Cytosolic Calcium Ion Research Articles

Interplay of Plasma Membrane and Vacuolar Ion Channels, Together with BAK1, Elicits Rapid Cytosolic Calcium Elevations in Arabidopsis during Aphid Feeding.

A transient rise in cytosolic calcium ion concentration is one of the main signals used by plants in perception of their environment. The role of calcium in the detection of abiotic stress is well documented; however, its role during biotic interactions remains unclear. Here, we use a fluorescent calcium biosensor (GCaMP3) in combination with the green peach aphid (Myzus persicae) as a tool to study Arabidopsis thaliana calcium dynamics in vivo and in real time during a live biotic interaction. We demonstrate rapid and highly localized plant calcium elevations around the feeding sites of M. persicae, and by monitoring aphid feeding behavior electrophysiologically, we demonstrate that these elevations correlate with aphid probing of epidermal and mesophyll cells. Furthermore, we dissect the molecular mechanisms involved, showing that interplay between the plant defense coreceptor BRASSINOSTEROID INSENSITIVE-ASSOCIATED KINASE1 (BAK1), the plasma membrane ion channels GLUTAMATE RECEPTOR-LIKE 3.3 and 3.6 (GLR3.3 and GLR3.6), and the vacuolar ion channel TWO-PORE CHANNEL1 (TPC1) mediate these calcium elevations. Consequently, we identify a link between plant perception of biotic threats by BAK1, cellular calcium entry mediated by GLRs, and intracellular calcium release by TPC1 during a biologically relevant interaction.

CBL-interacting protein kinase 6 negatively regulates immune response to Pseudomonas syringae in Arabidopsis.

Cytosolic calcium ion (Ca2+) is an essential mediator of the plant innate immune response. Here, we report that a calcium-regulated protein kinase Calcineurin B-like protein (CBL)-interacting protein kinase 6 (CIPK6) functions as a negative regulator of immunity against the bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana. Arabidopsis lines with compromised expression of CIPK6 exhibited enhanced disease resistance to the bacterial pathogen and to P. syringae harboring certain but not all avirulent effectors, while restoration of CIPK6 expression resulted in abolition of resistance. Plants overexpressing CIPK6 were more susceptible to P. syringae. Enhanced resistance in the absence of CIPK6 was accompanied by increased accumulation of salicylic acid and elevated expression of defense marker genes. Salicylic acid accumulation was essential for improved immunity in the absence of CIPK6. CIPK6 negatively regulated the oxidative burst associated with perception of pathogen-associated microbial patterns (PAMPs) and bacterial effectors. Accelerated and enhanced activation of the mitogen-activated protein kinase cascade in response to bacterial and fungal elicitors was observed in the absence of CIPK6. The results of this study suggested that CIPK6 negatively regulates effector-triggered and PAMP-triggered immunity in Arabidopsis.

XBP1-Mediated BiP/GRP78 Upregulation Copes with Oxidative Stress in Mosquito Cells during Dengue 2 Virus Infection.

Dengue viruses (DENVs) cause dengue fever which is an important mosquito-borne disease in tropical areas. Generally, DENV does not cause cellular damage in mosquito cells. However, alterations in cytosolic calcium ions ([Ca2+]cyt) and the mitochondrial membrane potential (MMP), as well as accumulated reactive oxygen species (ROS), including superoxide anions (O2∙−) and hydrogen peroxide (H2O2), can be detected in C6/36 cells with DENV2 infection. Evident upregulation of BiP/GRP78 also appeared at 24 h postinfection in DENV2-infected C6/36 cells. As expression of BiP/GRP78 mRNA was reduced when the transcription factor X-box-binding protein-1 (XBP1) was knocked down in C6/36 cells, it demonstrated that BiP/GRP78 is the target gene regulated by the XBP1 signal pathway. We further demonstrated that the expression and splicing activity of XBP1 were upregulated in parallel with DENV2 infection in C6/36 cells. In C6/36 cells with BiP/GRP78 overexpression, oxidative stress indicators including [Ca2+]cyt, MMP, O2∙−, and H2O2 were all pushed back to normal. Taken together, DENV2 activates XBP1 at earlier stage of infection, followed by upregulating BiP/GRP78 in mosquito cells. This regulatory pathway contributes a cascade in relation to oxidative stress alleviation. The finding provides insights into elucidating how mosquitoes can healthily serve as a vector of arboviruses in nature.

