Cyclosporin A Rescues Thymocytes from Apoptosis Induced by Very Low Concentrations of Thapsigargin: Effects on Mitochondrial Function

Raising intracellular calcium levels can induce apoptosis or programmed cell death in many cells. While early rises in intracellular calcium are not universally associated with apoptotic cell death, calcium clearly plays a key role in many of the biochemical events which occur during apoptosis. In this paper we have determined intracellular calcium rises induced by 2, 10, and 100 nMthapsigargin in mouse thymocytes. These concentrations cause increases in cytosolic calcium of 100–250, 400–600, and >1000 nM,respectively. These rises are sustained for at least 85 min and the ratio between the maximum rise caused by 10 nMcompared to 2 nMthapsigargin is 2.1 ± 0.4 (n= 6). Both 2 and 10 nMthapsigargin cause apoptosis at 24 h as shown by DNA fragmentation and morphology when examined by electron microscopy. Cyclosporin A (CsA) inhibits apoptosis caused by 2 nMthapsigargin but not that caused by 10 nMthapsigargin. Electron microscopy of thymocytes treated with 2 nMthapsigargin at 24 h shows intact mitochondria although with altered morphology. There is no loss of ATP or decrease in the ATP/ADP ratio in these cells over 12 h. Mitochondria in cells treated with 10 nMthapsigargin, however, are swollen by 6 h and many are lost by 24 h. These cells show greatly diminished ATP content by 12 h and a decrease in ATP/ADP ratio. Examination of the effects of PMA, an activator of the plasma membrane calcium ATPase pump, on cells treated with 10 nMthapsigargin suggests that two pools of calcium may be responsible for the differential effects of the two calcium levels in the cells. Probing of the mitochondrial membrane potential (MMP) by rhodamine 123 staining of live cells shows that the collapse of the MMP caused by 10 nMthapsigargin is unaffected by CsA. The MMP is also reduced in cells treated with 2 nMthapsigargin but this is restored by CsA. Cells are also rescued from apoptosis caused by 2 nMthapsigargin by incubation with FK506. This immunosuppressive agent has no effect on the membrane permeability transition induced in isolated mitochondria. These results suggest that very low rises in intracellular calcium in thymocytes cause activation-induced cell death inhibited by CsA and FK506 and are without effect on ATP levels and therefore do not involve irreversible mitochondrial damage. Exceeding these calcium levels by only twofold results in apoptosis accompanied by reduced ATP levels and mitochondrial damage, although apoptotic cell death in this instance is unaffected by the classic inhibitor of mitochondrial permeability transition, CsA.     

Arachidonate mobilization is coupled to depletion of intracellular calcium stores and influx of extracellular calcium in differentiated U937 cells

We have previously reported that dimethylsulfoxide-differentiation of U937 cells induced significant A23187-stimulatable arachidonate mobilization, consistent with characteristics of cytosolic phospholipase A₂ (Rzigalinski, B.A. and Rosenthal, M.D. (1994) Biochim. Biophys. Acta 1223, 219–225). The present report demonstrates that differentiated cells attained higher elevations of intracellular free calcium in response to A23187 and thapsigargin, consistent with enhancement of the capacitative calcium influx pathway. Differentiation induced a significant increase in the size of the intracellular calcium stores, as well as in the capacity for store-activated calcium influx. Alterations in the capacitative calcium influx pathway were coupled to differentiation-induced activation of cPLA₂ and mobilization of arachidonate in response to thapsigargin and fMLP stimulation. Although cPLA₂ activity is often associated with influx of extracellular calcium, arachidonate mobilization in response to thapsigargin or fMLP was not simply a consequence of calcium influx. Assessment of intracellular free calcium elevations during thapsigargin or fMLP-induced stimulation suggest that a low level of arachidonic acid release was initiated upon release of intracellular store calcium. This initial release of arachidonate was unaffected by inhibition of calcium influx with nickel, EGTA, or SKF96365. Arachidonate release was observed when extracellular calcium was replaced with extracellular strontium, suggesting activation of the cytosolic PLA₂ rather than secretory PLA₂. Inhibition of PLA₂ with prostaglandin B oligomer prevented both thapsigargin and fMLP-stimulated influx of extracellular calcium. Furthermore, exogenous free arachidonate stimulated influx of extracellular calcium in differentiated U937 cells. These results suggest that cPLA₂-mediated release of free arachidonate may participate in the formation of a calcium influx factor which controls influx of extracellular calcium through store-controlled channels in the plasma membrane.     

