Cytosolic Free Ca Concentration Research Articles

Calcium oscillations in HEK293 cells lacking SOCE suggest the existence of a balanced regulation of IP3 production and degradation

The concentration of free cytosolic Ca2+ is a critical second messenger in almost every cell type, with the signal often being carried by the period of oscillations, or spikes, in the cytosolic Ca2+ concentration. We have previously studied how Ca2+ influx across the plasma membrane affects the period and shape of Ca2+ oscillations in HEK293 cells. However, our theoretical work was unable to explain how the shape of Ca2+ oscillations could change qualitatively, from thin spikes to broad oscillations, during the course of a single time series. Such qualitative changes in oscillation shape are a common feature of HEK293 cells in which STIM1 and 2 have been knocked out. Here, we present an extended version of our earlier model that suggests that such time-dependent qualitative changes in oscillation shape might be the result of balanced positive and negative feedback from Ca2+ to the production and degradation of inositol trisphosphate.

Calcium-dependent subquantal peptide release from single docked lawn-resident vesicles of pituitary lactotrophs

Regulated exocytosis consists of the fusion between vesicles and the plasma membranes, leading to the formation of a narrow fusion pore through which secretions exit the vesicle lumen into the extracellular space. An increase in the cytosolic concentration of free Ca2+ ([Ca2+]i) is considered the stimulus of this process. However, whether this mechanism can be preserved in a simplified system of membrane lawns with docked secretory vesicles, devoid of cellular components, is poorly understood. Here, we studied peptide discharge from individual secretory vesicles docked at the plasma membrane, prepared from primary endocrine pituitary cells (the lactotrophs), releasing hormone prolactin. To label secretory vesicles, we transfected lactotrophs to express the fluorescent atrial natriuretic peptide (ANP.emd), previously shown to be expressed in and released from prolactin-containing vesicles. We used stimulating solutions containing different [Ca2+] to evoke vesicle peptide discharge, which appeared similar in membrane lawns and in intact stimulated lactotrophs. All vesicles examined discharged peptides in a subquantal manner, either exhibiting a unitary or sequential time course. In the membrane lawns, the unitary vesicle peptide discharge was predominant and slightly slower than that recorded in intact cells, but with a shorter delay with respect to the stimulation onset. This study revealed directly that Ca2+ triggers peptide discharge from docked single vesicles in the membrane lawns with a half-maximal response of ∼8 µM [Ca2+], consistent with previous whole-cell patch-clamp studies in endocrine cells where the rapid component of exocytosis, interpreted to represent docked vesicles, was fully activated at <10 µM [Ca2+]. Interestingly, the sequential subquantal peptide vesicle discharge indicates that fluctuations between constricted and dilated fusion pore states are preserved in membrane lawns and that fusion pore regulation appears to be an autonomously controlled process.

Cellular messengers involved in the inhibition of the Arabidopsis primary root growth by bacterial quorum-sensing signal N-decanoyl-L-homoserine lactone

BackgroundN-acyl-homoserine lactones (AHLs) are used as quorum-sensing signals by Gram-negative bacteria, but they can also affect plant growth and disease resistance. N-decanoyl-L-homoserine lactone (C10-HSL) is an AHL that has been shown to inhibit primary root growth in Arabidopsis, but the mechanisms underlying its effects on root architecture are unclear. Here, we investigated the signaling components involved in C10-HSL-mediated inhibition of primary root growth in Arabidopsis, and their interplay, using pharmacological, physiological, and genetic approaches.ResultsTreatment with C10-HSL triggered a transient and immediate increase in the concentrations of cytosolic free Ca2+ and reactive oxygen species (ROS), increased the activity of mitogen-activated protein kinase 6 (MPK6), and induced nitric oxide (NO) production in Arabidopsis roots. Inhibitors of Ca2+ channels significantly alleviated the inhibitory effect of C10-HSL on primary root growth and reduced the amounts of ROS and NO generated in response to C10-HSL. Inhibition or scavenging of ROS and NO neutralized the inhibitory effect of C10-HSL on primary root growth. In terms of primary root growth, the respiratory burst oxidase homolog mutants and a NO synthase mutant were less sensitive to C10-HSL than wild type. Activation of MPKs, especially MPK6, was required for C10-HSL to inhibit primary root growth. The mpk6 mutant showed reduced sensitivity of primary root growth to C10-HSL, suggesting that MPK6 plays a key role in the inhibition of primary root growth by C10-HSL.ConclusionOur results indicate that MPK6 acts downstream of ROS and upstream of NO in the response to C10-HSL. Our data also suggest that Ca2+, ROS, MPK6, and NO are all involved in the response to C10-HSL, and may participate in the cascade leading to C10-HSL-inhibited primary root growth in Arabidopsis.