Regulation of heat shock gene expression in response to stress

Heat shock (HS) genes, or stress genes, code for a number of proteins that collectively form the most ancient and universal stress defense system. The system determines the cell capability of adaptation to various adverse factors and performs a variety of auxiliary functions in normal physiological conditions. Common stress factors, such as higher temperatures, hypoxia, heavy metals, and others, suppress transcription and translation for the majority of genes, while HS genes are upregulated. Transcription of HS genes is controlled by transcription factors of the HS factor (HSF) family. Certain HSFs are activated on exposure to higher temperatures or other adverse factors to ensure stress-induced HS gene expression, while other HSFs are specifically activated at particular developmental stages. The regulation of the main mammalian stress-inducible factor HSF1 and Drosophila melanogaster HSF includes many components, such as a variety of early warning signals indicative of abnormal cell activity (e.g., increases in intracellular ceramide, cytosolic calcium ions, or partly denatured proteins); protein kinases, which phosphorylate HSFs at various Ser residues; acetyltransferases; and regulatory proteins, such as SUMO and HSBP1. Transcription factors other than HSFs are also involved in activating HS gene transcription; the set includes D. melanogaster GAF, mammalian Sp1 and NF-Y, and other factors. Transcription of several stress genes coding for molecular chaperones of the glucose-regulated protein (GRP) family is predominantly regulated by another stress-detecting system, which is known as the unfolded protein response (UPR) system and is activated in response to massive protein misfolding in the endoplasmic reticulum and mitochondrial matrix. A translational fine tuning of HS protein expression occurs via changing the phosphorylation status of several proteins involved in translation initiation. In addition, specific signal sequences in the 5'-UTRs of some HS protein mRNAs ensure their preferential translation in stress.

The neuroprotective action of dexmedetomidine on apoptosis, calcium entry and oxidative stress in cerebral ischemia-induced rats: Contribution of TRPM2 and TRPV1 channels.

Dexmedetomidine (DEX) may act as an antioxidant through regulation of TRPM2 and TRPV1 channel activations in the neurons by reducing cerebral ischemia-induced oxidative stress and apoptosis. The neuroprotective roles of DEX were tested on cerebral ischemia (ISC) in the cultures of rat primary hippocampal and DRG neurons. Fifty-six rats were divided into five groups. A placebo was given to control, sham control, and ISC groups, respectively. In the third group, ISC was induced. The DEX and ISC+DEX groups received intraperitoneal DEX (40 μg/kg) 3, 24, and 48 hours after ISC induction. DEX effectively reversed capsaicin and cumene hydroperoxide/ADP-ribose-induced TRPV1 and TRPM2 densities and cytosolic calcium ion accumulation in the neurons, respectively. In addition, DEX completely reduced ISC-induced oxidative toxicity and apoptosis through intracellular reactive oxygen species production and depolarization of mitochondrial membrane. The DEX and ISC+DEX treatments also decreased the expression levels of caspase 3, caspase 9, and poly (ADP-ribose) polymerase in the hippocampus and DRG. In conclusion, the current results are the first to demonstrate the molecular level effects of DEX on TRPM2 and TRPV1 activation. Therefore, DEX can have remarkable neuroprotective impairment effects in the hippocampus and DRG of ISC-induced rats.

Methylglyoxal: An Emerging Signaling Molecule in Plant Abiotic Stress Responses and Tolerance.

The oxygenated short aldehyde methylglyoxal (MG) is produced in plants as a by-product of a number of metabolic reactions, including elimination of phosphate groups from glycolysis intermediates dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. MG is mostly detoxified by the combined actions of the enzymes glyoxalase I and glyoxalase II that together with glutathione make up the glyoxalase system. Under normal growth conditions, basal levels of MG remain low in plants; however, when plants are exposed to abiotic stress, MG can accumulate to much higher levels. Stress-induced MG functions as a toxic molecule, inhibiting different developmental processes, including seed germination, photosynthesis and root growth, whereas MG, at low levels, acts as an important signaling molecule, involved in regulating diverse events, such as cell proliferation and survival, control of the redox status of cells, and many other aspects of general metabolism and cellular homeostases. MG can modulate plant stress responses by regulating stomatal opening and closure, the production of reactive oxygen species, cytosolic calcium ion concentrations, the activation of inward rectifying potassium channels and the expression of many stress-responsive genes. MG appears to play important roles in signal transduction by transmitting and amplifying cellular signals and functions that promote adaptation of plants growing under adverse environmental conditions. Thus, MG is now considered as a potential biochemical marker for plant abiotic stress tolerance, and is receiving considerable attention by the scientific community. In this review, we will summarize recent findings regarding MG metabolism in plants under abiotic stress, and evaluate the concept of MG signaling. In addition, we will demonstrate the importance of giving consideration to MG metabolism and the glyoxalase system, when investigating plant adaptation and responses to various environmental stresses.

