Modulation of intracellular calcium concentrations and T cell activation by prickly pear polyphenols

Opuntia ficus indica (prickly pear) polyphenolic compounds (OFPC) triggered an increase in [Ca2+]i in human Jurkat T-cell lines. Furthermore, OFPC-induced rise in [Ca2+]i was significantly curtailed in calcium-free buffer (0% Ca2+) as compared to that in 100% Ca2+ medium. Preincubation of cells with tyrphostin A9, an inhibitor of Ca2+ release-activated Ca2+ (CRAC) channels, significantly diminished the OFPC-induced sustained response on the increases in [Ca2+]i. Lanthanum and nifedipine, the respective inhibitors of voltage-dependent and L-type calcium channels, failed to curtail significantly the OFPC-induced calcium response. As OFPC still stimulated increases in [Ca2+]i in 0% Ca2+ medium, the role of intracellular calcium was investigated. Hence, addition of thapsigargin (TG), an inhibitor of Ca2+-ATPase of the endoplasmic reticulum (ER), during the OFPC-induced peak response exerted an additive effect, indicating that the mechanism of action of these two agents are different. Furthermore, U73122, an inhibitor of IP3 production, completely abolished increases in [Ca2+]i, induced by OFPC, suggesting that these polyphenols induce the production of IP3 that recruits calcium from ER pool. Polyphenolic compounds do act extracellularly as addition of fatty acid-free bovine serum albumin (BSA) significantly diminished the rise in [Ca2+]i evoked by the formers. OFPC also induced plasma membrane hyperpolarisation which was reversed by addition of BSA. OFPC were found to curtail the expression of IL-2 mRNA and T-cell blastogenesis. Together these results suggest that OFPC induce increases in [Ca2+]i via ER pool and opening of CRAC channels, and exert immunosuppressive effects in Jurkat T-cells.     

Regulation of calcium signalling by 1-O-alkylglycerols in human Jurkat T lymphocytes

We studied the role of natural occurring 1-O-alkylglycerols on the calcium signalling in Jurkat T-cells. Alkylglycerols evoked an increase in free intracellular calcium concentration [Ca2+]i, in a dose-dependent manner. When the experiments were performed in calcium-free buffer, the alkylglycerol response on the rise of [Ca2+]i was wholly abolished compared with the one in calcium-containing buffer, suggesting that these etherlipids induce a calcium influx by the opening of Ca2+ channels. We further employed inhibitors of voltage-gated calcium channels. We observed that omega-conotoxin, a blocker of N-type voltage-activated Ca2+ channels, but not verapamil, a blocker of L-type voltage-activated Ca2+ channels, curtailed significantly the calcium rise evoked by the lipid agents. Alkylglycerols also induced plasma membrane depolarisation, known to be involved in the opening of the voltage-gated calcium channels. Our study shows that alkylglycerols increase [Ca2+]i influx in human Jurkat T-cells possibly by modulating the permeability of calcium channels.     

Modulation of intracellular calcium concentrations and T cell activation by prickly pear polyphenols

Opuntia ficus indica(prickly pear) polyphenolic compounds (OFPC) triggered an increase in [Ca²⁺]_i in human Jurkat T-cell lines. Furthermore, OFPC-induced rise in [Ca²⁺]_i was significantly curtailed in calcium-free buffer (0% Ca²⁺) as compared to that in 100% Ca²⁺ medium. Preincubation of cells with tyrphostin A9, an inhibitor of Ca²⁺ release-activated Ca²⁺(CRAC) channels, significantly diminished the OFPC-induced sustained response on the increases in [Ca²⁺]_i. Lanthanum and nifedipine, the respective inhibitors of voltage-dependent and L-type calcium channels, failed to curtail significantly the OFPC-induced calcium response. As OFPC still stimulated increases in [Ca²⁺]_i in 0% Ca²⁺ medium, the role of intracellular calcium was investigated. Hence, addition of thapsigargin (TG), an inhibitor of Ca²⁺-ATPase of the endoplasmic reticulum (ER), during the OFPC-induced peak response exerted an additive effect, indicating that the mechanism of action of these two agents are different. Furthermore, U73122, an inhibitor of IP₃ production, completely abolished increases in [Ca²⁺]_i, induced by OFPC, suggesting that these polyphenols induce the production of IP₃ that recruits calcium from ER pool. Polyphenolic compounds do act extracellularly as addition of fatty acid-free bovine serum albumin (BSA) significantly diminished the rise in [Ca²⁺]_i evoked by the formers. OFPC also induced plasma membrane hyperpolarisation which was reversed by addition of BSA. OFPC were found to curtail the expression of IL-2 mRNA and T-cell blastogenesis. Together these results suggest that OFPC induce increases in [Ca²⁺]_i via ER pool and opening of CRAC channels, and exert immunosuppressive effects in Jurkat T-cells.     

