Induced Calcium Entry Research Articles

Piezo1 stretch-activated channel activity differs between murine bone marrow-derived and cardiac tissue-resident macrophages.

Macrophages (MΦ) play pivotal roles in tissue homeostasis and repair. Their mechanical environment has been identified as a key modulator of various cell functions, and MΦ mechanosensitivity is likely to be critical - in particular in a rhythmically contracting organ such as the heart. Cultured MΦ, differentiated in vitro from bone marrow (MΦBM), form a popular research model. This study explores the activity of mechanosensitive ion channels (MSC) in murine MΦBM and compares it to MSC activity in MΦ enzymatically isolated from cardiac tissue (tissue-resident MΦ; MΦTR). We show that MΦBM and MΦTR have stretch-induced currents, indicating the presence of functional MSC in their plasma membrane. The current profiles in MΦBM and in MΦTR show characteristics of cation non-selective MSC such as Piezo1 or transient receptor potential channels. While Piezo1 ion channel activity is detectable in the plasma membrane of MΦBM using the patch-clamp technique, or by measuring cytosolic calcium concentration upon perfusion with the Piezo1 channel agonist Yoda1, no Piezo1 channel activity was observed in MΦTR. The selective transient receptor potential vanilloid 4 (TRPV4) channel agonist GSK1016790A induces calcium entry in MΦTR and in MΦBM. In MΦ isolated from left-ventricular scar tissue 28days after cryoablation, stretch-induced current characteristics are not significantly different compared to non-injured control tissue, even though scarred ventricular tissue is expected to be mechanically remodelled and to contain an altered composition of pre-existing cardiac and circulation-recruited MΦ. Our data suggest that the in vitro differentiation protocols used to obtain MΦBM generate cells that differ from MΦ recruited from the circulation during tissue repair in vivo. Further investigations are needed to explore MSC identity in lineage-traced MΦ in scar tissue, and to compare mechanosensitivity of circulating monocytes with that of MΦBM. KEY POINTS: Bone marrow-derived (MΦBM) and tissue resident (MΦTR) macrophages have stretch-induced currents, indicating expression of functional mechanosensitive channels (MSC) in their plasma membrane. Stretch-activated current profiles show characteristics of cation non-selective MSC; and mRNA coding for MSC, including Piezo1 and TRPV4, is expressed in murine MΦBM and in MΦTR. Calcium entry upon pharmacological activation of TRPV4 confirms functionality of the channel in MΦTR and in MΦBM. Piezo1 ion channel activity is detected in the plasma membrane of MΦBM but not in MΦTR, suggesting that MΦBM may not be a good model to study the mechanotransduction of MΦTR. Stretch-induced currents, Piezo1mRNA expression and response to pharmacological activation are not significantly changed in cardiac MΦ 28days after cryoinjury compared to sham operated mice.

Effect of Distinct Induced Calcium Entry Pathways on Mitochondrial Respiration in Pulmonary Microvascular Endothelial Cells

