benzofurans and fura-red

Most chemical and, with only a few exceptions, all genetically encoded fluorimetric calcium (Ca(2+)) indicators (GECIs) emit green fluorescence. Many of these probes are compatible with red-emitting cell- or organelle markers. But the bulk of available fluorescent-protein constructs and transgenic animals incorporate green or yellow fluorescent protein (GFP and YFP respectively). This is, in part, not only heritage from the tendency to aggregate of early-generation red-emitting FPs, and due to their complicated photochemistry, but also resulting from the compatibility of green-fluorescent probes with standard instrumentation readily available in most laboratories and core imaging facilities. Photochemical constraints like limited water solubility and low quantum yield have contributed to the relative paucity of red-emitting Ca(2+) probes compared to their green counterparts, too. The increasing use of GFP and GFP-based functional reporters, together with recent developments in optogenetics, photostimulation and super-resolution microscopies, has intensified the quest for red-emitting Ca(2+) probes. In response to this demand more red-emitting chemical and FP-based Ca(2+)-sensitive indicators have been developed since 2009 than in the thirty years before. In this topical review, we survey the physicochemical properties of these red-emitting Ca(2+) probes and discuss their utility for biological Ca(2+) imaging. Using the spectral separability index Xijk (Oheim M., 2010. Methods in Molecular Biology 591: 3-16) we evaluate their performance for multi-color excitation/emission experiments, involving the identification of morphological landmarks with GFP/YFP and detecting Ca(2+)-dependent fluorescence in the red spectral band. We also establish a catalog of criteria for evaluating Ca(2+) indicators that ideally should be made available for each probe. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

Topics: Animals; Bacterial Proteins; Benzofurans; Boron Compounds; Calcium; Fluorescent Dyes; Green Fluorescent Proteins; Humans; Imidazoles; Luminescent Proteins; Molecular Imaging; Optogenetics; Photochemistry; Red Fluorescent Protein; Rhodamines; Spectrometry, Fluorescence; Thermodynamics

Our understanding of the underlying mechanisms of Ca2+ signaling as well as our appreciation for its ubiquitous role in cellular processes has been rapidly advanced, in large part, due to the development of fluorescent Ca2+ indicators. In this chapter, we discuss some of the most common chemical Ca2+ indicators that are widely used for the investigation of intracellular Ca2+ signaling. Advantages, limitations and relevant procedures will be presented for each dye including their spectral qualities, dissociation constants, chemical forms, loading methods and equipment for optimal imaging. Chemical indicators now available allow for intracellular Ca2+ detection over a very large range (<50 nM to >50 microM). High affinity indicators can be used to quantify Ca2+ levels in the cytosol while lower affinity indicators can be optimized for measuring Ca2+ in subcellular compartments with higher concentrations. Indicators can be classified into either single wavelength or ratiometric dyes. Both classes require specific lasers, filters, and/or detection methods that are dependent upon their spectral properties and both classes have advantages and limitations. Single wavelength indicators are generally very bright and optimal for Ca2+ detection when more than one fluorophore is being imaged. Ratiometric indicators can be calibrated very precisely and they minimize the most common problems associated with chemical Ca2+ indicators including uneven dye loading, leakage, photobleaching, and changes in cell volume. Recent technical advances that permit in vivo Ca2+ measurements will also be discussed.

Topics: Anesthesia; Aniline Compounds; Animals; Astrocytes; Benzofurans; Calcium; Calcium Signaling; Cell Compartmentation; Cytosol; Fluoresceins; Fluorescent Dyes; Fura-2; Glycine; Heterocyclic Compounds, 3-Ring; Imidazoles; Indicators and Reagents; Indoles; Mice; Organic Chemicals; Parietal Lobe; Xanthenes

Ca2+ release from intracellular stores was examined with the use of a confocal microscope in single, voltage-clamped myocytes from rat portal vein loaded with both Fluo-3 and Fura-red. Spontaneous local increases in [Ca2+]i from the sarcoplasmic reticulum, termed Ca2+ sparks, were observed in about 30% of the quiescent cells tested. Ca2+ sparks could be evoked by low concentrations of caffeine (1 mM) or ryanodine (1 microM). Both spontaneous and caffeine-evoked Ca2+ sparks were insensitive to blockers of voltage-dependent Ca2+ channels. Caffeine (10 mM) triggered propagating Ca2+ waves of large amplitude which started from the same site than spontaneous Ca2+ sparks in 73% of the cells, as expected if Ca2+ sparks were the elementary events that could account for the initiation of Ca2+ waves. Spontaneous Ca2+ sparks activated both Ca(2+)-dependent K+ and non-selective cation currents, whereas Ca2+ waves were able to evoke Ca(2+)-dependent chloride current. These results suggest that both inward cation current and outward K+ current activated by Ca2+ sparks may exert a key role in controlling the basal activity of vascular myocytes.

