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The source and dynamics of calcium is the key factor that regulates dendritic integration. Apart from the voltage-gated and ligand-gated calcium influx, an important source of calcium is from inner store of endoplasmic reticulum with a regenerative process of calcium-induced calcium release (CICR). To trigger this process, inositol 1,4,5-trisphosphate (IP3) and calcium are needed to satisfy certain requirements. The aim of our paper is to investigate how the CICR depends on the dynamics of membrane potential. We utilize one dimensional dendritic model to calculate membrane potential by Nernst–Planck Equation (NPE) and cable model and Pure Diffusion (PD) model, computational simulations are carried out to inject the calcium influx by synaptic stimulation and to predict subsequent CICR and calcium wave propagation. Our results demonstrate that CICR initiation and calcium wave propagation have much difference between electro-diffusion process of NPE and cable model. We find that cable model has lower threshold of IP3 stimulation to trigger CICR but is more difficult for calcium propagation than NPE, PD model requires even higher threshold of IP3 to initiate CICR process and calcium duration is shorter than NPE; the regenerative calcium wave propagates with faster speed in NPE than that in cable model and in PD model. Our work addresses the important role of electro-diffusion dynamics of charged ions in regulating CICR process in dendritic structure; and provides theoretical predictions for neurological process which requires sustaining calcium for downstream signaling processes. [ABSTRACT FROM AUTHOR]

Background and Purpose: Ca2+ influx via TRPV4 channels triggers Ca2+ release from the IP3‐sensitive internal store to generate repetitive oscillations. Although mitochondria are acknowledged regulators of IP3‐mediated Ca2+ release, how TRPV4‐mediated Ca2+ signals are regulated by mitochondria is unknown. We show that depolarised mitochondria switch TRPV4 signalling from relying on Ca2+‐induced Ca2+ release at IP3 receptors to being independent of Ca2+ influx and instead mediated by ATP release via pannexins. Experimental Approach TRPV4‐evoked Ca2+ signals were individually examined in hundreds of cells in the endothelium of rat mesenteric resistance arteries using the indicator Cal520. Key Results: TRPV4 activation with GSK1016790A (GSK) generated repetitive Ca2+ oscillations that required Ca2+ influx. However, when the mitochondrial membrane potential was depolarised, by the uncoupler CCCP or complex I inhibitor rotenone, TRPV4 activation generated large propagating, multicellular, Ca2+ waves in the absence of external Ca2+. The ATP synthase inhibitor oligomycin did not potentiate TRPV4‐mediated Ca2+ signals. GSK‐evoked Ca2+ waves, when mitochondria were depolarised, were blocked by the TRPV4 channel blocker HC067047, the SERCA inhibitor cyclopiazonic acid, the PLC blocker U73122 and the inositol trisphosphate receptor blocker caffeine. The Ca2+ waves were also inhibited by the extracellular ATP blockers suramin and apyrase and the pannexin blocker probenecid. Conclusion and Implications: These results highlight a previously unknown role of mitochondria in shaping TRPV4‐mediated Ca2+ signalling by facilitating ATP release. When mitochondria are depolarised, TRPV4‐mediated release of ATP via pannexin channels activates plasma membrane purinergic receptors to trigger IP3‐evoked Ca2+ release. [ABSTRACT FROM AUTHOR]

Background and Purpose: Coordinated endothelial control of cardiovascular function is proposed to occur by endothelial cell communication via gap junctions and connexins. To study intercellular communication, the pharmacological agents carbenoxolone (CBX) and 18β-glycyrrhetinic acid (18βGA) are used widely as connexin inhibitors and gap junction blockers.Experimental Approach: We investigated the effects of CBX and 18βGA on intercellular Ca2+ waves, evoked by inositol 1,4,5-trisphosphate (IP3 ) in the endothelium of intact mesenteric resistance arteries.Key Results: Acetycholine-evoked IP3 -mediated Ca2+ release and propagated waves were inhibited by CBX (100 μM) and 18βGA (40 μM). Unexpectedly, the Ca2+ signals were inhibited uniformly in all cells, suggesting that CBX and 18βGA reduced Ca2+ release. Localised photolysis of caged IP3 (cIP3 ) was used to provide precise spatiotemporal control of site of cell activation. Local cIP3 photolysis generated reproducible Ca2+ increases and Ca2+ waves that propagated across cells distant to the photolysis site. CBX and 18βGA each blocked Ca2+ waves in a time-dependent manner by inhibiting the initiating IP3 -evoked Ca2+ release event rather than block of gap junctions. This effect was reversed on drug washout and was unaffected by small or intermediate K+ -channel blockers. Furthermore, CBX and 18βGA each rapidly and reversibly collapsed the mitochondrial membrane potential.Conclusion and Implications: CBX and 18βGA inhibit IP3 -mediated Ca2+ release and depolarise the mitochondrial membrane potential. These results suggest that CBX and 18βGA may block cell-cell communication by acting at sites that are unrelated to gap junctions. [ABSTRACT FROM AUTHOR]

Oxytocin (OXT) is a neuropeptide that activates the oxytocin receptor (OXTR), a rhodopsin family G-protein coupled receptor. Our localization of OXTR to the retinal pigment epithelium (RPE), in close proximity to OXT in the adjacent photoreceptor neurons, leads us to propose that OXT plays an important role in RPE-retinal communication. An increase of RPE [Ca 2 + ] i in response to OXT stimulation implies that the RPE may utilize oxytocinergic signaling as a mechanism by which it accomplishes some of its many roles. In this study, we used an established human RPE cell line, a HEK293 heterologous OXTR expression system, and pharmacological inhibitors of Ca 2 + signaling to demonstrate that OXTR utilizes capacitative Ca 2 + entry (CCE) mechanisms to sustain an increase in cytoplasmic Ca 2 + . These findings demonstrate how multiple functional outcomes of OXT-OXTR signaling could be integrated via a single pathway. In addition, the activated OXTR was able to inhibit the Kir7.1 channel, an important mediator of sub retinal waste transport and K + homeostasis. [ABSTRACT FROM AUTHOR]