Release of Calcium from Mitochondrial and Nonmitochondrial Intracellular Stores in Mouse Pancreatic Acinar Cells by Hydrogen Peroxide

In the present study we have studied how [Ca²⁺] _i is influenced by H₂O₂ in collagenase-dispersed mouse pancreatic acinar cells and the mechanism underlying this effect by using a digital microspectrofluorimetric system. In the presence of normal extracellular calcium concentration, perfusion of pancreatic acinar cells with 1 mm H₂O₂ caused a slow sustained [Ca²⁺] _i increase, reaching a stable plateau after 10–15 min of perfusion. This increase induced by H₂O₂ was also observed in a nominally calcium-free medium, reflecting the release of calcium from intracellular store(s). Application of 1 mm H₂O₂ to acinar cells, in which nonmitochondrial agonist-releasable calcium pools had been previously depleted by a maximal concentration of CCK-8 (1 nm) or thapsigargin (0.5 μm) was still able to induce calcium release. Similar results were observed when thapsigargin was substituted for the mitochondrial uncoupler FCCP (0.5 μm). By contrast, simultaneous addition of thapsigargin and FCCP clearly abolished the H₂O₂-induced calcium increase. Interestingly, co-incubation of intact pancreatic acinar cells with CCK-8 plus thapsigargin and FCCP in the presence of H₂O₂ did not significantly affect the transient calcium spike induced by the depletion of nonmitochondrial and mitochondrial agonist-releasable calcium pools, but was followed by a sustained increase of [Ca²⁺] _i . In addition, H₂O₂ was able to block calcium efflux evoked by CCK and thapsigargin. Finally, the transient increase in [Ca²⁺] _i induced by H₂O₂ was abolished by an addition of 2 mm dithiothreitol (DTT), a sulfhydryl reducing agent. Our results show that H₂O₂ releases calcium from CCK-8- and thapsigargin-sensitive intracellular stores and from mitochondria. The action of H₂O₂ is likely mediated by oxidation of sulfhydryl groups of calcium-ATPases. Received: 15 May 2000/Revised: 4 October 2000     

Calcium influx and intracellular calcium release in anti-CD3 antibody-stimulated and thapsigargin-treated human T lymphoblasts

Summary Jurkat and MOLT-4 cultured T lymphoblasts were loaded with low concentrations (30–50 m) of indo-1 and with high concentrations (3.5–4.5mm) of quin-2, respectively, in order to follow the activation of calcium transport pathways after stimulation of the cells by a monoclonal antibody against the T cell antigen receptor (aCD3), or after the addition of thapsigargin, a presumed inhibitor of endoplasmic reticulum calcium pump. In the indo-1 loaded cells the dynamics of the intracellular calcium release and the calcium influx could be studied, while in the quin-2 overloaded cells the changes in cytoplasmic free calcium concentration ([Ca²⁺] _i ) were strongly buffered and the rate of calcium influx could be quantitatively determined. We found that in Jurkat lymphoblasts, in the absence of external calcium, both aCD3 and thapsigargin induced a rapid calcium release from internal stores, while upon the readdition of external calcium an increased rate of calcium influx could be observed in both cases, aCD3 and thapsigargin released calcium from the same intracellular pools. The calcium influx induced by either agent was of similar magnitude and had a nonadditive character if the two agents were applied simultaneously. As demonstrated in quin-2 overloaded cells, a significant initial rise in [Ca²⁺] _i or a pronounced depletion of internal calcium pools was not required to obtain a rapid calcium influx. The activation of protein kinase C by phorbol ester abolished the internal calcium release and the calcium influx induced by aCD3, while having only a small effect on these phenomena when evoked by thapsigargin. Membrane depolarization by gramicidin inhibited the rapid calcium influx in both aCD3- and thapsigargin-treated cells, although it did not affect the internal calcium release produced by either agent. In MOLT-4 cells, which have no functioning antigen receptors, aCD3 was ineffective in inducing a calcium signal, while thapsigargin produced similar internal calcium release and external calcium influx to those observed in Jurkat cells.     

