Cytosolic Ca Concentration Research Articles

Guard cells count the number of unitary cytosolic Ca2+ signals to regulate stomatal dynamics.

Transient stimulus-specific increases in the cytosolic Ca2+ concentration ("calcium signatures") of guard cells have been proposed to regulate the opening and closure of stomatal pores on plant leaves. However, the mechanism by which these Ca2+ signatures are generated and translated into stomatal movement is still largely unresolved. We used a light-gated, Ca2+-permeable variant of ChannelRhodopsin 2 (ChR2-XXM2.0) that was stimulated by tailored light pulses to investigate this phenomenon. We found that activation of the ChR2-XXM2.0 channel provoked characteristic increases in the cytosolic concentration of Ca2+. We also demonstrated that the endoplasmic reticulum (ER) was involved in the generation of these calcium signatures. Using ChR2-XXM2.0 technology, we showed that transient increases in Ca2+ activated S-type anion channels and determined the extent and speed of stomatal closure with their number and frequency. Our data reveal that guard cells are capable of counting Ca2+ transients in order to optimize stomatal aperture in the prevailing environmental conditions.

ATP and Formyl Peptides Facilitate Chemoattractant Leukotriene-B4 Synthesis and Drive Calcium Fluxes, Which May Contribute to Neutrophil Swarming at Sites of Cell Damage and Pathogens Invasion.

Here, we demonstrate that human neutrophil interaction with the bacterium Salmonella typhimurium fuels leukotriene B4 synthesis induced by the chemoattractant fMLP. In this work, we found that extracellular ATP (eATP), the amount of which increases sharply during tissue damage, can effectively regulate fMLP-induced leukotriene B4 synthesis. The vector of influence strongly depends on the particular stage of sequential stimulation of neutrophils by bacteria and on the stage at which fMLP purinergic signaling occurs. Activation of 5-lipoxygenase (5-LOX), key enzyme of leukotriene biosynthesis, depends on an increase in the cytosolic concentration of Ca2+. We demonstrate that eATP treatment prior to fMLP, by markedly reducing the amplitude of the fMLP-induced Ca2+ transient jump, inhibits leukotriene synthesis. At the same time, when added with or shortly after fMLP, eATP effectively potentiates arachidonic acid metabolism, including by Ca2+ fluxes stimulation. Flufenamic acid, glibenclamide, and calmodulin antagonist R24571, all of which block calcium signaling in different ways, all suppressed 5-LOX product synthesis in our experimental model, indicating the dominance of calcium-mediated mechanisms in eATP regulatory potential. Investigation into the adhesive properties of neutrophils revealed the formation of cell clusters when adding fMLP to neutrophils exposed to the bacterium Salmonella typhimurium. eATP added simultaneously with fMLP supported neutrophil polarization and clustering. A cell-derived chemoattractant such as leukotriene B4 plays a crucial role in the recruitment of additional neutrophils to the foci of tissue damage or pathogen invasion, and eATP, through the dynamics of changes in [Ca2+]i, plays an important decisive role in fMLP-induced leukotrienes synthesis during neutrophil interactions with the bacterium Salmonella typhimurium.

Calcium Signaling and the Response to Heat Shock in Crop Plants.

Climate change and the increasing frequency of high temperature (HT) events are significant threats to global crop yields. To address this, a comprehensive understanding of how plants respond to heat shock (HS) is essential. Signaling pathways involving calcium (Ca2+), a versatile second messenger in plants, encode information through temporal and spatial variations in ion concentration. Ca2+ is detected by Ca2+-sensing effectors, including channels and binding proteins, which trigger specific cellular responses. At elevated temperatures, the cytosolic concentration of Ca2+ in plant cells increases rapidly, making Ca2+ signals the earliest response to HS. In this review, we discuss the crucial role of Ca2+ signaling in raising plant thermotolerance, and we explore its multifaceted contributions to various aspects of the plant HS response (HSR).

Broadband Electrical Spectroscopy to Distinguish Single-Cell Ca2+ Changes Due to Ionomycin Treatment in a Skeletal Muscle Cell Line.