Pulmonary microvascular endothelial cells (PMVECs) are distributed in a unique vascular bed exposed to the entire cardiac output and thus are influenced by circulating molecules that regulate calcium influx. Despite recognition of mitochondria's ability to buffer calcium, disparate results assessing the role of mitochondrial calcium and oxidative respiration are reported. The impact of distinct calcium entry pathways on mitochondrial respiration in PMVEC is unknown. We hypothesize that calcium entry pathways originating in spatially distinct cellular regions lead to unique changes in PMVEC mitochondrial respiration. Calcium entry dynamic patterns were assessed using an Andor spinning disk confocal microscope in confluent PMVEC monolayers loaded with the cytosolic calcium sensor CAL520. Thapsigargin (1 μM), an alkaloid that inhibits the sarcoendoplasmic reticulum calcium ATPase and elicits store‐depletion‐induced calcium entry, resulted in a rapid, global increase in cytosolic calcium. In contrast, 4α‐phorbol 12,13‐didecanoate (4αPDD, 5 μM), an agonist of the transient receptor potential vanilloid 4 channel (TRPV4), resulted in a slower increase in cytosolic calcium when compared to thapsigargin and elicited repetitive, short‐lived dynamic calcium events that spread into local cellular microdomains. In parallel studies, mitochondrial respiration was assessed by measuring oxygen consumption rate (OCR) using the Seahorse XF24 analyzer. 40,000 PMVECs were seeded per well 24 hours prior to running the assay. Experiments followed a standardized sequential protocol to measure different mitochondrial states. At t=21 min, after obtaining three basal measurements, cells were exposed to substrates for mitochondrial complex I (pyruvate 1 mM) and complex II (succinate 1 mM), then either thapsigargin (1 μM, n=20) or 4αPDD (5 μM, n=20) were added to activate distinct calcium entry pathways. At t=38 min, the complex V inhibitor oligomycin (1 μM) was added. At t=55 min, the mitochondrial respiration uncoupler FCCP (1 μM) was added. At t=73 min, the complex I inhibitor rotenone (1 μM) and the complex III inhibitor antimycin A (1 μM) were added. Control groups (n=20) were exposed to a similar protocol with addition of vehicle instead of thapsigargin or 4αPDD. In the control group, under basal conditions OCR measured 195 pmol/min. Addition of oligomycin decreased OCR to 68 pmol/min, while addition of uncoupler FCCP induced maximal respiration of 389 pmol/min. Addition of rotenone and antimycin A ablated mitochondrial respiration as expected. In controls, the respiratory ratio, defined as the ratio of basal to oligomycin OCR, was 3.05 ± 0.08 (mean ± SEM). Addition of thapsigargin significantly decreased the respiratory ratio (2.27 ± 0.01), while in contrast, the ratio increased after exposure to 4αPDD (8.02 ± 0.89) (p<0.001, one‐way ANOVA). We conclude that the cytosolic calcium dynamics elicited by activation of store‐operated channels or TRPV4 in PMVECs differentially impact mitochondrial oxidative respiration.Support or Funding InformationNIH PO1 HL066299/Project 3 to M.I.T., NIH R01 HL118334 to D.F.A., and NIH S10OD02149 to USA microscopy core facility.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pyrtriazoles, a Novel Class of Store-Operated Calcium Entry Modulators: Discovery, Biological Profiling, and in Vivo Proof-of-Concept Efficacy in Acute Pancreatitis.

In recent years, channels that mediate store-operated calcium entry (SOCE, i.e., the ability of cells to sense a decrease in endoplasmic reticulum luminal calcium and induce calcium entry across the plasma membrane) have been associated with a number of disorders, spanning from immune disorders to acute pancreatitis and have been suggested to be druggable targets. In the present contribution, we exploited the click chemistry approach to synthesize a class of SOCE modulators where the arylamide substructure that characterizes most inhibitors so far described is substituted by a 1,4-disubstituted 1,2,3-triazole ring. Within this series, inhibitors of SOCE were identified and the best compound proved effective in an animal model of acute pancreatitis, a disease characterized by a hyperactivation of SOCE. Strikingly, two enhancers of the process were discovered, affording invaluable research tools to further explore the (patho)physiological role of capacitative calcium entry.

Deregulation of calcium homeostasis in Bcr-Abl-dependent chronic myeloid leukemia.

BackgroundChronic myeloid leukemia (CML) results from hematopoietic stem cell transformation by the bcr-abl chimeric oncogene, encoding a 210 kDa protein with constitutive tyrosine kinase activity. In spite of the efficiency of tyrosine kinase inhibitors (TKI; Imatinib), other strategies are explored to eliminate CML leukemia stem cells, such as calcium pathways.ResultsIn this work, we showed that Store-Operated Calcium Entry (SOCE) and thrombin induced calcium influx were decreased in Bcr-Abl expressing 32d cells (32d-p210). The 32d-p210 cells showed modified Orai1/STIM1 ratio and reduced TRPC1 expression that could explain SOCE reduction. Decrease in SOCE and thrombin induced calcium entry was associated to reduced Nuclear Factor of Activated T cells (NFAT) nucleus translocation in 32d-p210 cells. We demonstrated that SOCE blockers enhanced cell mobility of 32d-p210 cells and reduced the proliferation rate in both 32d cell lines. TKI treatment slightly reduced the thrombin-induced response, but imatinib restored SOCE to the wild type level. Bcr-Abl is also known to deregulate Protein Kinase C (PKC), which was described to modulate calcium entries. We showed that PKC enhances SOCE and thrombin induced calcium entries in control cells while this effect is lost in Bcr-Abl-expressing cells.ConclusionThe tyrosine kinase activity seems to regulate calcium entries probably not directly but through a global cellular reorganization involving a PKC pathway. Altogether, calcium entries are deregulated in Bcr-Abl-expressing cells and could represent an interesting therapeutic target in combination with TKI.

