Endomembrane Ca Research Articles

Guard cell endomembrane Ca2+-ATPases underpin a 'carbon memory' of photosynthetic assimilation that impacts on water-use efficiency.

Stomata of most plants close to preserve water when the demand for CO2 by photosynthesis is reduced. Stomatal responses are slow compared with photosynthesis, and this kinetic difference erodes assimilation and water-use efficiency under fluctuating light. Despite a deep knowledge of guard cells that regulate the stoma, efforts to enhance stomatal kinetics are limited by our understanding of its control by foliar CO2. Guided by mechanistic modelling that incorporates foliar CO2 diffusion and mesophyll photosynthesis, here we uncover a central role for endomembrane Ca2+ stores in guard cell responsiveness to fluctuating light and CO2. Modelling predicted and experiments demonstrated a delay in Ca2+ cycling that was enhanced by endomembrane Ca2+-ATPase mutants, altering stomatal conductance and reducing assimilation and water-use efficiency. Our findings illustrate the power of modelling to bridge the gap from the guard cell to whole-plant photosynthesis, and they demonstrate an unforeseen latency, or 'carbon memory', of guard cells that affects stomatal dynamics, photosynthesis and water-use efficiency.

The Cycling of Intracellular Calcium Released in Response to Fluid Shear Stress Is Critical for Migration-Associated Actin Reorganization in Eosinophils.

The magnitude of eosinophil mobilization into respiratory tissues drives the severity of inflammation in several airway diseases. In classical models of leukocyte extravasation, surface integrins undergo conformational switches to high-affinity states via chemokine binding activation. Recently, we learned that eosinophil integrins possess mechanosensitive properties that detect fluid shear stress, which alone was sufficient to induce activation. This mechanical stimulus triggered intracellular calcium release and hallmark migration-associated cytoskeletal reorganization including flattening for increased cell–substratum contact area and pseudopodia formation. The present study utilized confocal fluorescence microscopy to investigate the effects of pharmacological inhibitors to calcium signaling and actin polymerization pathways on shear stress-induced migration in vitro. Morphological changes (cell elongation, membrane protrusions) succeeded the calcium flux in untreated eosinophils within 2 min, suggesting that calcium signaling was upstream of actin cytoskeleton rearrangement. The inhibition of ryanodine receptors and endomembrane Ca2+-ATPases corroborated this idea, indicated by a significant increase in time between the calcium spike and actin polymerization. The impact of the temporal link is evident as the capacity of treated eosinophils to move across fibronectin-coated surfaces was significantly hampered relative to untreated eosinophils. Furthermore, we determined that the nature of cellular motility in response to fluid shear stress was nondirectional.

Dissecting blue light signal transduction pathway in leaf epidermis using a pharmacological approach.

Blue light signalling pathway in broad bean leaf epidermal cells includes key membrane transporters: plasma- and endomembrane channels and pumps of H (+) , Ca (2+) and K (+) ions, and plasma membrane redox system. Blue light signalling pathway in epidermal tissue isolated from the abaxial side of fully developed Vicia faba leaves was dissected by measuring the effect of inhibitors of second messengers on net K(+), Ca(2+) and H(+) fluxes using non-invasive ion-selective microelectrodes (the MIFE system). Switching the blue light on-off caused transient changes of the ion fluxes. The effects of seven groups of inhibitors were tested in this study: CaM antagonists, ATPase inhibitors, Ca(2+) anatagonists or chelators, agents affecting IP3 formation, redox system inhibitors, inhibitors of endomembrane Ca(2+) transport systems and an inhibitor of plasma membrane Ca(2+)-permeable channels. Most of the inhibitors had a significant effect on steady-state (basal) net fluxes, as well as on the magnitude of the transient ion flux responses to blue light fluctuations. The data presented in this study suggest that redox signalling and, specifically, plasma membrane NADPH oxidase and coupled Ca(2+) and K(+) fluxes play an essential role in blue light signal transduction.

