Stress-induced Ca2 Research Articles

Physiological roles and molecular mechanisms for stress-induced Ca^<2+> mobilization in plants

Physiological roles and molecular mechanisms for stress-induced Ca^<2+> mobilization in plants

Ca2+ sparks as a plastic signal for skeletal muscle health, aging, and dystrophy

Ca2+ sparks are the elementary units of intracellular Ca2+ signaling in striated muscle cells revealed as localized Ca2+ release events from sarcoplasmic reticulum (SR) by confocal microscopy. While Ca2+ sparks are well defined in cardiac muscle, there has been a general belief that these localized Ca2+ release events are rare in intact adult mammalian skeletal muscle. Several laboratories determined that Ca2+ sparks in mammalian skeletal muscle could only be observed in large numbers when the sarcolemmal membranes are permeabilized or the SR Ca2+ content is artificially manipulated, thus the cellular and molecular mechanisms underlying the regulation of Ca2+ sparks in skeletal muscle remain largely unexplored. Recently, we discovered that membrane deformation generated by osmotic stress induced a robust Ca2+ spark response confined in close spatial proximity to the sarcolemmal membrane in intact mouse muscle fibers. In addition to Ca2+ sparks, prolonged Ca2+ transients, termed Ca2+ bursts, are also identified in intact skeletal muscle. These induced Ca2+ release events are reversible and repeatable, revealing a plastic nature in young muscle fibers. In contrast, induced Ca2+ sparks in aged muscle are transient and cannot be re-stimulated. Dystrophic muscle fibers display uncontrolled Ca2+ sparks, where osmotic stress-induced Ca2+ sparks are not reversible and they are no longer spatially restricted to the sarcolemmal membrane. An understanding of the mechanisms that underlie generation of osmotic stress-induced Ca2+ sparks in skeletal muscle, and how these mechanisms are altered in pathology, will contribute to our understanding of the regulation of Ca2+ homeostasis in muscle physiology and pathophysiology.

Feed Forward Cycle of Hypotonic Stress-induced ATP Release, Purinergic Receptor Activation, and Growth Stimulation of Prostate Cancer Cells

ATP is released in many cell types upon mechanical strain, the physiological function of extracellular ATP is largely unknown, however. Here we report that ATP released upon hypotonic stress stimulated prostate cancer cell proliferation, activated purinergic receptors, increased intracellular [Ca(2+)](i), and initiated downstream signaling cascades that involved MAPKs ERK1/2 and p38 as well as phosphatidylinositol 3-kinase (PI3K). MAPK activation, the calcium response as well as induction of cell proliferation upon hypotonic stress were inhibited by preincubation with the ATP scavenger apyrase, indicating that hypotonic stress-induced signaling pathways are elicited by released ATP. Hypotonic stress increased prostaglandin E(2) (PGE(2)) synthesis. Consequently, ATP release was inhibited by antagonists of PI3K (LY294002 and wortmannin), phospholipase A(2) (methyl arachidonyl fluorophosphonate (MAFP)), cyclooxygenase-2 (COX-2) (indomethacin, etodolac, NS398) and 5,8,11,14-eicosatetraynoic acid (ETYA), which are involved in arachidonic acid metabolism. Furthermore, ATP release was abolished in the presence of the adenylate cyclase (AC) inhibitor MDL-12,330A, indicating regulation of ATP-release by cAMP. The hypotonic stress-induced ATP release was significantly blunted when the ATP-mediated signal transduction cascade was inhibited on different levels, i.e. purinergic receptors were blocked by suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), the Ca(2+) response was inhibited upon chelation of intracellular Ca(2+) by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and ERK1,2 as well as p38 were inhibited by UO126 and SB203580, respectively. In summary our data demonstrate that hypotonic stress initiates a feed forward cycle of ATP release and purinergic receptor signaling resulting in proliferation of prostate cancer cells.