Many skeletal muscle diseases such as muscular dystrophy, myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), and sarcopenia share the dysregulation of calcium (Ca2+) as a key mechanism of disease at a cellular level. Cytosolic concentrations of Ca2+ can signal dysregulation in organelles including the mitochondria, nucleus, and sarcoplasmic reticulum in skeletal muscle. In this work, a treatment is applied to mimic the Ca2+ increase associated with these atrophy-related disease states, and broadband impedance measurements are taken for single cells with and without this treatment using a microfluidic device. The resulting impedance measurements are fitted using a single-shell circuit simulation to show calculated electrical dielectric property contributions based on these Ca2+ changes. From this, similar distributions were seen in the Ca2+ from fluorescence measurements and the distribution of the S-parameter at a single frequency, identifying Ca2+ as the main contributor to the electrical differences being identified. Extracted dielectric parameters also showed different distribution patterns between the untreated and ionomycin-treated groups; however, the overall electrical parameters suggest the impact of Ca2+-induced changes at a wider range of frequencies.

Pro-Inflammatory Interactions of Dolutegravir with Human Neutrophils in an In Vitro Study.

There is increasing awareness of an association between the uptake of the HIV integrase inhibitor, dolutegravir, in first-line antiretroviral regimens with unusual weight gain and development of the metabolic syndrome, particularly in African women. Although seemingly unexplored, the development of systemic inflammation linked to the putative pro-inflammatory activity of dolutegravir represents a plausible pathophysiological mechanism of this unusual weight gain. This possibility was explored in the current study undertaken to investigate the effects of dolutegravir (2.5-20 μg/mL) on several pro-inflammatory activities of neutrophils isolated from the blood of healthy, adult humans. These activities included the generation of reactive oxygen species (ROS), degranulation (elastase release) and alterations in the concentrations of cytosolic Ca2+ using chemiluminescence, spectrophotometric and fluorimetric procedures, respectively. Exposure of neutrophils to dolutegravir alone resulted in the abrupt, dose-related, and significant (p < 0.0039-p < 0.0022) generation of ROS that was attenuated by the inclusion of the Ca2+-chelating agent, EGTA, or inhibitors of NADPH oxidase (diphenyleneiodonium chloride, DPI), phospholipase C (U733122), myeloperoxidase (sodium azide) and phosphoinositol-3-kinase (wortmannin). In addition, exposure to dolutegravir augmented the release of elastase by stimulus-activated neutrophils. These pro-inflammatory effects of dolutegravir on neutrophils were associated with significant, rapid, and sustained increases in the concentrations of cytosolic Ca2+ that appeared to originate from the extracellular compartment, seemingly consistent with an ionophore-like property of dolutegravir. These findings are preliminary and necessitate verification in the clinical setting of HIV infection. Nevertheless, given the complex link between inflammation and obesity, these pro-inflammatory interactions of dolutegravir with neutrophils may contribute to unexplained weight gain, possibly via the development of insulin resistance.

Calcium-sensing receptor acts as an antiviral factor for rotavirus infections and participates in cellular antiviral response.

Objective(s):Rotavirus (RV) is one of the most significant pathogens associated with childhood diarrhoeal deaths worldwide. Elevated cytoplasmic calcium is required for RV replication, but the underlying mechanisms responsible for calcium influx remain poorly understood. The Calcium-sensing receptor (CaSR) is an important Ca2+ sensor that regulates the transport of Ca2+ into or out of the extracellular space by affecting the status of Ca2+ ion channels on the membrane of cells. Currently, the function of CaSR in RV replication is unclear. Materials and Methods: We evaluated the mRNA and protein levels of CaSR in RV-infected cells using qRT-PCR and Western blotting, respectively. Furthermore, we silenced or overexpressed CaSR in Caco-2 cells using siRNA or a CaSR gene contained adenovirus (Adv-CaSR). qRT-PCR, plaque assay, and Western blotting were used to determine the synthesis of virus genomic RNA, production of progeny virion, and the levels of viral proteins. The content of Ca2+ in cells was observed under confocal microscopy. Results:Compared with control cells, the RV-infected cells presented significantly decreased CaSR expression. Moreover, adenoviral-mediated over-expression or induction of CaSR by R568 greatly inhibited the RV RNA synthesis, protein expression, and formation of viroplasm plaques, thereby suppressing RV replication. In contrast, CaSR-silenced cells exhibited significantly enhanced RV replication. Compared with the Adv-Control group, the concentration of cytosolic Ca2+ significantly decreased in the Adv-CaSR group. Conclusion:These findings demonstrated that CaSR is a potential target for inhibition of RV replication. Therefore, enhancing the expression of CaSR might protect hosts from RV infections.

