Fura Red Research Articles

Effects of disodium cromoglycate on cationic exchange of deoxygenated sickle cells

In the present work, we explored the way in which cromoglycate, a drug used to treat allergies acts on ion movements in sickle cells. Cells were either slowly deoxygenated by overnight exposure to nitrogen or acutely deoxygenated by exposure to metabisulfite, a strong reducing agent which induces sickling of red blood sickle cells. Flushing the cells with nitrogen increased the intracellular concentration of Na + and decreased the intracellular concentration of K + and the sum of the concentrations of the two cations. One hundred nM cromoglycate inhibited the decrease of intracellular K + and the increase of intracellular Na + induced by deoxygenation (n = 17). Metabisulfite (100 mM) increased the intracellular concentration of Ca 2+ (measured by Fura Red) (n = 15) and the shape of the cells (measured by light scattering) (n = 9). One μM cromoglycate partially inhibited these two responses. In conclusion, cromoglycate partially inhibits abnormal K + loss, Ca 2+ entry pathways or Ca 2+ channels opened by cell deoxygenation and ensuing membrane modifications and prevents cell sickling.

Kisspeptin-Evoked Calcium Signals in Isolated Primary Rat Gonadotropin- Releasing Hormone Neurones

Background: Kisspeptin and its cognate receptor GPR54 are the central driving forces in the hypothalamus-pituitary-gonadal axis essential for sexual maturation and reproduction. Kisspeptin/GPR54 signalling stimulates gonadotropin-releasing hormone (GnRH) neurones and induces pulsatile GnRH release. The molecular signalling pathway by which kisspeptin stimulates GnRH neurones is currently under investigation. Methods: Primary GnRH neurones were isolated from young adult rats and loaded with the calcium indicator Fura Red. Cytosolic calcium was measured while the cells were stimulated with kisspeptin. Results: GnRH neurones show a maintained increase of cytosolic calcium upon stimulation with 100 nM kisspeptin-10. The calcium elevation was inhibited 30% by 1 µM tetrodotoxin, a voltage-gated sodium channel blocker, and 76% by 30 µM SKF96365, an inhibitor of receptor-mediated calcium entry. Furthermore, removal of extracellular calcium completely abolished the kisspeptin-induced calcium elevation. Conclusion: Our results suggest that the major part of the kisspeptin-evoked calcium signal is generated by an action potential-independent calcium influx, possibly through channels of the classical transient receptor potential type, with an additional influx through voltage-gated calcium channels activated by sodium action potentials.

CAMP Mediators of Pulsatile Insulin Secretion from Glucose-stimulated Single β-Cells

Pulsatile insulin release from glucose-stimulated beta-cells is driven by oscillations of the Ca(2+) and cAMP concentrations in the subplasma membrane space ([Ca(2+)](pm) and [cAMP](pm)). To clarify mechanisms by which cAMP regulates insulin secretion, we performed parallel evanescent wave fluorescence imaging of [cAMP](pm), [Ca(2+)](pm), and phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) in the plasma membrane. This lipid is formed by autocrine insulin receptor activation and was used to monitor insulin release kinetics from single MIN6 beta-cells. Elevation of the glucose concentration from 3 to 11 mm induced, after a 2.7-min delay, coordinated oscillations of [Ca(2+)](pm), [cAMP](pm), and PIP(3). Inhibitors of protein kinase A (PKA) markedly diminished the PIP(3) response when applied before glucose stimulation, but did not affect already manifested PIP(3) oscillations. The reduced PIP(3) response could be attributed to accelerated depolarization causing early rise of [Ca(2+)](pm) that preceded the elevation of [cAMP](pm). However, the amplitude of the PIP(3) response after PKA inhibition was restored by a specific agonist to the cAMP-dependent guanine nucleotide exchange factor Epac. Suppression of cAMP formation with adenylyl cyclase inhibitors reduced already established PIP(3) oscillations in glucose-stimulated cells, and this effect was almost completely counteracted by the Epac agonist. In cells treated with small interfering RNA targeting Epac2, the amplitudes of the glucose-induced PIP(3) oscillations were reduced, and the Epac agonist was without effect. The data indicate that temporal coordination of the triggering [Ca(2+)](pm) and amplifying [cAMP](pm) signals is important for glucose-induced pulsatile insulin release. Although both PKA and Epac2 partake in initiating insulin secretion, the cAMP dependence of established pulsatility is mediated by Epac2.