Electrically induced changes in Ca2+ in Helisoma neurons: Regional and neuron-specific differences and implications for neurite outgrowth

The present experiments addressed the questions of how electrical stimulation influenced the magnitude, time course, and regional levels of free intracellular calcium of different identified neurons. The calcium concentration in the growth cones, neurites and cell bodies of Helisoma buccal neurons B4 and B19 was measured while somata were electrically stimulated via an intracellular electrode. The findings showed that calcium levels in B4 and B19 increased monotonically with increasing stimulation frequency. However, the range of calcium levels evoked by electrical stimulation differed significantly for each type of neuron. The greater increase in calcium concentration in B4 was correlated with its longer duration action potential compared to B19. The increase in calcium concentration was much smaller in the cell bodies than in the growth cones and neurites. Extending the duration of the B19 action potential produced a sixfold increase in the change in calcium concentration at 2 Hz stimulation. Under conditions where the electrical stimulation produced a calcium concentration of < 160 nM, the elevated level of free intracellular calcium remained constant. When calcium concentration increased above 200 nM in both identified neurons, an initial peak concentration was followed by a decline to a lower concentration suggesting increased calcium buffering occurring above 200 nM. By correlating the calcium concentration data herein with growth data from a previous study, we suggest that specific calcium levels that influence neurite outgrowth may differ widely between neurons. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 150–162, 1997.     

Divergent calcium signaling in RBCs from <Emphasis Type="Italic">Tropidurus torquatus</Emphasis> (Squamata – Tropiduridae) strengthen classification in lizard evolution

Background  

We have previously reported that a Teiid lizard red blood cells (RBCs) such as Ameiva ameiva and Tupinambis merianae controls intracellular calcium levels by displaying multiple mechanisms. In these cells, calcium stores could be discharged not only by: thapsigargin, but also by the Na⁺/H⁺ ionophore monensin, K⁺/H⁺ ionophore nigericin and the H⁺ pump inhibitor bafilomycin as well as ionomycin. Moreover, these lizards possess a P2Y-type purinoceptors that mobilize Ca²⁺ from intracellular stores upon ATP addition.     

Involvement of Intracellular or Extracellular Calcium in Activation of Tyrosine Hydroxylase Gene Expression in PC12 Cells

Abstract: The relationship between elevations in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) by different mechanisms and tyrosine hydroxylase (TH) gene expression was examined. Depolarization by an elevated K⁺ concentration triggered rapid and sustained increases in [Ca²⁺]_i from a basal level of ～50 to 110–150 nM and three- to fourfold elevations in TH mRNA levels, requiring extracellular calcium but not inositol 1,4,5-trisphosphate (IP₃). On the other hand, bradykinin or thapsigargin, both of which induce release of intracellular calcium stores via IP₃ or inhibition of Ca²⁺-ATPase, rapidly elevated [Ca²⁺]_i to >200 nM and increased TH gene expression (three-to fivefold). Confocal imaging showed that the elevations in [Ca²⁺]_i in each case occurred throughout the cyto- and nucleoplasm. The initial rise in [Ca²⁺]_i due to either bradykinin or thapsigargin, which did not require extracellular calcium, was sufficient to initiate the events leading to increased TH expression. Consistent with this, the effects of bradykinin on TH expression were inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester which chelates or inhibits the release of intracellular calcium, respectively. Bradykinin required a rise in [Ca²⁺]_i for <10 min, as opposed to 10–30 min for depolarization to increase TH mRNA levels. These results demonstrate that although each of these treatments increased TH gene expression by raising [Ca²⁺]_i, there are important differences among them in terms of the magnitude of elevated [Ca²⁺]_i, requirements for extracellular calcium or release of intracellular calcium stores, and duration of elevated [Ca²⁺]_i, indicating the involvement of different calcium signaling pathways leading to regulation of TH gene expression.     