Neutrophil priming that turns natural FFA2R agonists into potent activators of the superoxide generating NADPH-oxidase

Acetate, an agonist for the free fatty acid receptor 2 (FFA2R/GPR43), triggers an increase in the cytosolic concentration of free Ca2+ in neutrophils without any assembly of the superoxide generating NADPH-oxidase. We show that the phenylacetamide compound 58 (Cmp 58; (S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide), lacking a direct activating effect on neutrophils, acts as a positive FFA2R modulator that turns acetate into a potent activating agonist that triggers an assembly of the NADPH-oxidase. The NADPH-oxidase activity could be further increased in neutrophils treated with the pro-inflammatory cytokine TNF-α. Many neutrophil chemoattractant receptors are stored in secretory organelles but no FFA2R mobilization was induced in neutrophils treated with TNF-α. The receptor selectivity was demonstrated through the inhibition of the neutrophil response induced by the combined action of acetate and Cmp 58 by the FFA2R antagonist CATPB. Receptor modulators that positively co-operate with natural FFA2R agonists and prime neutrophils in their response to such agonists, may serve as good tools for further unraveling the physiological functions of FFA2R and its involvement in various diseases. In this study, we show that neutrophils primed with a presumed allosteric FFA2R modulator produce increased amounts of reactive oxygen species when activated by receptor specific agonists.

Circadian oscillations of cytosolic free calcium regulate the Arabidopsis circadian clock.

In the last decade, the view of circadian oscillators has expanded from transcriptional feedback to incorporate post-transcriptional, post-translational, metabolic processes and ionic signalling. In plants and animals, there are circadian oscillations in the concentration of cytosolic-free Ca2+ ([Ca2+]cyt), though their purpose has not been fully characterised. We investigated whether circadian oscillations of [Ca2+]cyt regulate the circadian oscillator of Arabidopsis thaliana. We report that in Arabidopsis, [Ca2+]cyt circadian oscillations can regulate circadian clock function through the Ca2+-dependent action of CALMODULIN-LIKE24 (CML24). Genetic analyses demonstrate a linkage between CML24 and the circadian oscillator, through pathways involving the circadian oscillator gene TIMING OF CAB2 EXPRESSION1 (TOC1).

Pathogenic role of ion channels in pulmonary arterial hypertension.

Pathogenic role of ion channels in pulmonary arterial hypertension.

Picomolar, selective, and subtype-specific small-molecule inhibition of TRPC1/4/5 channels

The concentration of free cytosolic Ca2+ and the voltage across the plasma membrane are major determinants of cell function. Ca2+-permeable non-selective cationic channels are known to regulate these parameters, but understanding of these channels remains inadequate. Here we focus on transient receptor potential canonical 4 and 5 proteins (TRPC4 and TRPC5), which assemble as homomers or heteromerize with TRPC1 to form Ca2+-permeable non-selective cationic channels in many mammalian cell types. Multiple roles have been suggested, including in epilepsy, innate fear, pain, and cardiac remodeling, but limitations in tools to probe these channels have restricted progress. A key question is whether we can overcome these limitations and develop tools that are high-quality, reliable, easy to use, and readily accessible for all investigators. Here, through chemical synthesis and studies of native and overexpressed channels by Ca2+ and patch-clamp assays, we describe compound 31, a remarkable small-molecule inhibitor of TRPC1/4/5 channels. Its potency ranged from 9 to 1300 pm, depending on the TRPC1/4/5 subtype and activation mechanism. Other channel types investigated were unaffected, including TRPC3, TRPC6, TRPV1, TRPV4, TRPA1, TRPM2, TRPM8, and store-operated Ca2+ entry mediated by Orai1. These findings suggest identification of an important experimental tool compound, which has much higher potency for inhibiting TRPC1/4/5 channels than previously reported agents, impressive specificity, and graded subtype selectivity within the TRPC1/4/5 channel family. The compound should greatly facilitate future studies of these ion channels. We suggest naming this TRPC1/4/5-inhibitory compound Pico145.

