Capacitative Ca2 Research Articles

Peroxynitrite Mediates Disruption of Ca2+ Homeostasis by Carbon Monoxide via Ca2+ ATPase Degradation

Sublethal carbon monoxide poisoning causes prolonged neurological damage involving oxidative stress. Given the central role of Ca(2+) homeostasis and its vulnerability to stress, we investigated whether CO disrupts neuronal Ca(2+) homeostasis. Cytosolic Ca(2+) transients evoked by muscarine in SH-SY5Y cells were prolonged by CO (applied via the donor CORM-2), and capacitative Ca(2+) entry (CCE) was dramatically enhanced. Ca(2+) store mobilization by cyclopiazonic acid was similarly augmented, as was the subsequent CCE, and that evoked by thapsigargin. Ca(2+) rises evoked by depolarization were also enhanced by CO, and Ca(2+) levels often did not recover in its presence. CO increased intracellular nitric oxide (NO) and all effects of CO were prevented by inhibiting NO formation. However, NO donors did not mimic the effects of CO. The antioxidant ascorbic acid inhibited effects of CO on Ca(2+) signaling, as did the peroxynitrite scavenger, FeTPPS, and CO increased peroxynitrite formation. Finally, CO caused significant loss of plasma membrane Ca(2+)ATPase (PMCA) protein, detected by Western blot, and this was also observed in brain tissue of rats exposed to CO in vivo. The cellular basis of CO-induced neurotoxicity is currently unknown. Our findings provide the first data to suggest signaling pathways through which CO causes neurological damage, thereby opening up potential targets for therapeutic intervention. CO stimulates formation of NO and reactive oxygen species which, via peroxynitrite formation, inhibit Ca(2+) extrusion via PMCA, leading to disruption of Ca(2+) signaling. We propose this contributes to the neurological damage associated with CO toxicity.

Immortalized hippocampal cell line H19‐7 express endothelin system genes

Previous studies indicate that endothelins (ETs) may play a neuromodulatory role in the central nervous system. However, there is a paucity of data regarding ETs’ action on specific subsets of neurons. We evaluated the hippocampal cell line H19‐7 (conditionally immortalized by a temperature‐sensitive SV40 large T antigen) as an in vitro model to investigate the role of ETs in neural activity. The relative abundance of endothelin system genes mRNA was assessed by real‐time PCR method using specific primers for endothelins (ET‐1 and ET‐3), endothelin receptors (ETA and ETB), and endothelin converting enzyme‐1 (ECE‐1). Under non‐differentiating conditions (34°C), we found a robust expression of ECE1, ETA and ET‐1, while ETB and ET‐3 mRNA levels were undetectable. The expression patterns of ET system genes were essentially similar when the cells were placed under differentiating condition (39°C) for 24–36 hours. When changes in cytosolic free Ca2+ levels were assessed by the Fura‐2 method, undifferentiated cells were unresponsive to ET1. However, 20 nM ET‐1 elicited a robust bi‐phasic increase of cytosolic free Ca2+ (a hallmark effect of ETA receptor activation) on differentiated cells, and markedly enhanced capacitative Ca2+ entry as well. Thus, differentiated H19‐7 cells seem suitable for mechanistic studies of ETs’ action on neuronal targets.

Severe disturbance in the Ca2+ signaling in astrocytes from mouse models of human infantile neuroaxonal dystrophy with mutated Pla2g6

Infantile neuroaxonal dystrophy (INAD; OMIM #no. 256600) is an inherited degenerative nervous system disorder characterized by nerve abnormalities in brain, spinal cord and peripheral nerves. About 85% of INAD patients carry mutations in the PLA2G6 gene that encodes for a Ca(2+)-independent phospholipase A(2) (VIA iPLA(2)), but how these mutations lead to disease is unknown. Besides regulating phospholipid homeostasis, VIA iPLA(2) is emerging with additional non-canonical functions, such as modulating store-regulated Ca(2+) entry into cells, and mitochondrial functions. In turn, defective Ca(2+) regulation could contribute to the development of INAD. Here, we studied possible changes in ATP-induced Ca(2+) signaling in astrocytes derived from two mutant strains of mice. The first strain carries a hypomorphic allele of the Pla2g6 that reduces transcript levels to 5-10% of that observed in wild-type mice. The second strain carries a point mutation in Pla2g6 that results in inactive VIA iPLA(2) protein with postulated gain in toxicity. Homozygous mice from both strains develop pathology analogous to that observed in INAD patients. The nucleotide ATP is the most important transmitter inducing Ca(2+) signals in astroglial networks. We demonstrate here a severe disturbance in Ca(2+) responses to ATP in astrocytes derived from both mutant mouse strains. The duration of the Ca(2+) responses in mutant astrocytes was significantly reduced when compared with values observed in control cells. We also show that the reduced Ca(2+) responses are probably due to a reduction in capacitative Ca(2+) entry (2.3-fold). Results suggest that altered Ca(2+) signaling could be a central mechanism in the development of INAD pathology.