Exposure to cardiomyogenic stimuli fails to transdifferentiate human umbilical cord blood-derived mesenchymal stem cells

The ability of human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) to transdifferentiate towards cardiomyocytes remains unclear. The aim of this study was to direct UCBMSCs to the cardiac lineage by exposure to: (1) 5-azacytidine (AZ) or dimethyl sulfoxide (DMSO); (2) a combination of growth factors involved in early cardiomyogenesis (BMP-2 + bFGF + IGF-1); (3) the Wnt signaling activators lithium chloride (LiCl) and phorbol-12-myristate-13-acetate (PMA); and (4) direct contact with neonatal rat cardiomyocytes. Expression of cardiomyocyte-specific proteins and beta-catenin were assessed by quantitative RT-PCR, immunofluorescence and Western blot. Cocultures of human UCBMSCs with neonatal rat cardiomyocytes were also analyzed for the presence of calcium oscillations and changes in electrical potential using Fura Red and di-4-ANEPPS confocal imaging, respectively. Induction of cardiac-specific proteins was not detected in 5-AZ- or DMSO-treated cells. Following DMSO addition, beta-catenin cytoplasmic expression increased, but did not translocate into cell nuclei to promote cardiac gene activation. Likewise, neither co-stimulation with BMP-2 + bFGF + IGF-1, nor exposure to LiCl and PMA resulted in the acquisition of a cardiac phenotype by UCBMSCs. Direct contact with neonatal rat cardiomyocytes promoted neither the expression of cardiomyocyte-specific proteins, nor the presence of calcium rhythmic oscillations and potential-dependent fluorescence emission in UCBMSCs. The cardiomyogenic stimuli investigated in this study failed to transdifferentiate human UCBMSCs. Alternative strategies or regulatory factors and signaling pathways may be better suited to recruit UCBMSCs into cardiac cell lineage.

Connexin 30 Deficiency Impairs Renal Tubular ATP Release and Pressure Natriuresis

In the renal tubule, ATP is an important regulator of salt and water reabsorption, but the mechanism of ATP release is unknown. Several connexin (Cx) isoforms form mechanosensitive, ATP-permeable hemichannels. We localized Cx30 to the nonjunctional apical membrane of cells in the distal nephron and tested whether Cx30 participates in physiologically important release of ATP. We dissected, partially split open, and microperfused cortical collecting ducts from wild-type and Cx30-deficient mice in vitro. We used PC12 cells as ATP biosensors by loading them with Fluo-4/Fura Red to measure cytosolic calcium and positioning them in direct contact with the apical surface of either intercalated or principal cells. ATP biosensor responses, triggered by increased tubular flow or by bath hypotonicity, were approximately three-fold greater when positioned next to intercalated cells than next to principal cells. In addition, these responses did not occur in preparations from Cx30-deficient mice or with purinergic receptor blockade. After inducing step increases in mean arterial pressure by ligating the distal aorta followed by the mesenteric and celiac arteries, urine output increased 4.2-fold in wild-type mice compared with 2.6-fold in Cx30-deficient mice, and urinary Na(+) excretion increased 5.2-fold in wild-type mice compared with 2.8-fold in Cx30-deficient mice. Furthermore, Cx30-deficient mice developed endothelial sodium channel-dependent, salt-sensitive elevations in mean arterial pressure. Taken together, we suggest that mechanosensitive Cx30 hemichannels have an integral role in pressure natriuresis by releasing ATP into the tubular fluid, which inhibits salt and water reabsorption.