RNA‐seq analysis identifies potential modulators of gravity response in spores of Ceratopteris (Parkeriaceae): Evidence for modulation by calcium pumps and apyrase activity

Gravity regulates the magnitude and direction of a trans-cell calcium current in germinating spores of Ceratopteris richardii. Blocking this current with nifedipine blocks the spore's downward polarity alignment, a polarization that is fixed by gravity ∼10 h after light induces the spores to germinate. RNA-seq analysis at 10 h was used to identify genes potentially important for the gravity response. The data set will be valuable for other developmental and phylogenetic studies. De novo Newbler assembly of 958 527 reads from Roche 454 sequencing was executed. The sequences were identified and analyzed using in silico methods. The roles of endomembrane Ca(2+)-ATPase pumps and apyrases in the gravity response were further tested using pharmacological agents. Transcripts related to calcium signaling and ethylene biosynthesis were identified as notable constituents of the transcriptome. Inhibiting the activity of endomembrane Ca(2+)-ATPase pumps with 2,5-di-(t-butyl)-1,4-hydroquinone diminished the trans-cell current, but increased the orientation of the polar axis to gravity. The effects of applied nucleotides and purinoceptor antagonists gave novel evidence implicating extracellular nucleotides as regulators of the gravity response in these fern spores. In addition to revealing general features of the transcriptome of germinating spores, the results highlight a number of calcium-responsive and light-receptive transcripts. Pharmacologic assays indicate endomembrane Ca(2+)-ATPases and extracellular nucleotides may play regulatory roles in the gravity response of Ceratopteris spores.

Endomembrane Ca2+-ATPases play a significant role in virus-induced adaptation to oxidative stress

Although the role of Ca2+ influx channels in oxidative stress signaling and cross-tolerance in plants is well established, little is known about the role of active Ca2+ efflux systems in this process. In our recent paper,17 we reported Potato Virus X (PVX)-induced acquired resistance to oxidative stress in Nicotiana benthamiana and showed the critical role of plasma membrane Ca2+/H+ exchangers in this process. The current study continues this research. Using biochemical and electrophysiological approaches, we reveal that both endomembrane P2A and P2B Ca2+-ATPases play significant roles in adaptive responses to oxidative stress by removing excessive Ca2+ from the cytosol, and that their functional expression is significantly altered in PVX-inoculated plants. These findings highlight the crucial role of Ca2+ efflux systems in acquired tolerance to oxidative stress and open up prospects for practical applications in agriculture, after in-depth comprehension of the fundamental mechanisms involved in common responses to environmental factors at the genomic, cellular and organismal levels.

Plant volatiles regulate the activities of Ca2+-permeable channels and promote cytoplasmic calcium transients in Arabidopsis leaf cells

A variety of plant species emit volatile compounds in response to mechanical stresses such as herbivore attack. Although these volatile compounds promote gene expression leading to anti-herbivore responses, the underlying transduction mechanisms are largely unknown. While indirect evidence suggests that the cytoplasmic free Ca2+ concentration ([Ca2+]c) plays a crucial role in the volatile-sensing mechanisms in plants, these roles have not been directly demonstrated. In the present study, we used Arabidopsis leaves expressing apoaequorin, a Ca2+-sensitive luminescent protein, in combination with a luminometer, to monitor [Ca2+]c transients that occur in response to a variety of volatile compounds and to characterized the pharmacological properties of the increase in [Ca2+]c. When leaves were exposed to volatiles, [Ca2+]c was transiently raised. The [Ca2+]c increases induced by acyclic compounds were disrupted by Ruthenium Red, a potential plasma-membrane and endo-membrane Ca2+-permeable channel inhibitor, but not by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), an extracellular Ca2+-chelator, suggesting that acyclic compounds promote Ca2+-release from intracellular stores. On the other hand, the electrophilic compound (E)-2-hexenal promoted Ca2+-influx via ROS production by natural oxidation at the aquarius phase. In a gpa1-2 mutant lacking a canonical Gα subunit, the [Ca2+]c transients induced by all tested volatiles were not attenuated, suggesting that G-protein coupled receptors are not involved in the volatile-induced [Ca2+]c transients in Arabidopsis leaves.

Interaction between Arabidopsis Ca2+/H+ Exchangers CAX1 and CAX3

In plants, high capacity tonoplast cation/H(+) antiport is mediated in part by a family of CAX (cation exchanger) transporters. Functional association between CAX1 and CAX3 has previously been inferred; however, the nature of this interaction has not been established. Here we analyze the formation of "hetero-CAX" complexes and their transport properties. Co-expressing both CAX1 and CAX3 mediated lithium and salt tolerance in yeast, and these phenotypes could not be recapitulated by expression of deregulated versions of either transporter. Coincident expression of Arabidopsis CAX1 and CAX3 occurs during particular stress responses, flowering, and seedling growth. Analysis of cax1, cax3, and cax1/3 seedlings demonstrated similar stress sensitivities. When plants expressed high levels of both CAXs, alterations in transport properties were evident that could not be recapitulated by high level expression of either transporter individually. In planta coimmunoprecipitation suggested that a protein-protein interaction occurred between CAX1 and CAX3. In vivo interaction between the CAX proteins was shown using a split ubiquitin yeast two-hybrid system and gel shift assays. These findings demonstrate cation exchange plasticity through hetero-CAX interactions.