Indole-3-acetic acid-induced oxidative burst and an increase in cytosolic calcium ion concentration in rice suspension culture.

Indole-3-acetic acid (IAA) is the major natural auxin involved in the regulation of a variety of growth and developmental processes such as division, elongation, and polarity determination in growing plant cells. It has been shown that dividing and/or elongating plant cells accompanies the generation of reactive oxygen species (ROS) and a number of reports have suggested that hormonal actions can be mediated by ROS through ROS-mediated opening of ion channels. Here, we surveyed the link between the action of IAA, oxidative burst, and calcium channel activation in a transgenic cells of rice expressing aequorin in the cytosol. Application of IAA to the cells induced a rapid and transient generation of superoxide which was followed by a transient increase in cytosolic Ca(2+) concentration ([Ca(2+)]c). The IAA-induced [Ca(2+)]c elevation was inhibited by Ca(2+) channel blockers and a Ca(2+) chelator. Furthermore, ROS scavengers effectively blocked the action of IAA on [Ca(2+)]c elevation.

Porcine Circovirus Type 2 Activates CaMMKβ to Initiate Autophagy in PK-15 Cells by Increasing Cytosolic Calcium.

Porcine circovirus type 2 (PCV2) induces autophagy via the 5′ adenosine monophosphate-activated protein kinase (AMPK)/extracellular signal-regulated kinase (ERK)/tuberous sclerosis complex 2 (TSC2)/mammalian target of rapamycin (mTOR) pathway in pig kidney PK-15 cells. However, the underlying mechanisms of AMPK activation in autophagy induction remain unknown. With specific inhibitors and RNA interference (RNAi), we show that PCV2 infection upregulated calcium/calmodulin-dependent protein kinase kinase-beta (CaMKKβ) by increasing cytosolic Ca2+ via inositol 1,4,5-trisphosphate receptor (IP3R). Elevation of cytosolic calcium ion (Ca2+) did not seem to involve inositol 1,4,5-trisphosphate (IP3) release from phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide phospholipase C-gamma (PLC-γ). CaMKKβ then activated both AMPK and calcium/calmodulin-dependent protein kinase I (CaMKI). PCV2 employed CaMKI and Trp-Asp (WD) repeat domain phosphoinositide-interacting protein 1 (WIPI1) as another pathway additional to AMPK signaling in autophagy initiation. Our findings could help better understanding of the signaling pathways of autophagy induction as part of PCV2 pathogenesis. Further research is warranted to study if PCV2 interacts directly with IP3R or indirectly with the molecules that antagonize IP3R activity responsible for increased cytosolic Ca2+ both in PK-15 cells and PCV2-targeted primary cells from pigs.

Induction of systemic disease resistance in Nicotiana benthamiana by the cyclodipeptides cyclo (l-Pro-l-Pro) and cyclo (d-Pro-d-Pro).

Cyclodipeptides, formed from two amino acids by cyclodehydration, are produced naturally by many organisms, and are known to possess a large number of biological activities. In this study, we found that cyclo (l-Pro-l-Pro) and cyclo (d-Pro-d-Pro) (where Pro is proline) could induce defence responses and systemic resistance in Nicotiana benthamiana. Treatment with the two cyclodipeptides led to a reduction in disease severity by Phytophthora nicotianae and Tobacco mosaic virus (TMV) infections compared with controls. Both cyclopeptides triggered stomatal closure, induced reactive oxygen species production and stimulated cytosolic calcium ion and nitric oxide production in guard cells. In addition, the application of cyclodipeptides significantly up-regulated the expression of the plant defence gene PR-1a and the PR-1a protein, and increased cellular salicylic acid (SA) levels. These results suggest that the SA-dependent defence pathway is involved in cyclodipeptide-mediated pathogen resistance in N. benthamiana. We report the systemic resistance induced by cyclodipeptides, which sheds light on the potential of cyclodipeptides for the control of plant diseases.