On the dynamical structure of calcium oscillations

Oscillations in the concentration of free cytosolic Ca2+ are an important and ubiquitous control mechanism in many cell types. It is thus correspondingly important to understand the mechanisms that underlie the control of these oscillations and how their period is determined. We show that Class I Ca2+ oscillations (i.e., oscillations that can occur at a constant concentration of inositol trisphosphate) have a common dynamical structure, irrespective of the oscillation period. This commonality allows the construction of a simple canonical model that incorporates this underlying dynamical behavior. Predictions from the model are tested, and confirmed, in three different cell types, with oscillation periods ranging over an order of magnitude. The model also predicts that Ca2+ oscillation period can be controlled by modulation of the rate of activation by Ca2+ of the inositol trisphosphate receptor. Preliminary experimental evidence consistent with this hypothesis is presented. Our canonical model has a structure similar to, but not identical to, the classic FitzHugh-Nagumo model. The characterization of variables by speed of evolution, as either fast or slow variables, changes over the course of a typical oscillation, leading to a model without globally defined fast and slow variables.

Effect of protein kinase C on glucose-mediated insulin secretion in HIT-T15 cells.

To clarify the regulation of protein kinase C on glucose-mediated insulin secretion. We examined the effect of protein kinase C on the cytosolic free Ca(2+) concentration ([Ca(2+)]i) and the activity of Ca(2+)-activated K(+) channels (K(Ca)-channel) in the insulinoma cell line, HIT-T15. Glucose at a concentration of 10 mmol/L increased the secretion of insulin. This increase was partly inhibited by 1 nmol/L staurosporine, a protein kinase C inhibitor. Staurosporine (1 nmol/L) also attenuated the glucose-induced elevations in [Ca(2+)]i. On the contrary, glibenclamide (100 nmol/L) specifically blocked ATP-sensitive K(+) channels, and increased both [Ca(2+)]i and insulin secretion, but staurosporine had no effect on them. Patch clamp studies showed that 10 mmol/L glucose almost completely blocked K(Ca) channel activity, an effect that was reversed by 1 nmol/L staurosporine. Phorbol 12-myristate 13-acetate (1 mmol/L), a protein kinase C activator, also decreased K(Ca) channel activity. These results indicate that the activation of protein kinase C is involved in the glucose-induced release of insulin by modulating K(+) channel function in HIT-T15 cells.

Ebselen alters cellular oxidative status and induces endoplasmic reticulum stress in rat hippocampal astrocytes.

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti-inflammatory effects. Because of its properties, it may be protective against injury to the nervous tissue. However, evidence suggests that its glutathione peroxidase activity could underlie certain deleterious actions on cell physiology. In this study we have analyzed the effect of ebselen on rat hippocampal astrocytes in culture. Cellular oxidative status, cytosolic free-Ca(2+) concentration ([Ca(2+)]c), setting of endoplasmic reticulum stress and phosphorylation of glial fibrillary acidic protein and major mitogen-activated protein kinases were analyzed. Our results show that ebselen induced a concentration-dependent increase in the generation of reactive oxygen species in the mitochondria. We observed a concentration-dependent increase in global cysteine oxidation and in the level of malondialdehyde in the presence of ebselen. We also detected increases in catalase, glutathione S-transferase and glutathione reductase activity. Ebselen also evoked a concentration-dependent increase in [Ca(2+)]c. Moreover, we observed a concentration-dependent increase in the phosphorylation of the unfolded protein response markers, eukaryotic translation initiation factor 2α and X-box binding protein 1. Finally, ebselen also induced an increase in the phosphorylation of glial fibrillary acidic protein, SAPK/JNK, p38 MAPK and p44/42 MAPK. Our results provide strong evidence that implicate endoplasmic reticulum stress and activation of crucial mitogen-activated protein kinases in an oxidative damage of cells in the presence of ebselen. The compound thus might exert deleterious actions on astrocyte physiology that could compromise their function.