Native STIM2 and ORAI1 proteins form a calcium‐sensitive and thapsigargin‐insensitive complex in cortical neurons

In non‐excitatory cells, stromal interaction molecule 1 (STIM1) and STIM2 mediate store‐operated calcium entry via an interaction with ORAI1 calcium channels. However, in neurons, STIM2 over‐expression appears to play a role in calcium homeostasis that is different from STIM1 over‐expression. The aim of this study was to establish the role and localization of native STIM2 in the neuronal cell. Co‐immunoprecipitation experiments revealed that the interaction between endogenous STIM2 and ORAI1 was greater in a low‐calcium medium than in a high‐calcium medium. Using a Proximity Ligation Assay (PLA), the number of apparent complexes of endogenous STIM2 with ORAI1 was quantified. No change in the number of PLA signals was observed in the presence of thapsigargin, which depletes calcium from the endoplasmic reticulum (ER). However, the number of apparent STIM2‐ORAI1 complexes increased when intracellular and subsequently ER calcium concentrations were decreased by BAPTA‐AM or a low‐calcium medium. Both Fura‐2 acetoxymethyl ester calcium imaging and PLA in the same neuronal cell indicated that the calcium responses correlated strongly with the number of endogenous STIM2‐ORAI1 complexes. The small drop in calcium levels in the ER caused by decreased intracellular calcium levels appeared to initiate the calcium‐sensitive and thapsigargin‐insensitive interaction between STIM2 and ORAI1.

     

Spatiotemporal calcium signaling in a<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis> cell line stably expressing a<Emphasis Type="Italic"> Drosophila</Emphasis> muscarinic acetylcholine receptor

A muscarinic acetylcholine receptor (mAChR), DM1, expressed in the nervous system of Drosophila melanogaster, has been stably expressed in a Drosophila S2 cell line (S2-DM1) and used to investigate spatiotemporal calcium changes following agonist activation. Carbamylcholine (CCh) and oxotremorine are potent agonists, whereas application of the vertebrate M1 mAChR agonist, McN-A-343, results in a weak response. Activation of S2-DM1 receptors using CCh resulted in an increase in intracellular calcium ([Ca²⁺]_i) that was biphasic. Two distinct calcium sources were found to contribute to calcium signaling: (1) internal stores that are sensitive to both thapsigargin and 2-aminoethoxydiphenyl borate and (2) capacitative calcium entry. Spatiotemporal imaging of individual S2-DM1 cells showed that the CCh-induced [Ca²⁺]_i transient resulted from a homogeneous calcium increase throughout the cell, indicative of calcium release from internal stores. In contrast, ionomycin induced the formation of a "calcium ring" at the cell periphery, consistent with external calcium influx.     

Endoplasmic Reticulum Stress Response of <Emphasis Type="Italic">Bombyx Mori</Emphasis> Calreticulin

We isolated a calreticulin cDNA from the silkworm, Bombyx mori. The cDNA encodes 398 amino acid residues of B. mori calreticulin, with an endoplasmic reticulum retentional HDEL motif at its C-terminus and a predicted molecular mass of 45,801 Da. The B. mori calreticulin shows high protein homology with calreticulin from G. mellonella (88%), A. aegypti (71%), D. melanogaster (69%) and H. sapiens (63%). The highest level of mRNA expression of B. mori calreticulin was exhibited in the fat body of this insect. Although expression of B. mori calreticulin was affected by disturbances in intracellular calcium levels, other ER stress conditions such as inhibition of intracellular protein transport, reduction of disulfide formation, glycosylation inhibition, heat shock and oxidative stress did not disrupt induction of B. mori calreticulin.     