Thapsigargin-stimulated MAP kinase phosphorylation via CRAC channels and PLD activation: inhibitory action of docosahexaenoic acid

This study was conducted on human Jurkat T-cells to investigate the role of depletion of intracellular Ca(2+) stores in the phosphorylation of two mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated kinase (ERK) 1 and ERK2, and their modulation by a polyunsaturated fatty acid, docosahexaenoic acid (DHA). We observed that thapsigargin (TG) stimulated MAPK activation by store-operated calcium (SOC) influx via opening of calcium release-activated calcium (CRAC) channels as tyrphostin-A9, a CRAC channel blocker, and two SOC influx inhibitors, econazole and SKF-96365, diminished the action of the former. TG-stimulated ERK1/ERK2 phosphorylation was also diminished in buffer containing EGTA, a calcium chelator, further suggesting the implication of calcium influx in MAPK activation in these cells. Moreover, TG stimulated the production of diacylglycerol (DAG) by activating phospholipase D (PLD) as propranolol (PROP) (a PLD inhibitor), but not U73122 (a phospholipase C inhibitor), inhibited TG-evoked DAG production in these cells. DAG production and protein kinase C (PKC) activation were involved upstream of MAPK activation as PROP and GF109203X, a PKC inhibitor, abolished the action of TG on ERK1/ERK2 phosphorylation. Furthermore, DHA seems to act by inhibiting PKC activation as this fatty acid diminished TG- and phorbol 12-myristate 13-acetate-induced ERK1/ERK2 phosphorylation in these cells. Together these results suggest that Ca(2+) influx via CRAC channels is implicated in PLD/PKC/MAPK activation which may be a target of physiological agents such as DHA.     

Low cytoplasmic [Ca(2+)] activates I(CRAC) independently of global Ca(2+) store depletion in RBL-1 cells

Release of Ca(2+) from inositol (1,4,5)-trisphosphate-sensitive Ca(2+) stores causes "capacitative calcium entry," which is mediated by the so-called "Ca(2+) release-activated Ca(2+) current" (I(CRAC)) in RBL-1 cells. Refilling of the Ca(2+) stores or high cytoplasmic [Ca(2+)] ([Ca(2+)](cyt)) inactivate I(CRAC). Here we address the question if also [Ca(2+)](cyt) lower than the resting [Ca(2+)](cyt) influences store-operated channels. We therefore combined patch clamp and mag fura-2 fluorescence methods to determine simultaneously both I(CRAC) and [Ca(2+)] within Ca(2+) stores of RBL-1 cells ([Ca(2+)](store)). We found that low [Ca(2+)](cyt) in the range of 30-50 nM activates I(CRAC) and Ca(2+) influx spontaneously and independently of global Ca(2+) store depletion, while elevation of [Ca(2+)](cyt) to the resting [Ca(2+)](cyt) (100 nM) resulted in store dependence of I(CRAC) activation. We conclude that spontaneous activation of I(CRAC) by low [Ca(2+)](cyt) could serve as a feedback mechanism keeping the resting [Ca(2+)](cyt) constant.     