NO-Mediated [Ca2+]cyt Increases Depend on ADP-Ribosyl Cyclase Activity in Arabidopsis

Cyclic ADP ribose (cADPR) is a Ca(2+)-mobilizing intracellular second messenger synthesized from NAD by ADP-ribosyl cyclases (ADPR cyclases). In animals, cADPR targets the ryanodine receptor present in the sarcoplasmic/endoplasmic reticulum to promote Ca(2+) release from intracellular stores to increase the concentration of cytosolic free Ca(2+) in Arabidopsis (Arabidopsis thaliana), and cADPR has been proposed to play a central role in signal transduction pathways evoked by the drought and stress hormone, abscisic acid, and the circadian clock. Despite evidence for the action of cADPR in Arabidopsis, no predicted proteins with significant similarity to the known ADPR cyclases have been reported in any plant genome database, suggesting either that there is a unique route for cADPR synthesis or that a homolog of ADPR cyclase with low similarity might exist in plants. We sought to determine whether the low levels of ADPR cyclase activity reported in Arabidopsis are indicative of a bona fide activity that can be associated with the regulation of Ca(2+) signaling. We adapted two different fluorescence-based assays to measure ADPR cyclase activity in Arabidopsis and found that this activity has the characteristics of a nucleotide cyclase that is activated by nitric oxide to increase cADPR and mobilize Ca(2.)

The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor.

The activity of calmodulin (CaM) is modulated not only by oscillations in the cytosolic concentration of free Ca(2+), but also by its phosphorylation status. In the present study, the role of tyrosine-phosphorylated CaM [P-(Tyr)-CaM] on the regulation of the epidermal growth factor receptor (EGFR) has been examined using invitro assay systems. We show that phosphorylation of CaM by rat liver solubilized EGFR leads to a dramatic increase in the subsequent phosphorylation of poly-L-(Glu:Tyr) (PGT) by the receptor in the presence of ligand, both in the absence and in the presence of Ca(2+). This occurred in contrast with assays where P-(Tyr)-CaM accumulation was prevented by the presence of Ca(2+), absence of a basic cofactor required for CaM phosphorylation and/or absence of CaM itself. Moreover, an antibody against CaM, which inhibits its phosphorylation, prevented the extra ligand-dependent EGFR activation. Addition of purified P-(Tyr)-CaM, phosphorylated by recombinant c-Src (cellular sarcoma kinase) and free of non-phosphorylated CaM, obtained by affinity-chromatography using an immobilized anti-phospho-(Tyr)-antibody, also increased the ligand-dependent tyrosine kinase activity of the isolated EGFR toward PGT. Also a CaM(Y99D/Y138D) mutant mimicked the effect of P-(Tyr)-CaM on ligand-dependent EGFR activation. Finally, we demonstrate that P-(Tyr)-CaM binds to the same site ((645)R-R-R-H-I-V-R-K-R-T-L-R-R-L-L-Q(660)) as non-phosphorylated CaM, located at the cytosolic juxtamembrane region of the EGFR. These results show that P-(Tyr)-CaM is an activator of the EGFR and suggest that it could contribute to the CaM-mediated ligand-dependent activation of the receptor that we previously reported in living cells.