Uncontrolled calcium sparks act as a dystrophic signal for mammalian skeletal muscle

Most excitable cells maintain tight control of intracellular Ca(2+) through coordinated interaction between plasma membrane and endoplasmic or sarcoplasmic reticulum. Quiescent sarcoplasmic reticulum Ca(2+) release machinery is essential for the survival and normal function of skeletal muscle. Here we show that subtle membrane deformations induce Ca(2+) sparks in intact mammalian skeletal muscle. Spontaneous Ca(2+) sparks can be reversibly induced by osmotic shock, and participate in a normal physiological response to exercise. In dystrophic muscle with fragile membrane integrity, stress-induced Ca(2+) sparks are essentially irreversible. Moreover, moderate exercise in mdx muscle alters the Ca(2+) spark response. Thus, membrane-deformation-induced Ca(2+) sparks have an important role in physiological and pathophysiological regulation of Ca(2+) signalling, and uncontrolled Ca(2+) spark activity in connection with chronic activation of store-operated Ca(2+) entry may function as a dystrophic signal in mammalian skeletal muscle.

CD45 Defines Signaling Thresholds Critical for Proliferation and Apoptosis in Myeloma Cells.

Expression of the common leukocyte antigen, CD45 is quite variable in human myeloma cells and cell lines, such as U266, and CD45+ U266 proliferates in response to a growth factor, interleukin-6 (IL-6). IL-6 as a mitogenic stimulus, activated a src family kinase, Lyn and phospholipase C (PLC)-g2 followed by Ca2+ influxes and protein kinase C (PKC) activation in CD45+ U266. Here we show that compared with CD45− U266, CD45+ U266 was more sensitive to various apoptotic stimuli, such as oxidative stress and endoplasmic reticulum (ER)-stress. Both release of apoptosis-inducing factor or cytochrome c and the activation of caspase-9 and caspase-3 were enhanced in CD45+ U266 more than in CD45− U266. Increased susceptibility to the stress-induced apoptosis was also observed in other CD45-expressing myeloma cell lines and the CD45-transfected U266. Reactive oxygen species (ROS) and calcium ion (Ca2+) seem to be involved in the susceptibility to apoptosis of CD45+ U266 because either an anti-oxidant, DTT or a Ca2+ chelating agent, BAPTA blocked the apoptosis. As the oxidative stress-induced Ca2+ influxes and apoptosis of CD45+ U266 were blocked by a src family kinase inhibitor, PP2, CD45 phosphatase regulating the src family kinase activity plays an important role in the augmented apoptosis in CD45+ U266 by oxidative stress. The intracellular accumulation of ROS may activate src associated with CD45, and the activated src kinases accelerate the Ca2+ influxes followed by calicineurin activation, mitochondrial translocation of Bad, and inhibition of Bcl-2 function. These results indicate that the CD45-expression renders myeloma cells competent with not only mitogenic but also apoptotic stimuli, resulting in either proliferation or apoptosis of CD45+ myeloma cells dependently upon the stimuli. Furthermore, voltage-dependent anion channel (VDAC)-1 was identified as a gene highly expressed in CD45+ U266 by cDNA subtraction. The increased expression of VDAC-1 seemed to augment the sensitivity to the ER-stress because the VDAC-1-transfected CD45− U266 was confirmed to be susceptible to the thapsigargin-induced apoptosis. Thus, CD45 may define the signaling thresholds that are critical for the IL-6-induced proliferation and the oxidative stress-induced apoptosis of myeloma cells, and the CD45 expression accompanied by the increased VDAC-1 expression sensitizes myeloma cells to the various extracellular stimuli triggering apoptosis via the mitochondrial pathways.