Pattern of rise in subplasma membrane Ca2+ concentration determines type of fusing insulin granules in pancreatic β cells

We simultaneously analyzed insulin granule fusion with insulin fused to green fluorescent protein and the subplasma membrane Ca2+ concentration ([Ca2+]PM) with the Ca2+ indicator Fura Red in rat β cells by dual-color total internal reflection fluorescence microscopy. We found that rapid and marked elevation in [Ca2+]PM caused insulin granule fusion mostly from previously docked granules during the high KCl-evoked release and high glucose-evoked first phase release. In contrast, the slow and sustained elevation in [Ca2+]PM induced fusion from newcomers translocated from the internal pool during the low KCl-evoked release and glucose-evoked second phase release. These data suggest that the pattern of the [Ca2+]PM rise directly determines the types of fusing granules.

Thromboxane A2 and ADP play synergistic roles in platelet activation by von Willebrand factor

High shear stress in a stenotic artery triggers interaction of von Willebrand factor (VWF) with its platelet receptor, glycoprotein Ib‐IX‐V (GpIb‐IX‐V). This leads to platelet activation by a poorly defined mechanism. We have examined the signaling mechanism on ristocetin‐mediated binding of VWF to human platelets. Intracellular calcium was monitored with Fura‐PE3 or Oregon Green 488 BAPTA‐1 and Fura Red. Platelet aggregation was assayed simultaneously from light transmittance, and activation through secretion of serotonin. Treatment of platelets with the cyclo‐oxygenase inhibitor aspirin or the thromboxane A2 receptor antagonist SQ 29,548 abolished the VWF‐induced calcium transient and platelet activation. Selective antagonists of each purinoceptor subtype were also examined. Antagonism of P2Y12 receptors with MRS 2395 suppressed both the calcium transient and platelet activation by VWF. In contrast, antagonism of the P2Y1 or P2X1 receptors had minimal effect. Blockade of either the thromboxane or ADP pathway eliminated the platelet responses, implying that they contribute synergistically to VWF signaling. This intrinsically weak mechanism and strict requirement for both secondary agonists may preclude inadvertent platelet activation when a little VWF binds to GpIb‐IX‐V during passage through a region of modestly elevated shear stress. [Supported by NSF and AHA (Greater Southeast).]

Coriaria Lactone Increased the Intracellular Level of Calcium through the Voltage-gated Calcium Channels in Rat Hippocampal Neurons

To investigate the effects of Coriaria Lactone (CL), an epileptogenic substance, on intracellular levels of calcium ([Ca(2+)](i)) and physiological properties of voltage-gated calcium channels (VGCCs). Ratiometric calcium imaging using Fura Red and whole-cell voltage patch-clamp technique were explored on freshly isolated rat hippocampal neurons exposed to CL. Coriaria Lactone increased [Ca(2+)](i) from 118 +/- 21 to 440 +/- 35 nM; VGCCs and calcium influx through NMDA receptor served as the main routes of entry. Coriaria Lactone could enhance both Low voltage activated (LVA) and High voltage activated calcium currents in a concentration-dependent way, and its effect on LVA current was more potent (about 60%). The increased calcium currents were accompanied by the shift of voltage-dependent steady-state inactivation to more positive potentials. These effects of CL, especially its impact on LVA current, could activate different calcium-dependent signaling pathways, and influence cellular excitable properties as well, which might play an important role in CL's epileptogenic process.

Flow Cytometric Quantification of Glucose-Stimulated β-Cell Metabolic Flux Can Reveal Impaired Islet Functional Potency

The objective of this study was to develop a multiparametric flow cytometry assay to simultaneously quantify isolated pancreatic islet cell viability, apoptosis, and glucose-induced metabolic flux. INS-1 and rat islet beta-cells were stained with fluorescent probes for cell viability (ToPro3), apoptosis (Annexin V and VADFMK), and intracellular calcium (Ca2+(i)) (Fura Red), stimulated with glucose, and analyzed on a FACS Vantage flow cytometer. Glucose-induced metabolic activity was indicated by changes in Fura Red fluorescence and the autofluorescence of the pyridine [NAD(P)H] and flavin (FAD/FMN) nucleotides. Rat islets cultured under conditions of proinflammatory cytokine-induced oxidative stress were evaluated by flow cytometry and transplantation into diabetic mice. INS-1 and rat islet beta-cell health and metabolic activity were quantified in response to elevated glucose dose and inhibitors of glycolysis and mitochondrial function. Changes in metabolite fluorescence were converted to an area under the curve (AUC) value. Rat islets cultured under oxidative stress conditions showed decreased viability, increased apoptosis, and decreased glucose-induced metabolic activity indicated by reduced AUC for pyridine and flavin nucleotides and Ca2+(i). Reduced metabolite AUC measured by flow cytometry correlated with the inability to reverse diabetes in mice. Single cell flow cytometry can simultaneously quantify both overall islet cell health and beta-cell glucose responsiveness as indicators of functional potency.