Salivary Peptides Regulate Intracellular Calcium in Rat Pancreatic Beta Cells

ObjectivesProline Rich Proteins (PRPs), secreted by parotid gland, have a variety of functions including anti‐caries and anti‐bacterial properties, but less is known about their actions outside the oral cavity. One of these proteins, called Parotid Hormone (PH) in Sus scrofa, is believed to play a significant role in the maintenance of healthy dentin and have effects on other organs including pancreas by having regulatory effects on maintaining the sensitivity of beta cells to circulating glucose. Basic Salivary Proline Rich Protein 2 and 3 (PRB2 & PRB3), in human, have the highest sequence homology with porcine Parotid Hormone. Changes in cytosolic calcium ion concentration constitute an important element of signal transduction that may lead to insulin release. We hypothesized that PH, PRB2 and PRB3 are involved in insulin stimulation by regulating the intracellular calcium ion levels in pancreatic beta cells.MethodsCultured RIN‐m beta cells from Rattus Norvegicus were used as an in vitro model for measuring intra‐cellular calcium levels using the fluorescent dye Fura2‐AM. Cells in calcium‐rich or calcium‐free medium were treated with PH (34nmoles/200 μL), PRB2 (11nmoles/200μL) or PRB3 (11nmoles/200μL). In some experiments cells were pretreated with the L‐type calcium channel blocker diltiazem (10μM) or with thapsigargin (2μM) to deplete internal calcium stores, before treatment with the peptides.ResultsPH, PRB2 and PRB3 increased intracellular calcium levels in cultured beta cells by 3‐, 10‐ and 3‐fold respectively. In calcium‐free medium, no change was observed in intracellular calcium level after treatment with PH or PRB2; however treatment with PRB3 caused an increase in intracellular calcium level. Interestingly, calcium uptake by cells following PH and PRB2 treatment was partially blocked by diltiazem (10μM), whereas thapsigargin (2μM) treatment blocked the response caused by PRB3.ConclusionSalivary peptides PH, PRB2 and PRB3 show differential regulation of intracellular calcium in RIN‐m beta cells. Our data indicates that PH and PRB2 regulate an influx of external calcium partially through L‐type calcium channels, while PRB3 potentially regulates an increase of calcium from internal stores. In summary, our results suggest that PH, PRB2 and PRB3 may have regulatory functions beyond those that have been described in the literature, and may play a role in a parotid‐pancreatic axis.Support or Funding InformationDepartmental funding from the Department of Diagnostic and Biomedical Sciences, University of Texas Health Science Center at Houston, School of Dentistry.

Eryptosis-inducing activity of bisphenol A and its analogs in human red blood cells (in vitro study)

Bisphenols are important chemicals that are widely used in the manufacturing of polycarbonates, epoxy resin and thermal paper, and thus the exposure of humans to these substances has been noted. The purpose of this study was to assess eryptotic changes in human erythrocytes exposed (in vitro) to bisphenol A (BPA) and its selected analogs, i.e.,bisphenol F (BPF), bisphenol S (BPS) and bisphenol AF (BPAF). The erythrocytes were incubated with compounds studied at concentrations ranging from 1 to 250μg/mL for 4, 12 or 24h. The results showed that BPA and its analogs increased cytosolic calcium ions level with the strongest effect noted for BPAF. It has also been revealed that all bisphenols analyzed, and BPAF and BPF in particular increased phosphatidylserine translocation in red blood cells, which confirmed that they exhibited eryptotic potential in this cell type. Furthermore, it was shown that BPA and its analogs caused significant increase in calpain and caspase-3 activities, while the strongest effect was noted for BPAF. BPS, which is the main substituent of bisphenol A in polymers and thermal paper production exhibited similar eryptotic potential to BPA. Eryptotic changes in human erythrocytes were provoked by bisphenols at concentrations, which may influence the human body during occupational exposure or subacute poisoning with these compounds.