An important step for successful fertilization and further development is the increase in intracellular Ca2+ in the activated oocyte. It has been known that starfish oocytes become increasingly sensitive to inositol 1,4,5-trisphosphate (IP3) during meiotic maturation to exhibit highly efficient IP3-induced Ca2+ release (IICR) by the time of germinal vesicle breakdown (GVBD). However, we noted that the peak level of intracellular Ca2+ increase after insemination is already high in the maturing oocytes before GVBD. Using maturing oocytes before GVBD, we investigated Ca2+ release mechanisms other than IICR. We report here that Ca2+-release mechanisms dependent on nicotinic acid adenine dinucleotide phosphate (NAADP) and nicotinamide adenine dinucleotide (NADP), the precursor of NAADP, became functional prior to the development of IICR mechanisms. As with IP3, but unlike NAADP, the Ca2+ stores responsive to NADP are sensitized during the meiotic maturation induced by 1-methyladenine (1-MA). This suggests that the process may represent a physiological response to the maturation hormone. NADP-dependent Ca2+ release in immature oocytes, however, did not induce oocyte maturation by itself, but was enhanced by the conditions mimicking the increases of intracellular Ca2+ and pH that take place in the maturing oocytes of starfish. [ABSTRACT FROM PUBLISHER]

Matured oocytes of the annelidan worm Pseudopotamilla occelata are fertilized at the first metaphase of the meiotic division. During the activation by fertilizing spermatozoa, the mature oocyte shows a two-step intracellular Ca2+ increase. Whereas the first Ca2+ increase is localized and appears to utilize the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores, the second Ca2+ increase is global and involves Ca2+ influx via voltage-gated Ca2+ channels on the entire surface of the oocyte. To study how sperm trigger the Ca2+ increases during fertilization, we prepared soluble sperm extract (SE) and examined its ability to induce Ca2+ increases in the oocyte. The SE could evoke a Ca2+ increase in the oocyte when it was added to the medium, but not when it was delivered by microinjection. However, the second-step Ca2+ increase leading to the resumption of meiosis did not follow in these eggs. Local application of SE induced a non-propagating Ca2+ increase and formed a cytoplasmic protrusion that was similar to that created by the fertilizing sperm at the first stage of the Ca2+ response, important for sperm incorporation into the oocyte. Our results suggest that the fertilizing spermatozoon may trigger the first-step Ca2+ increase before it fuses with the oocyte in a pathway that involves the G-protein-coupled receptor and phospholipase C. Thus, the first phase of the Ca2+ response in the fertilized egg of this species is independent of the second phase of the Ca2+ increase for egg activation. [ABSTRACT FROM PUBLISHER]

Phospholipase C-ε (PLCε) integrates signaling from G-protein coupled receptors (GPCRs) to downstream kinases to regulate a broad range of biological and pathophysiological responses. Relative to other PLCs, PLCε is unique in that it not only serves a catalytic function in phosphoinositide hydrolysis but also functions as an exchange factor small the low molecular weight G-protein Rap1. PLCε is selectively stimulated by agonists for GPCRs that couple to RhoA, which bind directly to the enzyme to regulate its activity. Rap1 also regulates PLCε activity by binding to its RA2 domain and this generates a feedback mechanism allowing sustained signaling. As a result of its regulation by inflammatory ligands for GPCRs and its ability to promote chronic signals, PLCε has been implicated in diseases ranging from cancer to ischemia/reperfusion injury. This review will discuss the regulation of PLCε, molecular mechanisms that contribute to sustained signaling, and the role of the enzyme in various disease contexts. [ABSTRACT FROM AUTHOR]

Abstract: The tomato [Solanum lycopersicum (Sl)] phosphatidylinositol-phospholipase C (PI-PLC) gene family is composed of six members, named SlPLC1 to SlPLC6, differentially regulated upon pathogen attack. We have previously shown that the fungal elicitor xylanase rapidly induces nitric oxide (NO), which is required for PI-PLCs activity and downstream defense responses in tomato cell suspensions. Here, we show that all six SlPLC genes are expressed in tomato cell suspensions. Treatment of the cells with xylanase induces an early increase in SlPLC5 transcript levels, followed by a raise of the amount of SlPLC2 transcripts. The production of NO is required to augment SlPLC5 transcript levels in xylanase-treated tomato cells. Xylanase also induces SlPLC2 and SlPLC5 transcript levels in planta. We knocked-down the expression of SlPLC2 and SlPLC5 by virus-induced gene silencing. We found that SlPLC2 is required for xylanase-induced expression of the defense-related genes PR1 and HSR203J. [Copyright &y& Elsevier]

Abstract: Introduction: Janus kinases (JAKs) are intracellular non-receptor tyrosine kinases that transduce cytokine-mediated signals through a pathway mediated by JAK and the signal transducer and activator of transcription (STAT) proteins. The JAK-STAT pathway is involved in immune response, inflammation, and tumorigenesis. Platelets are anuclear blood cells that play a central role in hemostasis. Methods: The aggregometry, immunoblotting, and platelet functional analysis used in this study. Results: We found that the JAK2 inhibitor AG490 (25 and 50μM) attenuated collagen-induced platelet aggregation and calcium mobilization in a concentration-dependent manner. In the presence of AG490, the phosphorylation of PLCγ2, protein kinase C (PKC), Akt or JNK in collagen-activated aggregation of human platelets was also inhibited. In addition, we found that various inhibitors, such as the PLCγ2 inhibitor U73122, the PKC inhibitor Ro318220, the phospoinositide 3-kinase inhibitor LY294002, the p38 mitogen-activated protein kinase inhibitor SB203580, the ERK inhibitor PD98059, and the JNK inhibitor SP600125, had no effects on collagen-induced JAK2 activity. However, U73122, Ro318220 and SP600125 significantly diminished collagen-induced STAT3 phosphorylation. These findings suggest that PLCγ2-PKC and JNK are involved in JAK2-STAT3 signaling in collagen-activated platelets. Conclusion: Our results demonstrate that the JAK2-STAT3 pathway is involved in collagen-induced platelet activation through the activation of JAK2-JNK/PKC-STAT3 signaling. The inhibition of JAK2 may represent a potential therapeutic strategy for the preventing or treating thromboembolic disorders. [Copyright &y& Elsevier]

Abstract: Cytoplasmic Ca2+ is a master regulator of airway physiology; it controls fluid, mucus, and antimicrobial peptide secretion, ciliary beating, and smooth muscle contraction. The focus of this review is on the role of cytoplasmic Ca2+ in fluid secretion by airway exocrine secretory cells. Airway submucosal gland serous acinar cells are the primary fluid secreting cell type of the cartilaginous conducting airways, and this review summarizes the current state of knowledge of the molecular mechanisms of serous cell ion transport, with an emphasis on their regulation by intracellular Ca2+. Many neurotransmitters that regulate secretion from serous acinar cells utilize Ca2+ as a second messenger. Changes in intracellular Ca2+ concentration regulate the activities of ion transporters and channels involved in transepithelial ion transport and fluid secretion, including Ca2+-activated K+ channels and Cl− channels. We also review evidence of interactions of Ca2+ signaling with other signaling pathways (cAMP, NO) that impinge upon different ion transport pathways, including the cAMP/PKA-activated cystic fibrosis (CF) transmembrane conductance regulator (CFTR) anion channel. A better understanding of Ca2+ signaling and its targets in airway fluid secretion may identify novel strategies to intervene in airway diseases, for example to enhance fluid secretion in CF airways. [Copyright &y& Elsevier]

