Increase In Intracellular Calcium Concentration Research Articles

Role of initial protein phosphorylation events and localized release-activated calcium influx in B cell antigen receptor signaling

An increase in intracellular calcium concentration is one of the major initial steps in B cell activation following antigen receptor (BCR) ligation. We show herein that in C57BL/6 murine B lymphocytes and in model cell lines, BCR-mediated calcium ion (Ca(2+)) influx occurs via highly selective Ca(2+) release-activated channels, and stromal interaction molecule 1 (STIM1) plays an important role in this pathway. We also demonstrate the temporal relation between Ca(2+)-dependent signaling events and formation of the immune synapse. Our data indicate that cytoplasmic Ca(2+) levels in areas adjacent to the immune synapse differ from those in the rest of the cytoplasm. Finally, a comparison of phosphorylation patterns of BCR-triggered signaling proteins in the presence or absence of Ca(2+) revealed the unanticipated finding that initial BCR-triggered, Ca(2+)-dependent tyrosine phosphorylation events involve predominantly Ca(2+) released from intracellular stores and that influx-derived Ca(2+) is not essential. This suggests a different role for this phase of Ca(2+) influx.

The ability of freeze-dried bull spermatozoa to induce calcium oscillations and resumption of meiosis

The objective was to investigate the ability of freeze-dried (FD) bull spermatozoa to induce calcium oscillations in mouse oocytes and meiosis resumption in in vitro-matured bovine oocytes after intracytoplasmic sperm injection (ICSI). Bull spermatozoa were freeze-dried and stored for 1 y at +25, +4, or −196 °C. In the first experiment, rehydrated sperm heads were microinseminated into hybrid mouse oocytes loaded with fluo-3/AM, and the kinetics of intracellular calcium concentration was monitored for 1 h. Repetitive increases of intracellular calcium concentration were recorded in the majority of injected oocytes, with exception of a few oocytes injected with FD sperm heads stored at +4 °C (11%) and +25 °C (8%) that exhibited a single increase or no response (non-oscillated). The proportion of oocytes that oscillated with high frequency (≥10 spikes/h) was higher in the non-dried control group (79%; P < 0.05) than in the FD groups (58, 55, and 54% for storage at −196, +4, and +25 °C, respectively). In the second experiment, control and FD spermatozoa were microinseminated into in vitro-matured, denuded bovine oocytes. The oocytes were fixed and stained 12 h after ICSI. A higher proportion of bovine oocytes injected with control spermatozoa (70%; P < 0.05) resumed meiosis than those injected with +25, +4 and −196 °C stored FD spermatozoa (53, 48, and 57%, respectively). The proportion of ICSI oocytes that developed to the pronuclear stage (complete activation) was higher in the control group (64%; P < 0.05) than those in all the FD groups (34, 27, and 28% for storage at −196, +4, and +25 °C, respectively). Thus, the ability of bull spermatozoa to induce frequent intracellular calcium spikes in mouse oocytes was impaired by the process of freeze-drying, without differences among storage at +25, +4 or −196 °C, probably resulting in a lower proportion of bovine oocytes that resumed meiosis and/or developed to the pronuclear stage.

Protein Kinase C-related Kinase and ROCK Are Required for Thrombin-induced Endothelial Cell Permeability Downstream from Gα12/13 and Gα11/q