The potassium channel KCa3.1 constitutes a pharmacological target for astrogliosis associated with ischemia stroke

BackgroundReactive astrogliosis is one of the significantly pathological features in ischemic stroke accompanied with changes in gene expression, morphology, and proliferation. KCa3.1 was involved in TGF-β-induced astrogliosis in vitro and also contributed to astrogliosis-mediated neuroinflammation in neurodegeneration disease.MethodsWild type mice and KCa3.1−/− mice were subjected to permanent middle cerebral artery occlusion (pMCAO) to evaluate the infarct areas by 2,3,5-triphenyltetrazolium hydrochloride staining and neurological deficit. KCa3.1 channels expression and cell localization in the brain of pMCAO mice model were measured by immunoblotting and immunostaining. Glia activation and neuron loss was measured by immunostaining. DiBAC4 (3) and Fluo-4AM were used to measure membrane potential and cytosolic Ca2+ level in oxygen-glucose deprivation induced reactive astrocytes in vitro.ResultsImmunohistochemistry on pMCAO mice infarcts showed strong upregulation of KCa3.1 immunoreactivity in reactive astrogliosis. KCa3.1−/− mice exhibited significantly smaller infarct areas on pMCAO and improved neurological deficit. Both activated gliosis and neuronal loss were attenuated in KCa3.1−/− pMCAO mice. In the primary cultured astrocytes, the expressions of KCa3.1 and TRPV4 were increased associated with upregulation of astrogliosis marker GFAP induced by oxygen-glucose deprivation. The activation of KCa3.1 hyperpolarized membrane potential and, by promoting the driving force for calcium, induced calcium entry through TRPV4, a cation channel of the transient receptor potential family. Double-labeled staining showed that KCa3.1 and TRPV4 channels co-localized in astrocytes. Blockade of KCa3.1 or TRPV4 inhibited the phenotype switch of reactive astrogliosis.ConclusionsOur data suggested that KCa3.1 inhibition might represent a promising therapeutic strategy for ischemia stroke.

Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models

Background & AimsSustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release–activated calcium modulator ORAI1 is the most abundant Ca2+ entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice.MethodsMouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects.ResultsGSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca2+ currents after Ca2+ release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis.ConclusionsCytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed for the treatment of patients with pancreatitis.

AP2M1 induced internalization of TRPC6 regulates calcium influx

Transient receptor canonical channel (TRPC)‐6, activated by second messenger DAG, induces calcium entry in endothelial cells independent of ER store depletion. We recently showed that deletion of TRPC6 in mice prevented lung endothelial permeability and inflammation in response to thrombin and LPS indicating targeting TRPC6 will be valuable in preventing uncontrolled increase in endothelial permeability. Here we determine the pathway that inhibits TRPC6 channel activity under physiological setting. Amino acid sequence analyses of TRPC6 reveals the presence of 4 ankyrin‐like repeat domains which are known to be important for protein‐protein interactions and activity. Thus, we performed mutational analysis on these repeats and assess channel activity. We found that deletion of Ist ankyrin repeat in TRPC6 blocked calcium influx in HEK cells. Further mutational analysis identified that 96‐111 residues within Ist ankyrin repeat was required for TRPC6 cell surface retention and calcium influx. Sequence analysis using ELM database showed that this domain has consensus site for AP2M1 (Adaptor‐related protein complex 2, mu 1 subunit) binding. AP2M1 is known to internalize transmembrane proteins via clathrin dependent endocytosis. Overexpression of AP2M1 with wt‐TRPC6 in HEK cells decreased calcium influx as well as expression level of TRPC6. Thus, designing a peptide against AP2M1 binding motif in TRPC6 will help in identifying targets for inhibiting TRPC6 activity.