Differential Dephosphorylation of the FcRγ Immunoreceptor Tyrosine-based Activation Motif Tyrosines with Dissimilar Potential for Activating Syk

The cell surface-expressed gamma chain of the high affinity receptor for IgE (FcepsilonRI) can be phosphorylated on two tyrosine residues of the immunoreceptor tyrosine-based activation motif (ITAM), leading to recruitment and activation of spleen tyrosine kinase (Syk), a kinase that is essential for mast cell signaling and allergic responses. However, it is not known whether preferential phosphorylation or dephosphorylation of the two individual FcRgamma tyrosines (the N-terminal Tyr47 and C-terminal Tyr58) could regulate Syk activation. Herein we report that phosphorylation of only Tyr58 was able to elicit Syk phosphorylation and a weak rise in intracellular calcium, suggesting that Tyr58 phosphorylation may be distinctively important for Syk activation. In vitro and in vivo studies revealed that both Tyr47 and Tyr58 could be similarly phosphorylated. However, mass spectrometric analysis of the phosphorylated FcepsilonRgamma from bone marrow-derived mast cells showed that phosphorylation at Tyr47 was at least 2-fold greater than at Tyr58. This suggested that, once phosphorylated, Tyr58 is preferentially dephosphorylated. In vitro studies demonstrated more efficient dephosphorylation of Tyr58 (by the receptor-associated phosphatases SHP-1 and SHP-2) than of Tyr47. Analysis of Syk binding to wild type and mutant phosphorylated FcepsilonRI revealed that mutation at Tyr58 almost completely ablated Syk binding, whereas mutation at Tyr47 moderately reduced Syk binding. The findings argue for a novel regulatory mechanism, where dephosphorylation of phospho-Tyr58 is likely to promote the down-regulation of Syk activation and suppression of mast cell responses.

Rapid downregulation of the Ca 2+-signal after exocytosis stimulation in Paramecium cells: Essential role of a centrin-rich filamentous cortical network, the infraciliary lattice

We analysed in Paramecium tetraurelia cells the role of the infraciliary lattice, a cytoskeletal network containing numerous centrin isoforms tightly bound to large binding proteins, in the re-establishment of Ca2+ homeostasis following exocytosis stimulation. The wild type strain d4-2 has been compared with the mutant cell line Delta-PtCenBP1 which is devoid of the infraciliary lattice ("Delta-PtCenBP1" cells). Exocytosis is known to involve the mobilization of cortical Ca2+-stores and a superimposed Ca2+-influx and was analysed using Fura Red ratio imaging. No difference in the initial signal generation was found between wild type and Delta-PtCenBP1 cells. In contrast, decay time was greatly increased in Delta-PtCenBP1 cells particularly when stimulated, e.g., in presence of 1mM extracellular Ca2+, [Ca2+]o. Apparent halftimes of f/f0 decrease were 8.5 s in wild type and approximately 125 s in Delta-PtCenBP1 cells, requiring approximately 30 s and approximately 180 s, respectively, to re-establish intracellular [Ca2+] homeostasis. Lowering [Ca2+]o to 0.1 and 0.01 mM caused an acceleration of intracellular [Ca2+] decay to t(1/2)=33 s and 28 s, respectively, in Delta-PtCenBP1 cells as compared to 8.1 and 5.6, respectively, for wild type cells. We conclude that, in Paramecium cells, the infraciliary lattice is the most efficient endogenous Ca2+ buffering system allowing the rapid downregulation of Ca2+ signals after exocytosis stimulation.