Increase in vascular permeability occurs under many physiological conditions such as wound repair, inflammation, and thrombotic reactions and is central in diverse human pathologies, including tumor-induced angiogenesis, ocular diseases, and septic shock. Thrombin is a pro-coagulant serine protease, which causes the local loss of endothelial barrier integrity thereby enabling the rapid extravasation of plasma proteins and the local formation of fibrin-containing clots. Available information suggests that thrombin induces endothelial permeability by promoting actomyosin contractility through the Rho/ROCK signaling pathway. Here we took advantage of pharmacological inhibitors, knockdown approaches, and the emerging knowledge on how permeability factors affect endothelial junctions to investigate in detail the mechanism underlying thrombin-induced endothelial permeability. We show that thrombin signals through PAR-1 and its coupled G proteins Galpha(12/13) and Galpha(11/q) to induce RhoA activation and intracellular calcium elevation, and that these events are interrelated. In turn, this leads to the stimulation of ROCK, which causes actin stress-fiber formation. However, this alone is not sufficient to account for thrombin-induced permeability. Instead, we found that protein kinase C-related kinase, a Rho-dependent serine/threonine kinase, is activated in endothelial cells upon thrombin stimulation and that its expression is required for endothelial permeability and the remodeling of cell-extracellular matrix and cell-cell adhesions. Our results demonstrate that the signal initiated by thrombin bifurcates at the level of RhoA to promote changes in the cytoskeletal architecture through ROCK, and the remodeling of focal adhesion components through protein kinase C-related kinase. Ultimately, both pathways converge to cause cell-cell junction disruption and provoke vascular leakage.

266 POSTER Apoptosis induction in acute myeloid leukemia by inhibition of MEK and MDM2 is strongly associated with the BH3-only proteins Puma and Bim

This study investigated the effects of C7 and C9 aliphatic (n-heptane, n-nonane), naphthenic (methylcyclohexane, 1,2,4-trimethylcyclohexane (TMCH)) and aromatic (toluene, 1,2,4-trimethylbenzene (TMB)) hydrocarbons on the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in rat brain synaptosome fraction. Methyl mercury (MeHg) was included as a positive control. Exposure of the synaptosomes to the hydrocarbons produced a concentration-dependent linear increase in the formation of the fluorescence of 2′,7′-dichlorofluorescein (DCF) as a measure of the production of ROS and RNS. Formation of RNS was demonstrated by preincubation of the synaptosome fraction with the neuronal nitric oxide synthase (nNOS) inhibitor Nω-nitro-l-arginine methyl ester (L-NAME), which reduced the MeHg and TMCH-stimulated fluorescence by 51% and 65%, respectively. The naphthenic hydrocarbon TMCH showed the strongest potential for ROS and RNS formation in rat brain synaptosomes, followed by TMB, toluene, n-nonane, n-heptane, and methylcyclohexane, respectively. TMCH was selected for mechanistic studies of the formation of ROS. Both MeHg and TMCH induced an increase in intracellular calcium concentration [Ca2+]i as measured with Fura-2. Blockade of voltage-dependent Ca2+ channels with lanthanum prior to stimulation with MeHg and TMCH led to a reduction in the ROS/RNS formation of 72% and 70%, respectively. Furthermore, addition of cyclosporin A (CSA), a blocker of the mitochondrial permeability transition pore (MTP), lowered both the MeHg and TMCH-elevated DCF fluorescence by 72% and 59%. Preincubation of the synaptosome fraction with the protein tyrosine kinase inhibitor genistein lowered the MeHg and TMCH-stimulated fluorescence by 85% and 91%, respectively. Addition of the extracellular signal-regulated protein kinase (MEK)-1 and -2 inhibitor U0126 reduced the fluorescence stimulated by MeHg and TMCH by 62% and 63%. Furthermore, the protein kinase C inhibitor bisindolylmaleimide reduced the fluorescence stimulated by MeHg and TMCH by 52% and 56%. The compound 1-(6-[17beta-3-methoxyestra- 1,3,5(10)-trien- 17-yl]-aminohexyl)-1H-pyrrole-2,5-dione (U73122), which inhibits phospholipase C, was shown to decrease the ROS and RNS formation induced by MeHg and TMCH by 49% and 64%, respectively. The phospholipase A2 (PLA2) inhibitor 7,7-dimethyl eicosadienoic acid (DEDA) reduced fluorescence in response to MeHg and TMCH by 49% and 54%. Simultaneous addition of L-NAME, CSA, and DEDA to the synaptosome fraction totally abolished the DCF fluorescence. In conclusion, C7 and C9 aliphatic, naphthenic, and aromatic hydrocarbons stimulated formation of ROS and RNS in rat brain synaptosomes. The naphthenic hydrocarbon TMCH stimulated formation of ROS and RNS in the synaptosomes through Ca2+-dependent activation of PLA2 and nNOS, and through increased transition permeability of the MTP. Exposure of humans to the naphthenic hydrocarbon TMCH may stimulate formation of free radicals in the brain, which may be a key factor leading to neurotoxicity.