Oxidative stress-induced calcium signalling in Aspergillus nidulans

The effects of oxidative stress on levels of calcium ion (Ca(2+)) in Aspergillus nidulans were measured using strains expressing aequorin in the cytoplasm (Aeq(cyt)) and mitochondria (Aeq(mt)). When oxidative stress was induced by exposure to 10-mM H(2)O(2), the mitochondrial calcium response (Ca(mt)(2+)) was greater than the change in cytoplasmic calcium (Ca(c)(2+)). The Ca(mt)(2+) response to H(2)O(2) was dose dependent, while the increase in [Ca(c)(2+)] did not change with increasing H(2)O(2). The increase in both [Ca(c)(2+)] and [Ca(mt)(2+)] in response to oxidative stress was enhanced by exposure of cells to Ca(2+). The presence of chelator in the external medium only partially inhibited the Ca(mt)(2+) and Ca(c)(2+) responses to oxidative stress. Reagents that alter calcium fluxes had varied effects on the Ca(mt)(2+) response to peroxide. Ruthenium red blocked the increase in [Ca(mt)(2+)], while neomycin caused an even greater increase in [Ca(mt)(2+)]. Treatment with ruthenium red and neomycin had no effect on the Ca(c)(2+) response. Bafilomycin A and oligomycin had no effect on either the mitochondrial or cytoplasmic response. Inhibitors of both voltage-regulated calcium channels and intracellular calcium release channels inhibited the Ca(2+)-dependent component of the Ca(mt)(2+) response to oxidative stress. We conclude that the more significant Ca(2+) response to oxidative stress occurs in the mitochondria and that both intracellular and extracellular calcium pools can contribute to the increases in [Ca(c)(2+)] and [Ca(mt)(2+)] induced by oxidative stress.     

Unitary Ca(2+) current through recombinant type 3 InsP(3) receptor channels under physiological ionic conditions

The ubiquitous inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) channel, localized primarily in the endoplasmic reticulum (ER) membrane, releases Ca(2+) into the cytoplasm upon binding InsP(3), generating and modulating intracellular Ca(2+) signals that regulate numerous physiological processes. Together with the number of channels activated and the open probability of the active channels, the size of the unitary Ca(2+) current (i(Ca)) passing through an open InsP(3)R channel determines the amount of Ca(2+) released from the ER store, and thus the amplitude and the spatial and temporal nature of Ca(2+) signals generated in response to extracellular stimuli. Despite its significance, i(Ca) for InsP(3)R channels in physiological ionic conditions has not been directly measured. Here, we report the first measurement of i(Ca) through an InsP(3)R channel in its native membrane environment under physiological ionic conditions. Nuclear patch clamp electrophysiology with rapid perfusion solution exchanges was used to study the conductance properties of recombinant homotetrameric rat type 3 InsP(3)R channels. Within physiological ranges of free Ca(2+) concentrations in the ER lumen ([Ca(2+)](ER)), free cytoplasmic [Ca(2+)] ([Ca(2+)](i)), and symmetric free [Mg(2+)] ([Mg(2+)](f)), the i(Ca)-[Ca(2+)](ER) relation was linear, with no detectable dependence on [Mg(2+)](f). i(Ca) was 0.15 +/- 0.01 pA for a filled ER store with 500 microM [Ca(2+)](ER). The i(Ca)-[Ca(2+)](ER) relation suggests that Ca(2+) released by an InsP(3)R channel raises [Ca(2+)](i) near the open channel to approximately 13-70 microM, depending on [Ca(2+)](ER). These measurements have implications for the activities of nearby InsP(3)-liganded InsP(3)R channels, and they confirm that Ca(2+) released by an open InsP(3)R channel is sufficient to activate neighboring channels at appropriate distances away, promoting Ca(2+)-induced Ca(2+) release.     

