Elevated Intracellular Calcium Levels Research Articles

Increased Lipolysis and Energy Expenditure in a Mouse Model with Severely Impaired Glucagon Secretion

BackgroundSecretion of insulin and glucagon is triggered by elevated intracellular calcium levels. Although the precise mechanism by which the calcium signal is coupled to insulin and glucagon granule exocytosis is unclear, synaptotagmin-7 has been shown to be a positive regulator of calcium-dependent insulin and glucagon secretion, and may function as a calcium sensor for insulin and glucagon granule exocytosis. Deletion of synaptotagmin-7 leads to impaired glucose-stimulated insulin secretion and nearly abolished Ca2+-dependent glucagon secretion in mice. Under non-stressed resting state, however, synaptotagmin-7 KO mice exhibit normal insulin level but severely reduced glucagon level.Methodology/Principal FindingsWe studied energy expenditure and metabolism in synaptotagmin-7 KO and control mice using indirect calorimetry and biochemical techniques. Synaptotagmin-7 KO mice had lower body weight and body fat content, and exhibited higher oxygen consumption and basal metabolic rate. Respiratory exchange ratio (RER) was lower in synaptotagmin-7 KO mice, suggesting an increased use of lipid in their energy production. Consistent with lower RER, gene expression profiles suggest enhanced lipolysis and increased capacity for fatty acid transport and oxidation in synaptotagmin-7 KO mice. Furthermore, expression of uncoupling protein 3 (UCP3) in skeletal muscle was approximately doubled in the KO mice compared with control mice.ConclusionsThese results show that the lean phenotype in synaptotagmin-7 KO mice was mostly attributed to increased lipolysis and energy expenditure, and suggest that reduced glucagon level may have broad influence on the overall metabolism in the mouse model.

The rotavirus enterotoxin (NSP4) promotes re-modeling of the intracellular microtubule network

Expression of the rotavirus enterotoxin (NSP4) in transfected monkey kidney cells was found to result in a dramatic re-modeling of the microtubule (MT) network. This important centrosome organized cytoskeletal element was dissolved by expression of NSP4 and re-formed in a ring array at the periphery of the cell, similar to that seen following normal virus infection. Site directed mutagenesis of the N-linked glycosylation sites in NSP4 was employed to show that glycosylation of NSP4 was not required for it to promote changes in the MT network. This result together with experiments using conventional inhibitors indicated that NSP4's ability to cause elevation of intracellular calcium levels was also not necessary to effect the changes in the MT network. Use of the centrosome function inhibitor nocodazole demonstrated that NSP4 based remodeling of the MT network was dominant over the normal organizational role of the centrosome. Finally the remodeling of the MT network was shown not to be linked to cellular apoptosis or necrosis. The potential importance of this newly recognised role for NSP4 in the overall process of intracellular pathogenesis by rotaviruses is discussed.

Extracellular ATP activates NFAT-dependent gene expression in neuronal PC12 cells via P2X receptors

BackgroundTreatment of neuronal PC12 cells with ATP induces depolarisation and increases intracellular calcium levels via purinergic receptors. In many cell types, sustained elevation of intracellular calcium levels cause changes in gene expression via activation of the transcription factor NFAT (nuclear factor of activated T cells). We have therefore characterised the signalling pathway by which ATP regulates NFAT-dependent gene expression in PC12 cells.ResultsThe activation of NFAT transcriptional activity by extracellular ATP was characterised with the help of reporter gene assays. Treatment of PC12 cells with ATP elicited a dose-dependent increase in luciferase activity (EC50 = 78 μM). UTP, 4-benzoylbenzoyl ATP and α,β-methylene ATP did not mimic the effect of ATP, which was abolished by treatment with the P2X receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS). This pharmacological characterisation provides evidence for a critical role of ionotropic P2X receptors. Blockade of L-type voltage-dependent calcium channels by nifedipine reduced the response of NFAT to ATP, indicating that a depolarisation-mediated calcium influx was required for maximal NFAT activation. Inhibition of store-operated calcium entry by the pyrazole derivative BTP2 also diminished ATP-dependent NFAT activation. Furthermore, ATP-induced NFAT activation was associated with the activation of the mitogen-activated protein kinases ERK1/2. Finally, treatment with ATP increased the levels of the NFAT target transcripts, RCAN1-4 (regulator of calcineurin) and BDNF (brain derived neurotrophic factor).ConclusionThe present data show that ATP induces NFAT-dependent changes in gene expression in PC12 cells by acting on P2X receptors. Maximal NFAT activation depends on both depolarisation-induced calcium influx and store-operated calcium entry and requires the activity of the protein phosphatase calcineurin and the mitogen-activated protein kinase cascade.