Size-Dependent Cytoprotective Effects of Selenium Nanoparticles during Oxygen-Glucose Deprivation in Brain Cortical Cells.

It is known that selenium nanoparticles (SeNPs) obtained on their basis have a pleiotropic effect, inducing the process of apoptosis in tumor cells, on the one hand, and protecting healthy tissue cells from death under stress, on the other hand. It has been established that SeNPs protect brain cells from ischemia/reoxygenation through activation of the Ca2+ signaling system of astrocytes and reactive astrogliosis. At the same time, for a number of particles, the limitations of their use, associated with their size, are shown. The use of nanoparticles with a diameter of less than 10 nm leads to their short life-time in the bloodstream and rapid removal by the liver. Nanoparticles larger than 200 nm activate the complement system and are also quickly removed from the blood. The effects of different-sized SeNPs on brain cells have hardly been studied. Using the laser ablation method, we obtained SeNPs of various diameters: 50 nm, 100 nm, and 400 nm. Using fluorescence microscopy, vitality tests, PCR analysis, and immunocytochemistry, it was shown that all three types of the different-sized SeNPs have a cytoprotective effect on brain cortex cells under conditions of oxygen-glucose deprivation (OGD) and reoxygenation (R), suppressing the processes of necrotic death and inhibiting different efficiency processes of apoptosis. All of the studied SeNPs activate the Ca2+ signaling system of astrocytes, while simultaneously inducing different types of Ca2+ signals. SeNPs sized at 50 nm- induce Ca2+ responses of astrocytes in the form of a gradual irreversible increase in the concentration of cytosolic Ca2+ ([Ca2+]i), 100 nm-sized SeNPs induce stable Ca2+ oscillations without increasing the base level of [Ca2+]i, and 400 nm-sized SeNPs cause mixed patterns of Ca2+ signals. Such differences in the level of astrocyte Ca2+ signaling can explain the different cytoprotective efficacy of SeNPs, which is expressed in the expression of protective proteins and the activation of reactive astrogliosis. In terms of the cytoprotective efficiency under OGD/R conditions, different-sized SeNPs can be arranged in descending order: 100 nm-sized > 400 nm-sized > 50 nm-sized.

Mn2+ modulates the production of mycophenolic acid in Penicillium brevicompactum NRRL864 via reactive oxygen species signaling and the investigation of pb-pho

Although Penicillium brevicompactum is a widely used commercial strain for the manufacture of mycophenolic acid, there are few findings of Ca2+, Mn2+, and reactive oxygen species (ROS) interaction. Metal ions play a crucial role in physiological metabolism. Calcium, as the important second messenger, influences fungus growth, virulence, and stress responses. The concentration of cytosolic Ca2+ was influenced by the Mn2+, which demonstrated the crosstalk between calcium and manganese. In the previous study, the crosstalk between calcium and ROS has been discovered and verified, which modified the secondary metabolism and enhanced the yield of MPA (Mycophenolic Acid). A higher concentration of Mn2+ in the fermentation broth causes an increase in cytoplasmic Ca2+ and ROS (Reactive Oxygen Species), enhancing the yield of MPA by about 20 % higher and disclosing the cascade regulation with the Mn2+, Ca2+, and ROS. To be more specific, the intracellular concentration of ROS at 6 mM Mn2+ is about 1.5 times higher than that at 0.6 mM. Furthermore, we identify an Mn2+ transport protein, designated as Pb–PHO, which shows 71.2 % identity to the inorganic phosphate transporter PHO84 (Q0CBJ6) from Aspergillus terreus. At the same time, the △Pb-pho exhibits damage to the cell wall integrity, while the OE-pho displays a more normal phenotype at high osmotic stress. The high-affinity Ca2+ channel, Pb–CCH, is examined via knockdown to demonstrate the crosstalk between Mn2+ and Ca2+. The results show that the addition of Mn2+ remits the negative influence of pb-cch knockdown and the addition of Ca2+ remits the negative influence of pb-pho knockdown, demonstrating the relationship between cytoplasmic Mn2+ and Ca2+. Taken together, our results demonstrate the mechanism of a manganese-induced cascade of manganese-calcium-ROS and reveal a signal pathway-relative method to illustrate the manganese-induced increase of MPA production in Penicillium brevicompactum. Furthermore, we discover and identify an Mn2+ transport protein, Pb–PHO, which is subcellular localized at the plasma membrane and proved to affect the cell wall integrity.