Stimulation of EAAC1 in C6 glioma cells by store-operated calcium influx

This study investigated how modulation of intracellular calcium alters the functional activity of the EAAC1 glutamate transporter in C6 glioma cells. Pre-incubation of C6 glioma cells with the endoplasmic reticulum Ca²⁺ ATP pump inhibitor, thapsigargin (10 μM) produced a time-dependent increase in the V_max for d-[³H]aspartate transport that reached a maximum at 15 min (143% of control; P < 0.001) that was accompanied by increased plasma membrane expression of EAAC1 and was blocked by inhibition of protein kinase C. Pre-incubation of C6 glioma cells with phorbol myristate-3-acetate (100 nM for 20 min) also caused a significant increase in the V_max of sodium-dependent d-[³H]aspartate transport (190% of control; P < 0.01). In contrast, in the absence of extracellular calcium, thapsigargin caused a significant inhibition in d-[³H]aspartate transport that was not mediated by protein kinase C. Blockade of store-operated calcium channels with 2-aminoethoxydiphenyl borate (50 μM) or SKF 96365 (10 μM) caused a net inhibition of d-[³H]aspartate uptake. Co-incubation of C6 glioma cells with both thapsigargin and 2-aminoethoxydiphenyl borate (but not SKF 96365) prevented the increase in d-[³H]aspartate transport that was observed in the presence of thapsigargin alone. Furthermore, 2-aminoethoxydiphenyl borate, but not SKF 96365, reduced the increase in intracellular calcium that occurred following pre-incubation of the cells with thapsigargin. It is concluded that, in C6 glioma cells, stimulation of EAAC1-mediated glutamate transport by thapsigargin is dependent on entry of calcium via the NSCC-1 subtype of store operated calcium channel and is mediated by protein kinase C. In contrast, in the absence of store operated calcium entry, thapsigargin inhibits transport.     

Mechanisms of calcium sequestration by isolated Malpighian tubules of the house cricket Acheta domesticus

Hemolymph calcium homeostasis in insects is achieved by the Malpighian tubules, primarily by sequestering excess Ca²⁺ within internal calcium stores (Ca‐rich granules) most often located within type I (principal) tubule cells. Using both the scanning ion‐selective electrode technique and the Ramsay secretion assay this study provides the first measurements of basolateral and transepithelial Ca²⁺ fluxes across the Malpighian tubules of an Orthopteran insect, the house cricket Acheta domesticus. Ca²⁺ transport was specific to midtubule segments, where 97% of the Ca²⁺ entering the tubule is sequestered within intracellular calcium stores and the remaining 3% is secreted into the lumen. Antagonists of voltage‐gated (L‐type) calcium channels decreased Ca²⁺ influx ≥fivefold in adenosine 3′,5′‐cyclic monophosphate (cAMP)‐stimulated tubules, suggesting basolateral Ca²⁺ influx is facilitated by voltage‐gated Ca²⁺ channels. Increasing fluid secretion through manipulation of intracellular levels of cAMP or Ca²⁺ had opposite effects on tubule Ca²⁺ transport. The adenylyl cyclase‐cAMP‐PKA pathway promotes Ca²⁺ sequestration whereas both 5‐hydroxytryptamine and thapsigargin inhibited sequestration. Our results suggest that the midtubules of Acheta domesticus are dynamic calcium stores, which maintain hemolymph calcium concentration by manipulating rates of Ca²⁺ sequestration through stimulatory (cAMP) and inhibitory (Ca²⁺) regulatory pathways.     

Effects of ocean warming and acidification on the energy budget of an excavating sponge