A Plant Ca2+ Pump, ACA2, Relieves Salt Hypersensitivity in Yeast: MODULATION OF CYTOSOLIC CALCIUM SIGNATURE AND ACTIVATION OF ADAPTIVE Na+HOMEOSTASIS

Stress responses in both plants and yeast utilize calcium-mediated signaling. A yeast strain, K616, which lacks Ca(2+) pumps, requires micromolar Ca(2+) for growth. In medium containing 100 microM Ca(2+), K616 can withstand osmotic stress (750 mM sorbitol) and ionic stress (300 mM KCl) but not hypersodic stress (300 mM NaCl). Heterologous expression of the endoplasmic reticulum-located Arabidopsis thaliana Ca(2+)-ATPase, ACA2, permits K616 to grow under NaCl stress even in Ca(2+)-depleted medium. All stresses tested generated transient elevation of cytosolic Ca(2+) in wild type yeast, K601, whereas NaCl alone induced prolonged elevation of cytosolic Ca(2+) in K616. Both the Ca(2+) transient and survival of cultures subjected to NaCl stress was similar for the ACA2 transformant and K601. However, whereas K601 maintained low cytosolic Na(+) predominantly by pumping it out across the plasma membrane, the transformant sequestered Na(+) in internal organelles. This sequestration requires the presence of an endomembrane Na(+)/H(+)-antiporter, NHX1, which does not play a significant role in salt tolerance of wild type yeast except at acidic pH. Transcript levels of the plasma membrane Na(+)-ATPase, ENA1, were strongly induced only in K601, whereas NHX1 was strongly induced in both K601 and the ACA2 transformant. The calmodulin kinase inhibitor KN62 significantly reduced the salt tolerance of the ACA2 transformant and the transcriptional induction of NHX1. Thus, the heterologous expression of a plant endomembrane Ca(2+) pump results in the rapid depletion of cytosolic Ca(2+) and the activation of an alternate mechanism for surviving saline stress.

Cytoplasmic calcium increases in response to changes in the gravity vector in hypocotyls and petioles of Arabidopsis seedlings.

Plants respond to a large variety of environmental signals, including changes in the gravity vector (gravistimulation). In Arabidopsis (Arabidopsis thaliana) seedlings, gravistimulation is known to increase the cytoplasmic free calcium concentration ([Ca(2+)](c)). However, organs responsible for the [Ca(2+)](c) increase and the underlying cellular/molecular mechanisms remain to be solved. In this study, using Arabidopsis seedlings expressing apoaequorin, a Ca(2+)-sensitive luminescent protein in combination with an ultrasensitive photon counting camera, we clarified the organs where [Ca(2+)](c) increases in response to gravistimulation and characterized the physiological and pharmacological properties of the [Ca(2+)](c) increase. When the seedlings were gravistimulated by turning 180 degrees, they showed a transient biphasic [Ca(2+)](c) increase in their hypocotyls and petioles. The second peak of the [Ca(2+)](c) increase depended on the angle but not the speed of rotation, whereas the initial peak showed diametrically opposite characters. This suggests that the second [Ca(2+)](c) increase is specific for changes in the gravity vector. The potential mechanosensitive Ca(2+)-permeable channel (MSCC) inhibitors Gd(3+) and La(3+), the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and the endomembrane Ca(2+)-permeable channel inhibitor ruthenium red suppressed the second [Ca(2+)](c) increase, suggesting that it arises from Ca(2+) influx via putative MSCCs in the plasma membrane and Ca(2+) release from intracellular Ca(2+) stores. Moreover, the second [Ca(2+)](c) increase was attenuated by actin-disrupting drugs cytochalasin B and latrunculin B but not by microtubule-disrupting drugs oryzalin and nocodazole, implying that actin filaments are partially involved in the hypothetical activation of Ca(2+)-permeable channels. These results suggest that the second [Ca(2+)](c) increase via MSCCs is a gravity response in the hypocotyl and petiole of Arabidopsis seedlings.