The Mechanisms Underlying the Protective Action of Selenium Nanoparticles against Ischemia/Reoxygenation Are Mediated by the Activation of the Ca2+ Signaling System of Astrocytes and Reactive Astrogliosis.

In recent years, much attention has been paid to the study of the therapeutic effect of the microelement selenium, its compounds, especially selenium nanoparticles, with a large number of works devoted to their anticancer effects. Studies proving the neuroprotective properties of selenium nanoparticles in various neurodegenerative diseases began to appear only in the last 5 years. Nevertheless, the mechanisms of the neuroprotective action of selenium nanoparticles under conditions of ischemia and reoxygenation remain unexplored, especially for intracellular Ca2+ signaling and neuroglial interactions. This work is devoted to the study of the cytoprotective mechanisms of selenium nanoparticles in the neuroglial networks of the cerebral cortex under conditions of ischemia/reoxygenation. It was shown for the first time that selenium nanoparticles dose-dependently induce the generation of Ca2+ signals selectively in astrocytes obtained from different parts of the brain. The generation of these Ca2+ signals by astrocytes occurs through the release of Ca2+ ions from the endoplasmic reticulum through the IP3 receptor upon activation of the phosphoinositide signaling pathway. An increase in the concentration of cytosolic Ca2+ in astrocytes leads to the opening of connexin Cx43 hemichannels and the release of ATP and lactate into the extracellular medium, which trigger paracrine activation of the astrocytic network through purinergic receptors. Incubation of cerebral cortex cells with selenium nanoparticles suppresses ischemia-induced increase in cytosolic Ca2+ and necrotic cell death. Activation of A2 reactive astrocytes exclusively after ischemia/reoxygenation, a decrease in the expression level of a number of proapoptotic and proinflammatory genes, an increase in lactate release by astrocytes, and suppression of the hyperexcitation of neuronal networks formed the basis of the cytoprotective effect of selenium nanoparticles in our studies.

Influenza A Viruses Enter Host Cells via Extracellular Ca2+ Influx-Involved Clathrin-Mediated Endocytosis.

Influenza A virus (IAV) is internalized into its host cells by endocytosis, which involves many cellular proteins and molecules. In this study, we focus on the function of calcium ion (Ca2+) in IAV endocytosis. We have found that IAV infection is accompanied by the increase in concentration of cytosolic Ca2+, which is mainly attributed to the influx of extracellular Ca2+. When Ca2+ influx is abolished, IAV internalization will be markedly suppressed, but the virus attachment to its host cells will be unaffected. Extracellular Ca2+ influx is essential to the clathrin-mediated endocytosis (CME) of IAVs but dispensable to the clathrin-independent endocytosis of the virus and is dispensable to the CME of transferrin or low-density lipoprotein as a control. Ca2+ influx might participate in the dynamin-promoted membrane fission in the CME of IAVs. Our study highlights that IAVs enter host cells via extracellular Ca2+ influx-involved clathrin- and dynamin-dependent endocytosis, which will facilitate better understanding of IAV infection and development of anti-influenza drugs.

Astrocytes: The Housekeepers and Guardians of the CNS.