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The mechanisms by which shear stress elevates intracellular Ca(2+) in endothelial cells (EC) are not fully understood. Here we report that endogenously released ATP contributes to shear stress-induced Ca(2+) responses. Application of a flow of Hank's balanced solution to human pulmonary artery EC (HPAEC) elicited shear stress-dependent increases in Ca(2+) concentration. Chelation of extracellular Ca(2+) with EGTA completely abolished the Ca(2+) responses, whereas the phospholipase C inhibitor U-73122 and the Ca(2+)-ATPase inhibitor thapsigargin had no effect, indicating that the response was due to the influx of extracellular Ca(2+). The Ca(2+) influx was significantly suppressed by apyrase, which degrades ATP, and by antisense oligonucleotide targeted to P2X4 receptors. A luciferase luminometric assay showed that shear stress induced dose-dependent release of ATP. When the ATP release was inhibited by the ATP synthase inhibitors angiostatin or oligomycin, the Ca(2+) influx was markedly suppressed but was restored by removal of these inhibitors or addition of extracellular ATP. These results suggest that shear stress stimulates HPAEC to release ATP, which activates Ca(2+) influx via P2X4 receptors.

Endogenously released ATP mediates shear stress-induced Ca2+ influx into pulmonary artery endothelial cells.

The mechanisms by which flow-imposed shear stress elevates intracellular Ca2+ in cultured endothelial cells (ECs) are not fully understood. Here we report finding that endogenously released ATP contributes to shear stress-induced Ca2+ responses. Application of flow of Hanks' balanced solution to human pulmonary artery ECs (HPAECs) elicited shear stress-dependent increases in Ca2+ concentrations. Chelation of extracellular Ca2+ with EGTA completely abolished the Ca2+ responses, whereas the phospholipase C inhibitor U-73122 or the Ca2+-ATPase inhibitor thapsigargin had no effect, which thereby indicates that the response was due to the influx of extracellular Ca2+. The Ca2+ influx was significantly suppressed by apyrase, which degrades ATP, or antisense oligonucleotide targeted to P2X4 purinoceptors. A luciferase luminometric assay showed that shear stress induced dose-dependent release of ATP. When the ATP release was inhibited by the ATP synthase inhibitors angiostatin or oligomycin, the Ca2+ influx was markedly suppressed but was restored by removal of these inhibitors or addition of extracellular ATP. These results suggest that shear stress stimulates HPAECs to release ATP, which activates Ca2+ influx via P2X4 receptors.

Expression of active c-Src disrupts cadherin-based cell adhesion, potentiates oxidative stress-induced disassembly and delays Ca2+-induced assembly of tight junction

Expression of active c-Src disrupts cadherin-based cell adhesion, potentiates oxidative stress-induced disassembly and delays Ca2+-induced assembly of tight junction

Molecular Dissection of the Hydrophobic Segments H3 and H4 of the Yeast Ca2+ Channel Component Mid1

The Saccharomyces cerevisiae MID1 gene product, Mid1, is composed of 548 amino acid residues, has four relatively hydrophobic segments named H1-H4, and functions as a Ca(2+)-permeable, stretch-activated channel when expressed in mammalian cells. In some conditions Mid1 cooperates with Cch1, a yeast homolog of the alpha1 subunit of mammalian voltage-gated channels. To identify the important regions or amino acid residues necessary for Mid1 function, we employed in vitro site-directed mutagenesis on H3 and H4 of Mid1 and expressed the resulting mutant genes in a mid1 null mutant to examine whether the mutant gene products are functional or not in vivo. Mutant Mid1 proteins lacking the whole H3 or H4 segment, H3De or H4De, did not complement the lethality and low Ca(2+) accumulation activity of the mid1 mutant, although their localization and contents appeared to be normal, indicating that H3 and H4 are required for Mid1 function itself. Single amino acid exchange experiments on individual amino acid residues of H3 and H4 showed that 10 of 20 residues in H3 and 14 of 23 residues in H4 were important for the normal function of Mid1. In particular, we found four severe loss-of-function mutations, D341E, F356S, C373D, and C373R, and two interesting mutations leading to a high level of Ca(2+) accumulation with a slightly low complementing activity, G342A and Y355A. The importance of these amino acid residues will be discussed.