P4.14 Investigating the role of calcium-independent phospholipase [formula omitted] in store-operated calcium entry in mdx muscle

Duchenne muscular dystrophy (DMD) is caused by lack of dystrophin. Characteristic signs of dystrophic muscles are membrane fragility and abnormally elevated levels of intracellular calcium, detrimental to the cells. Increased activity of store-operated calcium channels (SOC) has been proposed as a possible mechanism for the enhanced calcium concentration. Studies of SOC have indicated the enzyme calcium-independent phospholipase A2β (iPLA2β) as an important mediator of calcium influx. We have previously shown that SOC entry in FDB fibres isolated from mdx mice is reduced in the presence of bromoenol lactone (BEL), a suicide inhibitor of iPLA2.

Calcium-dependent mitochondrial extrusion in ciliated protozoa

Here we demonstrate that ciliated protozoa can jettison mitochondria as intact organelles, releasing their contents to the extracellular space either in a soluble form, or in association with membrane vesicles at the cell periphery. The response is triggered by lateral clustering of GPI-anchored surface antigens, or by heat shock. In the first instance, extrusion is accompanied by elevated levels of intracellular calcium and is inhibited by Verapamil and BAPTA-AM arguing strongly for the involvement of calcium in triggering the response. Cells survive mitochondrial discharge raising the interesting possibility that extrusion is an early evolutionary adaptation to cell stress.

Protective Effect of Chlorogenic Acid against Aβ-Induced Neurotoxicity

Beta-amyloid (A<TEX>${\beta}$</TEX>) is considered as one of the major causes of Alzheimer's disease. This study examined the neuroprotective effects of chlorogenic acid, a naturally occurring polyphenol which is distributed widely in plants, fruits and vegetables, against A<TEX>${\beta}$</TEX>-induced toxicity. A<TEX>${\beta}$</TEX> decreased significantly the viability of PC12 cells. This was accompanied by an increase in the intracellular calcium levels and cleaved caspase-3. In addition, A<TEX>${\beta}$</TEX> induced an increase in Bax, and a decrease in Bcl-2 compared to the controls. However, a pre-treatment with chlorogenic acid rescued the PC12 cells from A<TEX>${\beta}$</TEX> by attenuating the elevated intracellular calcium levels and reducing the levels of the apoptosis related proteins, including caspase-3, Bcl-2 and Bax. These results suggest that the protective effects of chlorogenic acid are, at least in parts, by attenuating the intracellular calcium influx and reducing apoptosis induced by A<TEX>${\beta}$</TEX>.

Stabilization of Nrf2 by tBHQ prevents LPS-induced apoptosis in differentiated PC12 cells

The inflammatory reaction plays an important role in the pathogenesis of the neurodegenerative disorders. tert-butylhydroquinone (tBHQ) exhibits a wide range of pharmacological activities including anti-oxidative and anti-inflammatory action. In this study, we tried to elucidate possible effects of tBHQ on lipopolysaccharide (LPS)-induced inflammatory reaction and its underlying mechanism in neuron-like PC12 cells. tBHQ inhibited LPS-induced generation of reactive oxygen species (ROS) and elevation of intracellular calcium level. It also inhibited LPS-induced cyclooxygenase 2 (COX-2), TNF-α, nuclear factor KappaB (NF-kB), and caspase-3 expression in a dose-dependent manner while stabilizing nuclear factor-erythroid 2 p45-related factor 2. Moreover, the phosphorylations of p38, ERK1/2, and JNK were suppressed by tBHQ. These results suggest that the anti-inflammatory properties of tBHQ might result from inhibition of COX-2 and TNF-α expression, inhibition of NF-kB nuclear translocation along with suppression of MAP kinases (p38, ERK1/2, and JNK) phosphorylation in PC12 cells, so may be a useful agent for prevention of inflammatory diseases.