Astroglia are a diverse group of cells in the central nervous system. They are of the ectodermal, neuroepithelial origin and vary in morphology and function, yet, they can be collectively defined as cells having principle function to maintain homeostasis of the central nervous system at all levels of organisation, including homeostasis of ions, pH and neurotransmitters; supplying neurones with metabolic substrates; supporting oligodendrocytes and axons; regulating synaptogenesis, neurogenesis, and formation and maintenance of the blood-brain barrier; contributing to operation of the glymphatic system; and regulation of systemic homeostasis being central chemosensors for oxygen, CO2 and Na+. Their basic physiological features show a lack of electrical excitability (inapt to produce action potentials), but display instead a rather active excitability based on variations in cytosolic concentrations of Ca2+ and Na+. It is expression of neurotransmitter receptors, pumps and transporters at their plasmalemma, along with transports on the endoplasmic reticulum and mitochondria that exquisitely regulate the cytosolic levels of these ions, the fluctuation of which underlies most, if not all, astroglial homeostatic functions.

Platelet Protease Activated Receptor 1 Is Involved in the Hemostatic Effect of 20(S)-Protopanaxadiol by Regulating Calcium Signaling.

Panax notoginseng (Burk.) F.H. Chen has long been used to stop bleeding for hundreds of years in China. At present, only dencichine, notoginsenoside Ft1, and 20(S)-protopanaxadiol (PPD) showed hemostatic effect. However, the molecular mechanism of PPD on the platelet aggragetion needs to be further investigated. The study aims to evaluate the hemostatic effect of PPD and reveal its interacting targets using a series of experiments. In this study, the bleeding time was measured in mouse tail amputation and liver scratch models to evaluate hemostatic effect of PPD. The routine blood and plasma coagulation parameters in NS, HC, and PPD (2, 4, and 8 mg/kg) groups were measured using a blood analyzer. Platelet aggregation rate and ATP release were analyzed by a platelet aggregometer. Subsequently, the degranulation marker CD62P and PAC-1, and the concentrations of cytosolic Ca2+ ([Ca2+]i), cAMP, cGMP, and PAC-1 expressions were also assessed. We found that PPD shorted the bleeding time on the mouse tail amputation and liver scratch models and mainly increased blood platelet count in the rats after subcutaneous injection for 4 h. Meanwhile, PPD decreased APTT, increased FIB content, and directly induced platelet aggregation in vitro. In the absence of Ca2+, PPD induced the increase of [Ca2+]i and slightly increased the levels of CD62P and PAC-1. After the addition of 1 mM Ca2+, PPD treatment markedly promoted platelet activation by promoting ATP level, releasing CD62P and increasing PAC-1 binding in washed platelets. Excitingly, PPD-induced changes including platelet aggregation, decreased cAMP content, and the increases of CD62P and PAC-1 were significantly reversed by protease-activated receptor 1 (PAR-1) antagonist, vorapaxar, which showed similar function as thrombin. In addition, molecular docking analysis and ELISA assay demonstrated that PPD had a promising docking score with -6.6 kcal/mol and increased PAR-1 expression in human platelets, which indicated that PAR-1 is involved in PPD-induced platelet aggregation by regulating calcium signaling. Collectively, our study could provide the new insights of PPD as an essential hemostatic ingredient in Panax notoginseng for the treatment of hemorrhagic disease.

Perturbation of calcium homeostasis and multixenobiotic resistance by nanoplastics in the ciliate Tetrahymena thermophila

Microplastics (MPs) are an environmental hazard of growing concern, including their potential toxic effects on the biota of different trophic levels. Nevertheless, the molecular mechanisms underlying MP-induced toxicity remain largely unknown. In the present study, Tetrahymena thermophila was exposed to polystyrene nanoplastics (PS-NPs) and the responses of this relatively sensitive ciliate were then followed using transcriptome analysis together with several other verification methods. The results showed that PS-NPs perturbed calcium (Ca) homeostasis, by inducing the inositol-1,4,5-trisphosphate-dependent liberation of Ca from the endoplasmic reticulum into the cytosol. The high cytosolic concentration of Ca induced Ca accumulation in mitochondria, which increased mitochondrial permeability and the generation of reactive oxygen species, finally leading to growth inhibition. Such toxicity is the so-called direct effects of PS-NPs. By contrast, PS-NPs also inhibited the activity of multixenobiotic resistance transporter, by down-regulating the ATP-binding cassette transporter genes Abcb15 and Abcc52. This additional effect may alter cellular responses to other pollutants and implicates PS-NPs in the risks to the organism posed by subsequent toxic exposures, which was named as the indirect effects of PS-NPs. Our study highlights the importance of considering both direct and indirect biological effects of MPs in evaluations of their environmental and health risks.