Taming the neutrophil: calcium clearance and influx mechanisms as novel targets for pharmacological control

Neutrophils are relatively insensitive to the anti-inflammatory actions of conventional chemotherapeutic agents, including corticosteroids, emphasizing the requirement for novel pharmacological strategies to control the potentially harmful proinflammatory activities of these cells. In the case of commonly-occurring inflammatory diseases of the airways, the neutrophil is the primary mediator of inflammation in conditions such as chronic obstructive pulmonary disease, cystic fibrosis, acute respiratory distress syndrome, bronchiectasis and non-eosinophilic bronchial asthma. Recent insights into the mechanisms utilized by neutrophils to restore Ca(2+) homeostasis following activation with Ca(2+)-mobilizing, proinflammatory stimuli have facilitated the identification of novel targets for anti-inflammatory chemotherapy in these cells. The most amenable of these from a chemotherapeutic perspective, is the cyclic AMP-dependent protein kinase-modulated endomembrane Ca(2+)-ATPase which promotes clearance of the cation from the cytosol of activated neutrophils. Second generation type 4 phosphodiesterase inhibitors and adenosine receptor agonists operative at the level of subtype A2A adenosine receptors, which are currently undergoing clinical and preclinical assessment respectively, hold promise as pharmacologic modulators during the restoration of Ca(2+) homeostasis. If this promise is realized, it may result in novel chemotherapeutic strategies for the control of hyperacute and chronic inflammatory conditions in which neutrophils are primary offenders. Alternative, potential future targets include the Na(+), Ca(2+)-exchanger and store-operated Ca(2+) channels, which cooperate in the refilling of intracellular Ca(2+) stores.

Investigation into the relationship between calyculin A-mediated potentiation of NADPH oxidase activity and inhibition of store-operated uptake of calcium by human neutrophils

The primary objective of the current study was to investigate possible relationships between calyculin A (CA)-mediated potentiation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and inhibition of store-operated uptake of Ca 2+ by chemoattractant-activated human neutrophils. Treatment of neutrophils with 100 nM CA, but not at lower concentrations (12.5–50 nM), prior to the addition of the N-formylated chemotactic tripeptide, N-formyl- l-methionyl- l-leucyl- l-phenylalanine (FMLP) (1 μM), both potentiated and prolonged the activity of NADPH oxidase which was accompanied by exaggerated membrane depolarisation, delayed and attenuated membrane repolarisation, and inhibition of store-operated Ca 2+ influx. Inclusion of diphenylene iodonium chloride (DPI, 10 μM), an inhibitor of NADPH oxidase, antagonised the effects of CA on NADPH oxidase activity and the membrane repolarisation responses of FMLP-activated neutrophils, but failed to restore store-operated influx of Ca 2+. Similarly, CA also inhibited store-operated influx of Ca 2+ into FMLP-activated neutrophils from a patient with chronic granulomatous disease, a primary immunodeficiency disorder characterised by the absence of a functional NADPH oxidase. CA also inhibited the store-operated influx of Ca 2+ into control neutrophils treated with 1 μM thapsigargin, a selective inhibitor of the endomembrane Ca 2+-ATPase, which does not activate NADPH oxidase. Taken together, these observations demonstrate that augmentation of NADPH oxidase activity is not primarily involved in CA-mediated inhibition of the store-operated influx of Ca 2+ into activated human neutrophils.

Nitric oxide regulates K+ and Cl- channels in guard cells through a subset of abscisic acid-evoked signaling pathways.

Abscisic acid (ABA) triggers a complex sequence of signaling events that lead to concerted modulation of ion channels at the plasma membrane of guard cells and solute efflux to drive stomatal closure in plant leaves. Recent work has indicated that nitric oxide (NO) and its synthesis are a prerequisite for ABA signal transduction in Arabidopsis and Vicia guard cells. Its mechanism(s) of action is not well defined in guard cells and, generally, in higher plants. Here we show directly that NO selectively regulates Ca2+-sensitive ion channels of Vicia guard cells by promoting Ca2+ release from intracellular stores to raise cytosolic-free [Ca2+]. NO-sensitive Ca2+ release was blocked by antagonists of guanylate cyclase and cyclic ADP ribose-dependent endomembrane Ca2+ channels, implying an action mediated via a cGMP-dependent cascade. NO did not recapitulate ABA-evoked control of plasma membrane Ca2+ channels and Ca2+-insensitive K+ channels, and NO scavengers failed to block the activation of these K+ channels evoked by ABA. These results place NO action firmly within one branch of the Ca2+-signaling pathways engaged by ABA and define the boundaries of parallel signaling events in the control of guard cell movements.