Ca2+ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane

Stromal interacting molecule 1 (STIM1), reported to be an endoplasmic reticulum (ER) Ca(2+) sensor controlling store-operated Ca(2+) entry, redistributes from a diffuse ER localization into puncta at the cell periphery after store depletion. STIM1 redistribution is proposed to be necessary for Ca(2+) release-activated Ca(2+) (CRAC) channel activation, but it is unclear whether redistribution is rapid enough to play a causal role. Furthermore, the location of STIM1 puncta is uncertain, with recent reports supporting retention in the ER as well as insertion into the plasma membrane (PM). Using total internal reflection fluorescence (TIRF) microscopy and patch-clamp recording from single Jurkat cells, we show that STIM1 puncta form several seconds before CRAC channels open, supporting a causal role in channel activation. Fluorescence quenching and electron microscopy analysis reveal that puncta correspond to STIM1 accumulation in discrete subregions of junctional ER located 10-25 nm from the PM, without detectable insertion of STIM1 into the PM. Roughly one third of these ER-PM contacts form in response to store depletion. These studies identify an ER structure underlying store-operated Ca(2+) entry, whose extreme proximity to the PM may enable STIM1 to interact with CRAC channels or associated proteins.     

Distinct properties of CRAC and MIC channels in RBL cells

In rat basophilic leukemia (RBL) cells and Jurkat T cells, Ca(2+) release-activated Ca(2+) (CRAC) channels open in response to passive Ca(2+) store depletion. Inwardly rectifying CRAC channels admit monovalent cations when external divalent ions are removed. Removal of internal Mg(2+) exposes an outwardly rectifying current (Mg(2+)-inhibited cation [MIC]) that also admits monovalent cations when external divalent ions are removed. Here we demonstrate that CRAC and MIC currents are separable by ion selectivity and rectification properties: by kinetics of activation and susceptibility to run-down and by pharmacological sensitivity to external Mg(2+), spermine, and SKF-96365. Importantly, selective run-down of MIC current allowed CRAC and MIC current to be characterized under identical ionic conditions with low internal Mg(2+). Removal of internal Mg(2+) induced MIC current despite widely varying Ca(2+) and EGTA levels, suggesting that Ca(2+)-store depletion is not involved in activation of MIC channels. Increasing internal Mg(2+) from submicromolar to millimolar levels decreased MIC currents without affecting rectification but did not alter CRAC current rectification or amplitudes. External Mg(2+) and Cs(+) carried current through MIC but not CRAC channels. SKF-96365 blocked CRAC current reversibly but inhibited MIC current irreversibly. At micromolar concentrations, both spermine and extracellular Mg(2+) blocked monovalent MIC current reversibly but not monovalent CRAC current. The biophysical characteristics of MIC current match well with cloned and expressed TRPM7 channels. Previous results are reevaluated in terms of separate CRAC and MIC channels.     

Calcium-channel activation and matrix protein upregulation in bone cells in response to mechanical strain

Femur-derived osteoblasts cultured from rat femora were loaded with Fluo-3 using the AM ester. A quantifiable stretch was applied and [Ca(2+)]i levels monitored by analysis of fluorescent images obtained using an inverted microscope and laser scanning confocal imaging system. Application of a single pulse of tensile strain via an expandable membrane resulted in immediate increase in [Ca(2+)]i in a proportion of the cells, followed by a slow and steady decrease to prestimulation levels. Application of parathyroid hormone (10(-6) M) prior to mechanical stimulation potentiated the load-induced elevation of [Ca(2+)]i. Mechanically stimulating osteoblasts in Ca(2+)-free media or in the presence of either nifedipine (10 microM; L-type Ca(2+)-channel blocker) or thapsigargin (1 microM; depletes intracellular Ca(2+) stores) reduced strain-induced increases in [Ca(2+) ]i. Furthermore, strain-induced increases in [Ca(2+)]i were enhanced in the presence of Bayer K 8644 (500 nm), an agonist of L-type calcium channels. The effects of mechanical strain with and without inhibitors and agonists are described on the total cell population and on single cell responses. Application of strain and strain in the presence of the calcium-channel agonist Bay K 8644 to periosteal-derived osteoblasts increased levels of the extracellular matrix proteins osteopontin and osteocalcin within 24 h postload. This mechanically induced increase in osteopontin and osteocalcin was inhibited by the addition of the calcium-channel antagonist, nifedipine. Our results suggest an important role for L-type calcium channels and a thapsigargin-sensitive component in early mechanical strain transduction pathways in osteoblasts.     

Inhibition of the calcium release-activated calcium (CRAC) current in Jurkat T cells by the HIV-1 envelope protein gp160.