Changes In Mitochondrial Permeability Initiate Platelet Phosphatidylserine Exposure In Response to Elevated Cytoplasmic Calcium Levels

AbstractAbstract 2024Platelet phosphatidylserine (PS) exposure and procoagulant activity occur in a subset of activated platelets following stimulation with multiple or high levels of agonists. Key among the upstream regulators of agonist-initiated PS exposure is elevated cytoplasmic calcium levels. Additionally, the importance of mitochondrial permeability transition pore (mPTP) formation in regulating PS exposure has been highlighted by recent studies using cyclophilin D null (CypD -/-) platelets. CypD is a critical regulatory protein in the initiation of mPTP formation, and in the absence of CypD agonist-initiated PS exposure is markedly impaired. An alternative, slower pathway of PS exposure, initiated by the BH3 mimetic ABT-737, is regulated by members of the Bcl-2 protein pathway.It is currently unclear how mitochondrial and calcium-signaling pathways interact to regulate PS exposure in platelets. mPTP formation has been reported as both a consequence and a cause of elevated intracellular calcium levels in other cells. To clarify the relative roles of these two upstream signals, we examined the sequence of events that occur upon platelet activation using calcium and mitochondrial transmembrane potential (ΔΨm) sensitive fluorescent probes. In isolated platelets studied by flow cytometry and among adherent platelets evaluated by confocal microscopy, elevated intracellular calcium levels were observed prior to mPTP formation (as determined by loss of ΔΨm). A rise in mitochondrial calcium levels as measured by rhod-2 occurred coincidentally with elevations in cytoplasmic calcium levels. Similar increases in intracellular calcium levels were observed in CypD -/- and CypD +/+ platelets, despite the almost complete absence of mPTP formation and PS exposure in the CypD -/- platelets. These results are consistent with calcium acting as an upstream signal regulating mPTP formation.Ionomycin (3 μM) stimulation can overcome the defect in PS exposure in CypD -/- platelets. This result has been interpreted to indicate that calcium activates effector pathways downstream of the mPTP. However, an alternative explanation is that ionomycin raises cytoplasmic calcium to an elevated level that is sufficient to initiate mPTP formation even in CypD's absence. Dose response studies indicated that the calcium sensitivity of the mPTP was decreased in CypD -/- platelets, but ionomycin (3uM) caused mPTP opening and PS externalization in both CypD +/+ and CypD -/- platelets. Furthermore, in both CypD +/+ and CypD -/- platelets, mPTP formation and PS exposure were closely correlated at all doses of ionomycin utilized. These results indicate that high doses of ionomycin overcome the CypD -/- defect in PS exposure by initiating non-CypD-dependent mPTP formation.The roles of the mitochondrial and calcium pathways in regulating ABT-737-mediated PS exposure were examined. Extracellular calcium was not required for PS exposure initiated by ABT-737, distinct from its importance in agonist-initiated PS exposure. In contrast to previously reported results (Schoenwaelder et al, Blood 2009) we observed that ABT-737 stimulation causes a loss of ΔΨm coincident with PS exposure. Since disruptions in oxidative metabolism could also cause the observed effect on ΔΨm, ABT-737's effects on mitochondrial permeability were confirmed using a calcein-cobalt quenching assay. Unlike agonist-initiated PS exposure, both increased mitochondrial permeability and PS exposure were not dependent on CypD.Together these results indicate a central role for alterations in mitochondrial membrane permeability in mediating PS exposure. In agonist-stimulated platelets increases in cytoplasmic calcium result in mPTP formation and mitochondrial permeabilization, triggering downstream events that result in PS exposure. In ABT-737 stimulated platelets non-CypD-dependent mitochondrial permeability changes mediated by Bcl-2 proteins may also act as an important initiator of the events that result in PS exposure. Disclosures:No relevant conflicts of interest to declare.