Electromagnetic radiation (Wi-Fi) and epilepsy induce calcium entry and apoptosis through activation of TRPV1 channel in hippocampus and dorsal root ganglion of rats.

Incidence rates of epilepsy and use of Wi-Fi worldwide have been increasing. TRPV1 is a Ca(2+) permeable and non-selective channel, gated by noxious heat, oxidative stress and capsaicin (CAP). The hyperthermia and oxidant effects of Wi-Fi may induce apoptosis and Ca(2+) entry through activation of TRPV1 channel in epilepsy. Therefore, we tested the effects of Wi-Fi (2.45 GHz) exposure on Ca(2+) influx, oxidative stress and apoptosis through TRPV1 channel in the murine dorsal root ganglion (DRG) and hippocampus of pentylentetrazol (PTZ)-induced epileptic rats. Rats in the present study were divided into two groups as controls and PTZ. The PTZ groups were divided into two subgroups namely PTZ + Wi-Fi and PTZ + Wi-Fi + capsazepine (CPZ). The hippocampal and DRG neurons were freshly isolated from the rats. The DRG and hippocampus in PTZ + Wi-Fi and PTZ + Wi-Fi + CPZ groups were exposed to Wi-Fi for 1 hour before CAP stimulation. The cytosolic free Ca(2+), reactive oxygen species production, apoptosis, mitochondrial membrane depolarization, caspase-3 and -9 values in hippocampus were higher in the PTZ group than in the control although cell viability values decreased. The Wi-Fi exposure induced additional effects on the cytosolic Ca(2+) increase. However, pretreatment of the neurons with CPZ, results in a protection against epilepsy-induced Ca(2+) influx, apoptosis and oxidative damages. In results of whole cell patch-clamp experiments, treatment of DRG with Ca(2+) channel antagonists [thapsigargin, verapamil + diltiazem, 2-APB, MK-801] indicated that Wi-Fi exposure induced Ca(2+) influx via the TRPV1 channels. In conclusion, epilepsy and Wi-Fi in our experimental model is involved in Ca(2+) influx and oxidative stress-induced hippocampal and DRG death through activation of TRPV1 channels, and negative modulation of this channel activity by CPZ pretreatment may account for the neuroprotective activity against oxidative stress.

ROS Sensitive Calcium Channel TRPM2 Regulates VEGF Induced Angiogenesis

TRPM2 is a cation channel involved in calcium influx in response to cellular oxidative stress. We hypothesized that TRPM2 regulates VEGF induced vascular angiogenesis secondary to ROS production. We show that TRPM2 knockdown suppressed VEGF induced human lung endothelial cell (hLEC) hyper‐permeability, which is essential for angiogenesis, as measured by trans‐endothelial resistance and albumin influx (EBA). VEGF induced phosphorylation of VE cadherin at Tyr658 residue was inhibited by TRPM2 knockdown. Additionally, VEGF stimulation increased ROS production in hLEC, and application of apocynin (ROS scavenger) inhibited VEGF induced phosphorylation of Tyr658 of VE cadherin. TRPM2 knockdown reduced VEGF induced calcium entry in hLEC, and delayed migration of HUVEC. VEGF induced angiogenesis measured by in vivo matrigel plug assay was markedly reduced in TRPM2 KO mice compared to wild type. We conclude that calcium influx from ROS activated TRPM2 channels on VEGF stimulation is a key mechanism signaling angiogenesis.Research support: NIH grants RO1 HL45638‐23 and P01 HL077806

Hyposmotic Stress‐induced Cytoplasmic Calcium Increase Involves TRPV4 and Src Family Kinase Activation