Eryptosis as an Underlying Mechanism in Systemic Lupus Erythematosus-Related Anemia

Background: The progression of systemic lupus erythematosus (SLE) leads to anemia in patients, adversely affecting prognosis. The diverse causes of anemia may include excessive eryptosis or premature suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine (PS) exposure on the cell surface. The present study explored if SLE enhances eryptosis and the underlying mechanisms. Materials and Methods: Eryptosis was assessed using flow cytometry in healthy volunteers (n = 20) and anemic patients hospitalized for SLE (n = 22), for parameters including PS exposure, cell volume, cytosolic calcium ion (Ca²⁺) levels and reactive oxygen species (ROS) and ceramide abundance. These indicators were measured in erythrocytes of experimental subjects and erythrocytes treated with plasma from healthy volunteers or SLE patients. Results: The hemoglobin and hematocrit levels were significantly lower in anemic SLE patients than in healthy volunteers (***p<0.001, p<0.001, respectively). The percentage of PS-exposing erythrocytes was significantly higher in SLE patients than in healthy volunteers (p<0.001), accompanied by an increase in cytosolic Ca²⁺ levels, oxidative stress. The measurements of PS and Ca²⁺ levels were significantly higher in the erythrocytes of healthy volunteers following incubation in plasma of SLE patients than in plasma of healthy volunteers for 24h (***p<0.001, *p<0.05 respectively). Conclusion: Eryptosis is enhanced in SLE and may contribute to anemia. The probable underlying mechanisms may be an excessive formation of ROS in erythrocytes. Also, some plasma components may trigger eryptosis by increasing the cytosolic Ca²⁺ concentration.

Phytoplankton calcification as an effective mechanism to alleviate cellular calcium poisoning

Abstract. Marine phytoplankton have developed the remarkable ability to tightly regulate the concentration of free calcium ions in the intracellular cytosol at a level of ~ 0.1 μmol L−1 in the presence of seawater Ca2+ concentrations of 10 mmol L−1. The low cytosolic calcium ion concentration is of utmost importance for proper cell signalling function. While the regulatory mechanisms responsible for the tight control of intracellular Ca2+ concentration are not completely understood, phytoplankton taxonomic groups appear to have evolved different strategies, which may affect their ability to cope with changes in seawater Ca2+ concentrations in their environment on geological timescales. For example, the Cretaceous (145 to 66 Ma), an era known for the high abundance of coccolithophores and the production of enormous calcium carbonate deposits, exhibited seawater calcium concentrations up to 4 times present-day levels. We show that calcifying coccolithophore species (Emiliania huxleyi, Gephyrocapsa oceanica and Coccolithus braarudii) are able to maintain their relative fitness (in terms of growth rate and photosynthesis) at simulated Cretaceous seawater calcium concentrations, whereas these rates are severely reduced under these conditions in some non-calcareous phytoplankton species (Chaetoceros sp., Ceratoneis closterium and Heterosigma akashiwo). Most notably, this also applies to a non-calcifying strain of E. huxleyi which displays a calcium sensitivity similar to the non-calcareous species. We hypothesize that the process of calcification in coccolithophores provides an efficient mechanism to alleviate cellular calcium poisoning and thereby offered a potential key evolutionary advantage, responsible for the proliferation of coccolithophores during times of high seawater calcium concentrations. The exact function of calcification and the reason behind the highly ornate physical structures of coccoliths remain elusive.

TRPC6 is the endothelial calcium channel that regulates leukocyte transendothelial migration during the inflammatory response.

Leukocyte transendothelial migration (TEM) is a tightly regulated, multistep process that is critical to the inflammatory response. A transient increase in endothelial cytosolic free calcium ion concentration (↑[Ca(2+)]i) is required for TEM. However, the mechanism by which endothelial ↑[Ca(2+)]i regulates TEM and the channels mediating this ↑[Ca(2+)]i are unknown. Buffering ↑[Ca(2+)]i in endothelial cells does not affect leukocyte adhesion or locomotion but selectively blocks TEM, suggesting a role for ↑[Ca(2+)]i specifically for this step. Transient receptor potential canonical 6 (TRPC6), a Ca(2+) channel expressed in endothelial cells, colocalizes with platelet/endothelial cell adhesion molecule-1 (PECAM) to surround leukocytes during TEM and clusters when endothelial PECAM is engaged. Expression of dominant-negative TRPC6 or shRNA knockdown in endothelial cells arrests neutrophils apically over the junction, similar to when PECAM is blocked. Selectively activating endothelial TRPC6 rescues TEM during an ongoing PECAM blockade, indicating that TRPC6 functions downstream of PECAM. Furthermore, endothelial TRPC6 is required for trafficking of lateral border recycling compartment membrane, which facilitates TEM. Finally, mice lacking TRPC6 in the nonmyeloid compartment (i.e., endothelium) exhibit a profound defect in neutrophil TEM with no effect on leukocyte trafficking. Our findings identify endothelial TRPC6 as the calcium channel mediating the ↑[Ca(2+)]i required for TEM at a step downstream of PECAM homophilic interactions.