Role of dynamics of intracellular calcium in aluminium-toxicity syndrome.

This review is concentrating on the role of aluminium (Al)-calcium (Ca) interactions in Al toxicity syndrome in plants. Disruption of cytoplasmic Ca2+ homeostasis has been suggested as a primary trigger of Al toxicity. Aluminium causes an increase in cytosolic Ca2+ activity, potentially disrupting numerous biochemical and physiological processes, including those involved in the root growth. The source of Ca2+ for the increase in cytosolic Ca2+ activity under Al exposure is partly extracellular (likely to be due to the Al-resistant portion of the flux through depolarization-activated Ca2+ channels and fluxes through Ca2+ -permeable nonselective cation channels in the plasma membrane) as well as intracellular (increased cytosolic Ca2+ activity enhances the activity of Ca2+ release channels in the tonoplast and the endoplasmic reticulum membrane). The effect on increased cytosolic Ca2+ activity of possible Al-related inhibition of the plasma membrane and endo-membrane Ca2+ -ATPases and Ca2+ exchangers (CaX) that sequester Ca2+ out of the cytosol is insufficiently documented at present. The relationship between Al toxicity, cytoplasmic Ca2+ homeostasis and cytoplasmic pH needs to be elucidated. Technical improvements that would allow measurements of cytosolic Ca2+ activity within the short time after exposure to Al (seconds or shorter) are eagerly awaited. Contents I. Introduction 296 II. Symptoms of aluminium toxicity 296 III. Calcium - aluminium interactions 297 IV. The role of electrical properties of the plasma membrane in calcium-aluminium interactions 306 V. Oxidative stress 307 VI. Callose 308 VII. Cytoskeleton 308 VIII. Conclusions 309 Acknowledgements 309 References 309.

Endogenous adenosine regulates neutrophil pro-inflammatory activities by cyclic AMP-dependent accelerated clearance of cytosolic calcium.

To identify the involvement of adenosine in restoration of Ca2+ homeostasis to activated human neutrophils. Neutrophils were isolated from venous blood taken from healthy, adult, human volunteers. The cells were exposed to adenosine deaminase (ADA, 0.1-2 units/ml) for 10 min at 37 degrees C prior to activation with N-formyl-L-methionyl-L-leucyl-L-phenylala-nine (FMLP, 1 microM). Cytosolic Ca2+ concentrations and transmembrane fluxes of the cation in FMLP-activated neutrophils +/- ADA were measured using spectrofluorimetric and radiometric procedures respectively, while intracellular cAMP and inositol triphosphate were measured by radioassay, and superoxide production and elastase release by, chemiluminescence and colourimetric methods respectively. Levels of statistical significance were calculated using the Mann-Whitney U-test and ANOVA. Although FMLP-activated generation of inositol triphosphate and mobilisation of Ca2+ from neutrophil internal stores, as well as the magnitude of the subsequent efflux and store-operated influx of the cation were unaffected by ADA, there was a prolonged elevation in cytosolic Ca2+ in the presence of the enzyme, which was associated with failure to activate adenylate cyclase and with increased production of superoxide and release of elastase. These effects of ADA were attenuated by dibutyryl cAMP (4 mM), CGS 21680 (1 microM) and rolipram (0.5 microM), as well as by EGTA (10 mM). These results are compatible with a physiological role for adenosine in promoting deactivation of neutrophils, possibly by promoting cAMP-dependent clearance of Ca2+ from the cytosol of the cells by the endo-membrane Ca2+-ATPase.

Cyclic AMP-mediated inhibition of 5-lipoxygenase translocation and leukotriene biosynthesis in human neutrophils.