Activation of Cytosolic Phospholipase A2α in Resident Peritoneal Macrophages by Listeria monocytogenes Involves Listeriolysin O and TLR2

Eicosanoid production by macrophages is an early response to microbial infection that promotes acute inflammation. The intracellular pathogen Listeria monocytogenes stimulates arachidonic acid release and eicosanoid production from resident mouse peritoneal macrophages through activation of group IVA cytosolic phospholipase A2 (cPLA2alpha). The ability of wild type L. monocytogenes (WTLM) to stimulate arachidonic acid release is partially dependent on the virulence factor listeriolysin O; however, WTLM and L. monocytogenes lacking listeriolysin O (DeltahlyLM) induce similar levels of cyclooxygenase 2. Arachidonic acid release requires activation of MAPKs by WTLM and DeltahlyLM. The attenuated release of arachidonic acid that is observed in TLR2-/- and MyD88-/- macrophages infected with WTLM and DeltahlyLM correlates with diminished MAPK activation. WTLM but not DeltahlyLM increases intracellular calcium, which is implicated in regulation of cPLA2alpha. Prostaglandin E2, prostaglandin I2, and leukotriene C4 are produced by cPLA2alpha+/+ but not cPLA2alpha-/- macrophages in response to WTLM and DeltahlyLM. Tumor necrosis factor (TNF)-alpha production is significantly lower in cPLA2alpha+/+ than in cPLA2alpha-/- macrophages infected with WTLM and DeltahlyLM. Treatment of infected cPLA2alpha+/+ macrophages with the cyclooxygenase inhibitor indomethacin increases TNFalpha production to the level produced by cPLA2alpha-/- macrophages implicating prostaglandins in TNFalpha down-regulation. Therefore activation of cPLA2alpha in macrophages may impact immune responses to L. monocytogenes.

Aldosterone Enhances Ligand-Stimulated Nitric Oxide Production in Endothelial Cells

Chronic and acute actions of aldosterone have been shown recently to directly affect the cardiovascular system. However, it is unclear whether the acute effects of aldosterone on vasculature are constrictive or dilatory. Here, to clarify the nongenomic effects of aldosterone on endothelial function, we examined the effects of aldosterone on nitric oxide (NO) production in cultured endothelial cells (ECs) and on vascular tone. The intracellular NO production of bovine aortic ECs loaded with DAF-2 was determined using confocal microscopy. Accumulated NO in the culture medium was quantified by a microplate reader using membrane-impermeable DAF-2. Phosphorylation of endothelial NO synthase (eNOS) at Ser(1179) was assessed by Western blotting. Changes in intracellular Ca(2+) ([Ca(2+)](i)) were determined by confocal microscopy in ECs doubly loaded with fluo-4 and Fura Red. The effects of aldosterone, acetylcholine (ACh), and other signaling molecules on the tension of phenylephrine (PE)-contracted aortas of Sprague-Dawley rats were examined in an ex vivo organ bath chamber system. Short-term pre-exposure to aldosterone (1 x 10(-7) mol/L) enhanced ATP-induced NO production in ECs with increased phosphorylation of eNOS at Ser(1179). These effects were blocked by eplerenone, a mineralocorticoid receptor (MR) antagonist, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Notably, aldosterone alone did not affect ATP-induced [Ca(2+)](i) changes or the Ser(1179) phosphorylation. Similarly, aldosterone (1 x 10(-8) to 1 x 10(-7) mol/L) did not affect the tone of rat aortas pre-contracted by PE, but enhanced ACh-induced vasorelaxation, which was again reversed by eplerenone or LY29400. In contrast, sodium nitroprusside-induced vasorelaxation in endothelium-denuded aortas was not affected by aldosterone. Thus, aldosterone acutely enhances ligand-mediated endothelial NO production by eplerenone-sensitive mechanisms involving a PI3K that may synergize Ca(2+)-dependent eNOS phosphorylation at Ser(1179).