Summary: Both in vacuolar and plasma membranes, in addition to truly K+-selective channels there is a variety of non-selective channels, which conduct K+ and other ions with little preference. Many non-selective channels in the plasma membrane are active at depolarized potentials, thus, contributing to K+ efflux rather than to K+ uptake. They may play important roles in xylem loading or contribute to a K+ leak, induced by salt or oxidative stress. Here, three currents, expressed in root cells, are considered: voltage-insensitive cation current, non-selective outwardly rectifying current, and low-selective conductance, activated by reactive oxygen species. The latter two do not only poorly discriminate between different cations (like K+ vs Na+), but also conduct anions. Such solute channels may mediate massive electroneutral transport of salts and might be involved in osmotic adjustment or volume decrease, associated with cell death. In the tonoplast two major currents are mediated by SV (slow) and FV (fast) vacuolar channels, respectively, which are virtually impermeable for anions. SV channels conduct mono- and divalent cations indiscriminately and are activated by high cytosolic Ca2+ and depolarized voltages. FV channels are inhibited by micromolar cytosolic Ca2+, Mg2+, and polyamines, and conduct a variety of monovalent cations, including K+. Strikingly, both SV and FV channels sense the K+ content of vacuoles, which modulates their voltage dependence, and in case of SV, also alleviates channel's inhibition by luminal Ca2+. Therefore, SV and FV channels may operate as K+-sensing valves, controlling K+ distribution between the vacuole and the cytosol. [ABSTRACT FROM AUTHOR]

Highlights: [•] Micronutrients in the one carbon cycle play a vital role in mediating epigenetic processes. [•] An imbalance in micronutrients reduced mRNA levels of BDNF, NGF, TrkB and CREB. [•] Prenatal omega-3 supplementation to an imbalanced diet normalized all mRNA levels. [•] An imbalanced diet adversely programmes brain development/function in the offspring. [ABSTRACT FROM AUTHOR]

Highlights: [•] A melatonin receptor antagonist blocked Ca2+ oscillation in P. falciparum and inhibited parasite growth. [•] P. falciparum development is controlled by Ca2+- and cAMP-signaling pathways. [•] The cAMP-signaling pathway at ring form and late trophozoite stages governs parasite growth of P. falciparum. [ABSTRACT FROM AUTHOR]

Abstract: We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC-γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca] i ). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1min after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca] i and other fertilization events. As compared to 14 other lipids, PA specifically bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca] i , PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca] i release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca] i release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm–egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization. [Copyright &y& Elsevier]

Abstract: Background: The triterpene glycoside actein from the herb black cohosh preferentially inhibits the growth of breast cancer cells and activates the ER stress response. The ER IP3 receptor and Na,K-ATPase form a signaling microdomain. Since actein is lipophilic, its action may be limited by bioavailability. Purpose: To develop actein to prevent and treat cancer, we examined the primary targets and combinations with chemotherapy agents, as well as the ability of nanoparticles to enhance the activity. Materials and methods: To reveal signaling pathways, we treated human breast and colon cancer, as well as 293T and 293T (NF-κB), cells with actein, and measured effects using the MTT, luciferase promoter, Western blot and histology assays. To assess effects on calcium release, we preloaded cells with the calcium sensitive dye Fura-2. To enhance bioavailability, we conjugated actein to nanoparticle liposomes. Results: Actein strongly inhibited the growth of human breast cancer cells and induced a dose dependent release of calcium into the cytoplasm. The ER IP3 receptor antagonist heparin blocked this release, indicating that the receptor is required for activity. Heparin partially blocked the growth inhibitory effect, while the MEK inhibitor U0126 enhanced it. Consistent with this, actein synergized with the ER mobilizer thapsigargin. Further, actein preferentially inhibited the growth of 293T (NF-κB) cells. Nanoparticle liposomes increased the growth inhibitory activity of actein. Conclusions: Actein alters the activity of the ER IP3 receptor and Na,K-ATPase, induces calcium release and modulates the NF-κB and MEK pathways and may be worthwhile to explore to prevent and treat breast cancer. [Copyright &y& Elsevier]

Abstract: Intracellular Ca2+ levels are tightly regulated in the neuronal system. The loss of Ca2+ homeostasis is associated with many neurological diseases and neuropsychiatric disorders such as Parkinson's, Alzheimer's, and schizophrenia. We investigated the mechanisms involved in intracellular Ca2+ signaling in PC-12 cells. The stimulation of NGF-differentiated PC-12 cells with 3μM ATP caused an early Ca2+ release followed by a delayed Ca2+ release. The delayed Ca2+ release was dependent on prior ATP priming and on dopamine secretion by PC-12 cells. Delayed Ca2+ release was abolished in the presence of spiperone, suggesting that it is due to the activation of D2 dopamine receptors (D2R) by dopamine secreted by PC-12 cells. This was shown to be independent of PKA activation but dependent on PLC activity. An endocytosis step was required for inducing the delayed Ca2+ release. Given the importance of calcyon in clathrin-mediated endocytosis, we verified the role of this protein in the delayed Ca2+ release phenomenon. siRNA targeting of calcyon blocked the delayed Ca2+ release, decreased ATP-evoked IP3R-mediated Ca2+ release, and impaired subsequent Ca2+ oscillations. Our results suggested that calcyon is involved in an unknown mechanism that causes a delayed IP3R-mediated Ca2+ release in PC-12 cells. In schizophrenia, Ca2+ dysregulation may depend on the upregulation of calcyon, which maintains elevated Ca2+ levels as well as dopamine signaling. [Copyright &y& Elsevier]