The influence of chronic lithium administration on deafferentation-induced cellular changes in the chick cochlear nucleus

The avian brainstem serves as a useful model system to address the question of how afferent activity influences viability of target neurons. Approximately 20–30% of neurons in the chick cochlear nucleus, nucleus magnocellularis (NM) die following deafferentation (i.e. deafness produced by cochlea removal). Previous studies have identified cellular events that occur within hours following cochlea removal, which are thought to lead to the ultimate death of NM neurons. We have recently shown that chronic lithium treatment increases neuronal survival following deafferentation. To assess where in the cell death cascade lithium is having its effect, we evaluated some of the early deafferentation-induced cellular changes in NM neurons. Lithium did not affect deafferentation-induced changes that occur across the entire population of NM neurons. There were still deafferentation-induced increases in intracellular calcium concentrations and early changes in the ribosomes, as indicated by Y10b immunolabeling. Lithium did, however, affect changes that are believed to be indicative of the subpopulation of NM neurons that will eventually die. Ribosomes recovered in all of the deafferented NM neurons (as assessed by Y10b labeling) by 10 h following cochlea removal in subjects pretreated with lithium, while a subpopulation of the NM neurons in saline-treated subjects showed dramatic reduction in Y10b labeling at that time. Lithium treatment also prevented the robust upregulation of b cell leukemia/lymphoma-2 (Bcl-2) mRNA that is observed in a subpopulation of deafferented NM neurons 6 h following cochlea removal.

Characterization of subcellular localization and Ca2+ modulation of calsenilin/DREAM/KChIP3

Earlier reports found that calsenilin is a transcriptional repressor or a subunit of plasma membrane channel, and indicated that calsenilin was present in the nucleus or plasma membrane. Immunohistochemical and subcellular fractionation analysis, however, revealed that calsenilin/DREAM/KChIP3 was distributed throughout the cytoplasm of SK-N-BE2(C), Jurkat, and HeLa cells. In addition, the expression of calsenilin suppressed the ATP-induced increase in intracellular Ca2+ concentrations. By increase in intracellular calcium concentration, calsenilin was translocated into the nucleus.

Activation of P2Y receptors by ATP and by its analogue, ADPβS, triggers two calcium signal pathways in the longitudinal muscle of mouse distal colon

Our previous research showed that ATP and adenosine 5′-O-2-thiodiphosphate (ADPβS) induce contractile effects in the longitudinal muscle of mouse distal colon via activation of P2Y receptors which are not P2Y 1 or P2Y 12 subtypes. This study investigated the nature of the P2Y receptor subtype(s) and the mechanisms leading to the intracellular calcium concentration increase necessary to trigger muscular contraction. Motor responses of mouse colonic longitudinal muscle to P2Y receptor agonists were examined in vitro as changes in isometric tension. ATP or ADPβS induced muscular contraction, which was not affected by P2Y 11 or P2Y 13 selective antagonists. Calcium-free solution or the calcium channel blocker, nifedipine, failed to modify the contractile responses to ATP or ADPβS, which were virtually abolished by depletion of calcium intracellular stores after repetitive addition of carbachol in calcium-free medium with addition of cyclopiazonic acid. Neomycin or U-73122, phospholipase C inhibitors, or 2-aminoethoxy-diphenylborate (2-APB), membrane-permeant IP 3 receptor inhibitor reduced the response to ATP, whilst ryanodine or ruthenium red, inhibiting calcium release from ryanodine-sensitive stores, abolished the response to ADPβS. Responses to maximally effective concentrations of ATP and ADPβS were not fully additive. Desensitisation with ADPβS antagonized the contractile effects of ATP, as desensitisation with ATP antagonized the response to ADPβS. In the longitudinal muscle of mouse distal colon, ATP and ADPβS induce muscular contraction via a P2Y receptor, coupled to differential signal pathways leading to intracellular calcium increase.