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7. The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

Patch-clamp capacitance measurements and Ca²⁺ imaging at single nerve terminals in retinal slices.

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 μm diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca(2+) currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABA(A) and GABA(C) receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied. The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Human umbilical artery smooth muscle exhibits a 2-apb- sensitive capacitative contractile response evoked by vasoactive substances and exprsses mrnas for stim, orai and trpc channelsa

After depletion of intracellular Ca2+ stores the capacitative response triggers an extracellular Ca2+ influx through store-operated channels (SOCs) which refills these stores. Our objective was to explore if human umbilical artery smooth muscle presented this response and if it was involved in the mechanism of serotonin- and histamine-induced contractions. Intracellular Ca2+ depletion by a Ca(2+)-free extracellular solution followed by Ca2+ readdition produced a contraction in artery rings which was inhibited by the blocker of Orai and TRPC channels 2-aminoethoxydiphenyl borate (2-APB), suggesting a capacitative response. In presence of 2-APB the magnitude of a second paired contraction by serotonin or histamine was significantly less than a first one, likely because 2-APB inhibited store refilling by capacitative Ca2+ entry. 2-APB inhibition of sarcoplasmic reticulum Ca2+ release was excluded because this blocker did not affect serotonin force development in a Ca(2+)-free solution. The PCR technique showed the presence of mRNAs for STIM proteins (1 and 2), for Orai proteins (1, 2 and 3) and for TRPC channels (subtypes 1, 3, 4 and 6) in the smooth muscle of the human umbilical artery. Hence, this artery presents a capacitative contractile response triggered by stimulation with physiological vasoconstrictors and expresses mRNAs for proteins and channels previously identified as SOCs.

Constitutive lysosome exocytosis releases ATP and engages P2Y receptors in human monocytes

Elucidating mechanisms by which Ca(2+) signals are generated by monocytes is important for understanding monocyte function in health and disease. We have investigated mechanisms underlying Ca(2+) signals generated following disruption of lysosomes by exposure to the cathepsin C substrate glycyl-L-phenylalanine-β-napthylamide (GPN). Exposure to 0.2 mM GPN resulted in robust increases in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in the absence of extracellular Ca(2+). The response was antagonised by thapsigargin and evoked capacitative Ca(2+) entry. Dantrolene-sensitive Ca(2+) responses were observed at higher concentrations of GPN (0.4 mM) but not at 0.2 mM. Strikingly, GPN-evoked Ca(2+) responses and β-hexosaminidase secretion were inhibited by the ATPase/ADPase apyrase. Simultaneous measurement of [Ca(2+)](i) and extracellular ATP revealed a concomitant secretion of ATP during GPN-evoked Ca(2+) signalling. Furthermore, the ability of GPN to raise [Ca(2+)](i) was inhibited by P2Y receptor antagonists or by inhibiting vesicular exocytosis with N-ethylmaleimide (NEM). NEM treatment was associated with an inability of GPN to trigger ATP secretion, a drop in baseline [Ca(2+)](i) and reduction in extracellular ATP concentration. Antagonism of purinergic signalling also caused a reduction in baseline [Ca(2+)](i). In summary, these data suggest that P2Y receptor activation contributes significantly to GPN-evoked Ca(2+) signalling, and that constitutive secretion of lysosomal ATP is a major determinant of Ca(2+) homeostasis in monocytes. Lysosomal Ca(2+) stores can communicate with ER Ca(2+) stores either directly through activation of ryanodine receptors, or indirectly through release of ATP and engagement of P2Y receptors.