Ratiometric calcium concentration estimation using LED excitation during mechanotransduction in single sensory neurons

In a previous study using Oregon Green BAPTA-1 fluorescence we found that intracellular calcium concentration in spider mechanoreceptor neurons rose during mechanical stimulation. We also showed that calcium elevation required the opening of voltage-dependent calcium channels by action potentials, and could not be produced by the receptor potential alone. While evidence for mechanisms of calcium elevation in these neurons was clear, our estimates of actual calcium concentration depended on properties of the fluorescent dye in the neuron cytoplasm that could not be verified. We have now developed a method for ratiometric estimation of calcium concentration in these neurons using Fura Red dye, excitation by two light emitting diodes (LEDs) of different wavelengths, and an avalanche photodiode fluorescence detector. The method is simple and economical to implement, allows concentration changes to be measured in the millisecond time range, and could easily be applied to a wide range of preparations. Resting calcium concentration in these neurons was about 70nM and rose to a maximum of about 400nM at firing rates above 20 action potentials per second.

Cytohesin Binder and Regulator Augments T Cell Receptor-induced Nuclear Factor of Activated T Cells·AP-1 Activation through Regulation of the JNK Pathway

Cytohesin binder and regulator (Cybr; also known as CYTIP, CASP, and PSCDBP) is a cytokine-induced gene preferentially expressed in hematopoietic tissues and in T helper 1 cells. Cybr protein associates with members of the cytohesin family, which are known ADP-ribosylation factors-GDP/GTP exchange factors, and its functions appear to regulate lymphocyte adhesion and cell-cell contact. Here we show that Cybr mRNA and protein levels are increased upon T cell receptor engagement. Cybr expression then influences T cell receptor-dependent signaling events, such as nuclear factor of activated T cells and AP-1 transcriptional activity. In addition, expression of Cybr results in increased T cell receptor-mediated activation of the Rho/Rac exchange factor Vav and of the JNK-p38 MAPK signaling pathway. The effects of Cybr on nuclear factor of activated T cells and AP-1 are dependent on MAPK activation, and enhanced activation of this cascade results in cooperation between the two transcription factors in the regulation of gene expression. These findings provide the first evidence that the adaptor protein Cybr not only regulates lymphocyte adhesion and cell-cell interaction but also contributes to the regulation of the signaling cascade and of the genetic program downstream of the T cell receptor.

Regulation of the Transient Receptor Potential Channel TRPM2 by the Ca2+ Sensor Calmodulin

TRPM2, a member of the transient receptor potential (TRP) superfamily, is a Ca(2+)-permeable channel activated by oxidative stress or tumor necrosis factoralpha involved in susceptibility to cell death. TRPM2 activation is dependent on the level of intracellular Ca(2+). We explored whether calmodulin (CaM) is the Ca(2+) sensor for TRPM2. HEK 293T cells were transfected with TRPM2 and wild type CaM or mutant CaM (CaM(MUT)) with substitutions of all four EF hands. Treatment of cells expressing TRPM2 with H(2)O(2) or tumor necrosis factor alpha resulted in a significant increase in intracellular calcium ([Ca(2+)](i)). This was not affected by coexpression of CaM, suggesting that endogenous CaM levels are sufficient for maximal response. Cotransfection of CaM(MUT) with TRPM2 dramatically inhibited the increase in [Ca(2+)](i), demonstrating the requirement for CaM in TRPM2 activation. Immunoprecipitation confirmed direct interaction of CaM and CaM(MUT) with TRPM2, and the Ca(2+) dependence of this association. CaM bound strongly to the TRPM2 N terminus (amino acids 1-730), but weakly to the C terminus (amino acids 1060-1503). CaM binding to an IQ-like motif (amino acids 406-416) in the TRPM2 N terminus was demonstrated utilizing gel shift, immunoprecipitation, biotinylated CaM overlay, and pull-down assays. A substitution mutant of the IQ-like motif of TRPM2 (TRPM2-IQ(MUT1)) reduced but did not eliminate CaM binding to TRPM2, suggesting the presence of at least one other CaM binding site. The functional importance of the TRPM2 IQ-like motif was demonstrated by treatment of TRPM2-IQ(MUT1)-expressing cells with H(2)O(2). The increase in [Ca(2+)](i) observed with wild type TRPM2 was absent and cell viability was preserved. These data demonstrate the requirement for CaM in TRPM2 activation. They suggest that Ca(2+) entering through TRPM2 enhances interaction of CaM with TRPM2 at the IQ-like motif in the N terminus, providing crucial positive feedback for channel activation.