Abstract: Amyotrophic Lateral Sclerosis (ALS) is a devastating progressive neurodegenerative disease, resulting in selective motor neuron degeneration and paralysis. Patients die approximately 3–5years after diagnosis. Disease pathophysiology is multifactorial, including excitotoxicity, but is not yet fully understood. Genetic analysis has proven fruitful in the past to further understand genes modulating the disease and increase knowledge of disease mechanisms. Here, we revisit a previously performed microsatellite analysis in ALS and focus on another hit, PLCD1, encoding phospholipase C delta 1 (PLCδ1), to investigate its role in ALS. PLCδ1 may contribute to excitotoxicity as it increases inositol 1,4,5-trisphosphate (IP3) formation, which releases calcium from the endoplasmic reticulum through IP3 receptors. We find that expression of PLCδ1 is increased in ALS mouse spinal cord and in neurons from ALS mice. Furthermore, genetic ablation of this protein in ALS mice significantly increases survival, but does not affect astrogliosis, microgliosis, aggregation or the amount of motor neurons at end stage compared to ALS mice with PLCδ1. Interestingly, genetic ablation of PLCδ1 prevents nuclear shrinkage of motor neurons in ALS mice at end stage. These results indicate that PLCD1 contributes to ALS and that PLCδ1 may be a new target for future studies. [Copyright &y& Elsevier]

Abstract: RhoA/Rho-kinase pathway plays a pivotal role in numerous fundamental cellular functions including contraction, motility, proliferation, differentiation and apoptosis. The pathway is also involved in the development of many diseases such as vasospasm, pulmonary hypertension, cancer and central nervous systems (CNS) disorders. The inhibitors of Rho kinase have been extensively studied since the Rho/Rho-kinase pathway was verified as a target for a number of diseases. Herein, we reviewed the advances in the studies of the roles of Rho/Rho-kinase in diseases and the development of Rho-kinase inhibitors in recent five years. [Copyright &y& Elsevier]

Abstract: Ca2+-regulating and Ca2+-dependent molecules enriched in caveolae are typically shaped as plasmalemmal invaginations or vesicles. Caveolae structure and subcellular distribution are critical for Ca2+ release from endoplasmic reticulum Ca2+ stores and for Ca2+ influx from the extracellular space into the cell. However, Ca2+ dynamics inside caveolae have never been directly measured and remain uncharacterized. To target the fluorescence resonance energy transfer (FRET)-based Ca2+ sensing protein D1, a mutant of cameleon, to the intra-caveolar space, we made a cDNA construct encoding a chimeric protein of lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) and D1 (LOXD1). Immunofluorescence and immunoelectron microscopy confirmed that a significant portion of LOXD1 was localized with caveolin-1 at morphologically apparent caveolar vesicles in endothelial cells. LOXD1 detected ATP-induced transient Ca2+ decreases by confocal FRET imaging in the presence or absence of extracellular Ca2+. This ATP-induced Ca2+ decrease was abolished following knockdown of caveoin-1, suggesting an association with caveolae. The X-ray spectra obtained by the spot analysis of electron-opaque pyroantimonate precipitates further confirmed that ATP-induced calcium decreases in intra-caveolar vesicles. In conclusion, subplasmalemmal caveolae function as Ca2+-releasable Ca2+ stores in response to ATP. This intracellular local Ca2+ delivery system may contribute to the complex spatiotemporal organization of Ca2+ signaling. [Copyright &y& Elsevier]

Highlights: [•] Glucocorticoids exert their antiallergic action by inhibiting signaling pathways in mast cells. [•] Some antiallergic actions of gluccorticoids are mediated by protein tyrosine phosphatase. [•] Glucocorticoids exhibit rapid antiallergic effect on mast cell activation. [ABSTRACT FROM AUTHOR]

Abstract: Lysophosphatidylethanolamine (LPE) is a lyso-type metabolite of phosphatidylethanolamine (a plasma membrane component), and its intracellular Ca2+ ([Ca2+]i) increasing actions may be mediated through G-protein-coupled receptor (GPCR). However, GPCRs for lysophosphatidic acid (LPA), a structurally similar representative lipid mediator, have not been implicated in LPE-mediated activities in SK-OV3 or OVCAR-3 ovarian cancer cells or in receptor over-expression systems. In the present study, LPE-induced [Ca2+]i increase was observed in MDA-MB-231 cells but not in other breast cancer cell lines. In addition, LPE- and LPA-induced responses showed homologous and heterologous desensitization. Furthermore, VPC32183 and Ki16425 (antagonists of LPA1 and LPA3) inhibited LPE-induced [Ca2+]i increases, and knockdown of LPA1 by transfection with LPA1 siRNA completely inhibited LPE-induced [Ca2+]i increases. Furthermore, the involvement of CD97 (an adhesion GPCR) in the action of LPA1 in MDA-MB-231 cells was demonstrated by siRNA transfection. Pertussis toxin (a specific inhibitor of Gi/o proteins), edelfosine (an inhibitor of phospholipase C), or 2-APB (an inhibitor of IP3 receptor) completely inhibited LPE-induced [Ca2+]i increases, whereas HA130, an inhibitor of autotaxin/lysophospholipase D, did not. Therefore, LPE is supposed to act on LPA1-CD97/Gi/o proteins/phospholipase C/IP3/Ca2+ rise in MDA-MB-231 breast cancer cells. [Copyright &y& Elsevier]

Abstract: σ2 Receptors are promising biomarkers for cancer diagnosis given the relationship between the proliferative status of tumors and their density. With the aim of contributing to the research of σ2 receptor Positron Emission Tomography (PET) probes, we developed 2-[3-[6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl]propyl]-3,4-dihydroisoquinolin-1(2H)-one (3), with optimal σ2 pharmacological properties and appropriate lipophilicity. Hence, 3 served as the lead compound for the development of a series of dihydroisoquinolinones amenable to radiolabeling. Radiosynthesis for compound 26, which displayed the most appropriate σ2 profile, was developed and σ2 specific binding for the corresponding [18F]-26 was confirmed by in vitro autoradiography on rat brain slices. Despite the excellent in vitro properties, [18F]-26 could not successfully image σ2 receptors in the rat brain in vivo, maybe because of its interaction with P-gp. Nevertheless, [18F]-26 may still be worthy of further investigation for the imaging of σ2 receptors in peripheral tumors devoid of P-gp overexpression. [Copyright &y& Elsevier]

Highlights: [•] PPARβ/δ is a possible nexus of PPARα- and PPARγ. [•] PPARβ/δ controls molecular pathways in neurodegenerative diseases. [•] PPARβ/δ has a central role in the “PPAR triad”. [•] PPARβ/δ is involved in regulation of myelination, and ROS/NO/Ca2+ signaling pathways. [Copyright &y& Elsevier]

Highlights: [•] Review behavioral sensitization in response to noxious stimulation or injury. [•] Review functional, synaptic, and cellular mechanisms of sensitization. [•] Review epigenetic mechanisms in learning- and pain-related sensitization. [ABSTRACT FROM AUTHOR]