An improved method for patch clamp recording and calcium imaging of neurons in the intact dorsal root ganglion in rats

The properties of dorsal root ganglion (DRG) neurons have been mostly investigated in culture of dissociated cells, and it is uncertain whether these cells maintain the electrophysiological properties of the intact DRG neurons. Few attempts have been made to record from DRG neurons in the intact ganglion using the patch clamp technique. In this study, rat DRGs were dissected and incubated for at least 1h at 37°C in collagenase (10mg/ml). We used oblique epi-illumination to visualize DRG neurons and perform patch clamp recordings. All DRG neurons exhibited strong delayed rectifier potassium current and a high threshold for spike generation (−15mV) that rendered the cells very weakly excitable, generating only one action potential upon strong current injection (>300pA). It is therefore possible that cultured DRG neurons, commonly used in studies of pain processing, may be hyperexcitable because they acquired “neuropathic” properties due to the injury induced by their dissociation. Electrical stimulation of the attached root produced an antidromic spike in the soma that could be blocked by intracellular hyperpolarization or high frequency stimulation. Imaging intracellular calcium concentration with Oregon Green BAPTA-1 indicates that antidromic stimulation caused a long-lasting increase in intracellular calcium concentration mostly near the cell membrane. This study describes a simple approach to examine the electrophysiological and pharmacological properties and intracellular calcium signaling in DRG neurons in the intact ganglion where the effects of somatic spike invasion can be studied as well.

Elucidation of a TRPC6-TRPC5 Channel Cascade That Restricts Endothelial Cell Movement

Canonical transient receptor potential (TRPC) channels are opened by classical signal transduction events initiated by receptor activation or depletion of intracellular calcium stores. Here, we report a novel mechanism for opening TRPC channels in which TRPC6 activation initiates a cascade resulting in TRPC5 translocation. When endothelial cells (ECs) are incubated in lysophosphatidylcholine (lysoPC), rapid translocation of TRPC6 initiates calcium influx that results in externalization of TRPC5. Activation of this TRPC6-5 cascade causes a prolonged increase in intracellular calcium concentration ([Ca(2+)](i)) that inhibits EC movement. When TRPC5 is down-regulated with siRNA, the lysoPC-induced rise in [Ca(2+)](i) is shortened and the inhibition of EC migration is lessened. When TRPC6 is down-regulated or EC from TRPC6(-/-) mice are studied, lysoPC has minimal effect on [Ca(2+)](i) and EC migration. In addition, TRPC5 is not externalized in response to lysoPC, supporting the dependence of TRPC5 translocation on the opening of TRPC6 channels. Activation of this novel TRPC channel cascade by lysoPC, resulting in the inhibition of EC migration, could adversely impact on EC healing in atherosclerotic arteries where lysoPC is abundant.

Nucleolus and c-Myc: potential targets of cardenolide-mediated antitumor activity