5-Lipoxygenase (5-LO) catalyzes the transformation of arachidonic acid to leukotrienes (LT). In stimulated human PMN, activation of 5-LO involves calcium, p38 MAP kinase (p38) phosphorylation, and translocation of 5-LO from the cytosol to nuclear membranes containing the 5-LO activating protein (FLAP). In this study, cAMP-elevating agents such as isoproterenol, prostaglandin E(2), CGS-21680 (an adenosine A(2a) receptor agonist), the type IV phosphodiesterase inhibitor RO 20-1724, the adenylate cyclase activator forskolin, and the Gs-protein activator cholera toxin all inhibited LT biosynthesis and 5-LO translocation to the nucleus in cytokine-primed human PMN stimulated with platelet-activating factor and in human PMN stimulated with the endomembrane Ca(2+)-ATPase blocker thapsigargin. Furthermore, monophosphorothioate analogs of cAMP, which activate protein kinase A (PKA), also inhibited LT biosynthesis and 5-LO translocation in stimulated cells. Treatment of PMN with CGS-21680 also prevented the phosphorylation of p38 by thapsigargin. Treatment of PMN with the PKA inhibitors H-89 and KT-5720 prevented the inhibitory effect of cAMP-elevating agents on LT biosynthesis, 5-LO translocation, and p38 phosphorylation, whereas the p38 inhibitor SB 203,580 dose-dependently inhibited arachidonic acid-induced LT biosynthesis. The 5-LO translocation was also inhibitable by the FLAP antagonist MK-0591 and correlated with LT biosynthesis in all experimental conditions tested. These results indicate that cAMP-mediated PKA activation in PMN results in the concomitant inhibition of 5-LO translocation and LT biosynthesis and support a role of p38 in the signaling pathway involved. This represents the first physiological down-regulation mechanism of 5-LO translocation in human PMN.

Regulation of Calcium Homeostasis in Activated Neutrophils and its Relevance to Inflammatory Airway Disorders

Neutrophils are relatively resistant to the anti-inflammatory actions of conventional chemotherapeutic agents, including corticosteroids, underscoring the requirement for novel pharmacologic strategies to control the potentially harmful proinflammatory activities of these cells. Recent insights into the mechanisms by which neutrophils handle Ca2+ have enabled identification of novel targets for anti-inflammatory chemotherapy. The most amenable of these is the cAMP-dependent protein kinase-modulated endomembrane Ca2+-ATPase. Pharmacologic up-regulation of this Ca2+ pump accelerates clearance of the cation from the cytosol of activated neutrophils, resulting in deactivation of the proinflammatory activities of these cells. Type 4 phosphodiesterase inhibitors and adenosine receptor agonists operative at the level of subtype A2A adenosine receptors, which are currently undergoing clinical and preclinical assessment, respectively, are the most promising pharmacologic modulators of Ca2+ clearance in activated neutrophils. If this promise is realized, it may result in novel chemotherapeutic strategies for the control of hyperacute and chronic inflammatory conditions of the airways in which neutrophils are the primary offenders. This may be particularly important in those inflammatory disorders associated with dysregulation of Ca2+ homeostasis and consequent hyperactivity of neutrophils.

Possible participation of intracellular platelet-activating factor in NF-κB activation in rat peritoneal macrophages

As we had found previously that thapsigargin, an endomembrane Ca 2+-ATPase inhibitor, induces production of intracellular platelet-activating factor (PAF) [Br. J. Pharmacol. 116 (1995) 2141], we decided to investigate the possible roles of intracellular PAF in nuclear factor (NF)-κB activation of thapsigargin-stimulated rat peritoneal macrophages. When rat peritoneal macrophages were stimulated with thapsigargin, the level of inhibitory protein of NF-κB-α (IκB-α) was decreased and the nuclear translocation of NF-κB was increased. The thapsigargin-induced activation of NF-κB was inhibited by the PAF synthesis inhibitor SK&F 98625 and the PAF antagonist E6123. Structurally unrelated PAF antagonists such as E5880 and L-652,731 also inhibited the thapsigargin-induced activation of NF-κB. Lipopolysaccharide (LPS)-induced activation of NF-κB was also suppressed by these drugs. In a culture of rat peritoneal macrophages, exogenously added PAF did not induce degradation of IκB-α. These findings suggest that the intracellular PAF produced by the stimulation with thapsigargin or LPS is involved in activation of the NF-κB pathway.