Abstract: Store-operated Ca2+ channels (SOCs) are activated by depletion of intracellular Ca2+ stores following agonist-mediated Ca2+ release. Previously we demonstrated that Ca2+ influx through SOCs elicits exocytosis efficiently in pancreatic duct epithelial cells (PDEC). Here we describe the biophysical, pharmacological, and molecular properties of the duct epithelial SOCs using Ca2+ imaging, whole-cell patch-clamp, and molecular biology. In PDEC, agonists of purinergic, muscarinic, and adrenergic receptors coupled to phospholipase C activated SOC-mediated Ca2+ influx as Ca2+ was released from intracellular stores. Direct measurement of [Ca2+] in the ER showed that SOCs greatly slowed depletion of the ER. Using IP3 or thapsigargin in the patch pipette elicited inwardly rectifying SOC currents. The currents increased ∼8-fold after removal of extracellular divalent cations, suggesting competitive permeation between mono- and divalent cations. The current was completely blocked by high doses of La3+ and 2-aminoethoxydiphenyl borate (2-APB) but only partially depressed by SKF-96365. In polarized PDEC, SOCs were localized specifically to the basolateral membrane. RT-PCR screening revealed the expression of both STIM and Orai proteins for the formation of SOCs in PDEC. By expression of fluorescent STIM1 and Orai1 proteins in PDEC, we confirmed that colocalization of the two proteins increases after store depletion. In conclusion, basolateral Ca2+ entry through SOCs fills internal Ca2+ stores depleted by external stimuli and will facilitate cellular processes dependent on cytoplasmic Ca2+ such as salt and mucin secretion from the exocrine pancreatic ducts. [Copyright &y& Elsevier]

Highlights: [•] We developed a new method for evaluating properties of IP3R ligands using fluorescent biosensors. [•] This method revealed a diversity in subtype selectivity of adenophostin A and its analogs. [•] We also found a compound showing a partial agonistic effect on IP3R1. [Copyright &y& Elsevier]

Abstract: Endothelin receptors are present on the nuclear membranes in adult cardiac ventricular myocytes. The objectives of the present study were to determine 1) which endothelin receptor subtype is in cardiac nuclear membranes, 2) if the receptor and ligand traffic from the cell surface to the nucleus, and 3) the effect of increased intracellular ET-1 on nuclear Ca2+ signaling. Confocal microscopy using fluorescently-labeled endothelin analogs confirmed the presence of ETB at the nuclear membrane of rat cardiomyocytes in skinned-cells and isolated nuclei. Furthermore, in both cardiac myocytes and aortic endothelial cells, endocytosed ET:ETB complexes translocated to lysosomes and not the nuclear envelope. Although ETA and ETB can form heterodimers, the presence or absence of ETA did not alter ETB trafficking. Treatment of isolated nuclei with peptide: N-glycosidase F did not alter the electrophoretic mobility of ETB. The absence of N-glycosylation further indicates that these receptors did not originate at the cell surface. Intracellular photolysis of a caged ET-1 analog ([Trp-ODMNB21]ET-1) evoked an increase in nucleoplasmic Ca2+ ([Ca2+]n) that was attenuated by inositol 1,4,5-trisphosphate receptor inhibitor 2-aminoethoxydiphenyl borate and prevented by pre-treatment with ryanodine. A caged cell-permeable analog of the ETB-selective antagonist IRL-2500 blocked the ability of intracellular cET-1 to increase [Ca2+]n whereas extracellular application of ETA and ETB receptor antagonists did not. These data suggest that 1) the endothelin receptor in the cardiac nuclear membranes is ETB, 2) ETB traffics directly to the nuclear membrane after biosynthesis, 3) exogenous endothelins are not ligands for ETB on nuclear membranes, and 4) ETB associated with the nuclear membranes regulates nuclear Ca2+ signaling. [Copyright &y& Elsevier]

Abstract: Great progress has recently been made in structural and functional research of phospholipase C (PLC)-β. We now understand how PLC-β isoforms (β1-β4) are activated by GTP-bound Gαq downstream of G protein-coupled receptors. Numerous studies indicate that PLC-βs participate in the differentiation and activation of immune cells that control both the innate and adaptive immune systems. The PLC-β3 isoform also interplays with tyrosine kinase-based signaling pathways, to inhibit Stat5 activation by recruiting the protein-tyrosine phosphatase SHP-1, with which PLC-β3 and Stat5 form a multi-molecular signaling platform, named SPS complex. The SPS complex has important regulatory roles in tumorigenesis and immune cell activation. [Copyright &y& Elsevier]

Highlights: [•] Somatostatin is a wide-range antisecretory peptide that binds to five receptors (SSTR1–5). [•] SSTR1, 2, 3 and 5 exert their antiproliferative actions and SSTR4 its pro-proliferative action through unique pathways. [•] SSTR2 targeting sensitizes tumor cells to antitumor activity of mammalian target of rapamycin (mTOR) inhibitors. [•] Co-targeting of SSTR1, 2, 3 and 5 and dopamine receptor type 2 (D2DR) yields potent therapeutic outcomes. [•] SSTR signaling factors are putative biomarkers of somatostatin analog response. [ABSTRACT FROM AUTHOR]

Abstract: The Na+ gradient produced across the cardiac sarcolemma by the ATP-dependent Na+-pump is a constant source of energy for Na+-dependent transporters. The plasma membrane Na+/H+ exchanger (NHE) is one such secondary active transporter, regulating intracellular pH, Na+ concentration, and cell volume. NHE1, the major isoform found in the heart, is activated in response to a variety of stimuli such as hormones and mechanical stress. This important characteristic of NHE1 is intimately linked to heart diseases, including maladaptive cardiac hypertrophy and subsequent heart failure, as well as acute ischemic-reperfusion injury. NHE1 activation results in elevation of pH and intracellular Na+ concentration, which potentially enhance downstream signaling cascades in the myocardium. Therefore, in addition to determining the mechanism underlying regulation of NHE1 activity, it is important to understand how the ionic signal produced by NHE1 is transmitted to the downstream targets. Extensive studies have identified many accessory factors that interact with NHE1. Here, we have summarized the recent progress on understanding the molecular mechanism underlying NHE1 regulation and have shown a possible signaling pathway leading to cardiac remodeling, which is initiated from NHE1. This article is part of a Special Issue entitled “Na+ Regulation in Cardiac Myocytes”. [Copyright &y& Elsevier]