Many cells respond to osmotic stress by releasing ATP that acts as an extracellular signal for purinergic receptors. Receptor‐triggered increases of calcium and activation of Src family tyrosine kinases (SFKs) have previously been observed in lens epithelium. Here we examined the effect of hyposmotic stress on cytoplasmic calcium [(Ca2+)i] response.Hyposmotic solution caused an immediate increase in (Ca2+)i. The selective Src Family Kinase (SFK) inhibitor PP2 suppressed the calcium increase. Hyposmotic solution was found to cause SFK activation which is significantly suppressed by RN 1734, a selective TRPV4 antagonist. The hypotonicity‐induced (Ca2+)i increase also was prevented by RN 1734 or HC 067047. TRPV4 agonist, GSK1016790A or 4á‐PDD caused similar increase in (Ca2+)i as that caused by hyposmotic stress. Expression of TRPV4 was confirmed by western blot. Pertussis toxin pretreatment for 24h blocked the calcium response. The calcium response points to the involvement of P2‐purinoceptors and G protein. Consistent with this notion, intact lenses released ATP when exposed to hyposmotic solution.The results suggest that the hyposmotic stress‐induced (Ca2+)i increase is the result of an interaction between purinergic P2 receptors and SFK. TRPV4 activation induced calcium entry could be the early mechanism for the complex signaling pathways in the lenses response to osmotic stress.

Sulfhydryl Modification Induces Calcium Entry through IP3-Sensitive Store-Operated Pathway in Activation-Dependent Human Neutrophils

As the first line of host defense, neutrophils are stimulated by pro-inflammatory cytokines from resting state, facilitating the execution of immunomodulatory functions in activation state. Sulfhydryl modification has a regulatory role in a wide variety of physiological functions through mediation of signaling transductions in various cell types. Recent research suggested that two kinds of sulfhydryl modification, S-nitrosylation by exogenous nitric oxide (NO) and alkylation by N-ethylmaleimide (NEM), could induce calcium entry through a non-store-operated pathway in resting rat neutrophils and DDT1MF-2 cells, while in active human neutrophils a different process has been observed by us. In the present work, data showed that NEM induced a sharp rising of cytosolic calcium concentration ([Ca2+]c) without external calcium, followed by a second [Ca2+]c increase with readdition of external calcium in phorbol 12-myristate 13-acetate (PMA)-activated human neutrophils. Meanwhile, addition of external calcium did not cause [Ca2+]c change of Ca2+-free PMA-activated neutrophils before application of NEM. These data indicated that NEM could induce believable store-operated calcium entry (SOCE) in PMA-activated neutrophils. Besides, we found that sodium nitroprusside (SNP), a donor of exogenous NO, resulted in believable SOCE in PMA-activated human neutrophils via S-nitrosylation modification. In contrast, NEM and SNP have no effect on [Ca2+]c of resting neutrophils which were performed in suspension. Furthermore, 2-Aminoethoxydiphenyl borate, a reliable blocker of SOCE and an inhibitor of inositol 1,4,5-trisphosphate (IP3) receptor, evidently abolished SNP and NEM-induced calcium entry at 75 µM, while preventing calcium release in a concentration-dependent manner. Considered together, these results demonstrated that NEM and SNP induced calcium entry through an IP3-sensitive store-operated pathway of human neutrophils via sulfhydryl modification in a PMA-induced activation-dependent manner.

Natural Forms of Vitamin E and 13□Œ‐Carboxychromanol, a Long‐Chain Vitamin E Metabolite, Inhibit Leukotriene Generation from Stimulated Neutrophils by Blocking Calcium Influx and Suppressing 5‐Lipoxygenase Activity, Respectively