Ca2+ -independent and voltage-dependent exocytosis in mouse chromaffin cells.

It is widely accepted that the exocytosis of synaptic and secretory vesicles is triggered by Ca2+ entry through voltage-dependent Ca2+ channels. However, there is evidence of an alternative mode of exocytosis induced by membrane depolarization but lacking Ca2+ current and intracellular Ca2+ increase. In this work we investigated if such a mechanism contributes to secretory vesicle exocytosis in mouse chromaffin cells. Exocytosis was evaluated by patch-clamp membrane capacitance measurements, carbon fibre amperometry and TIRF. Cytosolic Ca2+ was estimated using epifluorescence microscopy and fluo-8 (salt form). Cells stimulated by brief depolatizations in absence of extracellular Ca+2 show moderate but consistent exocytosis, even in presence of high cytosolic BAPTA concentration and pharmacological inhibition of Ca+2 release from intracellular stores. This exocytosis is tightly dependent on membrane potential, is inhibited by neurotoxin Bont-B (cleaves the v-SNARE synaptobrevin), is very fast (saturates with time constant <10ms), it is followed by a fast endocytosis sensitive to the application of an anti-dynamin monoclonal antibody, and recovers after depletion in <5s. Finally, this exocytosis was inhibited by: (i) ω-agatoxin IVA (blocks P/Q-type Ca2+ channel gating), (ii) in cells from knock-out P/Q-type Ca2+ channel mice, and (iii) transfection of free synprint peptide (interferes in P/Q channel-exocytic proteins association). We demonstrated that Ca2+ -independent and voltage-dependent exocytosis is present in chromaffin cells. This process is tightly coupled to membrane depolarization, and is able to support secretion during action potentials at low basal rates. P/Q-type Ca2+ channels can operate as voltage sensors of this process.

Eryptosis Is Altered in Peritoneal Dialysis Patients

Background: Red blood cells (RBCs) undergo programmed cell death known as eryptosis. Triggers of eryptosis include increased cytosolic Ca(2+) concentration, oxidative stress, osmotic shock, energy depletion and several uremic toxins. Little is known about the pathogenesis of eryptosis in peritoneal dialysis (PD) patients; furthermore, its relevance in worsening clinical conditions in these patients is still not completely defined. Objectives: We investigated eryptosis levels in PD patients and its association with inflammatory and clinical parameters. Material and Methods: A total of 46 PD patients and 17 healthy subjects (CTR) were enrolled. All eryptosis measurements were made in freshly isolated RBCs using the flow cytometer. Results: Eryptosis was significantly higher in PD patients than that in CTR (p < 0.001). Eryptosis levels did not differ significantly between PD patients with and without diabetes, with and without hypertension, and with and without cardiovascular disease. Eryptosis showed no significant differences between patients treated with continuous ambulatory PD/automated PD, with Kt/Vurea value ≤1.7 and >1.7, with a negative or positive history of peritonitis. On the contrary, eryptosis showed significantly lower levels in PD patients with weekly creatinine clearance ≥45 L/week/1.73 m² (2.8%, 1.7–4.9 vs. 5.6%, 5.0–13.5; p= 0.049). Eryptosis showed significantly lower levels in PD patients with residual diuresis (n = 23) than that in patients without (3.7%, 2.6–5.6 vs. 5%, 3.1–16; p = 0.03). In these 23 patients, significant negative correlations between percentage of eryptosis and residual glomerular filtration rate (rGFR; Spearman’s rho = –0.51, p = 0.01) and diuresis volume (Spearman’s rho = –0.43, p = 0.05) were found. Conclusions: The present study demonstrated higher eryptosis levels in PD patients compared to corresponding levels in CTR. Furthermore, important PD comorbidity and main PD parameters do not influence eryptosis. Importantly, our data have reported an increase in eryptosis levels with progressive residual diuresis and rGFR loss, probably due to decreased uremic toxins clearance.