The use of cardenolides like ouabain, digitoxin, or oleandrin has been reported previously many times as a means of potentially combating human refractory prostate cancer by inducing apoptosis through an increase in intracellular calcium concentrations. The aims of the current study were to investigate if part of the antitumor effects mediated by cardenolides concerned disorganization of nucleolar structure and whether this was further associated with a marked decrease in c-Myc expression. Accordingly, the antitumor activity of a novel hemisynthetic cardenolide [1R,3aS,3bR,5aS,6aR,7aS,9R,12aR,13aR,15aR]-3a,11a-dihydroxy-13a-(hydroxymethyl)-9,15a-dimethyl-1-(5-oxo-2,5-dihydrofuran-3-yl)icosahydro-1H,4'H-spiro[cyclopenta [7,8]phenanthro[2,3-b]pyrano[3,2-e][1,4]dioxine-11,2'-[1,3]thiazolidin]-4'-one (UNBS1450)] was compared with that of classic cardenolides and reference anticancer agents in prostate cancer cell lines in vitro and in vivo following s.c. and orthotopic prostate cancer cell grafting into mice. The present study indicates that UNBS1450 markedly decreases the in vitro viability/proliferation of human prostate cancer cell lines but not of normal cells. The induced effects are not linked to an increase in intracellular calcium concentrations and subsequent induction of apoptosis. Rather, they appear to relate to the compound's capacity to disorganize nucleolar structure and function (through an impairment of cyclin-dependent kinase and c-Myc expression and related signaling pathways; paralleled by the disorganization of cancer cell-specific perinucleolar bodies as revealed by disruption of Sam68). This nonapoptotic cancer cell death mediated by severe nucleolar targeting and down-regulation of c-Myc expression is a completely new cardenolide-induced mechanism of antitumor action.

Modulation by BNP of GABA receptors on ON-type rod bipolar cells is dependent on subcellular sites

Brain natriuretic peptide (BNP) suppresses GABA A receptor-mediated current of ON-type rod-dominant bipolar cells (RBCs) in the rat retina. Here we report that such BNP-induced modulation is dependent on subcellular sites. Whole-cell currents could be induced by GABA focally applied to both dendrites/somata and axon terminals of isolated ON-type RBCs. Whilst the GABA currents induced at the axon terminal were significantly suppressed by BNP (50 nM), those at the dendrites/somata were hardly changed or slightly suppressed. Similar results were obtained when such experiments were performed in rat retinal slices. Calcium imaging showed that application of BNP (50 nM) caused a prominent increase in intracellular calcium concentrations ([Ca 2+] i) at the axon terminal, and the increase monotonically decayed when the acting site of BNP was moved away from the axon terminal along the cell: more distant, less significant. No detectable increase in [Ca 2+] i was found at the dendrites. Such increase in [Ca 2+] i could be completely blocked by pre-incubation with anantin, an antagonist of the NP-receptor-A (NPR-A). On the other hand, caffeine, an agonist of the ryanodine receptor, caused a similar subcellular site-dependent changes in [Ca 2+] i, thus mimicking the BNP effect. All these results suggest that BNP-induced modulation of the activity of GABA receptors may be largely restricted to the inner retina.

Chemotransport contributes to the effect of oscillatory fluid flow on human bone marrow stromal cell proliferation

Mechanical loads produce a diverse set of biophysical signals that may regulate bone cell activity, but accumulating evidence suggests that interstitial fluid flow is the primary signal that bone cells perceive. Because we previously demonstrated that oscillatory fluid flow increases human bone marrow stromal cell proliferation, we investigated the contribution of fluid shear stress and chemotransport, two stimuli induced by interstitial fluid flow. Alterations in flow rate at a constant peak shear stress were associated with decreases in oscillatory fluid flow-induced marrow stromal cell proliferation, while variations in peak fluid shear stress had no significant effect. Modulation of marrow stromal cell proliferation by flow rate may be attributed to changes in the release of ATP and intracellular calcium signaling. We found that if the flow rate is decreased while maintaining a constant peak fluid shear stress, marrow stromal cells release less ATP into the extracellular environment. Moreover, as the flow rate decreased fewer cells respond to fluid flow with an increase in intracellular calcium concentration. These data suggest that chemotransport is a prerequisite for marrow stromal cells to respond to interstitial fluid flow.