Leukotrienes generated by 5‐lipoxygenase (5‐LOX)‐catalyzed reaction are key regulators of inflammation. In ionophore A23187‐stimulated human neutrophils or differentiated HL60 cells, vitamin E forms differentially inhibited leukotriene B4 (LTB4) with IC50 of 5–20μM for γ‐tocopherol, δ‐tocopherol (δT) and γ‐tocotrienol, but much higher for α‐tocopherol. 13′‐carboxychromanol, a long‐chain metabolite of δT, suppressed neutrophil‐ and HL60 cell‐generated LTB4 with IC50 of 4–7μM and potently inhibited human recombinant 5‐LOX activity with IC50 of 1.5±1μM, but other metabolites, such as 9′‐carboxychromanol and 7,8‐dimethyl‐2‐(β‐carboxyethyl)‐6‐hydroxychroman (CEHC) did not show promising effects. In contrast, vitamin E forms had no effect on human 5‐LOX activity, but impaired ionophore‐induced intracellular calcium increase and calcium influx as well as the subsequent signaling including ERK1/2 phosphorylation and 5‐LOX translocation. Further investigation showed that δT suppressed cytosolic Ca2+ increase and/or LTB4 formation triggered by ionophores, sphingosine 1‐phosphate and lysophosphatidic acid, but not by fMLP or thapsigargin, indicating that δT does not likely affect fMLP receptor‐mediated signaling or storage‐depletion induced calcium entry. Instead, δT prevented ionophore‐caused cytoplasmic membrane disruption, which may account for its blocking of calcium influx. These activities by vitamin E forms and long‐chain carboxychromanol provide potential molecular bases for the differential anti‐inflammatory effects of vitamin E forms in vivo.

An emerging problem in clinical practice: how to treat chronic headache patients

In this issue, an interesting article by Farinelli and coworkers [1] debates an important emerging and not yet resolved problem: how to treat the chronic headaches complicated by medication overuse. Headache is the most common neurological disease in clinical practice. In Europe, this affects about 51% of the population, of which 31% are tension-type headache and 14% are migraine sufferers; 2% of these patients become chronic sufferers, more than 15/day/month, and chronic daily headache (CDH). 4–5% of general population suffers from a chronic form (CDH), with prevalence between 1.7 and 2.1% in men and between 2.8 and 6.8% in women [2]; within this group of patients in the US general population ranges around 1.3–2% of chronic migraine (CM). A number of possible pathophysiological mechanisms for the transformation from episodic to chronic headache have been proposed including a progressive damage of the central nociceptive system. One physiopathological hypothesis is the activation of N-methyl-D-aspartate (NMDA) and nonNMDA receptors glutamate, released by central nociceptive terminations, induces calcium entry in dorsal horn neurons, as well as in the trigeminal nucleus caudalis. Calcium entry leads to the activation of nitric oxide (NO) synthetase causing NO synthesis. These neurotransmitters produce the release of sensory neuropeptides, such as CGRP and substance P, which support the development of hyperalgesia and maintain central sensitization [3–5]. Medication near-daily use and subsequent medication overuse headache (MOH) have been described by the revised International Classification of Headache Disorders (ICHDs)-IIR criteria as the use of each drug for at least 3 months, for a certain number of days per month, and is one of the most critical parameters in the process of

S1P triggers cerebral vessel constriction through the store‐operated calcium entry pathway

ObjectiveTo characterize signaling pathways by which sphingosine‐1‐phosphate (S1P) modulates vascular tone.MethodsThe role of store‐operated calcium entry (SOCE) in S1P‐induced Ca2+ entry in vascular smooth muscle cells (VSMCs) and vessel constriction was assessed using ratiometric Ca2+ measurements, quantitative RT‐PCR and isolated arteries.ResultsIn VSMCs, S1P triggered STIM1 puncta formation, and SOCE activated by S1P was inhibited by 2‐aminoethoxydiphenylborate (2‐APB), diethylstilbestrol and gadolinium. In VSMCs prepared from S1P3 receptor knockout mice, the initial calcium rise triggered by S1P was abolished, but SOCE was still present suggesting that S1P acts via multiple signaling pathways. S1P‐induced SOCE was larger in proliferative than in contractile VSMCs, correlating with increases in STIM1, Orai1, S1P1 and S1P3 receptor mRNA. STIM1, S1P1 and S1P3 receptor mRNA expression were larger in freshly isolated brain vessels than in aorta. These data, and the fact that 2‐APB inhibits S1P‐induced cerebral artery constriction, suggest that SOCE modulates S1P‐induced vasoconstriction in vivo.ConclusionThese data support a role for SOCE in S1P induced calcium entry and vessel constriction. The relationship between increased S1P signaling and SOCE in proliferative VSMCs suggests a role of S1P in disorders associated with vascular proliferation, such as atherosclerosis or vasospasm.