Enhanced airway smooth muscle cell thromboxane receptor signaling via activation of JNK MAPK and extracellular calcium influx

Thromboxane is a key inflammatory mediator and potent airway constrictor. It acts on thromboxane A 2 (TP) receptors and contributes to airway inflammation and airway hyperresponsiveness that is the characteristic feature of asthma. The present study was designed to study TP receptor signaling in airway smooth muscle cells by using an organ culture model and a set of selective pharmacological inhibitors for mitogen-activated protein kinase (MAPK) and calcium signal pathways. Western-blot, immunohistochemistry, myograph and a selective TP receptor agonist U46619 were used for examining TP receptor signal proteins and function. Organ culture of rat bronchial segments for up to 48 h induces a time-dependently increased airway contractile response to U46619. This indicates that organ culture increases TP receptor signaling in the airway smooth muscle cells. The enhanced bronchial contraction was attenuated by the inhibition of c-Jun N-terminal kinase (JNK) MAPK activity, chelation of extracellular calcium and calcium channel blocker nifedipine, suggesting that JNK MAPK activity and elevated intracellular calcium level are required for the TP receptor signaling. In conclusion, airway smooth muscle cell TP receptor signaling occurs via JNK MAPK activity and the elevation of extracellular calcium influx, which may provide knowledge for understanding the signaling pathway responsible for the modulation of TP receptor mediated airway hyperresponsiveness to thromboxane.

Calcium Regulation of EGF-Induced ERK5 Activation: Role of Lad1-MEKK2 Interaction

The ERK5 cascade is a MAPK pathway that transmits both mitogenic and stress signals, yet its mechanism of activation is not fully understood. Using intracellular calcium modifiers, we found that ERK5 activation by EGF is inhibited both by the depletion and elevation of intracellular calcium levels. This calcium effect was found to occur upstream of MEKK2, which is the MAP3K of the ERK5 cascade. Co-immunoprecipitation revealed that EGF increases MEKK2 binding to the adaptor protein Lad1, and this interaction was reduced by the intracellular calcium modifiers, indicating that a proper calcium concentration is required for the interactions and transmission of EGF signals to ERK5. In vitro binding assays revealed that the proper calcium concentration is required for a direct binding of MEKK2 to Lad1. The binding of these proteins is not affected by c-Src-mediated phosphorylation on Lad1, but slightly affects the Tyr phosphorylation of MEKK2, suggesting that the interaction with Lad1 is necessary for full Tyr phosphorylation of MEKK2. In addition, we found that changes in calcium levels affect the EGF-induced nuclear translocation of MEKK2 and thereby its effect on the nuclear ERK5 activity. Taken together, these findings suggest that calcium is required for EGF-induced ERK5 activation, and this effect is probably mediated by securing proper interaction of MEKK2 with the upstream adaptor protein Lad1.

Discovery and Development of Endocannabinoid-Hydrolyzing Enzyme Inhibitors

Fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGL) are hydrolytic enzymes which degrade the endogenous cannabinoids (endocannabinoids) N-arachidonoylethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG), respectively. Endocannabinoids are an important class of lipid messenger molecules that are produced on demand in response to elevated intracellular calcium levels. They recognize and activate the cannabinoid CB(1) and CB(2) receptors, the molecular targets for Delta(9)-tetrahydrocannabinol (Delta(9)-THC) in marijuana evoking several beneficial therapeutic effects. However, in vivo the cannabimimetic effects of AEA and 2-AG remain weak owing to their rapid inactivation by FAAH and MGL, respectively. The inactivation of FAAH and MGL by specific enzyme inhibitors increases the levels of AEA and 2-AG, respectively, producing therapeutic effects such as pain relief and depression of anxiety.