Modulation of STIM1 and capacitative Ca2+ entry by the endoplasmic reticulum luminal oxidoreductase ERp57

STIM1 is an endoplasmic reticulum (ER) membrane Ca(2+) sensor responsible for activation of store-operated Ca(2+) influx. We discovered that STIM1 oligomerization and store-operated Ca(2+) entry (SOC) are modulated by the ER oxidoreductase ERp57. ERp57 interacts with the ER luminal domain of STIM1, with this interaction involving two conserved cysteine residues, C(49) and C(56). SOC is accelerated in the absence of ERp57 and inhibited in C(49) and C(56) mutants of STIM1. We show that ERp57, by ER luminal interaction with STIM1, has a modulatory role in capacitative Ca(2+) entry. This is the first demonstration of a protein involved in ER intraluminal regulation of STIM1.

34 Mitochondrial morphology affects cardiomyocyte differentiation of mouse embryonic stem cells through calcineurin and notch signalling pathways

Mitochondria play essential and versatile roles in cardiomyocyte pathophysiology: they are the main source of ATP; they control apoptosis, and Ca2+ signalling, impacting on almost any process, like outcome of...

Baicalein, isolated from Scutellaria baicalensis, protects against endothelin-1-induced pulmonary artery smooth muscle cell proliferation via inhibition of TRPC1 channel expression

Ethnopharmacological relevanceWe investigated the antiproliferative effects of baicalein, isolated from Scutellaria baicalensis (Huang-qin), on ET-1-mediated pulmonary artery smooth muscle cells (PASMCs) proliferation and the mechanisms underlying these effects. Materials and methodsIntrapulmonary artery smooth muscle cells were isolated and cultured from female Sprague-Dawley rats and used during passages 3–6. The proliferation of PASMCs was quantified by cell counting and XTT assay. The protein expression of TRPC1 and PKCα were determined by western blotting. The cell cycle pattern was assayed by flow cytometry. The intracellular calcium concentrations ([Ca2+]i) were measured using the fluorescent indicator fura-2-AM and flow cytometry. ResultsBaicalein (0.3–3μM) inhibited PASMCs proliferation, promoted cell cycle progression, enhanced [Ca2+]i levels, increased capacitative Ca2+ entry (CCE), upregulated the canonical transient receptor potential 1 (TRPC1) channel and membrane protein kinase Cα (PKCα) expression induced by ET-1 (0.1μM). The PKC activator PMA (1μM) reversed the inhibitory effects of baicalein on ET-1-induced upregulation of TRPC1 expression and S phase accumulation, while the PKC inhibitor chelerythrine (1μM) potentiated baicalein-mediated G2/M phase arrest and TRPC1 channel inhibition. ConclusionOur findings suggest that baicalein protects against ET-1-induced PASMCs proliferation via modulation of the PKC-mediated TRPC channel.

Trpc2 depletion protects red blood cells from oxidative stress-induced hemolysis

Transient receptor potential (TRP) channels Trpc2 and Trpc3 are expressed on normal murine erythroid precursors, and erythropoietin stimulates an increase in intracellular calcium ([Ca(2+)](i)) through TRPC2 and TRPC3. Because modulation of [Ca(2+)](i) is an important signaling pathway in erythroid proliferation and differentiation, Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were utilized to explore the roles of these channels in erythropoiesis. Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were not anemic, and had similar red blood cell counts, hemoglobins, and reticulocyte counts as wild-type littermate controls. Although the erythropoietin-induced increase in [Ca(2+)](i) was reduced, these knockout mice showed no defects in red cell production. The major phenotypic difference at steady state was that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrit of red cells were significantly greater in Trpc2 and Trpc2/Trpc3 double knockout mice, and mean corpuscular hemoglobin concentration was significantly reduced. All hematological parameters in Trpc3 knockout mice were similar to controls. When exposed to phenylhydrazine, unlike the Trpc3 knockouts, Trpc2 and Trpc2/Trpc3 double knockout mice showed significant resistance to hemolysis. This was associated with a significant reduction in hydrogen peroxide-induced calcium influx in erythroblasts. Although erythropoietin-induced calcium influx through TRPC2 or TRPC3 is not critical for erythroid production, these data demonstrate that TRPC2 plays an important role in oxidative stress-induced hemolysis, which may be related to reduced calcium entry in red cells in the presence of Trpc2 depletion.