P2Y2 receptor signaling in neutrophils is regulated from inside by a novel cytoskeleton-dependent mechanism

Functional selectivity, a process by which G-protein coupled receptors (GPCRs) can activate one signaling route while avoiding another, is regulated by ligand-mediated stabilization of specific receptor states that modulate different downstream signaling events. We propose a novel mechanism for functional selectivity, induced by the endogenous P2Y2R agonist ATP and regulated at the signaling interface by the cytoskeleton. Upon ATP stimulation of human neutrophils, a transient rise in the cytosolic concentration of free Ca2+ was not followed by activation of the superoxide anion-generating NADPH-oxidase. This was in contrast to signals generated through the formyl peptide receptor 1 (FPR1), as its activation was accompanied by both a mobilization of Ca2+ and activation of the NADPH-oxidase. The phospholipase C/Ca2+ signaling route is not modulated by the cytoskeleton-disrupting drug latrunculin A, but this drug was able to launch a new signaling route downstream of P2Y2R that led to NADPH-oxidase activation. The signaling downstream of P2Y2R was rapidly terminated and the receptors were desensitized; however, in contrast to desensitized FPR1, no P2Y2 receptor reactivation could be induced by latrunculin A. Thus, P2Y2R desensitization does not appear to involve the cytoskeleton, contrary to FPR1 desensitization. In summary, we hereby describe how ATP regulates functional selectivity via the cytoskeleton, leading to intracellular Ca2+ increase, alone or with simultaneous NADPH-oxidase activation in neutrophils.

Hypericum perforatum Attenuates Spinal Cord Injury-Induced Oxidative Stress and Apoptosis in the Dorsal Root Ganglion of Rats: Involvement of TRPM2 and TRPV1 Channels.

Oxidative stress and cytosolic Ca(2+) overload have important roles on apoptosis in dorsal root ganglion (DRG) neurons after spinal cord injury (SCI). Hypericum perforatum (HP) has an antioxidant property in the DRGs due to its ability to modulate NADPH oxidase and protein kinase C pathways. We aimed to investigate the protective property of HP on oxidative stress, apoptosis, and Ca(2+) entry through transient receptor potential melastatin 2 (TRPM2) and transient receptor potential vanilloid 1 (TRPV1) channels in SCI-induced DRG neurons of rats. Rats were divided into four groups as control, HP, SCI, and SCI + HP. The HP groups received 30mg/kg HP for three concessive days after SCI induction. The SCI-induced TRPM2 and TRPV1 currents and cytosolic free Ca(2+) concentration were reduced by HP. The SCI-induced decrease in glutathione peroxidase and cell viability values were ameliorated by HP treatment, and the SCI-induced increase in apoptosis, caspase 3, caspase 9, cytosolic reactive oxygen species (ROS) production, and mitochondrial membrane depolarization values in DRG of SCIgroup were overcome by HP treatment. In conclusion, we observed a protective role of HP on SCI-induced oxidative stress, apoptosis, and Ca(2+) entry through TRPM2 and TRPV1 in the DRG neurons. Our findings may be relevant to the etiology and treatment of SCI by HP. Graphical Abstract Possible molecular pathways of involvement of Hypericum perforatum (HP) on apoptosis, oxidative stress, and calcium accumulation through TRPM2 and TRPV1 channels in DRG neurons of SCI-induced rats. The TRPM2 channel is activated by ADP-ribose and oxidative stress through activation of ADP-ribose pyrophosphate although it was inhibited by N-(p-amylcinnamoyl) anthranilic acid (ACA) and 2-aminoethyl diphenylborinate (2APB). The TRPV1 channel is activated by oxidative stress and capsaicin and it is blocked by capsazepine. Injury in the DRG can result in augmented ROS release, leading to Ca(2+) uptake through TRPM2 and TRPV1 channels. Mitochondria were reported to accumulate Ca(2+), provided intracellular Ca(2+) rises, thereby leading to depolarization of mitochondrial membranes and release of apoptosis-inducing factors such as caspase 3 and caspase 9. HP via regulation of NADPH oxidase and PKC inhibits TRPM2 and TRPV1 channels. The molecular pathway may be a cause of SCI-induced pain and neuronal death, and the subject should be urgently investigated.

Ca2+/Calmodulin and Apo-Calmodulin Both Bind to and Enhance the Tyrosine Kinase Activity of c-Src.

Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca2+ signal generated in cells by different effectors, where the Ca2+-receptor protein calmodulin (CaM) plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca2+-dependent and Ca2+-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR), in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca2+/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca2+-free CaM (apo-CaM) exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca2+/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca2+ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred.