Effect of GABAA receptor activation on UT-coupled signaling pathways in rat cortical astrocytes

Cultured rat cortical astrocytes express two types of urotensin II (UII) binding sites: a high affinity site corresponding to the UT (GPR14) receptor and a low affinity site that has not been fully characterized. Activation of the high affinity site in astroglial cells stimulates polyphosphoinositide (PIP) turnover and provokes an increase in intracellular calcium concentration. We have hypothesized that the existence of distinct affinity sites for UII in rat cortical astrocytes could be accounted for by a possible cross-talk between UT and the ligand-gated ion channel GABA(A) receptor (GABA A R). Exposure of cultured astrocytes to UII provoked a bell-shaped increase in cAMP production, with an EC50 stimulating value of 0.83+/-0.04 pM, that was totally blocked in the presence of the adenylyl cyclase inhibitor SQ 22,536. In contrast, UII was found to inhibit forskolin-induced cAMP formation. In the presence of the specific PKA inhibitor H89, UII provoked a sustained stimulation of cAMP formation. Inhibition of PKA by H89 strongly reduced the stimulatory effect of UII on PIP metabolism. GABA and the GABA A R agonist isoguvacine provoked a marked inhibition of UII-induced cAMP synthesis and a significant reduction of UII-evoked PIP turnover. These data suggest that functional interaction between UT and GABA(A)R negatively regulates coupling of UT to the classical PLC/IP(3) signaling cascade as well as to the adenylyl cyclase/PKA pathway.

Rapid effects of 1α,25(OH) 2D3 in resting human peripheral blood mononuclear cells

The steroid hormone 1α,25(OH) 2D3 produces biological responses via both genomic and nongenomic mechanisms. Stimulation of rapid, nongenomic responses by 1α,25(OH) 2D3 has been postulated to result from interaction of the ligand with cell membrane 1α,25(OH) 2D3 receptors and to involve membrane receptors. We examined the rapid effects of 1α,25(OH) 2D3 on calcium mobilization and calcium entry into resting human peripheral blood mononuclear cells isolated from healthy volunteers. We also investigated the possible involvement of purinergic receptors in this action. 1α,25(OH) 2D3 induced a time-dependent increase in intracellular calcium concentration ([Ca 2+] i ). The initial 1α,25(OH) 2D3-stimulated calcium increment was sensitive to thapsigargin (Tg), indicating its origins in calcium release from intracellular stores. 2-Aminoethyldiphenyl borate (2APB), an inhibitor of capacitative calcium entry, caused a significant [Ca 2+] i decrease in human cells treated with 1α,25(OH) 2D3. Furthermore, in contrast to observations in osteoblasts and skeletal muscle cells, nifedipine had no effect on 1α,25(OH) 2D3-induced calcium entry, suggesting that L-type calcium channels were not implicated in this action. Besides, 1α,25(OH) 2D3 prevented the calcium entry induced by 3′- O-(4-benzoyl)benzoyl-adenosine 5′-triphosphate (BzATP), a specific agonist of purinergic P2X 7 receptors. This finding was further confirmed by 1α,25(OH) 2D3-induced reduction of BzATP- and 4-aminopyridine (4AP)-stimulated ethidium bromide fluorescence. The presented results demonstrate, for the first time in healthy, resting human peripheral blood mononuclear cells that 1α,25(OH) 2D3 is capable of exerting a rapid, nongenomic effect on [Ca 2+] i , while inhibiting of the P2X 7 channel permeability.