Effects of chronic treatment with fluoxetine on receptor-stimulated increase of [Ca 2+] i in astrocytes mimic those of acute inhibition of TRPC1 channel activity

Primary cultures of mouse astrocytes were used to investigate effects by chronic treatment (3–21 days) with fluoxetine (0.5–10 μM) on capacitative Ca 2+ influx after treatment with the SERCA inhibitor thapsigargin and on receptor agonist-induced increases in free cytosolic Ca 2+ concentration [Ca 2+] i, determined with Fura-2. The agonists were the 5-HT 2B agonist fluoxetine, the α 2-adrenergic agonist dexmedetomidine, and ryanodine receptor (RyR) and IP 3 receptor (IP 3R) agonists. In untreated sister cultures each agonist distinctly increased [Ca 2+] i, but in cultures treated for sufficient length of time or with sufficiently high doses of fluoxetine, acute administration of fluoxetine, dexmedetomidine, or RyR or IP 3R agonists elicited reduced, in some cases abolished, effects. Capacitative Ca 2+ entry, meditated by TRPC1 channels, was sufficiently inhibited to cause a depletion of Ca 2+ stores, which could explain the reduced agonist effects. All effects of chronic fluoxetine administration could be replicated by TRPC1 channel antibody or siRNA. Since increases in astrocytic [Ca 2+] i regulate release of gliotransmitters, these effects may have profound effects on brain function. They may be important for therapeutic effects of all 5 conventional ‘serotonin-specific reuptake inhibitors’ (SSRIs), which at concentrations used therapeutically (∼1 μM) share other of fluoxetine's chronic effects (Zhang et al., Neuron Glia Biol. 16 (2010) 1–13).

Sildenafil decreases rat tracheal hyperresponsiveness to carbachol and changes canonical transient receptor potential gene expression after antigen challenge

Inhibition of type-5 phosphodiesterase by sildenafil decreases capacitative Ca2+ entry mediated by transient receptor potential proteins (TRPs) in the pulmonary artery. These families of channels, especially the canonical TRP (TRPC) subfamily, may be involved in the development of bronchial hyperresponsiveness, a hallmark of asthma. In the present study, we evaluated i) the effects of sildenafil on tracheal rings of rats subjected to antigen challenge, ii) whether the extent of TRPC gene expression may be modified by antigen challenge, and iii) whether inhibition of type-5 phosphodiesterase (PDE5) may alter TRPC gene expression after antigen challenge. Sildenafil (0.1 µM to 0.6 mM) fully relaxed carbachol-induced contractions in isolated tracheal rings prepared from naive male Wistar rats (250-300 g) by activating the NO-cGMP-K+ channel pathway. Rats sensitized to antigen by intraperitoneal injections of ovalbumin were subjected to antigen challenge by ovalbumin inhalation, and their tracheal rings were used to study the effects of sildenafil, which more effectively inhibited contractions induced by either carbachol (10 µM) or extracellular Ca2+ restoration after thapsigargin (1 µM) treatment. Antigen challenge increased the expression of the TRPC1 and TRPC4 genes but not the expression of the TRPC5 and TRPC6 genes. Applied before the antigen challenge, sildenafil increased the gene expression, which was evaluated by RT-PCR, of TRPC1 and TRPC6, decreased TRPC5 expression, and was inert against TRPC4. Thus, we conclude that PDE5 inhibition is involved in the development of an airway hyperresponsive phenotype in rats after antigen challenge by altering TRPC gene expression.

Sildenafil decreases rat tracheal hyperresponsiveness to carbachol and changes canonical transient receptor potential gene expression after antigen challenge

Inhibition of type-5 phosphodiesterase by sildenafil decreases capacitative Ca2+ entry mediated by transient receptor potential proteins (TRPs) in the pulmonary artery. These families of channels, especially the canonical TRP (TRPC) subfamily, may be involved in the development of bronchial hyperresponsiveness, a hallmark of asthma. In the present study, we evaluated i) the effects of sildenafil on tracheal rings of rats subjected to antigen challenge, ii) whether the extent of TRPC gene expression may be modified by antigen challenge, and iii) whether inhibition of type-5 phosphodiesterase (PDE5) may alter TRPC gene expression after antigen challenge. Sildenafil (0.1 µM to 0.6 mM) fully relaxed carbachol-induced contractions in isolated tracheal rings prepared from naive male Wistar rats (250-300 g) by activating the NO-cGMP-K+ channel pathway. Rats sensitized to antigen by intraperitoneal injections of ovalbumin were subjected to antigen challenge by ovalbumin inhalation, and their tracheal rings were used to study the effects of sildenafil, which more effectively inhibited contractions induced by either carbachol (10 µM) or extracellular Ca2+ restoration after thapsigargin (1 µM) treatment. Antigen challenge increased the expression of the TRPC1 and TRPC4 genes but not the expression of the TRPC5 and TRPC6 genes. Applied before the antigen challenge, sildenafil increased the gene expression, which was evaluated by RT-PCR, of TRPC1 and TRPC6, decreased TRPC5 expression, and was inert against TRPC4. Thus, we conclude that PDE5 inhibition is involved in the development of an airway hyperresponsive phenotype in rats after antigen challenge by altering TRPC gene expression.