Radiation Induced Bystander effects in Mice given Low Doses of Radiation in vivo

The 'bystander effect' phenomenon has challenged the traditional framework for assessing radiation damage by showing radiation induced changes in cells which have not been directly targeted, but are neighbors to or receive medium from directly hit cells. Our group performed a range of single and serial low dose irradiations on two genetically distinct strains of mice. Bladder explants established from these mice were incubated in culture medium, which was used to measure death responses in a keratinocyte reporter system. The study revealed that the medium harvested from bladder tissues' (ITCM) from acutely irradiated C57BL6 but not Balb/c mice, was able to induce clonogenic death. Administration of a priming dose(s) before a challenge dose to both C57BL6 and Balb/c mice stimulated reporter cell survival irrespective of the time interval between dose(s) delivery. When ITCM corresponding to both strains of mice was measured for its calcium mobilization inducing ability, results showed an elevation in intracellular calcium levels that was strain dependent. This indicates that genotype determined the type of bystander signal/response that was produced after exposure to low and acute doses of radiation. However, serial exposure conditions modified bystander signal production to induce similar effects that were characterized by excessive growth.

Peptide gomesin triggers cell death through L-type channel calcium influx, MAPK/ERK, PKC and PI3K signaling and generation of reactive oxygen species

Gomesin is an antimicrobial peptide isolated from hemocytes of a common Brazilian tarantula spider named Acanthoscurria gomesiana. This peptide exerts antitumor activity in vitro and in vivo by an unknown mechanism. In this study, the cytotoxic mechanism of gomesin in human neuroblastoma SH-SY5Y and rat pheochromocytoma PC12 cells was investigated. Gomesin induced necrotic cell death and was cytotoxic to SH-SY5Y and PC12 cells. The peptide evoked a rapid and transient elevation of intracellular calcium levels in Fluo-4-AM loaded PC12 cells, which was inhibited by nimodipine, an L-type calcium channel blocker. Preincubation with nimodipine also inhibited cell death induced by gomesin in SH-SY5Y and PC12 cells. Gomesin-induced cell death was prevented by the pretreatment with MAPK/ERK, PKC or PI3K inhibitors, but not with PKA inhibitor. In addition, gomesin generated reactive oxygen species (ROS) in SH-SY5Y cells, which were blocked with nimodipine and MAPK/ERK, PKC or PI3K inhibitors. Taken together, these results suggest that gomesin could be a useful anticancer agent, which mechanism of cytotoxicity implicates calcium entry through L-type calcium channels, activation of MAPK/ERK, PKC and PI3K signaling as well as the generation of reactive oxygen species.

Zinc Depletion‐induced Apoptosis in Human Breast Cancer MDA‐MB‐231 Cells Is Calcium‐dependent

We have previously observed an association between zinc depletion‐induced apoptosis and an elevated expression of eNOS in MDA‐MB‐231 cells. Since eNOS is sensitive to changes in intracellular [Ca2+]i, we hypothesized that zinc depletion‐induced apoptosis is calcium‐dependent. To test this hypothesis, MDA‐MB‐231 cells were cultured in DMEM supplemented with FBS (10%) for 3 days. Zinc depletion was achieved by incubating the cells with TPEN (0 or 20 μM) for 0–6 h. Zinc depletion resulted in a sustained elevation in intracellular calcium level after 3 h and a 20‐fold increase in caspase‐3 activity at 6 h (p &lt; 0.05). Chelating intracellular Ca2+ with BAPTA‐AM (10–50 μM) reduced caspase‐3 activity by 82%–96% (p &lt; 0.05). Further, zinc depletion also resulted in a 2‐fold increase in mitochondrial superoxide level (p &lt; 0.05). Blocking mitochondria calcium transport with Ruthenium red (25 μM) reduced caspase‐3 activity by 50% (p &lt; 0.05). Inhibiting intracellular Ca2+ channel with 2‐APB (50–150 μM) and U‐73122 (2–20 μM) reduced caspase‐3 activity by 30–50% and 20–70%, respectively (p &lt; 0.05). In summary, these results showed that zinc depletion altered cellular calcium homeostasis resulting in an elevated intracellular calcium level required for apoptosis in MDA‐MB‐231 cells. (Supported by NSERC).