An RGS for PI3K

Immunoglobulin E (IgE) triggers the activation of the FcεRI immune receptor, which signals through immunoreceptor-based tyrosine activation motifs, in response to allergens. FcεRI activation on mast cells produces an increase in intracellular calcium concentration, which causes degranulation and the release of inflammatory mediators such as histamine. The signaling pathway from the FcεRI to calcium involves the tyrosine kinases Lyn and Syk, the adaptor proteins Gab2 and Grb2, phosphoinositide 3-kinase (PI3K), and phospholipase C-mediated production of inositol 1,4,5-trisphosphate (IP 3 ). G protein-coupled receptors, such as adenosine or prostaglandin receptors, may amplify IgE-mediated mast cell activation. Bansal et al . found that RGS13, a member of the R4 subfamily of regulator of G protein signaling (RGS) proteins, provided an inhibitory input into IgE-mediated mast cell degranulation through a mechanism involving the p85 subunit of PI3K instead of a G protein. RGS proteins are best known for attenuating G protein signaling through their actions as GTPase-activating proteins (GAPs) for specific Gα subunits and for interfering with the interactions between Gα subunits and downstream effectors. Bansal et al . studied bone marrow-derived mast cells (BMMCs) from Rgs13 knockout ( Rgs13 -KO) mice and found that antigen-elicited degranulation was greater and inhibition of G proteins with pertussis toxin did not alter this enhanced response, suggesting that G proteins were not mediating the effect. Furthermore, reconstitution of the Rgs13 -deficient BMMCs with mutant forms of RGS13 that could not interact with G proteins prevented the enhanced degranulation response. The Rgs13 -KO mice also exhibited elevated cutaneous allergic response compared with wild-type mice, yet did not show altered mast cell numbers or morphology. Using glutathione S -transferase fused to RGS13, the p85 subunit of PI3K was identified from lysates of BMMCs as a tyrosine-phosphorylated protein that interacted with RGS13. The interaction between RGS13 and p85 was direct and occurred with recombinant proteins, although it did require p85 tyrosine phosphorylation (achieved by incubation with Lyn). Rgs13 -deficient BMMCs showed increased production of IP 3 , phosphorylation of Akt, and increased intracellular calcium concentrations in response to antigen. Transduction of wild-type BMMCs with a cell-permeable RGS13 construct inhibited antigen-stimulated calcium flux and PI3K activity. Immunoprecipitation experiments with BMMCS transduced with the cell-permeable RGS13 indicated that RGS13 interfered with the recruitment of p85 to Gab2 of the FcεRI signaling complex. G. Bansal, Z. Xie, S. Rao, K. H. Nocka, K. M. Druey, Suppression of immunoglobulin E-mediated allergic responses by regulator of G protein signaling 13. Nat. Immunol. 9 , 73-80 (2008). [PubMed]

Takotsubo Cardiomyopathy With Marked ST-Segment Elevation and Electrical Alternans Complicated With Hyperglycemic Hyperosmolar State

This is the first report of a case of Takotsubo cardiomyopathy with a hyperglycemic hyperosmolar state (HHS). This case presented with marked ST-segment elevation and electrical alternans, uncommon findings in Takotsubo cardiomyopathy. We believe that hyperosmolarity-induced myocardial dehydration and consequent increase in intracellular calcium concentration may be the mechanism of Takotsubo cardiomyopathy and electrical alternans in HHS.

Disrupted intracellular calcium regulates BACE1 gene expression via nuclear factor of activated T cells 1 (NFAT 1) signaling

Beta-site APP-cleaving enzyme 1 (BACE1) expression is elevated in the brains of Alzheimer's disease (AD) patients and in aged-animal models. Because both AD and aging are associated with disrupted calcium homeostasis, we investigated the role of nuclear factor of activated T cells (NFAT) - a transcription factor regulated by the calcium- and calmodulin-dependent phosphatase calcineurin - in BACE1 expression. BACE1 expression was stimulated by a calcium ionophore in primary cortical cultures, and by SH-SY5Y neuroblastoma cells, which was both blocked by pretreatment with either cyclosporin A, an inhibitor of calcineurin, or ethyleneglycotetraacetic acid, a calcium chelator. Gel shift assays revealed direct binding of NFAT1 to specific DNA sequences within the BACE1 gene promoter region. Treatment with amyloid beta (Abeta), one of the major factors in AD pathogenesis, stimulated activation and nuclear translocation of NFAT1 following up-regulation of BACE1 expression. In addition, primary cortical cultures from Tg2576 mouse brains generated more Abeta by ionophore stimulation, which was reversed by cyclosporin A treatment. Furthermore, NFAT1 activation was observed in Tg2576 mouse brains. These results suggest that calcium ionophore- or Abeta-induced increases in intracellular calcium concentration stimulate BACE1 expression, resulting in accelerated Abeta generation, and that this process is mediated through the calcineurin-NFAT1 signaling pathway. This process may play a significant role in the pathogenesis of AD and aging.