Evidence in Favor of a Novel Excitation‐Contraction Calcium Release Mechanism in Skeletal Muscle L6 Cells

Our laboratory has provided evidence for the first half of a model to explain both store‐operated or capacitative Ca2+ entry (CCE) as well as excitation‐contraction coupled Ca2+ release (ECC) in skeletal muscle. Here we offer evidence in favor of a model that proposes ECC acts by a relative displacement of the sarcolemmal L‐channel (DHPR) with respect to the sarcoplasmic reticulum‐localized Ryanodine Receptor (RyR). We titrated cells with ryanodine (Ry) which is known to both active (low concentrations) and inhibit (high concentrations) the RyR. We then repeated this titration after two types of treatments: conditions to depolarize the sarcolemma; and alterations in cytosolic Ca2+ levels under non‐depolarizing conditions. Depolarizing conditions, including high extracellular [K+] and field depolarization with solution electrodes, caused a shift in the Ry titration such that lower concentrations elicited both stimulation and inhibition. Addition of the DHPR agonist BAY K‐8644 caused a similar shift, although of smaller magnitude. On the other hand, altering intracellular Ca2+ through direct activation of RyR with the caffeine‐like xx did not cause a shift in Ry sensitivity. Similarly, alterations in exogenous Ca2+ concentration did not change Ry sensitivity. We suggest that these findings are consistent with our “relative displacement” hypothesis for ECC, whereby excitation moves the DHPR to unoccupied RyR, releasing Ca2+ into the cytosol from previously occluded RyR channels.

Minocycline Suppresses Activation of Nuclear Factor of Activated T Cells 1 (NFAT1) in Human CD4+ T Cells

Minocycline is a tetracycline family antibiotic that has anti-inflammatory and immunomodulatory properties. These properties have shown promise in the treatment of conditions such as rheumatoid arthritis, Huntington disease, and multiple sclerosis. As lymphocyte activation is involved in the pathogenesis of many of these diseases, T cells are postulated to be a primary target in minocycline therapy. Previous studies have demonstrated attenuation of CD4(+) T cell activation by minocycline, but a specific mechanism has not been elucidated. In this study, we investigated the effect of minocycline on the activity of three key transcription factors regulating CD4(+) T cell activation: NF-κB, AP-1 (activator protein 1), and NFAT (nuclear factor of activated T) cells. Our data demonstrate that minocycline selectively impairs NFAT-mediated transcriptional activation, a result of increased phosphorylation and reduced nuclear translocation of the isoform NFAT1. Minocycline increased the activity of the NFAT kinase GSK3 and decreased intracellular Ca(2+) flux, both of which facilitate NFAT1 phosphorylation. These findings provide a novel mechanism for minocycline induced suppression of CD4(+) T cell activation and may better inform the application of minocycline as an immunomodulatory agent.

Mechanisms of Glucosamine‐Mediated Relaxing Responses in Isolated Rat Aortic Rings