Measurement of stress-induced Ca2+pulses in single aequorin-transformed tobacco cells

Signaling patterns measured in large cell populations are the sum of differing signals from separate cells, and thus, the detailed kinetics of Ca2+pulses can often be masked. In an effort to evaluate whether the cytosolic Ca2+pulses previously reported in populations of elicitor- and stress-stimulated tobacco cells accurately represent the pulses that occur in individual cells, a study of single cell Ca2+fluxes in stress-stimulated tobacco cells was undertaken. Individual aequorin-transformed cells were isolated from a tobacco suspension culture and placed directly on a sensitive photo-multiplier tube mounted in a dark chamber. Ca2+-dependent luminescence was then monitored after stimulation with hypo- or hyper-osmotic shock, cold shock, or defense elicitors (oligogalacturonic acid and harpin). Hypo-osmotic shock induced a biphasic Ca2+transient in 67% of the single cells tested that exhibited similar kinetics to the biphasic pulses measured repeatedly in 1ml cell suspensions. In contrast, 33% of the stimulated cells displayed Ca2+flux patterns that were not previously seen in cell suspension studies. Additionally, because only 29% of the cells tested responded with measurable Ca2+pulses to oligogalacturonic acid and 33% to the harpin protein, we conclude that not all cells in a suspension are simultaneously sensitive to stimulation with defense elicitors. In contrast, all cells tested responded with an immediate Ca2+influx after cold or hyperosmotic shock. We conclude that in many cases the Ca2+signaling patterns of single cells are accurately represented in the signaling patterns of large populations, but that single cell measurements are still required to characterize the Ca2+fluxes of the less prominent cell populations.

Involvement of Rho‐kinase and tyrosine kinase in hypotonic stress‐induced ATP release in bovine aortic endothelial cells

Hypotonic stress induces ATP release followed by Ca2+ oscillations in bovine aortic endothelial cells (BAECs). We have investigated the cellular mechanism of the hypotonic stress-induced ATP release. Hypotonic stress induced tyrosine phosphorylation of at least two proteins, of 110 and 150 kDa. Inhibition of tyrosine kinase by the tyrosine kinase inhibitors herbimycin A and tyrphostin 46 prevented ATP release and ATP-mediated Ca2+ oscillations induced by hypotonic stress. ATP release was also inhibited by the pretreatment of the cells with botulinum toxin C3, and augmented by lysophosphatidic acid. Furthermore, pre-treating the cells with Y-27632, a selective inhibitor of Rho-kinase, also suppressed the hypotonic stress-induced ATP release and Ca2+ oscillations, indicating that Rho-mediated activation of Rho-kinase may be involved in the hypotonic ATP release. Hypotonic stress also induced a transient rearrangement of the actin cytoskeleton, which was suppressed by the tyrosine kinase inhibitors Y-27632 and cytochalasin B. However, pretreatment of the cell with cytochalasin B inhibited neither the hypotonic stress-induced ATP release nor the Ca2+ oscillations. These results indicate that tyrosine kinase and the Rho-Rho-kinase pathways are involved in hypotonic stress-induced ATP release and actin rearrangement, but actin polymerization is not required for ATP release in BAECs.

Osmotic swelling-induced ATP release: a new role for tyrosine and Rho-kinases?