Recent research efforts have demonstrated increased bioerosion rates under experimentally elevated partial pressures of seawater carbon dioxide (pCO₂) with or without increased temperatures, which may lead to net erosion on coral reefs in the future. However, this conclusion clearly depends on the ability of the investigated bioeroding organisms to survive and grow in the warmer and more acidic future environments, which remains unexplored. The excavating sponge Cliona orientalis Thiele, 1900 is a widely distributed bioeroding organism and symbiotic with dinoflagellates of the genus Symbiodinium. Using C. orientalis, an energy budget model was developed to calculate amounts of carbon directed into metabolic maintenance and growth. This model was tested under a range of CO₂ emission scenarios (temperature + pCO₂) appropriate to an Austral early summer. Under a pre‐industrial scenario, present day (control) scenario, or B1 future scenario (associated with reducing the rate of CO₂ emissions over the next few decades), C. orientalis maintained a positive energy budget, where metabolic demand was likely satisfied by autotrophic carbon provided by Symbiodinium and heterotrophic carbon via filter‐feeding, suggesting sustainability. Under B1, C. orientalis likely benefited by a greater supply of photosynthetic products from its symbionts, which increased by up to 56% per unit area, and displayed an improved condition with up to 52% increased surplus carbon available for growth. Under an A1FI future scenario (associated with ‘business‐as‐usual’ CO₂ emissions) bleached C. orientalis experienced the highest metabolic demand, but carbon acquired was insufficient to maintain the sponge, as indicated by a negative energy budget. These metabolic considerations suggest that previous observations of increased bioerosion under A1FI by C. orientalis may not last through the height of future A1FI summers, and survival of individual sponges may be dependent on the energy reserves (biomass) they have accumulated through the rest of the year.     

Inhibitors of cell division and protoplasmic streaming fail to cause a detectable effect on intracellular calcium levels in stamen-hair cells of Tradescantia virginiana L.

The herbicides amiprophos-methyl (APM) and oryzalin disrupt mitosis and cytokinesis in plant cells by causing the depolymerization of microtubules. These drugs have also been shown to affect calcium sequestration by mitochondria. Controversy thus exists as to whether microtubule depolymerization occurs as a result of direct interaction between the drug and tubulin, or because of elevated intracellular calcium levels resulting from drug interference with calcium regulation. In order to clarify this issue we have directly measured the effect of these herbicides and other cell-motility-altering drugs on intracellular calcium levels in stamen-hair cells of Tradescantia. The results indicate that low levels (1–3 M) of APM and oryzalin can act within 3–7 min causing disorganization of mitosis. Studies using the calcium indicator indo-1 injected into stamen-hair cells to monitor internal levels of calcium, show that at drug concentrations where inhibitory effects on mitosis and-or cytokinesis are clearly seen, APM, oryzalin, isopropyl-N-phenyl carbamate, caffeine and cytochalasin D produce no change in intracellular calcium levels. Furthermore, except for cytochalasin D, these drugs do not inhibit cytoplasmic streaming, a calcium-sensitive process. We conclude that the mode of action of these drugs on the cytoskeleton is independent of an effect on intracellular calcium.Abbreviations and Symbols APM amiprophos-methyl - [Ca²⁺]_i free intracellular calcium ion concentration - CD cytochalasin D - DMSO dimethylsulfoxide - IPC isopropyl N-phenylcarbamate - MT(s) microtubule(s) To whom correspondence should be addressedWe thank Dr. L.C. Morejohn, University of Texas, Austin, for encouraging us to perform this study and for his gift of amiprophosmethyl and oryzalin. We also thank our colleagues at the University of Massachusetts for many helpful discussions. This work has been supported by grants from the U.S. Department of Agriculture (88-37261-3727) and the National Science Foundation (DCB-88-01-01750).     

Interaction between thapsigargin and ATP4− in the regulation of the intracellular calcium in rat submandibular glands

Rat submandibular glands were digested with crude collagenase, and the intracellular calcium concentration of the cellular suspension was measured using fura-2. In the absence of extracellular magnesium and calcium ([Ca²⁺]_o), ATP had no effect; the response to ATP peaked at 1–2.5 mM [Ca²⁺]_o and was inhibited at 5 mM. One millimolar (mM) extracellular ATP did not increase the leak of LDH or fura-2; 10 m?M Coomassie brilliant blue G specifically inhibited the effect of ATP on [Ca²⁺]_in. Depleting intracellular calcium pools with thapsigargin did not affect the response to ATP. Using a Ca²⁺-free/Ca²⁺ reintroduction protocol, it was shown that ATP and thapsigargin increase the uptake of extracellular calcium. The effect of the two agonists was synergistic. Removal of extracellular sodium inhibited the effect of carbachol on [Ca²⁺]_in and the calcium uptake but potentiated the response to ATP. These results suggest that, after binding to purinergic receptors, extracellular ATP^4- increases [Ca²⁺]_in. ATP^4- does not mobilize thapsigargin-sensitive intracellular calcium pools (among which is the IP₃-sensitive calcium pool) but stimulates the uptake of extracellular calcium by a mechanism inhibited by extracellular sodium, probably by opening a nonselective cation channel. © 1994 Wiley-Liss, Inc.     