Intermediate-conductance Ca2+-activated K+ channels (IKCa1) regulate human prostate cancer cell proliferation through a close control of calcium entry

Accumulating data point to K(+) channels as relevant players in controlling cell cycle progression and proliferation of human cancer cells, including prostate cancer (PCa) cells. However, the mechanism(s) by which K(+) channels control PCa cell proliferation remain illusive. In this study, using the techniques of molecular biology, biochemistry, electrophysiology and calcium imaging, we studied the expression and functionality of intermediate-conductance calcium-activated potassium channels (IK(Ca1)) in human PCa as well as their involvement in cell proliferation. We showed that IK(Ca1) mRNA and protein were preferentially expressed in human PCa tissues, and inhibition of the IK(Ca1) potassium channel suppressed PCa cell proliferation. The activation of IK(Ca1) hyperpolarizes membrane potential and, by promoting the driving force for calcium, induces calcium entry through TRPV6, a cation channel of the TRP (Transient Receptor Potential) family. Thus, the overexpression of the IK(Ca1) channel is likely to promote carcinogenesis in human prostate tissue.

Two Chromogranin A-Derived Peptides Induce Calcium Entry in Human Neutrophils by Calmodulin-Regulated Calcium Independent Phospholipase A2

BackgroundAntimicrobial peptides derived from the natural processing of chromogranin A (CgA) are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides.Methodology/Principal FindingsPMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR) or Catestatin (CAT) induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate), a store operated channels (SOCs) blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ) and that the two sequences can be aligned with the two CaM-binding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity.Conclusions/SignificanceFor the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated iPLA2 pathway. Our study highlights the role of two CgA-derived peptides in the active communication between neuroendocrine and immune systems.

STIMulating Calcium Entry at ER-Plasma Membrane Junctions

Store-operated calcium (SOC) entry is essential for maintaining endoplasmic reticulum (ER) functions and generating the sustained calcium signals crucial for gene expression, secretion, cell motility, and cell proliferation. To understand the molecular mechanism of how receptor-evoked ER calcium store depletion induces calcium entry through SOC channels at the plasma membrane, we screened a small interference RNA (siRNA) library targeting the human signaling proteome and identified several regulators of SOC entry including STIM1 and STIM2.

Kihi-to, a herbal traditional medicine, improves Abeta(25–35)-induced memory impairment and losses of neurites and synapses

BackgroundWe previously hypothesized that achievement of recovery of brain function after the injury requires the reconstruction of neuronal networks, including neurite regeneration and synapse reformation. Kihi-to is composed of twelve crude drugs, some of which have already been shown to possess neurite extension properties in our previous studies. The effect of Kihi-to on memory deficit has not been examined. Thus, the goal of the present study is to determine the in vivo and in vitro effects of Kihi-to on memory, neurite growth and synapse reconstruction.MethodsEffects of Kihi-to, a traditional Japanese-Chinese traditional medicine, on memory deficits and losses of neurites and synapses were examined using Alzheimer's disease model mice. Improvements of Aβ(25–35)-induced neuritic atrophy by Kihi-to and the mechanism were investigated in cultured cortical neurons.ResultsAdministration of Kihi-to for consecutive 3 days resulted in marked improvements of Aβ(25–35)-induced impairments in memory acquisition, memory retention, and object recognition memory in mice. Immunohistochemical comparisons suggested that Kihi-to attenuated neuritic, synaptic and myelin losses in the cerebral cortex, hippocampus and striatum. Kihi-to also attenuated the calpain increase in the cerebral cortex and hippocampus. When Kihi-to was added to cells 4 days after Aβ(25–35) treatment, axonal and dendritic outgrowths in cultured cortical neurons were restored as demonstrated by extended lengths of phosphorylated neurofilament-H (P-NF-H) and microtubule-associated protein (MAP)2-positive neurites. Aβ(25–35)-induced cell death in cortical culture was also markedly inhibited by Kihi-to. Since NF-H, MAP2 and myelin basic protein (MBP) are substrates of calpain, and calpain is known to be involved in Aβ-induced axonal atrophy, expression levels of calpain and calpastatin were measured. Treatment with Kihi-to inhibited the Aβ(25–35)-evoked increase in the calpain level and decrease in the calpastatin level. In addition, Kihi-to inhibited Aβ(25–35)-induced calcium entry.ConclusionIn conclusion Kihi-to clearly improved the memory impairment and losses of neurites and synapses.