Nucleotides such as adenosine and uridine di- and triphosphates are important extracellular signalling molecules that participate in intercellular communication and regulate a broad range of physiological responses including vascular tone, muscle contraction, cell proliferation, mucociliary clearance, platelet aggregation and neurotransmission. Once released, they interact with G-protein coupled purinergic receptors (P2Y1, 2, 4, 6, 11) and ligand-gated ion channel receptors (P2X1-7), all of which can trigger rapid changes in cytosolic [Ca2+]. Exocytotic release of ATP occurs at synapses and from activated blood platelets, chromaffin cells and mast cells. Mechanical perturbations such as shear stress, membrane stretch, media change or hypo-osmotic cell swelling induce significant ATP release from endothelial and epithelial cells, from smooth muscle, fibroblasts, erythrocytes and hepatocytes through mechanisms that remain poorly understood. Except for certain pathological conditions that lead to irreversible membrane damage, increasing evidence suggests that mechanically induced release of ATP is a regulated process that does not involve cellular lysis. Stretch-induced ATP release by urinary bladder epithelium was proposed as a mechanism for sensing when the bladder is full (Ferguson et al. 1997). ATP release from airway epithelial cells is exquisitely sensitive to mechanical perturbations and can be evoked by gentle mixing of the bath solution (Grygorczyk & Hanrahan, 1997). Such mechanically induced release could have a host-defence role in vivo, since luminal ATP could trigger fluid secretion that helps clear the epithelial surface of particulates. While mechanically induced release of ATP is an intriguing phenomenon, the intracellular pool of ATP used, efflux mechanisms and regulatory pathways remain obscure. The report by Koyama et al. in this issue of The Journal of Physiology provides the first evidence that tyrosine kinase and Rho-kinase signalling pathways are involved in hypotonic stress-induced ATP release (Fig. 1). Osmotic swelling of bovine aortic endothelial cells stimulated release of ATP, which, by autocrine/ paracrine action on purinergic receptors, induced oscillations of intracellular Ca2+. Both effects were prevented by the tyrosine kinase inhibitors herbimycin A and tyrphostin 46, although inhibition of ATP release was only partial. This may indicate that additional tyrosine kinase-independent mechanisms are also involved. Indeed, osmotic cell swelling and mechanical load are known to activate multiple signalling cascades including tyrosine and MAP kinase, phosphatidylinositol 3-kinase (PI 3-kinase) and Rho, a monomeric GTPase involved in organising the actin cytoskeleton, endo/exocytosis and in forming focal adhesions. Koyama et al. (2001) found that inhibiting Rho directly with Clostridium botulinum C3 exoenzyme, or downstream at the level of Rho-kinase with Y-27632, diminished osmotic stress-induced ATP release and Ca2+ oscillations. Interestingly, the PI 3-kinase inhibitor wortmannin did not suppress hypotonic stress-induced, ATP-mediated oscillations of intracellular Ca2+. Although data showing a direct effect of wortmannin on ATP release are not presented, this result contrasts with earlier studies on liver cells that indicated volume-sensitive ATP release requires activation of PI 3-kinase (Ferenchak et al. 1998). This difference suggests the existence of distinct, cell-specific pathways for regulating ATP release. Another important phenomenon noted by Koyama et al. (2001) is the basal release of ATP by resting, unperturbed endothelial cells, which confirms similar observations with other cell types (Lazarowski et al. 2000). Resting levels of extracellular nucleotide tonically activate purinergic receptors and establish the set point for signal transduction pathways (Ostrom et al. 2000). Figure 1 Schematic model of signalling pathways implicated in swelling-induced ATP release The present findings provide no clues as to the pathway of ATP release, and several may operate in the same cell. For example, basal ATP release could result from exocytosis during constitutive membrane recycling. Stimulated release could, in addition, involve ATP transporter(s) analogous to those of the inner mitochondrial membrane, or perhaps ATP-permeable channels analogous to mitochondrial porin VDAC. Indeed, ATP channels have been implicated in cellular ATP release, but remain to be identified (Braunstein et al. 2001). The tyrosine kinase and Rho-kinase pathways studied by Koyama et al. (2001) are also known to regulate volume-sensitive Cl− channels (Nilius et al. 1999). Cl− channels are unlikely to provide the pathway for swelling-induced ATP release, however, since Hazama et al. (1999) clearly showed that, at least in intestinal cells, the ATP release pathway was distinct from volume-sensitive Cl− channels. This work by Koyama et al. (2001) greatly strengthens the notion that mechanically induced ATP efflux is an important, cell-regulated process. Identifying the ATP efflux pathway at the molecular level should provide mechanistic insight into how these signalling pathways control ATP release.