The Functional Role of Calcineurin in Hypertrophy, Regeneration, and Disorders of Skeletal Muscle

Skeletal muscle uses calcium as a second messenger to respond and adapt to environmental stimuli. Elevations in intracellular calcium levels activate calcineurin, a serine/threonine phosphatase, resulting in the expression of a set of genes involved in the maintenance, growth, and remodeling of skeletal muscle. In this review, we discuss the effects of calcineurin activity on hypertrophy, regeneration, and disorders of skeletal muscle. Calcineurin is a potent regulator of muscle remodeling, enhancing the differentiation through upregulation of myogenin or MEF2A and downregulation of the Id1 family and myostatin. Foxo may also be a downstream candidate for a calcineurin signaling molecule during muscle regeneration. The strategy of controlling the amount of calcineurin may be effective for the treatment of muscular disorders such as DMD, UCMD, and LGMD. Activation of calcineurin produces muscular hypertrophy of the slow-twitch soleus muscle but not fast-twitch muscles.

Autism Spectrum Disorder is related to endoplasmic reticulum stress induced by mutations in the synaptic cell adhesion molecule, CADM1

Astaxanthin (AST) is a carotenoid that has been shown to have neuroprotective effects. In this study, it was found that AST significantly inhibited glutamate-induced loss of cell viability and apoptosis. AST pretreatment attenuated glutamate-induced activation of caspase-3, reduction of anti-apoptotic protein Bcl-2, and increase of pro-apoptotic protein Bak. In addition, AST pretreatment suppressed the production of intracellular reactive oxygen species. AST treatment also prevented glutamate-induced increase of the level of activated p38 mitogen-activated protein kinase (MAPK), which has been shown to promote apoptotic events. Furthermore, AST treatment greatly reduced the elevation of intracellular calcium level induced by glutamate and inhibited the activity of calpain, a calcium-dependent protease that plays an important role in mediating apoptosis stimulated by calcium overload in cytoplasm. Both oxidative stress and calcium overload can lead to endoplasmic reticulum (ER) stress. C/EBP-homologous protein (CHOP) is a bZIP transcription factor that can be activated by ER stress and promotes apoptosis. Here we found that AST attenuated glutamate-induced elevation of CHOP and ER chaperone glucose-regulated protein (GRP78). Overall, these results suggested that AST might protect cells against glutamate-induced apoptosis through maintaining redox balance and inhibiting glutamate-induced calcium influx and ER stress.

Neuregulin β1 enhances peak glutamate-induced intracellular calcium levels through endoplasmic reticulum calcium release in cultured hippocampal neuronsThis article is one of a selection of papers published in a special issue celebrating the 125th anniversary of the Faculty of Medicine at the University of Manitoba.

Modulation of intracellular free calcium levels is the primary second messenger system of the neuronal glutamatergic system, playing a role in regulation of all major cellular processes. The protein neuregulin (NRG) beta1 acts as an extracellular signaling ligand in neurons, rapidly regulating currents through ionotropic glutamate receptors. The effect NRG may have on glutamate-induced changes in intracellular free calcium concentrations has not been examined, however. In this study, cultured embryonic rat hippocampal neurons were treated with NRGbeta1 to determine a possible effect on glutamate-induced intracellular calcium levels. Long-term (24 h), but not short-term (1 h), incubation with NRGbeta1 resulted in a significantly greater glutamate-mediated acute peak elevation of intracellular calcium levels than occurred in vehicle-treated neurons. Long-term NRGbeta1 incubation significantly enhanced calcium increase induced by specific stimulation of metabotropic glutamate receptors, but did not significantly alter the N-methyl D-aspartate (NMDA)- or KCl-induced calcium increase and paradoxically decreased the effect of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) treatment on intracellular calcium. Metabotropic glutamate receptors cause increased intracellular free calcium via release of calcium from intracellular stores; thus this system was examined in more detail. NRGbeta1 treatment significantly (greater than 2-fold) enhanced calcium release from endoplasmic reticulum stores after stimulation of ryanodine receptors with caffeine, but did not significantly increase calcium release from endoplasmic reticulum mediated by inositol trisphosphate (IP3) receptors. In addition, ryanodine receptor inhibition with ruthenium red prevented the glutamate-induced increase in intracellular calcium levels in NRGbeta1-treated neurons. These data show that long-term NRGbeta1 treatment can enhance glutamate-induced peak intracellular calcium levels through metabotropic glutamate receptor activation by increasing endoplasmic reticulum calcium release through ryanodine receptors.

