP or AMP-PNP prevented the effects of P_i on Ca²⁺ uptake. The results show that anion transport imposes an upper limit on mitochondrial Ca²⁺ uptake and modifies the [Ca²⁺]_mito response in a complex manner.     

Oxidative stress disruption of receptor-mediated calcium signaling mechanisms

Background

Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins. In this study, oxidative stress was produced by exposure to tert-butyl hydroperoxide (tBHP); cell viability was assessed using a dye reduction assay; receptor binding was characterized using [³H]N-methylscopolamine ([³H]MS); and cytosolic and luminal endoplasmic reticulum (ER) calcium concentrations ([Ca²⁺]_i and [Ca²⁺]_L, respectively) were measured by fluorescent imaging.

Results

Activation of M3 muscarinic receptors induced a biphasic increase in [Ca²⁺]_i: an initial, inositol trisphosphate (IP3)-mediated release of Ca²⁺ from endoplasmic reticulum (ER) stores followed by a sustained phase of Ca²⁺ entry (i.e., store-operated calcium entry; SOCE). Under non-cytotoxic conditions, tBHP increased resting [Ca²⁺]_i; a 90 minute exposure to tBHP (0.5-10 mM ) increased [Ca²⁺]_i from 26 to up to 127 nM and decreased [Ca²⁺]_L by 55%. The initial response to 10 μM carbamylcholine was depressed by tBHP in the absence, but not the presence, of extracellular calcium. SOCE, however, was depressed in both the presence and absence of extracellular calcium. Acute exposure to tBHP did not block calcium influx through open SOCE channels. Activation of SOCE following thapsigargin-induced depletion of ER calcium was depressed by tBHP exposure. In calcium-free media, tBHP depressed both SOCE and the extent of thapsigargin-induced release of Ca²⁺ from the ER. M3 receptor binding parameters (ligand affinity, guanine nucleotide sensitivity, allosteric modulation) were not affected by exposure to tBHP.

Conclusions

Oxidative stress induced by tBHP affected several aspects of M3 receptor signaling pathway in CHO cells, including resting [Ca²⁺]_i, [Ca²⁺]_L, IP3 receptor mediated release of calcium from the ER, and calcium entry through the SOCE. tBHP had little effect on M3 receptor binding or G protein coupling. Thus, oxidative stress affects multiple aspects of calcium homeostasis and calcium dependent signaling.