Low concentrations of 17β-estradiol protect single cardiac cells against metabolic stress-induced Ca2+ loading

OBJECTIVESThe main objective of the present study was to determine whether low physiological levels of estrogen directly protect cardiac cells against metabolic stress.BACKGROUNDThe beneficial effect of estrogens on the cardiovascular system has been traditionally ascribed to decrease in peripheral vascular resistance and to an antiatherogenic action. Whether physiological concentrations of 17β-estradiol (E2) are also able to protect cardiomyocytes against metabolic insult directly is unknown.METHODSIsolated ventricular cardiomyocytes were loaded with the Ca2+-sensitive fluorescent dye Fluo-3 and imaged by a digital epifluorescence imaging system. In cardiac cells preincubated with hormones and/or drugs for 8 h, metabolic stress was induced by addition and removal of 2,4-dinitrophenol (DNP).RESULTSIn cardiomyocytes, a 3-min-long exposure to chemical hypoxia, followed by reoxygenation, produced intracellular Ca2+ loading independently of gender (female: 729 ± 88 nmol/liter; male: 778 ± 97 nmol/liter). Pretreatment with E2 (10 nmol/liter) significantly reduced the magnitude of hypoxia/reoxygenation-induced Ca2+ loading in female (E2-treated: 298 ± 39 nmol/liter; untreated: 729 ± 88 nmol/liter), but not in male (E2-treated: 1029 ± 177 nmol/liter; untreated: 778 ± 97 nmol/liter) cardiac cells. The protective action of E2 was not mimicked by the inactive estrogen stereoisomer, 10 nmol/liter 17α estradiol (17α estradiol-treated: 886 ± 122 nmol/liter; untreated: 729 ± 88 nmol/liter), and was abolished by tamoxifen (1 μmol/liter), which acts as an antagonist of E2 on estrogen receptors (E2 plus tamoxifen-treated: 702 ± 98 nmol/liter; untreated: 729 ± 88 nmol/liter).CONCLUSIONSIn a gender-dependent manner, E2 directly protects cardiac cells against hypoxia-reoxygenation injury through an estrogen receptor–mediated mechanism. Such property of E2 may contribute to cardioprotection in the female gender.

Hypotonic stress-induced dual Ca(2+) responses in bovine aortic endothelial cells.

We have investigated the effects of hypotonic stress on intracellular calcium concentration ([Ca(2+)](i)) in bovine aortic endothelial cells. Reducing extracellular osmolarity by 5% to 40% elicited a steep Ca(2+) transient both in normal Krebs and Ca(2+)-free solutions. The hypotonic stress-induced Ca(2+) transient was inhibited by phospholipase C inhibitors (neomycin and U-73122), a P(2)-receptor antagonist (suramin), and an ATP-hydrolyzing enzyme (apyrase), suggesting that the hypotonic stress-induced Ca(2+) transient is mediated by ATP. A luciferin-luciferase assay confirmed that 40% hypotonic stress released 91.0 amol/cell of ATP in 10 min. When the hypotonic stress-induced fast Ca(2+) transient was inhibited by neomycin, suramin, or apyrase, a gradual [Ca(2+)](i) increase was observed instead. This hypotonic stress-induced gradual [Ca(2+)](i) increase was inhibited by a phospholipase A(2) inhibitor, 4-bromophenacyl bromide. Furthermore, exogenously applied arachidonic acid induced a gradual [Ca(2+)](i) increase with an ED(50) of 13.3 microM. These observations indicate that hypotonic stress induces a dual Ca(2+) response in bovine aortic endothelial cells, i.e., an ATP-mediated fast Ca(2+) transient and an arachidonic acid-mediated gradual Ca(2+) increase, the former being the predominant response in normal conditions.