(209) Characterization of the Na+/Ca2+ Exchanger (NCS) in Human DRG Neurons

Because of the importance of intracellular Ca2+ to processes ranging from protein-interactions to cell death, the concentration of cytosolic Ca2+ ([Ca2+]c) is tightly regulated by a complex array of pumps, transporters, exchangers, and binding proteins. We have recently demonstrated that the Na+/Ca2+ exchanger (NCX), in particular NCX3, plays an important role in attenuating large-increases in [Ca2+]c in a subpopulation of putative nociceptive rat dorsal root ganglion (DRG) neurons. Interestingly, we subsequently demonstrated that inflammation-induced dysregulation of [Ca2+]c in this subpopulation of putative nociceptors was due, at least in part, to a decrease in NCX activity in the sensory neuron cell body. Given the potential importance of this exchanger to nociceptive processing as well as injury-induced changes in nociceptive processing, in the face of a growing list of differences between rodent and human sensory neurons, the purpose of the present study was to characterize NCX activity in human sensory neurons. We employed electrical field stimulation to evoke Ca2+ transients in the presence and absence of LiCl or Choline-Cl bath to block NCX activity. Interestingly, electrical field stimulation of human DRG neurons, at the same parameters used in rat, evoked a much larger Ca2+ transient in both magnitude and duration. We obtained evidence of NCX activity in the majority of neurons tested. Similar to the role of NCX in rat DRG neurons, block of NCX in human DRG neurons increased the duration, but not the magnitude of the electrically evoked Ca2+ transient. However, in contrast to the rat, there appeared to be constitutive NCX activity. Consistent with a number of recent studies, these results highlight potentially important similarities and differences between rodent and human sensory neurons, underscoring the importance of human sensory neurons as a pre-clinical screen of novel therapeutics targeting the primary afferent. Research funded by NIH T32NS073548, 1R01NS083347, and R01DK107966.

An allosteric mechanism of inactivation in the calcium-dependent chloride channel BEST1.

Bestrophin proteins are calcium (Ca2+)-activated chloride channels. Mutations in bestrophin 1 (BEST1) cause macular degenerative disorders. Whole-cell recordings show that ionic currents through BEST1 run down over time, but it is unclear whether this behavior is intrinsic to the channel or the result of cellular factors. Here, using planar lipid bilayer recordings of purified BEST1, we show that current rundown is an inherent property of the channel that can now be characterized as inactivation. Inactivation depends on the cytosolic concentration of Ca2+, such that higher concentrations stimulate inactivation. We identify a C-terminal inactivation peptide that is necessary for inactivation and dynamically interacts with a receptor site on the channel. Alterations of the peptide or its receptor dramatically reduce inactivation. Unlike inactivation peptides of voltage-gated channels that bind within the ion pore, the receptor for the inactivation peptide is on the cytosolic surface of the channel and separated from the pore. Biochemical, structural, and electrophysiological analyses indicate that binding of the peptide to its receptor promotes inactivation, whereas dissociation prevents it. Using additional mutational studies we find that the "neck" constriction of the pore, which we have previously shown to act as the Ca2+-dependent activation gate, also functions as the inactivation gate. Our results indicate that unlike a ball-and-chain inactivation mechanism involving physical occlusion of the pore, inactivation in BEST1 occurs through an allosteric mechanism wherein binding of a peptide to a surface-exposed receptor controls a structurally distant gate.