PKC-mediated inhibitory feedback of the cholecystokinin 1 receptor controls the shape of oscillatory Ca²⁺ signals.

Translation of extracellular hormonal input into cellular responses is often mediated by repetitive increases in cytosolic free Ca(2+) concentration ([Ca(2+) ]c ). Amplitude, duration and frequency of these so-called [Ca(2+) ]c oscillations then carry information about the nature and concentration of the extracellular signalling molecule. At present, there are different hypotheses concerning the induction and control of these oscillations. Here, we investigated the role of agonist-induced receptor phosphorylation in this process using Chinese hamster ovary cells stably expressing a variant of the cholecystokinin 1 receptor (CCK1R) lacking the four consensus sites for protein kinase C (PKC) phosphorylation and deficient in CCK-induced receptor phosphorylation (CCK1R-mt cells). In the presence of cholecystokinin-(26-33)-peptide amide (CCK-8), these cells displayed Ca(2+) oscillations with a much more pronounced bursting dynamics rather than the dominant spiking dynamics observed in Chinese hamster ovary cells stably expressing the wild-type CCK1R. The bursting behaviour returned to predominantly spiking behaviour following removal of extracellular Ca(2+) , suggesting that CCK-8-induced, PKC-mediated CCK1R phosphorylation inhibits Ca(2+) influx across the plasma membrane. To gain mechanistic insight into the underlying mechanism we developed a mathematical model able to reproduce the experimental observations. From the model we conclude that binding of CCK-8 to the CCK1R leads to activation of PKC which subsequently phosphorylates the receptor to inhibit the receptor-mediated influx of Ca(2+) across the plasma membrane. Receptor-specific differences in this feedback mechanism may, at least in part, explain the observation that different agonists evoke [Ca(2+) ]c oscillations with different kinetics in the same cell type.

Endoplasmic Reticulum Stress and Store-Operated Calcium Entry Contribute to Usnic Acid-Induced Toxicity in Hepatic Cells.

The use of usnic acid as a weight loss agent is a safety concern due to reports of acute liver failure in humans. Previously we demonstrated that usnic acid induces apoptosis and cytotoxicity in hepatic HepG2 cells. We also demonstrated that usnic acid induces autophagy as a survival mechanism against its cytotoxicity. In this study, we investigated and characterized further molecular mechanisms underlying the toxicity of usnic acid in HepG2 cells. We found that usnic acid causes endoplasmic reticulum (ER) stress demonstrated by the increased expression of typical ER stress markers, including CHOP, ATF-4, p-eIF2α, and spliced XBP1. Usnic acid inhibited the secretion of Gaussia luciferase measured by an ER stress reporter assay. An ER stress inhibitor 4-phenylbutyrate attenuated usnic acid-induced apoptosis. Moreover, usnic acid significantly increased the cytosolic free Ca(2+) concentration. Usnic acid increased the expression of calcium release-activated calcium channel protein 1 (CRAM1 or ORAI1) and stromal interaction molecule 1, two key components of store-operated calcium entry (SOCE), which is the major Ca(2+) influx pathway in non-excitable cells, this finding was also confirmed in primary rat hepatocytes. Furthermore, knockdown of ORAI1 prevented ER stress and ATP depletion in response to usnic acid. In contrast, overexpression of ORAI1 increased ER stress and ATP depletion caused by usnic acid. Taken together, our results suggest that usnic acid disturbs calcium homeostasis, induces ER stress, and that usnic acid-induced cellular damage occurs at least partially via activation of the Ca(2+) channel of SOCE.

Aequorin-based luminescence imaging reveals differential calcium signalling responses to salt and reactive oxygen species in rice roots.