Providing a Calcium Source

Gliomas--highly migratory brain malignancies derived from glia--express an unusual splice variant of BK channels (large conductance potassium channels independently activated by depolarization and calcium) whose activity may support migration of these diffusely infiltrating tumors. Noting that gliomas are not excitable and that global increases in intracellular calcium concentration ([Ca 2+ ] i ) of sufficient magnitude to activate BK channels would be expected to influence numerous other Ca 2+ -dependent processes, Weaver et al . investigated the possibility that activation of glioma BK channels might depend on a nearby Ca 2+ source. Immunofluorescence analysis of two glioma cell lines revealed that BK channels localized with lipid raft markers in "hot spots" at the protruding edges of cells. Furthermore, Western analysis of fractionated cell lysates indicated that BK channels were associated with a lipid raft fraction defined by the presence of caveolin-1. Treatment of U251 cells with methyl-β-cyclodextrin to disrupt lipid rafts led to BK channel redistribution and rapidly decreased whole-cell BK currents. This reduction in BK current was not associated with channel internalization, nor was it associated with a loss of BK channel sensitivity to a global increase in [Ca 2+ ] i . Pharmacological analysis indicated that inositol 1,4,5-trisphosphate receptors (IP 3 Rs), which were present in BK-containing hot spots and lipid raft fractions but did not coimmunoprecipitate with BK channels, provided the calcium source for BK activation. Thus, the authors propose that the localization of BK channels to lipid rafts allows IP 3 -mediated signaling pathways to activate BK channels and suggest that disruption of such pathways may provide a novel approach to inhibiting glioma migration. A. K. Weaver, M. L. Olsen, M. B. McFerrin, H. Sontheimer, BK channels are linked to inositol 1,4,5-triphosphate receptors via lipid rafts. J. Biol. Chem. 282 , 31558-31568 (2007). [Abstract] [Full Text]

Ammonium-induced calcium mobilization in 1321N1 astrocytoma cells

High blood levels of ammonium/ammonia (NH 4 +/NH 3) are associated with severe neurotoxicity as observed in hepatic encephalopathy (HE). Astrocytes are the main targets of ammonium toxicity, while neuronal cells are less vulnerable. In the present study, an astrocytoma cell line 1321N1 and a neuroblastoma glioma hybrid cell line NG108-15 were used as model systems for astrocytes and neuronal cells, respectively. Ammonium salts evoked a transient increase in intracellular calcium concentrations ([Ca 2+] i) in astrocytoma (EC 50 = 6.38 mM), but not in NG108-15 cells. The ammonium-induced increase in [Ca 2+] i was due to an intracellular effect of NH 4 +/NH 3 and was independent of extracellular calcium. Acetate completely inhibited the ammonium effect. Ammonium potently reduced calcium signaling by G q protein-coupled receptors (H 1 and M3) expressed on the cells. Ammonium (5 mM) also significantly inhibited the proliferation of 1321N1 astrocytoma cells. While mRNA for the mammalian ammonium transporters RhBG and RhCG could not be detected in 1321N1 astrocytoma cells, both transporters were expressed in NG108-15 cells. RhBG and RhBC in brain may promote the excretion of NH 3/NH 4 + from neuronal cells. Cellular uptake of NH 4 +/NH 3 was mainly by passive diffusion of NH 3. Human 1321N1 astrocytoma cells appear to be an excellent, easily accessible human model for studying HE, which can substitute animal studies, while NG108-15 cells may be useful for investigating the role of the recently discovered Rhesus family type ammonium transporters in neuronal cells. Our findings may contribute to the understanding of pathologic ammonium effects in different brain cells, and to the treatment of hyperammonemia.

Formation of systemic structural trace in rat kidneys after cold adaptation

We studied the adaptive response of rats to cold exposure. It was found that the development of the adaptive response and formation of systemic structural trace in the kidneys occurred within 1 month after the start of adaptation. Activation of proliferative processes in the kidneys was accompanied by an increase in intracellular calcium concentration.