The HIV-1 envelope glycoprotein gp120/160 has pleiotropic effects on T cell function. We investigated whether Ca(2+) signaling, a crucial step for T cell activation, was altered by prolonged exposure of Jurkat T cells to gp160. Microfluorometric measurements showed that Jurkat cells incubated with gp160 had smaller (approximately 40%) increases in [Ca(2+)](i) in response to phytohemagglutinin and had a reduced Ca(2+) influx (approximately 25%). gp160 had similar effects on Jurkat cells challenged with thapsigargin. We used the patch clamp technique to record the Ca(2+) current, which is responsible for Ca(2+) influx and has properties of the calcium release-activated Ca(2+) current (I(CRAC)). gp160 reduced I(CRAC) by approximately 40%. The inhibitory effects of gp160 were antagonized by staurosporine (0.1 microm), an inhibitor of protein-tyrosine kinases and protein kinase Cs (PKCs), and by G? 6976 (5 microm), an inhibitor acting especially on PKC alpha and PKC beta I. 12-O-Tetradecanoyl phorbol 13-acetate (16 nm), a PKC activator, reproduced the effects of gp160 in untreated cells. A Western blotting analysis of PKC isoforms alpha, beta I, delta, and zeta showed that only the cellular distribution of PKC alpha and -beta I were significantly modified by gp160. In addition, gp160 was able to modify the subcellular distribution of PKC alpha and PKC beta I caused by phytohemagglutinin. Therefore the reduction in I(CRAC) caused by prolonged incubation with gp160 is probably mediated by PKC alpha or -beta I.     

The elementary unit of store-operated Ca2+ entry: local activation of CRAC channels by STIM1 at ER-plasma membrane junctions

The activation of store-operated Ca(2+) entry by Ca(2+) store depletion has long been hypothesized to occur via local interactions of the endoplasmic reticulum (ER) and plasma membrane, but the structure involved has never been identified. Store depletion causes the ER Ca(2+) sensor stromal interacting molecule 1 (STIM1) to form puncta by accumulating in junctional ER located 10-25 nm from the plasma membrane (see Wu et al. on p. 803 of this issue). We have combined total internal reflection fluorescence (TIRF) microscopy and patch-clamp recording to localize STIM1 and sites of Ca(2+) influx through open Ca(2+) release-activated Ca(2+) (CRAC) channels in Jurkat T cells after store depletion. CRAC channels open only in the immediate vicinity of STIM1 puncta, restricting Ca(2+) entry to discrete sites comprising a small fraction of the cell surface. Orai1, an essential component of the CRAC channel, colocalizes with STIM1 after store depletion, providing a physical basis for the local activation of Ca(2+) influx. These studies reveal for the first time that STIM1 and Orai1 move in a coordinated fashion to form closely apposed clusters in the ER and plasma membranes, thereby creating the elementary unit of store-operated Ca(2+) entry.     

pH-dependent effect of mitochondria on calcium influx into Jurkat cells; a novel mechanism of cell protection against calcium entry during energy stress

Loss of the mitochondrial membrane potential results in a significant inhibition of calcium influx through calcium release-activated channels (CRAC) in Jurkat cells suspended in the medium of pH lower than 7.4. This effect disappears when the medium pH increases. Alkalinisation of the cytosol achieved by the addition of NH(4)Cl to the cells pretreated with thapsigargin, CCCP and CaCl(2), suspended in the medium of pH 7.2, does not affect CRAC activity, while alkalisation of the extracellular milieu by NaOH results in a strong stimulation of calcium entry. Thus, the mitochondrial effect on CRAC is exclusively related to the extracellular pH.Coupled mitochondria are able to take up Ca(2+) accumulated in the close proximity of CRAC. This protects these channels against feedback inhibition exerted by high [Ca(2+)](c). We conclude that CRAC may exist in two conformations: inhibitable and not inhibitable by cytosolic Ca(2+). Lower extracellular pH promotes the former one. This explains a much higher inhibitory effect of mitochondrial uncouplers on the calcium influx into the cells exposed to pH 7.2 than that observed in the cells suspended in the medium of pH 7.8. This phenomenon may provide an additional mechanism protecting cells against calcium overloading in transient episodes of energy stress.     