The anti-inflammatory interactions of epinephrine with human neutrophils in vitro are achieved by cyclic AMP-mediated accelerated resequestration of cytosolic calcium

This study was designed to evaluate the effects of epinephrine (0.01–1 μM) on superoxide production by, and release of elastase from human neutrophils activated with the chemotactic tripeptide, N-formyl- l-methionyl- l-leucyl- l-phenylalanine (FMLP) (1 μM) in vitro, and to relate alterations in these responses to changes in adenosine 3,5′ cyclic monophosphate (cAMP) and cytosolic free Ca 2+. Cyclic AMP, superoxide production and elastase release were measured by radioimmunoassay, lucigenin-enhanced chemiluminescence, and a colorimetric procedure respectively. Cytosolic Ca 2+ fluxes were measured by fura-2 spectrofluorimetry in combination with radiometric procedures that enable distinction between net efflux and influx of the cation. Epinephrine treatment of neutrophils resulted in increased cAMP and dose-related inhibition of both superoxide production and elastase release, which was potentiated by the type 4 phosphodiesterase inhibitor, rolipram, and attenuated by propranolol, but not by selective β 1-, α 1- or α 2-adrenoreceptor antagonists. Although epinephrine did not affect the FMLP-activated abruptly-occurring increase in fura-2 fluorescence intensity, indicating no effects on the release of Ca 2+ from neutrophil intracellular stores, this agent accelerated the rate of decline in fluorescence in the setting of decreased efflux and a reduction in store-operated influx of Ca 2+. These effects of epinephrine on the clearance of Ca 2+ from the cytosol of FMLP-activated neutrophils were attenuated by propranolol, and are compatible with enhancement of the activity of the cAMP-dependent Ca 2+ sequestering/resequestering endo-membrane Ca 2+-ATPase. We conclude that epinephrine down-regulates the pro-inflammatory activities of neutrophils by cAMP-mediated enhancement of the clearance of cytosolic Ca 2+.

Regulation of the arachidonic acid-stimulated respiratory burst in neutrophils by intracellular and extracellular calcium

The respiratory burst is an important physiological function of the neutrophils in killing the bacteria invading in human body. We used chemiluminescence method to measure the exogenous arachidonic acid-stimulated respiratory burst, and measured the cytosolic free calcium concentration in neutrophils by the fluorescence method. It was found that, on one hand, the arachidonic acid-stimulated respiratory burst was enhanced by elevating the cytosolic free calcium concentration in neutrophils with a potent endomembrane Ca2+-ATPase inhibitor, Thapsgargin; on the other hand, chelating the intracellular or extracellular calcium by EGTA or BAPTA inhibited the respiratory burst. Results showed that calcium plays an important regulatory role in the signaling pathway involved in the exogenous arachidonic acid-stimulated respiratory burst of neutrophils.

Accelerated resequestration of cytosolic calcium and suppression of the pro-inflammatory activities of human neutrophils by CGS 21680 in vitro.

We have investigated the effects of the adenosine A(2A) receptor agonist CGS 21680 (0.01 - 1 microM) on reactive oxidant production by, and elastase release from FMLP-activated human neutrophils, as well as on cytosolic Ca(2+) fluxes and intracellular concentrations of cyclic AMP. Oxidant production, elastase release and cyclic AMP were assayed using lucigenin-enhanced chemiluminescence, colourimetric and radioimmunoassay procedures respectively, while cytosolic Ca(2+) fluxes were measured by fura-2 spectrofluorimetry in combination with radiometric procedures which distinguish between net efflux and influx of the cation. Treatment of neutrophils with CGS 21680 did not affect the FMLP-activated release of Ca(2+) from intracellular stores, but resulted in dose-related acceleration of the rate of decline in fura-2 fluorescence, as well as decreases in both efflux and store-operated influx of Ca(2+), compatible with enhancement of resequestration of the cation by the endo-membrane Ca(2+)-ATPase. These effects on neutrophil Ca(2+) handling were associated with increased intracellular cyclic AMP and with inhibition of oxidant production and release of elastase. In contrast, treatment of neutrophils with the selective A(2A) receptor antagonist, ZM 241385 (2.5 microM), prevented the transient increase in cyclic AMP in FMLP-activated neutrophils which was associated with delayed sequestration of incoming Ca(2+) during store-operated influx. The CGS 21680-mediated reduction of Ca(2+) efflux from FMLP-activated neutrophils was also antagonized by pretreatment of the cells with ZM 241385 (2.5 microM), as well as by thapsigargin (1 microM), an inhibitor of the endo-membrane Ca(2+)-ATPase. ZM 241385 also neutralized the cyclic AMP-elevating and anti-inflammatory interactions of CGS 21680 with neutrophils. We conclude that A(2A) receptors regulate the pro-inflammatory activities of human neutrophils by promoting cyclic AMP-dependent sequestration of cytosolic Ca(2+).