Modulation of calmodulin by UV and X-rays in primary human endothelial cell cultures

Purpose : Previous studies by the present authors and others have shown that the expression of many genes is modulated by radiation. The purpose of this study is to identify additional genes that are affected by UV and X-radiation. Identification of specific genes affected by radiation may allow the determination of pathways important in radiation responses as well as an examination of transcriptional elements that are involved in the process. Materials and methods : A modified differential display approach coupled with sequencing was used to identify genes that are modulated in response to UV and ionizing radiation, and Northern blot analysis was used to confirm specific gene modulation. Results : Treatment of human primary umbilical vein endothelial cells with UV radiation resulted in the differential expression of several genes. Sequencing of the bands revealed that one of these was calmodulin. There was a 30% reduction in accumulation of calmodulin-specific mRNA 1h post UV exposure, and a 50% decrease 3h after treatment. X-rays also repressed accumulation of calmodium mRNA. Radiation exposure of HeLa cells also resulted in a decrease in expression of this gene. Conclusions : UV and ionizing radiations cause a decrease in accumulation of calmodulin transcripts in the first 1-3 h following exposure. Repression of calmodium mRNA levels may be one mechanism of stress-induced intracellular Ca 2+ modulation.

Mechanical stress-induced Ca2+ entry and Cl- current in cultured human aortic endothelial cells.

A fluid stream through a microtube was applied to cultured human aortic endothelial cells to investigate the endothelial responses of both the ionic currents and intracellular Ca2+ concentration ([Ca2+]i) to mechanical stimulation. The fluid stream induced an increase in [Ca2+]i that was dependent on both the flow rate and the extracellular Ca2+ concentration. Gd3+ and niflumic acid inhibited the fluid stream-induced increase in [Ca2+]i, whereas Ba2+ and tetraethylammonium ion exhibited no effect. The fluid stream-induced [Ca2+]i increase was accompanied by the activation of an inward current at -52.8 mV. The reversal potential of the fluid stream-induced current shifted to positive potentials when the external Cl- concentration was reduced but was not affected by variation of the external Na+ concentration. During the exposure to the fluid stream, [Ca2+]i was voltage dependent, i.e., depolarization decreased [Ca2+]i. We therefore conclude that the fluid stream-induced current is largely carried by Cl- and that the Cl- current may thus play a role in modulating the Ca2+ influx by altering the membrane potential of endothelial cells.

A history of stress alters drought calcium signalling pathways in Arabidopsis.

Environmental stresses commonly encountered by plants lead to rapid transient elevations in cytosolic free calcium concentration ([Ca2+]cyt) (Bush, 1995; Knight et al., 1991). These cellular calcium (Ca2+) signals lead ultimately to the increased expression of stress-responsive genes, including those encoding proteins of protective function (Knight et al., 1996; Knight et al., 1997). The kinetics and magnitude of the Ca2+ signal, or 'calcium signature', differ between different stimuli and are thought to contribute to the specificity of the end response (Dolmetsch et al., 1997; McAinsh and Hetherington, 1998). We measured [Ca2+]cyt changes during treatment with mannitol (to mimic drought stress) in whole intact seedlings of Arabidopsis thaliana. The responses of plants which were previously exposed to osmotic and oxidative stresses were compared to those of control plants. We show here that osmotic stress-induced Ca2+ responses can be markedly altered by previous encounters with either osmotic or oxidative stress. The nature of the alterations in Ca2+ response depends on the identity and severity of the previous stress: oxidative stress pre-treatment reduced the mannitol-induced [Ca2+]cyt response whereas osmotic stress pretreatment increased the [Ca2+]cyt response. Therefore, our data show that different combinations of environmental stress can produce novel Ca2+ signal outputs. These alterations are accompanied by corresponding changes in the patterns of osmotic stress-induced gene expression and, in the case of osmotic stress pre-treatment, the acquisition of stress-tolerance. This suggests that altered Ca2+ responses encode a 'memory' of previous stress encounters and thus may perhaps be involved in acclimation to environmental stresses.