Bisphenol A and its analogs exhibit different apoptotic potential in peripheral blood mononuclear cells (in vitro study)

There are only a few studies that have assessed the effect of bisphenol A (BPA) on human blood cells and no study has been conducted to analyze the impact of BPA analogs on human leucocytes. In this study, we have investigated the effect of BPA and its analogs like bisphenol F (BPF), bisphenol S (BPS) and bisphenol AF (BPAF) on apoptosis induction in human peripheral blood mononuclear cells (PBMCs). In order to clarify the mechanism of bisphenols-induced programmed cell death, changes in various signaling molecules of this process have been assessed. We observed an increase in cytosolic calcium ions (Ca2+) level and reduction of transmembrane mitochondrial potential (ΔΨm) in PBMCs incubated with all compounds examined, and particularly BPA and BPAF. All compounds studied changed PBMCs membrane permeability, activated caspase-8, -9, -3 and induced PARP-1 cleavage and chromatin condensation, which confirmed that they were capable of inducing apoptosis both via intrinsic and extrinsic pathway. Moreover, we have found that modus operandi of bisphenols studied was different. We noticed that BPAF and BPS caused mainly necrotic and apoptotic changes, respectively, whereas BPA induced comparable apoptotic and necrotic effects in the incubated cells.

VEGF-A isoform-specific regulation of calcium ion flux, transcriptional activation and endothelial cell migration.

Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology such as cell migration, proliferation, tubulogenesis and cell-cell interactions. Numerous isoforms of VEGF-A exist but their physiological significance is unclear. Here we evaluated two different VEGF-A isoforms and discovered differential regulation of cytosolic calcium ion flux, transcription factor localisation and endothelial cell response. Analysis of VEGF-A isoform-specific stimulation of VEGFR2-dependent signal transduction revealed differential capabilities for isoform activation of multiple signal transduction pathways. VEGF-A165 treatment promoted increased phospholipase Cγ1 phosphorylation, which was proportional to the subsequent rise in cytosolic calcium ions, in comparison to cells treated with VEGF-A121. A major consequence of this VEGF-A isoform-specific calcium ion flux in endothelial cells is differential dephosphorylation and subsequent nuclear translocation of the transcription factor NFATc2. Using reverse genetics, we discovered that NFATc2 is functionally required for VEGF-A-stimulated endothelial cell migration but not tubulogenesis. This work presents a new mechanism for understanding how VEGF-A isoforms program complex cellular outputs by converting signal transduction pathways into transcription factor redistribution to the nucleus, as well as defining a novel role for NFATc2 in regulating the endothelial cell response.

A novel, rapid method to quantify intraplatelet calcium dynamics by ratiometric flow cytometry.

Cytosolic free calcium ions represent important second-messengers in platelets. Therefore, quantitative measurement of intraplatelet calcium provides a popular and very sensitive tool to evaluate platelet activation and reactivity. Current protocols for determination of intracellular calcium concentrations in platelets have a number of limitations. Cuvette-based methods do not allow measurement of calcium flux in complex systems, such as whole blood, and therefore require isolation steps that potentially interfere with platelet activation. Flow cytometry has the potential to overcome this limitation, but to date the application of calibrated, quantitative readout of calcium kinetics has only been described for Indo-1. As excitation of Indo-1 requires a laser in the ultraviolet range, such measurements cannot be performed with a standard flow cytometer. Here, we describe a novel, rapid calibration method for ratiometric calcium measurement in platelets using both Ar+-laser excited fluorescence dyes Fluo-4 and Fura Red. We provide appropriate equations that allow rapid quantification of intraplatelet calcium fluxes by measurement of only two standardisation buffers. We demonstrate that this method allows quantitative calcium measurement in platelet rich plasma as well as in whole blood. Further, we show that this method prevents artefacts due to platelet aggregate formation and is therefore an ideal tool to determine basal and agonist induced calcium kinetics.

Psychiatric Disorders and TRP Channels: Focus on Psychotropic Drugs.