Glucosamine (GlcN) has been shown to block capacitative Ca2+ entry, also referred to as store‐operated Ca2+ entry (SOCE), in rat cardiomyocytes in response to phenylephrine (PHE). We hypothesized that GlcN would suppress SOCE in smooth muscle cells and lower intracellular [Ca2+] in PHE‐contracted rat aortic rings leading to vasorelaxation. In the wire‐myograph, relaxing responses to GlcN (0.01 – 30 mM) were assessed in PHE‐contracted rat aortic rings. GlcN relaxed endothelium‐denuded (−E) aortic rings with a sensitivity (pEC50) of 2.73 ± 0.09. The cell impermeable and osmotic control L‐ glucose (L‐Glc; 0.01 – 30 mM) did not result in arterial relaxations. Inhibition of the sarcoendoplasmic reticulum (ER) Ca2+‐ATPase (SERCA) results in Ca2+ depletion in the ER, which activates SOCE. The selective SERCA inhibitors thapsigargin (10 μM) and cyclopiazonic acid (50 μM) both significantly suppressed relaxations to GlcN in −E aortic rings. In Ca2+ depleted –E aortic rings, re‐titration of CaCl2 (0.01 – 5 mM) resulted in a lower sensitivity to CaCl2 in the presence of 5 mM GlcN compared to L‐Glc. Pre‐incubation with the SOCE inhibitor SKF96365 (10 μM) reduced sensitivity to CaCl2to comparable levels for both GlcN and L‐Glc. The present findings demonstrate that GlcN acts via an endothelium‐independent and cGMP‐dependent mechanism that inhibits SOCE to lower smooth muscle cell [Ca2+]i with subsequent vasorelaxation.

TRPC1, STIM1 and Orai1 Mediate Capacitative Calcium Entry Activated by Acute Hypoxia in Mouse Pulmonary Arterial Smooth Muscle Cells

Previous studies in pulmonary artery smooth muscle cells (PASMCs) showed that acute hypoxia activates capacitative Ca2+ entry (CCE) but the molecular channel(s) and the molecular signal(s) mediating CCE caused by acute hypoxia remain unclear. The present study aimed to determine if TRPC1, STIM1 and Orai1 mediate CCE caused by acute hypoxia in mouse PASMCs. In primary cultured PASMCs loaded with fura-2, acute hypoxia caused a transient followed by a sustained rise in intracellular Ca2+ concentration ([Ca2+]i). The transient but not sustained rise in [Ca2+]i was partially inhibited by nifedipine. In addition, acute hypoxia increased the rate of Mn2+ quench of fura-2 fluorescence that was inhibited by SKF 96365, Ni2+, La3+ and Gd3, exhibiting pharmacological properties characteristic of CCE. The nifedipine-insensitive rise in [Ca2+]i and the increase in Mn2+ quench rate were both inhibited in cells pretreated with antibodies raised against extracellular epitopes of TRPC1, in cells transfected with STIM1 siRNA and in cells transfected with Orai1 siRNA. Taken together, acute hypoxia causes activation of voltage-operated Ca2+ entry and CCE. These data suggest that TRPC1, STIM1 and Orai1 mediate CCE activated by acute hypoxia in mouse PASMCs. [Supported by HL49254, NCRR P20RR15581 (JR Hume) and AHA Scientist Development Grant (LC Ng)]

Proinflammatory treatment of astrocytes with lipopolysaccharide results in augmented Ca2+ signaling through increased expression of VIA phospholipase A2 (iPLA2)

Many Ca(2+)-regulated intracellular processes are involved in the development of neuroinflammation. However, the changes of Ca(2+) signaling in the brain under inflammatory conditions were hardly studied. ATP-induced Ca(2+) signaling is a central event of signal transmission in astrocytic networks. We investigated primary astrocytes after proinflammatory stimulation with lipopolysaccharide (LPS; 100 ng/ml) for 6-24 h. We reveal that Ca(2+) responses to purinergic ATP stimulation are significantly increased in amplitude and duration after stimulation with LPS. We detected that increased amplitudes of Ca(2+) responses to ATP in LPS-treated astrocytes can be explained by substantial increase of Ca(2+) load in stores in endoplasmic reticulum. The mechanism implies enhanced Ca(2+) store refilling due to the amplification of capacitative Ca(2+) entry. The reason for the increased duration of Ca(2+) responses in LPS-treated cells is also the amplified capacitative Ca(2+) entry. Next, we established that the molecular mechanism for the LPS-induced amplification of Ca(2+) responses in astrocytes is increased expression and activity of VIA phospholipase A(2) (VIA iPLA(2)). Indeed, both gene silencing with specific small interfering RNA and pharmacological inhibition of VIA iPLA(2) with S-bromoenol lactone reduced the load of the Ca(2+) stores and caused a decrease in the amplitudes of Ca(2+) responses in LPS-treated astrocytes to values, which were comparable with those in untreated cells. Our findings highlight a novel regulatory role of VIA iPLA(2) in development of inflammation in brain. We suggest that this enzyme might be a possible target for treatment of pathologies related to brain inflammation.