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.     

Release of intracellular calcium stores leads to retraction of membrane sheets and cell death in mature mouse oligodendrocytes

The ability of mature oligodendrocytes (OLs) to recover from insult is important in repair of damage following demyelination. Since regulation of Ca²⁺ levels within cells plays a critical role in function and survival, this study investigates the effects of changes in cytoplasmic Ca²⁺ on the viability of cultured mouse OLs and their ability to maintain membrane sheets. Mature OLs in culture respond rapidly to the calcium ionophore A23187 and promptly return to resting Ca²⁺ levels when the ionophore is removed. Longer exposure to 0.1–1.0 μM A23187 leads to microtubule disruption, membrane sheet retraction and eventual cell death; nuclear lysis occurs in many of the OLs, as reported by Scolding, et al. (1) for rat OLs. In our cultures, mature OLs were more susceptible to nuclear lysis than were immature OLs or astroglia. Release of intracellular Ca²⁺ stores with thapsigargin at 5–10 μM also leads to retraction of membrane sheets. Following 6 hours of continuous exposure to thapsigargin, the effects on membrane sheets are reversed over the next 12 hours. After 18 hours of continuous exposure to thapsigargin, only occasional nuclear lysis is observed, but a number of the mature OLs show signs of DNA fragmentation, indicating that apoptotic death is occurring. Our results suggest that mature OLs cannot survive a prolonged influx of extracellular calcium as readily as immature OLs and astroglia, but have mechanisms to withstand similar increases in cytoplasmic Ca²⁺ following sustained release of intracellular stores. Special issue dedicated to Dr. Marion E. Smith.     

Larval excretory-secretory products from the parasite <Emphasis Type="Italic">Schistosoma mansoni</Emphasis> modulate HSP70 protein expression in defence cells of its snail host, <Emphasis Type="Italic">Biomphalaria glabrata</Emphasis>

Synthesis of heat shock proteins (HSPs) following cellular stress is a response shared by many organisms. Amongst the HSP family, the ∼70 kDa HSPs are the most evolutionarily conserved with intracellular chaperone and extracellular immunoregulatory functions. This study focused on the effects of larval excretory-secretory products (ESPs) from the parasite Schistosoma mansoni on HSP70 protein expression levels in haemocytes (defence cells) from its snail intermediate host Biomphalaria glabrata. S. mansoni larval stage ESPs are known to interfere with haemocyte physiology and behaviour. Haemocytes from two different B. glabrata strains, one which is susceptible to S. mansoni infection and one which is resistant, both showed reduced HSP70 protein levels following 1 h challenge with S. mansoni ESPs when compared to unchallenged controls; however, the reduction observed in the resistant strain was less marked. The decline in intracellular HSP70 protein persisted for at least 5 h in resistant snail haemocytes only. Furthermore, in schistosome-susceptible snails infected by S. mansoni for 35 days, haemocytes possessed approximately 70% less HSP70. The proteasome inhibitor, MG132, partially restored HSP70 protein levels in ESP-challenged haemocytes, demonstrating that the decrease in HSP70 was in part due to intracellular degradation. The extracellular signal-regulated kinase (ERK) signalling pathway appears to regulate HSP70 protein expression in these cells, as the mitogen-activated protein-ERK kinase 1/2 (MEK1/2) inhibitor, U0126, significantly reduced HSP70 protein levels. Disruption of intracellular HSP70 protein expression in B. glabrata haemocytes by S. mansoni ESPs may be a strategy employed by the parasite to manipulate the immune response of the intermediate snail host.