Psychiatric and neurological disorders are mostly associated with the changes in neural calcium ion signaling pathways required for activity-triggered cellular events. One calcium channel family is the TRP cation channel family, which contains seven subfamilies. Results of recent papers have discovered that calcium ion influx through TRP channels is important. We discuss the latest advances in calcium ion influx through TRP channels in the etiology of psychiatric disorders.Activation of TRPC4, TRPC5, and TRPV1 cation channels in the etiology of psychiatric disorders such as anxiety, fear-associated responses, and depression modulate calcium ion influx. Evidence substantiates that anandamide and its analog (methanandamide) induce an anxiolytic-like effect via CB1 receptors and TRPV1 channels. Intracellular calcium influx induced by oxidative stress has an significant role in the etiology of bipolar disorders (BDs), and studies recently reported the important role of TRP channels such as TRPC3, TRPM2, and TRPV1 in converting oxidant or nitrogen radical signaling to cytosolic calcium ion homeostasis in BDs. The TRPV1 channel also plays a function in morphine tolerance and hyperalgesia. Among psychotropic drugs, amitriptyline and capsazepine seem to have protective effects on psychiatric disorders via the TRP channels. Some drugs such as cocaine and methamphetamine also seem to have an important role in alcohol addiction and substance abuse via activation of the TRPV1 channel.Thus, we explore the relationships between the etiology of psychiatric disorders and TRP channel-regulated mechanisms. Investigation of the TRP channels in psychiatric disorders holds the promise of the development of new drug treatments.

Cytosolic calcium ions exert a major influence on the firing rate and maintenance of pacemaker activity in guinea-pig sinus node.

The sino-atrial node (SAN) provides the electrical stimulus to initiate every heart beat. Cellular processes underlying this activity have been debated extensively, especially with regards to the role of intracellular calcium. We have used whole-cell application of 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), a rapid calcium chelator, to guinea pig isolated SAN myocytes to assess the effect of rapid reduction of intracellular calcium on SAN cell electrical activity. High-dose (10 mM) BAPTA induced rapid and complete cessation of rhythmic action potential (AP) firing (time to cessation 5.5 ± 1.7 s). Over a range of concentrations, BAPTA induced slowing of action potential firing and disruption of rhythmic activity, which was dose-dependent in its time of onset. Exposure to BAPTA was associated with stereotyped action potential changes similar to those previously reported in the presence of ryanodine, namely depolarization of the most negative diastolic potential, prolongation of action potentials and a reduction in action potential amplitude. These experiments are consistent with the view that cytosolic calcium is essential to the maintenance of rhythmic pacemaker activity.

Regulation of ryanodine receptor-mediated calcium signaling by presenilins

Ca 2+ dyshomeostasis is a critical causative mechanism underlying the functional impairment of the central nervous system seen in ‘healthy’ aging [1] , specifically in normal brain aging processes in the absence of disease-causing mutations or external causes. It is generally thought that such impairments result from the gradual accumulation of small changes at the molecular level, such as oxidative damage to the structure and function of synapses, that ultimately cause the loss of biological function [2] . Alzheimer’s disease (AD) is a multifactorial pathology directly related to aging and with age as a primary risk factor [3] . Neurons affected by AD have an elevated cytosolic calcium ion concentration [4] . It has not yet been determined if the observed elevation of cytosolic calcium is contributing to or results from AD pathology, but there is mounting evidence in support of the Calcium Hypothesis of brain aging [4-9] . The two presenilin genes found in vertebrates are ubiquitously expressed as the transmembrane proteins presenilin-1 (PS1) and presenilin-2 (PS2), respectively, in the endoplasmic reticulum (ER). They are part of the γ-secretase enzyme complex catalyzing the processing of amyloid precursor protein (APP), which - if aberrant under disease conditions - can result in the generation of neurotoxic amyloid-beta (Aβ) peptides [10] (Fig. 1). Approximately 80% of the mutations linked to familial AD have been found in PS1 [11] and clinically relevant PS1 mutations exert effects on cytosolic calcium concentrations [10, 12] . While both presenilins affect cytosolic calcium concentrations, the PS1 isoform binds to several proteins essential to the regulation of intracellular calcium signaling, e.g. the inositol 1,4,5-trisphosphate receptor (IP 3 R) [13] , the N -methyl-D-aspartate (NMDA) receptor (NMDAR) [14] , and the ryanodine receptor (RyR) [15-17] . Here, the interaction of presenilins with RyRs will be reviewed. PMID: 25646163 [PubMed]