Abstract: The endoplasmic reticulum (ER) performs multiple functions in the cell: it is the major site of protein and lipid synthesis as well as the most important intracellular Ca2+ reservoir. Adverse conditions, including a decrease in the ER Ca2+ level or an increase in oxidative stress, impair the formation of new proteins, resulting in ER stress. The subsequent unfolded protein response (UPR) is a cellular attempt to lower the burden on the ER and to restore ER homeostasis by imposing a general arrest in protein synthesis, upregulating chaperone proteins and degrading misfolded proteins. This response can also lead to autophagy and, if the stress can not be alleviated, to apoptosis. The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and IP3-induced Ca2+ signaling are important players in these processes. Not only is the IP3R activity modulated in a dual way during ER stress, but also other key proteins involved in Ca2+ signaling are modulated. Changes also occur at the structural level with a strengthening of the contacts between the ER and the mitochondria, which are important determinants of mitochondrial Ca2+ uptake. The resulting cytoplasmic and mitochondrial Ca2+ signals will control cellular decisions that either promote cell survival or cause their elimination via apoptosis. This article is part of a Special Issue entitled: 12th European Symposium on Calcium. [Copyright &y& Elsevier]

Abstract: Research conducted over the past two decades has provided convincing evidence that cell death, and more specifically apoptosis, can exceed single cell boundaries and can be strongly influenced by intercellular communication networks. We recently reported that gap junctions (i.e. channels directly connecting the cytoplasm of neighboring cells) composed of connexin43 or connexin26 provide a direct pathway to promote and expand cell death, and that inositol 1,4,5-trisphosphate (IP3) diffusion via these channels is crucial to provoke apoptosis in adjacent healthy cells. However, IP3 itself is not sufficient to induce cell death and additional factors appear to be necessary to create conditions in which IP3 will exert proapoptotic effects. Although IP3-evoked Ca2+ signaling is known to be required for normal cell survival, it is also actively involved in apoptosis induction and progression. As such, it is evident that an accurate fine-tuning of this signaling mechanism is crucial for normal cell physiology, while a malfunction can lead to cell death. Here, we review the role of IP3 as an intracellular and intercellular cell death messenger, focusing on the endoplasmic reticulum-mitochondrial synapse, followed by a discussion of plausible elements that can convert IP3 from a physiological molecule to a killer substance. Finally, we highlight several pathological conditions in which anomalous intercellular IP3/Ca2+ signaling might play a role. This article is part of a Special Issue entitled:12th European Symposium on Calcium. [Copyright &y& Elsevier]

Highlights: [•] Autophagic degradation is impaired in the neurons of AD brain. [•] Impaired autophagy affects mitochondrial clearance and APP processing. [•] Beclin 1/Vps34 complex is the major inducer of autophagy. [•] Beclin 1 function is disturbed in AD brains and transgenic AD mice. [•] Mechanisms impairing the function of Beclin 1 interactome in AD will be discussed. [ABSTRACT FROM AUTHOR]

Abstract: Protein activities are generally regulated by intramolecular allosteric interactions, by which spatially separated sites in a protein molecule communicate. Intramolecular allosteric interactions in the phospholipase C (PLC)-δ1 pleckstrin homology (PH) domain were investigated by solution NMR spectroscopy for selectively [α-15N]Lys-labeled proteins. The results of NMR analyses indicated that the binding of inositol 1,4,5-trisphosphate (IP3) to the protein induces local environmental changes at all lysine residues, including residues such as Lys-43 spatially separated from the specific IP3 binding site consisting of Lys-30, Lys-32, and Lys-57. IP3 binding also induces conformational stabilization of a characteristic short α-helix (α2) from residues 82 to 87. Mutational analyses indicated that an interaction network mainly consisting of the side chains of Lys-30, Lys-32, and Lys-43 exists in the ligand-free protein, and it was therefore predicted that binding of IP3 to the specific site modifies the interaction network, resulting in formation of a new interaction network, in which the side chains of Lys-57 and Phe-87 contribute to stable IP3 binding. These results provide evidence for intramolecular interactions in the PLC-δ1 PH domain, the function of which could be allosterically regulated by modifications at sites spatially separated from the ligand-binding site through the intramolecular interaction network. [Copyright &y& Elsevier]

Highlights: [•] Curcumin significantly increases GLP-1 secretion in GLUTag cells. [•] One OCH3 moiety and β-diketone structure are essential for the GLP-1 secretion. [•] Curcumin-induced GLP-1 secretion involves the Ca2+-CaMKII pathway. [•] Curcumin-induced GLP-1 secretion is not regulated by the PKA, PKCζ, or ERK pathways. [ABSTRACT FROM AUTHOR]

Abstract: Hinokitiol is a tropolone-related bioactive compound that has been used in hair tonics, cosmetics, and food as an antimicrobial agent. Recently, hinokitiol has attracted considerable interest because of its anticancer activities. Platelet activation plays a crucial role in atherothrombotic processes. We examined the effects of hinokitiol treatment on platelet activation using human platelets. In the present study, hinokitiol (1 and 2μM) inhibited the collagen-induced aggregation of human platelets, but did not inhibit the activation of platelets by other agonists, including thrombin, arachidonic acid, and ADP. Hinokitiol inhibited the phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and Akt in collagen-activated human platelets, and significantly reduced intracellular calcium mobilization and hydroxyl radical (OH ) formation. Hinokitiol also reduced the PKC activation and platelet aggregation stimulated by PDBu. In addition, hinokitiol significantly prolonged thrombogenesis in mice. Hinokitiol did not influence the binding of a fluorescent triflavin probe to the αIIbβ3 integrin on platelet membrane, and neither ODQ nor SQ22536 significantly reversed the hinokitiol-mediated inhibition of platelet aggregation. In conclusion, hinokitiol may inhibit platelet activation by inhibiting the PLCγ2-PKC cascade and hydroxyl radical formation, followed by suppressing the activation of MAPKs and Akt. Our study suggests that hinokitiol may represent a potential therapeutic agent for the prevention or treatment of thromboembolic disorders. [Copyright &y& Elsevier]

Abstract: G protein-coupled receptors (GPCRs) are the largest class of integral membrane protein receptors in the human genome. We examined here the reports whether the GnRH receptor (GnRHR) interacts with a single or multiple types of G proteins. It seems that the GnRHR, as other GPCRs, alternates between various conformations and is stabilized by its ligands, other modulators and intracellular partners in selective conformations culminating in coupling with a single type or multiple G proteins in a cell- and context-specific manner. [Copyright &y& Elsevier]