Delphinidin Ameliorates Beta-Amyloid-Induced Neurotoxicity by Inhibiting Calcium Influx and Tau Hyperphosphorylation

Beta-amyloid (Abeta) has been suggested to induce neurotoxicity in Alzheimer's disease. We evaluated the neuroprotective effects of delphinidin, an anthocyanidin commonly present in pigmented fruits and vegetables, against Abeta-induced toxicity. Abeta (25-35) significantly decreased the viability of PC12 cells, and this was accompanied by an increase in intracellular calcium levels and tau phosphorylation. However, treatment with delphinidin rescued PC12 cells from Abeta by attenuating the elevation of intracellular calcium levels and tau phosphorylation. Taken together, these results suggest that delphinidin protects PC12 cells against Abeta-induced toxicity by attenuating intracellular calcium influx and tau hyperphosphorylation.

Differentiation dependent expression of TRPA1 and TRPM8 channels in IMR‐32 human neuroblastoma cells

TRPA1 and TRPM8 are transient receptor potential (TRP) channels involved in sensory perception. TRPA1 is a non-selective calcium permeable channel activated by irritants and proalgesic agents. TRPM8 reacts to chemical cooling agents such as menthol. The human neuroblastoma cell line IMR-32 undergoes a remarkable differentiation in response to treatment with 5-bromo-2-deoxyuridine. The cells acquire a neuronal morphology with increased expression of N-type voltage gated calcium channels and neurotransmitters. Here we show using RT-PCR, that mRNA for TRPA1 and TRPM8 are strongly upregulated in differentiating IMR-32 cells. Using whole cell patch clamp recordings, we demonstrate that activators of these channels, wasabi, allyl-isothiocyanate (AITC) and menthol activate membrane currents in differentiated cells. Calcium imaging experiments demonstrated that AITC mediated elevation of intracellular calcium levels were attenuated by ruthenium red, spermine, and HC-030031 as well as by siRNA directed against the channel. This indicates that the detected mRNA level correlate with the presence of functional channels of both types in the membrane of differentiated cells. Although the differentiated IMR-32 cells responded to cooling many of the cells showing this response did not respond to TRPA1/TRPM8 channel activators (60% and 90% for AITC and menthol respectively). Conversely many of the cells responding to these activators did not respond to cooling (30%). This suggests that these channels have also other functions than cold perception in these cells. Furthermore, our results suggest that IMR-32 cells have sensory characteristics and can be used to study native TRPA1 and TRPM8 channel function as well as developmental expression.

Regulation of Intestinal Electroneutral Sodium Absorption and the Brush Border Na+/H+ Exchanger by Intracellular Calcium

The intestinal electroneutral Na(+) absorptive processes account for most small intestinal Na(+) absorption in the period between meals and also for the great majority of the increase in ileal Na(+) absorption that occurs postprandially. In most diarrheal diseases, there is inhibition of neutral NaCl absorption. Elevated levels of intracellular calcium ([Ca(2+)](i)) are known to inhibit NaCl absorption and involve multiple components of the Ca(2+) signaling pathway. The BB Na(+)/H(+) exchanger NHE3 accounts for most of the recognized digestive changes in neutral NaCl absorption, as well as most of the changes in Na(+) absorption that occur in diarrheal diseases. Previous studies have examined several aspects of Ca(2+) regulation of NHE3 activity. These include phosphorylation, protein trafficking, and multiprotein complex formation. In addition, recent studies have demonstrated the role of the NHERF family of PDZ domain-containing proteins in Ca(2+) regulation of NHE3 activity, thereby adding a new level of complexity to understanding Ca(2+)-dependent inhibition of Na(+) absorption. In this article, we will review the current understanding of (1) Ca(2+) signaling events in intestinal epithelial cells; (2) Ca(2+) regulation of intestinal electroneutral sodium absorption, which includes NHE3; and (3) the role of the NHERF family of PDZ domain-containing proteins in Ca(2+) regulation of NHE3 activity. We will also present new data on using advanced imaging showing rapid BB NHE3 endocytosis in response to elevated [Ca(2+)](i).