P-428 - Mechanical stress-induced Ca2+ influx from local region of basement membrane in the presence of lysophosphatidic acid in cultured lens epithelial cells

P-428 - Mechanical stress-induced Ca2+ influx from local region of basement membrane in the presence of lysophosphatidic acid in cultured lens epithelial cells

Lysophosphatidic acid sensitizes mechanical stress-induced Ca 2+ response via activation of phospholipase C and tyrosine kinase in cultured smooth muscle cells

We previously reported that lysophosphatidic acid (LPA) sensitized mechanical stress-induced intracellular free Ca 2+ concentration response (Biochem. Biophys. Res. Commun. 208, 19–25, 1995). In the present study, the signal transduction pathway of the sensitizing effect of LPA was investigated in cultured longitudinal muscle cells from guinea pig ileum. Suramin, a putative LPA receptor antagonist, did not affect the response in the presence of 30 nM LPA, suggesting that the response is induced via activation of suramin-insensitive LPA receptor. Neither pertussis toxin nor wortmannin inhibited the LPA-sensitized response, indicating that G i/o − and phosphatidylinositol 3-kinase (PI3-kinase)-mediated pathways are not involved in the sensitizing effect. C3 ADP ribosyltransferase had no effect on the response, whereas formation of actin-stress fiber in the presence of LPA was completely inhibited, suggesting rho-related cytoskeletal change is not involved in the response. In contrast, a phospholipase C (PLC) inhibitor, U73122, completely inhibited the response, but broad spectrum kinase inhibitors, staurosporine and H7, had no effect on the response. In addition, tyrosine kinase inhibitor, genistein, but not tyrphostin partially inhibited the response. These results suggest that LPA sensitizes the mechanical stress-induced response via activation of PLC, but not protein kinase C. Additionally, tyrphostin-insensitive tyrosine kinase, which is related to other pathway than G i/o − and rho-mediated pathways, may be involved in the response.

Syk-dependent and -independent Signaling Cascades in B Cells Elicited by Osmotic and Oxidative Stress

It was found that Syk protein-tyrosine kinase is rapidly activated in B cells after H2O2 treatment (oxidative stress) or increased extracellular NaCl concentration (osmotic stress) as well as in response to B cell receptor activation. In this study we examined the involvement of Syk in responses elicited by these types of extracellular stress, particularly Ca2+ responses and c-Jun amino-terminal kinase (JNK) activation, using a chicken B cell line, DT40, as well as the DT40-derived mutant DT40/Syk(-), which does not express Syk. Osmotic stress evokes increases in [Ca2+]i by stimulating an extracellular Ca2+ influx in both DT40 and DT40/Syk(-) cells. In comparison, oxidative stress elicits an increase in [Ca2+]i by stimulating both an extracellular Ca2+ influx and Ca2+ release from internal stores in DT40 cells, but this Ca2+ response is partially abolished in DT40/Syk(-) cells, indicating that the oxidative stress-induced Ca2+ response is at least partly dependent on Syk. Interestingly, the depletion of Ca2+ results in a significantly decreased level of Syk activation in DT40 cells stimulated by oxidative but not osmotic stress. Furthermore, JNK is activated to different extents by these two types of stress. The extent of JNK activation in DT40/Syk(-) cells in response to osmotic stress is comparable to that observed in DT40 cells. Intriguingly, oxidative stress-induced JNK activation is significantly compromised in DT40/Syk(-) cells. Collectively, these results indicate that both the Ca2+ response and JNK activity induced by oxidative stress are partly dependent on Syk, whereas those induced by osmotic stress are independent of Syk.