Disruption of the cortical actin cytoskeleton does not affect store operated Ca2+ channels in human T-cells

Lymphocyte signaling and activation leads to the influx of extracellular Ca(2+) via the activation of Ca(2+) release activated Ca(2+) (CRAC) channels in the plasma membrane. Activation of CRAC channels occurs following emptying of the endoplasmic reticulum intracellular Ca(2+) stores. One model to explain the coupling of store-emptying to CRAC activation is the secretion-like conformational coupling model. This model proposes that store depletion increases junctions between the endoplasmic reticulum and the plasma membrane in a manner that could be regulated by the cortical actin cytoskeleton. Here, we show that stabilization or depolymerization of the actin cytoskeleton failed to affect CRAC activation. We therefore conclude that rearrangement of the actin cytoskeleton is dispensable for store-operated Ca(2+) entry in T-cells.     

Sorting of calcium signals at the junctions of endoplasmic reticulum and mitochondria

Calcium signal transmission between endoplasmic reticulum (ER) and mitochondria is supported by a local [Ca(2+)] control that operates between IP(3)receptor Ca(2+)release channels (IP(3)R) and mitochondrial Ca(2+)uptake sites, and displays functional similarities to synaptic transmission. Activation of IP(3)R by IP(3)is known to evoke quantal Ca(2+)mobilization that is associated with incremental elevations of mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)). Here we report that activation of IP(3)R by adenophostin-A (AP) yields non-quantal Ca(2+)mobilization in mast cells. We also show that the AP-induced continuous Ca(2+)release causes relatively small [Ca(2+)](m)responses, in particular, the sustained phase of Ca(2+)release is not sensed by the mitochondria. Inhibition of ER Ca(2+)pumps by thapsigargin slightly increases IP(3)-induced [Ca(2+)](m)responses, but augments AP-induced [Ca(2+)](m)responses in a large extent. In adherent permeabilized cells exposed to elevated [Ca(2+)], ER Ca(2+)uptake fails to affect global cytosolic [Ca(2+)], but attenuates [Ca(2+)](m)responses. Moreover, almost every mitochondrion exhibits a region very close to ER Ca(2+)pumps visualized by BODIPY-FL-thapsigargin or SERCA antibody. Thus, at the ER-mitochondrial junctions, localized ER Ca(2+)uptake provides a mechanism to attenuate the mitochondrial response during continuous Ca(2+)release through the IP(3)R or during gradual Ca(2+)influx to the junction between ER and mitochondria.     

Decreased agonist-stimulated mitochondrial ATP production caused by a pathological reduction in endoplasmic reticulum calcium content in human complex I deficiency

Although a large number of mutations causing malfunction of complex I (NADH:ubiquinone oxidoreductase) of the OXPHOS system is now known, their cell biological consequences remain obscure. We previously showed that the bradykinin (Bk)-induced increase in mitochondrial [ATP] ([ATP](M)) is significantly reduced in primary skin fibroblasts from a patient with an isolated complex I deficiency. The present work addresses the mechanism(s) underlying this impaired response. Luminometry of fibroblasts from 6 healthy subjects and 14 genetically characterized patients expressing mitochondria targeted luciferase revealed that the Bk-induced increase in [ATP](M) was significantly, but to a variable degree, decreased in 10 patients. The same variation was observed for the increases in mitochondrial [Ca(2+)] ([Ca(2+)](M)), measured with mitochondria targeted aequorin, and cytosolic [Ca(2+)] ([Ca(2+)](C)), measured with fura-2, and for the Ca(2+) content of the endoplasmic reticulum (ER), calculated from the increase in [Ca(2+)](C) evoked by thapsigargin, an inhibitor of the ER Ca(2+) ATPase. Regression analysis revealed that the increase in [ATP](M) was directly proportional to the increases in [Ca(2+)](C) and [Ca(2+)](M) and to the ER Ca(2+) content. Our findings provide evidence that a pathological reduction in ER Ca(2+) content is the direct cause of the impaired Bk-induced increase in [ATP](M) in human complex I deficiency.