Abstract: Autophagy and apoptosis are crucial cellular housekeeping and tissue survival mechanisms. There is emerging evidence of important crosstalk between apoptosis and autophagy which can be linked to inflammasome activation. Beclin 1 is a platform protein which assembles an interactome consisting of diverse proteins which control the initiation of autophagocytosis and distinct phases in endocytosis. Recent studies have demonstrated that the anti-apoptotic Bcl-2 family members can interact with Beclin 1 and inhibit autophagy. Consequently, impaired autophagy can trigger inflammasome activation. Interestingly, the hallmarks of the ageing process include a decline in autophagy, increased resistance to apoptosis and a low-grade inflammatory phenotype. Age-related stresses, e.g. genotoxic, metabolic and environmental insults, enhance the expression of NF-κB-driven anti-apoptotic Bcl-2 proteins which repress the Beclin 1-dependent autophagy. Suppression of autophagocytosis provokes inflammation including NF-κB activation which further potentiates anti-apoptotic defence. In a context-dependent manner, this feedback defence mechanism can enhance the aging process or provoke tumorigenesis or cellular senescence. We will review the role of Beclin 1 interactome in the crosstalk between apoptosis, autophagy and inflammasomes emphasizing that disturbances in Beclin 1-dependent autophagy can have a crucial impact on the aging process. [Copyright &y& Elsevier]

IL (interleukin)-1 signalling in anchorage-dependent cells involves focal-adhesion-restricted and Ca2+ -dependent Ras and ERK (extracellular-signal-regulated kinase) activation that leads to MMP (matrix metalloproteinase) release and extracellular matrix remodelling. Ras activity is regulated, in part, by the Ca2+ - responsive Ras GRFs (guanine-nucleotide-releasing factors) 1 and 2, but the mechanisms that link and localize IL-1-induced Ca2+ signalling to focal adhesions are not defined. In the present study we characterized the role of Ras-GRF1/2 in Ca2+ and Ras?ERK signalling after IL-1 stimulation. By immunoprecipitation we found that Ras-GRF1/2 associates with PLC? 1 (phospholipase C? 1). This association enables PLC? 1 recruitment to focal adhesions and is required for Ras signalling, ERK activation and MMP-3 release downstream of IL-1 stimulation. Depletion of PLC? 1 by siRNA (small interfering RNA) abolished IL-1-induced Ras activation and MMP-3 expression. Buffering of cytosolic Ca2+ reduced Ras interactions with Ras-GRF1/2 and blocked MMP-3 release. The results of the present study showthat, in addition to their functions as Ras-exchange factors, Ras-GRF1 and -GRF2 may act as adaptors that bind PLC? 1 and restrict Ca2+ signalling to the vicinity of focal adhesions, indicating a new role for these GRFs that is required for IL-1 induction of the Ras?ERK pathway and MMP-3 expression. [ABSTRACT FROM AUTHOR]

Abstract: LIBRA is a fluorescent biosensor of inositol 1,4,5-trisphosphate (IP3) and is composed of the ligand-binding domain of the rat type 3 IP3 receptor and cyan and yellow fluorescent proteins. We examined the responses of LIBRA and its IP3-insensitive mutant LIBRA-N to compounds known to inhibit IP3-induced Ca2+ release. Heparin, a competitive antagonist of IP3 receptors, increased the emission ratio of LIBRA but not that of LIBRA-N. In contrast, 2-aminoethoxydiphenyl borate, a known non-competitive inhibitor of IP3 receptor, decreased the emission ratios of both LIBRA and LIBRA-N. Thus, the concurrent use of LIBRA-N with LIBRA identifies nonspecific responses. These results indicate that LIBRA and its mutant control can be used to detect specific agonists and antagonists of IP3 receptors. We also demonstrate the utility of LIBRA and LIBRA-N in discriminating between specific and nonspecific responses in intact cells. [Copyright &y& Elsevier]

Abstract: Glutamate is the main excitatory neurotransmitter in the cerebral cortex. Altered glutamatergic transmission has been suggested as having a central role in many neurodegenerative diseases. Metabotropic glutamate receptors (mGluRs) are coupled to intracellular signal transduction via G proteins, and they mediate slower responses than ionotropic glutamate receptors. Group I mGluRs are positively coupled to phospholipase C β1 (PLCβ1). Creutzfeldt-Jakob disease (CJD) is a human transmissible spongiform encephalopathy associated with a dysfunction in the membrane glycoprotein PrP which is converted into an abnormal isoform, with a predominant β-sheet structure, that is pathogenic and partially resistant to protease digestion. Proteins associated with the signal transduction of group I mGluRs were examined in the frontal cortex (area 8) of 12 cases with sCJD and four age-matched controls, by means of gel electrophoresis and Western blotting of total homogenates. Densitometric analysis of the bands demonstrated decreased expression levels of PLCβ1 and PLCγ, a non-related phospholipase which is a substrate of tyrosine kinase, in CJD cases when compared with controls. Novel protein kinase C δ (nPKCδ) has also been found to be significantly decreased in CJD cases. However, no modifications in mGluR1 cPKCα expression levels are found in CJD when compared with controls. No modifications in PLCβ1 solubility in PBS-, deoxycholate- and sodium dodecylsulphate-soluble fractions have been observed in CJD when compared with controls. Finally, no interactions between PLCβ1 and PrP, as revealed by immunoprecipitation assays, have been found in CJD and controls. The present results show, for the first time, reduced expression levels of phospholipases, particularly PLCβ1, which may interfere with group I mGluR signaling in the cerebral cortex in CJD. These abnormalities are not the result of abnormal PLC solubility or interactions with PrP. Selective involvement of group I mGluRs may have functional effects on glutamatergic transmission modulation and processing in CJD. [Copyright &y& Elsevier]

Abstract: Mutations in the presenilin (PS) genes are linked to the development of early-onset Alzheimer''s disease by a gain-of-function mechanism that alters proteolytic processing of the amyloid precursor protein (APP). Recent work indicates that Alzheimer''s-disease-linked mutations in presenilin1 and presenilin2 attenuate calcium entry and augment calcium release from the endoplasmic reticulum (ER) in different cell types. However, the regulatory mechanisms underlying the altered profile of Ca2+ signaling are unknown. The present study investigated the influence of two familial Alzheimer''s-disease-linked presenilin2 variants (N141I and M239V) and a loss-of-function presenilin2 mutant (D263A) on the activity of the transient receptor potential canonical (TRPC)6 Ca2+ entry channel. We show that transient coexpression of Alzheimer''s-disease-linked presenilin2 mutants and TRPC6 in human embryonic kidney (HEK) 293T cells abolished agonist-induced TRPC6 activation without affecting agonist-induced endogenous Ca2+ entry. The inhibitory effect of presenilin2 and the Alzheimer''s-disease-linked presenilin2 variants was not due to an increase in amyloid β-peptides in the medium. Despite the strong negative effect of the presenilin2 and Alzheimer''s-disease-linked presenilin2 variants on agonist-induced TRPC6 activation, conformational coupling between inositol 1,4,5-trisphosphate receptor type 3 (IP3R3) and TRPC6 was unaffected. In cells coexpressing presenilin2 or the FAD-linked presenilin2 variants, Ca2+ entry through TRPC6 could still be induced by direct activation of TRPC6 with 1-oleoyl-2-acetyl-sn-glycerol (OAG). Furthermore, transient coexpression of a loss-of-function PS2 mutant and TRPC6 in HEK293T cells enhanced angiotensin II (AngII)- and OAG-induced Ca2+ entry. These results clearly indicate that presenilin2 influences TRPC6-mediated Ca2+ entry into HEK293 cells. [Copyright &y& Elsevier]