CK2β regulates thrombopoiesis and Ca2+-triggered platelet activation in arterial thrombosis

AbstractPlatelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-)physiology has remained elusive. The present study explored the impact of the CK2 regulatory β-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2β (ck2β−/−) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2β−/− MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2β−/− platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-triphosphate receptor–dependent intracellular calcium (Ca2+) release. Presumably due to a blunted increase in the concentration of cytosolic Ca2+, activation-dependent increases of α and dense-granule secretion and integrin αIIbβ3 activation, and aggregation were abrogated in ck2β−/− platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo was significantly blunted in ck2β−/− mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2β−/− mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2βfl/fl mice. The present observations reveal CK2β as a novel powerful regulator of thrombopoiesis, Ca2+-dependent platelet activation, and arterial thrombosis in vivo.

Phospholipases AtPLDζ1 and AtPLDζ2 function differently in hypoxia.

Besides hydrolyzing different membrane phospholipids, plant phospholipases D and molecular species of their byproducts phosphatidic acids (PLDs/PAs) are involved in diverse cellular events such as membrane-cytoskeleton dynamics, hormone regulation and biotic and/or abiotic stress responses at cellular or subcellular levels. Among the 12 Arabidopsis PLD genes, PLDζ1 and PLDζ2 uniquely possess Ca2+ -independent phox (PX) and pleckstrin (PH) homology domains. Here, we report that mutants deficient in these PLDs, pldζ1 and pldζ2, show differential sensitivities to hypoxia stimulus. In the present study, we used protoplasts of wild type and mutants and compared the hypoxia-induced changes in the levels of three major signaling mediators such as cytoplasmic free calcium [Ca2+cyt. ], hydrogen peroxide (H2 O2 ) and PA. The concentrations of cytosolic Ca2+ and H2 O2 were determined by fluorescence microscopy and the fluorescent dyes Fura 2-AM and CM-H2 DCFDA, specific for calcium and H2 O2 , respectively, while PA production was analyzed by an enzymatic method. The study reveals that AtPLDζ1 is involved in reactive oxygen species (ROS) signaling, whereas AtPLDζ2 is involved in cytosolic Ca2+ signaling pathways during hypoxic stress. Hypoxia induces an elevation of PA level both in Wt and pldζ1, while the PA level is unchanged in pldζ2. Thus, it is likely that AtPLDζ2 is involved in PA production by a calcium signaling pathway, while AtPLDζ1 is more important in ROS signaling.

Transient Receptor Potential Melastatin 2 Regulates Phagosome Maturation and Is Required for Bacterial Clearance in Escherichia coli Sepsis.

Transient receptor potential melastatin 2 is a Ca-permeable cation channel abundantly expressed in macrophages. Trpm2 mice showed exacerbated infection and mortality during polymicrobial sepsis, which is associated with inefficient bacterial killing in macrophages. However, the mechanism of transient receptor potential melastatin 2 regulating bacterial killing remains unknown. Trpm2 mice were intraperitoneally injected with Escherichia coli. The survival rate (n = 21) and bacterial burden (n = 5) were assessed. The processes of phagosome maturation and phagosome-lysosome fusion in peritoneal macrophages were extensively studied. The impact of increasing intracellular Ca concentration on bacterial clearance in macrophages (n = 3) and on survival rate of Trpm2 mice infected with E. coli (n = 21) was investigated. Trpm2 mice exhibited increased mortality (85% vs. 54%; P < 0.01) and aggravated bacterial burden during E. coli sepsis. Trpm2 peritoneal macrophages infected with E. coli showed dampened recruitment of lysosomal-associated membrane protein 1 and impaired phagosome maturation evidenced by a decrease in the accumulation of early endosome antigen 1, whereas a normal acquisition of Ras-related protein in brain 5. Increasing the cytosolic Ca concentration in Trpm2 peritoneal macrophages via ionomycin treatment facilitated early endosome antigen 1 recruitment to Ras-related protein in brain 5 and phagosomal localization of lysosomal-associated membrane protein 1 and consequently enhanced bactericidal activity. Adoptive transfer of ionomycin-treated Trpm2 peritoneal macrophages improved bacterial clearance and survival (67% vs. 29%; P < 0.01) in Trpm2 mice challenged with E. coli. Transient receptor potential melastatin 2 plays a critical role in host defense against invading bacteria via promoting phagosome maturation through facilitation of early endosome antigen 1 recruitment.