High Glucose Enhances Transient Receptor Potential Channel Canonical Type 6–Dependent Calcium Influx in Human Platelets via Phosphatidylinositol 3-Kinase–Dependent Pathway

Transient receptor potential canonical type 6 (TRPC6) channels mediating 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced calcium entry have been identified on human platelets. In the present study we tested the hypothesis that hyperglycemia increases the expression of TRPC6 channels. Platelets from healthy control subjects and patients with type 2 diabetes mellitus were incubated with glucose and calcium influx was measured using the fluorescent dye technique. TRPC channel protein expression was investigated using immunofluorescence and fluorescence microscopy of single platelets. Administration of 25 mmol/L glucose significantly enhanced the OAG-induced calcium influx, which was attenuated by inhibitors of the phosphatidylinositol 3-kinase, wortmannin or LY294002. The glucose-enhanced and OAG-induced calcium influx was concentration- and time-dependent. Glucose significantly increased the TRPC6 protein expression in platelets to 131+/-12% (n=33; P<0.05), whereas the expression of TRPC1, TRPC3, TRPC4, or TRPC5 were unchanged. The glucose-induced TRPC6 expression was significantly attenuated in the presence of wortmannin or LY294002. Platelets from patients with type 2 diabetes mellitus showed increased TRPC6 expression compared to nondiabetic individuals (P<0.05). The study indicates that high glucose increases TRPC6 channel protein expression on the platelet surface which is mediated by a phosphatidylinositol 3-kinase-dependent pathway.

Capacitative calcium entry and transient receptor potential canonical 6 expression control human hepatoma cell proliferation

Store-operated calcium entry (SOCE) is the main Ca(2+) influx pathway involved in controlling proliferation of the human hepatoma cell lines Huh-7 and HepG2. However, the molecular nature of the calcium channels involved in this process remains unknown. Huh-7 and HepG2 cells express transient receptor potential canonical 1 (TRPC1) and TRPC6, as well as STIM1 and Orai1, and these 4 channels are the most likely candidates to account for the SOCE in these cells. We generated stable TRPC6-overexpressing or TRPC6-knockdown Huh-7 clones, in which we investigated correlations between the presence of the protein, the rate of cell proliferation, and SOCE amplitude. TRPC6-overexpressing Huh-7 cells proliferated 80% faster than did untransfected cells and their SOCE amplitude was 160% higher. By contrast, proliferation rate was 50% lower and SOCE amplitude 85% lower in TRPC6-knockdown clones than in untransfected cells. OAG (olyl acetyl glycerol)-induced calcium entry was similar in all cells, and small interfering RNA (siRNA) against TRPC1 had no effect on SOCE amplitude, highlighting the relationship among SOCE, TRPC6 and cell proliferation in Huh-7 cells. SOCE amplitude was reduced by STIM1 and Orai1 knockdowns, suggesting possible cooperation between these proteins and TRPC6 in these cells. Endothelial growth factor and hepatocyte growth factor increased TRPC6 expression and SOCE amplitude in Huh-7 cells, and cyclin D1 expression was decreased by STIM1, Orai1, and TRPC6 knockdowns. TRPC6 was very weakly expressed in isolated hepatocytes from healthy patients and expressed more strongly in tumoral samples from the liver of a cancer patient, strongly supporting a role for these calcium channels in liver oncogenesis.