Abstract: Expression of constitutively active Src protein tyrosine kinase in Xenopus oocytes has been shown to accelerate oocyte maturation suggesting that Src may be involved in meiotic progression. However, meiotic regulation of endogenous Src kinase in oocytes has not been investigated in detail. To address this problem, we measured the activity, expression level, and phosphorylation state of the endogenous Xenopus Src (xSrc) and overexpressed xSrc mutants in the process of progesterone-induced oocyte maturation. We found that the enzyme is first transiently activated in the plasma membrane-containing fraction of oocytes within 3 min of progesterone administration. This event represents one of the earliest responses of oocytes to the hormone and should be related to triggering some early signaling pathways of maturation. Thereafter, xSrc activity increases again at the time of germinal vesicle breakdown (GVBD) and remains elevated till the completion of maturation. This elevation of xSrc activity is associated with a 2-fold increase of xSrc protein content in the absence of change in its specific activity and xSrc mRNA content. No significant changes in the phosphorylation state of C-terminal regulatory phosphotyrosine can be registered either in endogenous xSrc or in overexpressed kinase-negative and wild-type xSrc proteins during maturation. Altogether, these results indicate that upregulation of xSrc in the meiotic metaphase occurs at the translation level. We also demonstrate here that the expression of constitutively active xSrc in Xenopus oocytes is accompanied by the activation of mitogen-activated protein kinase (MAPK). Our data suggest that the Src kinase acts through the MAPK pathway to accelerate oocyte maturation. [Copyright &y& Elsevier]

Abstract: PKCε has been strongly linked to cell activation and proliferation in many cell types, including leukemic T-cell lines. In particularly, an essential role of PKCε has been established in the IKK-β/I-κB/NF-κB transactivation cascade. To study the physiological function of PKCε in primary T-cells, we used our newly established PKCε null mice. Unexpectedly, however, we did not reveal any defect in the development and function of CD3+ T-cells. Proliferative responses as well as IL-2 cytokine secretion of PKCε-deficient T-cells induced by allogenic MHC, plate-bound anti-CD3 antibodies (with or without anti-CD28 costimulation), or mitogenic stimuli such as phorbol ester and Ca2+ ionophore were comparable with wild-type controls. Consistently, after CD3/CD28 engagement, deficiency of PKCε did not impair NF-κB transactivation as well as CD25, CD44 and CD69 induction. Thus, PKCε-deficient T-cells had similar physiological thresholds for activation in vitro. This finding suggests that PKCε plays a redundant role in TCR-induced regulation of T-cell proliferation. [Copyright &y& Elsevier]

The effects of cyclic ADP-ribose (cADPR) and the immunosuppressant drug FK506 on microsomal Ca2+ release through a ryanodine-sensitive mechanism were investigated in rat pancreatic acinar cells. After a steady state of 45Ca2+ uptake into the microsomal vesicles, ryanodine or caffeine was added. Preincubation of the vesicles with cADPR (0.5 μM) shifted the dose–response curve of ryanodine- or caffeine-induced 45Ca2+ release from the vesicles to the left. Preincubation with cADPR shifted the dose–response curve of the FK506-induced 45Ca2+ release upward. Preincubation with FK506 (3 μM) shifted the dose–response curve of the ryanodine- or caffeine-induced 45Ca2+ release to the left by the same extent as that in the case of cADPR. FK506 shifted the dose–response curve of the cADPR-induced 45Ca2+ release upward. The presence of both cADPR and FK506 enhanced the ryanodine (30 μM)- or caffeine (10 mM)-induced 45Ca2+ release by the same extent as that in the case of cADPR alone or FK506 alone.These results indicate that cADPR and FK506 modulate the ryanodine-sensitive Ca2+ release mechanism of rat pancreatic acinar cells by increasing the ryanodine or caffeine sensitivity to the mechanism. In addition, there is a possibility that the mechanisms of modulation by cADPR and FK506 are the same. [Copyright &y& Elsevier]

The activated proteinase inhibitor α2-macroglobulin (α2M*) binds to two receptors, the low density lipoprotein receptor-related protein (LRP-1) and the α2M* signalling receptor (α2MSR). Silencing LRP-1 gene expression in macrophages by RNA interference does not block α2M* activation of signalling cascades. We now demonstrate that transfection of macrophages with a double-stranded RNA homologous in sequence to the Grp78 gene markedly decreased induction of inositol 1,4,5-trisphosphate (IP3) and subsequent IP3-dependent elevation of [Ca2+]i induced by α2M*. Concomitantly, α2M*-induced increase in [3H]thymidine uptake was abolished in these transfected cells. Insulin treatment significantly upregulates α2MSR and it also caused a marked increase in Grp78 expression which could be blocked by RNA interference. α2M* treatment of cells activates the Ras- and PI 3-kinase-dependent signalling pathways. Suppressing Grp78 expression leads to the loss of these activation events in transfected macrophages. We thus conclude that Grp78 is the α2M* signalling receptor. [Copyright &y& Elsevier]

In just a few years, the discovery and subsequent characterization of several members of the TRPM family of cation channels have provided us with surprising new insights into unknown aspects of cellular ion-homeostasis regulation. This includes reports about ADP-ribose functioning as a novel intracellular second messenger and gating molecule of the Ca2+-permeable TRPM2 channel, studies demonstrating the central role of mouse TRPM5 in taste signaling, as well as the unexpected involvement of TRPM6 and TRPM7 in regulating Mg2+-homeostasis, or the cool properties of TRPM8 acting as a cold and menthol sensor in sensory neurons. At least four of the eight known TRPM proteins have been shown to be present in the immune context: TRPM1 (melastatin), TRPM2, TRPM4 and TRPM7. Although we currently lack animal models allowing a detailed assessment of the potential involvement of TRPM family members in modulating the immune response, the powerful combination of molecular and cellular biology, biochemistry, and electrophysiology have provided the first clues as to how these molecules could contribute to immunity. [Copyright &y& Elsevier]