A flow cytometric comparison of Indo-1 to fluo-3 and Fura Red excited with low power lasers for detecting Ca 2+ flux

Indo-1 and high-power water-cooled lasers have been the standard for flow cytometric based Ca 2+ flux measurements. With advances in technology and the availability of low-power air-cooled lasers, there is interest in alternative protocols. Here, we have compared Indo-1 with the combination of fluo-3 and Fura Red calcium indicator dyes using low-power air-cooled lasers as the excitation source. The reagents were examined in parallel to detect Ca 2+ flux in peripheral blood T lymphocytes and in a T lymphoblastoid cell line. Ca 2+ flux was detected with a FACSVantage SE equipped with an Omnichrome Series 74 Helium–Cadmium, or a Spectra Physics 177-G1202 Argon ion air-cooled laser. Following determination of optimal loading conditions, Ca 2+ flux was examined in response to membrane receptor stimulation or intracellular Ca 2+ mobilization. Dose dependent Ca 2+ flux to anti-CD3 and thapsigargin was detected with either Indo-1 or with fluo-3 and Fura Red. The profile of the Ca 2+ flux detected by Indo-1 or with fluo-3 and Fura Red appeared similar, with the combination of fluo-3 and Fura Red more sensitive under the particular test conditions. The results clearly demonstrated that Indo-1 could be usefully excited with a low-power air-cooled laser. The alternative use of fluo-3 and Fura Red does not require the availability of a UV capable laser and produced equivalent data.

Fatty Acid Ethyl Esters Cause Pancreatic Calcium Toxicity via Inositol Trisphosphate Receptors and Loss of ATP Synthesis

Fatty acid ethyl esters are ethanol metabolites inducing sustained, toxic elevations of the acinar cytosolic free calcium ion concentration ([Ca(2+)](C)) implicated in pancreatitis. We sought to define the mechanisms of this elevation. Isolated mouse pancreatic acinar cells were loaded with fluorescent dyes for confocal microscopy to measure [Ca(2+)](C) (Fluo 4, Fura Red), endoplasmic reticulum calcium ion concentration ([Ca(2+)](ER), Mg Fluo 4), mitochondrial membrane potential (TMRM), ADP:ATP ratio (Mg Green), and NADH autofluorescence in response to palmitoleic acid ethyl ester and palmitoleic acid (10-100 micromol/L). Whole-cell patch clamp was used to measure the calcium-activated chloride current and apply ethanol metabolites and/or ATP intracellularly. Intracellular delivery of ester induced oscillatory increases of [Ca(2+)](C) and calcium-activated currents, inhibited acutely by caffeine (20 mmol/L), but not atropine, indicating involvement of inositol trisphosphate receptor channels. The stronger effect of extracellular ester or acid caused depletion of [Ca(2+)](ER), not prevented by caffeine, but associated with depleted ATP, depleted NADH autofluorescence, and depolarized mitochondria, suggesting calcium-ATPase pump failure because of lack of ATP. Intracellular ATP abolished the sustained rise in [Ca(2+)](C), although oscillatory signals persisted that were prevented by caffeine. Inhibition of ester hydrolysis markedly reduced its calcium-releasing effect and consequent toxicity. Fatty acid ethyl ester increases [Ca(2+)](C) through inositol trisphosphate receptors and, following hydrolysis, through calcium-ATPase pump failure from impaired mitochondrial ATP production. Lowering cellular fatty acid substrate concentrations may reduce cell injury in pancreatitis.