It is well established that both salt and reactive oxygen species (ROS) stresses are able to increase the concentration of cytosolic free Ca(2+) ([Ca(2+)]i), which is caused by the flux of calcium (Ca(2+)). However, the differences between these two processes are largely unknown. Here, we introduced recombinant aequorin into rice (Oryza sativa) and examined the change in [Ca(2+)]i in response to salt and ROS stresses. The transgenic rice harbouring aequorin showed strong luminescence in roots when treated with exogenous Ca(2+). Considering the histological differences in roots between rice and Arabidopsis, we reappraised the discharging solution, and suggested that the percentage of ethanol should be 25%. Different concentrations of NaCl induced immediate [Ca(2+)]i spikes with the same durations and phases. In contrast, H₂O₂ induced delayed [Ca(2+)]i spikes with different peaks according to the concentrations of H₂O₂. According to the Ca(2+) inhibitor research, we also showed that the sources of Ca(2+) induced by NaCl and H₂O₂ are different. Furthermore, we evaluated the contribution of [Ca(2+)]i responses in the NaCl- and H₂O₂-induced gene expressions respectively, and present a Ca(2+)- and H₂O₂-mediated molecular signalling model for the initial response to NaCl in rice.

Multiple cellular roles of Neurospora crassa plc-1, splA2, and cpe-1 in regulation of cytosolic free calcium, carotenoid accumulation, stress responses, and acquisition of thermotolerance.

Phospholipase C1 (PLC1), secretory phospholipase A2 (sPLA2) and Ca(2+)/H(+) exchanger proteins regulate calcium signaling and homeostasis in eukaryotes. In this study, we investigate functions for phospholipase C1 (plc-1), sPLA2 (splA2) and a Ca(2+)/H(+) exchanger (cpe-1) in the filamentous fungus Neurospora crassa. The Δplc-1, ΔsplA2, and Δcpe-1 mutants exhibited a growth defect on medium supplemented with the divalent ionophore A23187, suggesting that these genes might play a role in regulation of cytosolic free Ca(2+) concentration ([Ca(2+)](c)) in N. crassa. The strains lacking plc-1, splA2, and cpe-1 possessed higher carotenoid content than wild type at 8°C, 22°C, and 30°C, and showed increased ultraviolet (UV)-survival under conditions that induced carotenoid accumulation. Moreover, Δplc-1, ΔsplA2, and Δcpe-1 mutants showed reduced survival rate under hydrogen peroxide-induced oxidative stress and induced thermotolerance after exposure to heat shock temperatures. Thus, this study revealed multiple cellular roles for plc-1, splA2, and cpe-1 genes in regulation of [Ca(2+)](c), carotenoid accumulation, survival under stress conditions, and acquisition of thermotolerance induced by heat shock.

Notch Activation of Ca(2+) Signaling in the Development of Hypoxic Pulmonary Vasoconstriction and Pulmonary Hypertension.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological response that optimizes the ventilation/perfusion ratio. Chronic hypoxia causes vascular remodeling, which is central to the pathogenesis of hypoxia-induced pulmonary hypertension (HPH). We have previously shown that Notch3 is up-regulated in HPH and that activation of Notch signaling enhances store-operated Ca(2+) entry (SOCE), an important mechanism that contributes to pulmonary arterial smooth muscle cell (PASMC) proliferation and contraction. Here, we investigate the role of Notch signaling in HPV and hypoxia-induced enhancement of SOCE. We examined SOCE in human PASMCs exposed to hypoxia and pulmonary arterial pressure in mice using the isolated perfused/ventilated lung method. Wild-type and canonical transient receptor potential (TRPC) 6(-/-) mice were exposed to chronic hypoxia to induce HPH. Inhibition of Notch signaling with a γ-secretase inhibitor attenuates hypoxia-enhanced SOCE in PASMCs and hypoxia-induced increase in pulmonary arterial pressure. Our results demonstrate that hypoxia activates Notch signaling and up-regulates TRPC6 channels. Additionally, treatment with a Notch ligand can mimic hypoxic responses. Finally, inhibition of TRPC6, either pharmacologically or genetically, attenuates HPV, hypoxia-enhanced SOCE, and the development of HPH. These results demonstrate that hypoxia-induced activation of Notch signaling mediates HPV and the development of HPH via functional activation and up-regulation of TRPC6 channels. Understanding the molecular mechanisms that regulate cytosolic free Ca(2+) concentration and PASMC proliferation is critical to elucidation of the pathogenesis of HPH. Targeting Notch regulation of TRPC6 will be beneficial in the development of novel therapies for pulmonary hypertension associated with hypoxia.