FATTY ACID ETHYL ESTERS INDUCE PANCREATIC CA2+ TOXICITY THROUGH INOSITOL TRISPHOSPHATE RECEPTORS AND LOSS OF MITOCHONDRIAL ATP PRODUCTION

Background & Aims: Fatty acid ethyl esters are ethanol metabolites that induce sustained, toxic elevations of acinar cytosolic Ca2+ concentrations ([Ca2+]c) implicated in pancreatitis. We sought to determine the mechanisms of this elevation. Methods: Isolated mouse pancreatic acinar cells were loaded with fluorescent dyes for confocal microscopy to measure [Ca2+]c (Fluo4, Fura Red), endoplasmic reticulum Ca2+ concentrations ([Ca2+]ER, Mg Fluo4), mitochondrial membrane potential (TMRM) and NADH autofluorescence in response to palmitoleic acid ethyl ester (POAEE) and palmitoleic acid (POA) (10-100 μM). Whole-cell patch clamp was used to measure the Ca2+-activated Cl− current and apply ethanol metabolites and/or ATP intracellularly. Results: Intracellular delivery of ester induced oscillatory increases of [Ca2+]c and Ca2+-activated currents, inhibited acutely by caffeine (20 mM, n = 11), but not atropine (n = 4), indicating involvement of inositol trisphosphate receptor channels. The stronger effect of extracellular application of ester or acid induced depletion of [Ca2+]ER (n = 15-22), which was not prevented by caffeine (n = 10-11), but was associated with decreased NADH autofluorescence and depolarised mitochondria (n = 9-16), suggesting Ca2+-ATPase pump failure due to lack of ATP. Intracellular ATP abolished the sustained rise in [Ca2+]c (10 of 11 cells), although oscillatory signals persisted that were prevented by caffeine. Inhibition of ester hydrolysis by BNPP (200 μM) markedly reduced its Ca2+ releasing effect (6 of 8 cells) and consequent toxicity (assessed by propidium iodide staining, n = 6). Conclusions: Fatty acid ethyl ester increases [Ca2+]c through inositol trisphosphate receptors and, following hydrolysis, through Ca2+-ATPase pump failure from impaired mitochondrial ATP production. Lowering intracellular fatty acid substrate concentrations may reduce cell injury in pancreatitis.

Feedback activation of phospholipase C via intracellular mobilization and store-operated influx of Ca2+ in insulin-secreting β-cells

Phospholipase C (PLC) regulates various cellular processes by catalyzing the formation of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol from phosphatidylinositol-4,5-bisphosphate (PIP2). Here, we have investigated the influence of Ca2+ on receptor-triggered PLC activity in individual insulin-secreting beta-cells. Evanescent wave microscopy was used to record PLC activity using green fluorescent protein (GFP)-tagged PIP2/IP3-binding pleckstrin homology domain from PLCdelta1, and the cytoplasmic Ca2+ concentration ([Ca2+]i) was simultaneously measured using the indicator Fura Red. Stimulation of MIN6 beta-cells with the muscarinic-receptor agonist carbachol induced rapid and sustained PLC activation. By contrast, only transient activation was observed after stimulation in the absence of extracellular Ca2+ or in the presence of the non-selective Ca2+ channel inhibitor La3+. The Ca2+-dependent sustained phase of PLC activity did not require voltage-gated Ca2+ influx, as hyperpolarization with diazoxide or direct Ca2+ channel blockade with nifedipine had no effect. Instead, the sustained PLC activity was markedly suppressed by the store-operated channel inhibitors 2-APB and SKF96365. Depletion of intracellular Ca2+ stores with the sarco(endo)plasmic reticulum Ca2+-ATPase inhibitors thapsigargin or cyclopiazonic acid abolished Ca2+ mobilization in response to carbachol, and strongly suppressed the PLC activation in Ca2+-deficient medium. Analogous suppressions were observed after loading cells with the Ca2+ chelator BAPTA. Stimulation of primary mouse pancreatic beta-cells with glucagon elicited pronounced [Ca2+]i spikes, reflecting protein kinase A-mediated activation of Ca2+-induced Ca2+ release via IP3 receptors. These [Ca2+]i spikes were found to evoke rapid and transient activation of PLC. Our data indicate that receptor-triggered PLC activity is enhanced by positive feedback from Ca2+ entering the cytoplasm from intracellular stores and via store-operated channels in the plasma membrane. Such amplification of receptor signalling should be important in the regulation of insulin secretion by hormones and neurotransmitters.