Topics: Aniline Compounds; Animals; Benzofurans; Caffeine; Calcium; Calcium Channels; Evoked Potentials; Fluorescent Dyes; Heart; Imidazoles; Microscopy, Confocal; Muscle, Smooth, Vascular; Myocardium; Portal Vein; Rats; Rats, Wistar; Xanthenes

Topics: Aniline Compounds; Animals; Benzofurans; Calcium; Cations, Divalent; Flow Cytometry; Fluorescent Dyes; Humans; Imidazoles; Indoles; Xanthenes

Topics: Animals; Benzofurans; Embryo, Nonmammalian; Female; Fluorescent Dyes; Image Processing, Computer-Assisted; Imidazoles; Microscopy, Fluorescence; Mitosis; Sea Urchins

The search for new antimalarial drugs with unexplored mechanisms of action is currently one of the main objectives to combat the resistance already in the clinic. New drugs should target specific mechanisms that once initiated lead inevitably to the parasite's death and clearance and cause minimal toxicity to the host. One such new mode of action recently characterized is to target the parasite's calcium dynamics. Disruption of the calcium homeostasis is associated with compromised digestive vacuole membrane integrity and release of its contents, leading to programmed cell death-like features characterized by loss of mitochondrial membrane potential and DNA degradation. Intriguingly, chloroquine (CQ)-treated parasites were previously reported to exhibit such cellular features. Using a high-throughput phenotypic screen, we identified 158 physiological disruptors (hits) of parasite calcium distribution from a small subset of approximately 3000 compounds selected from the GSK TCAMS (Tres Cantos Anti-Malarial Set) compound library. These compounds were then extensively profiled for biological activity against various CQ- and artemisinin-resistant

Topics: Antimalarials; Benzofurans; Calcium; Cytosol; DNA; High-Throughput Screening Assays; Homeostasis; Imidazoles; Mitochondria; Plasmodium falciparum; Structure-Activity Relationship

The skin is exposed to various external stimuli. Keratinocytes, which are the main cell type in the epidermis, interact with peripheral sensory neurons and modulate neuronal activity. Recent studies have revealed that keratinocytes play crucial roles in nociception, and that ATP is one of the main mediators of signal transduction from keratinocytes to sensory neurons. However, no quantitative cellular level analyses of ATP-mediated information flow from keratinocytes to sensory dorsal root ganglion (DRG) neurons have been conducted. In this study, we performed simultaneous imaging of cell surface ATP and intracellular Ca

Topics: Adenosine Triphosphate; Animals; Benzofurans; Calcium; Cations, Divalent; Cell Membrane; Coculture Techniques; Epidermis; Ganglia, Spinal; Genes, Reporter; Humans; Imidazoles; Infant, Newborn; Keratinocytes; Mechanotransduction, Cellular; Molecular Probes; Nociception; Rats; Rats, Wistar; Sensory Receptor Cells; Time-Lapse Imaging

The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (1 nM to 25.6 μM) and a specified dye concentration (12 μM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye molecules [Formula: see text] is determined to -17.8 ml mol-1 for Fluo-4 and -21.3 ml mol-1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence.

Topics: Aniline Compounds; Benzofurans; Buffers; Calcium; Egtazic Acid; Fluorescent Dyes; Hydrogen-Ion Concentration; Hydrostatic Pressure; Imidazoles; Kinetics; Osmolar Concentration; Spectrometry, Fluorescence; Temperature; Xanthenes

Calcium flux is a rapid and sensitive measure of cell activation whose utility could be enhanced with better techniques for data extraction. We describe a technique to monitor calcium flux by flow cytometry, measuring Fura Red calcium dye by ratiometric analysis. This technique has several advantages: 1) using a single calcium dye provides an additional channel for surface marker characterization, 2) allows robust detection of calcium flux by minority cell populations within a heterogeneous population of primary T cells and monocytes 3) can measure total calcium flux and additionally, the proportion of responding cells, 4) can be applied to studying the effects of drug treatment, simultaneously stimulating and monitoring untreated and drug treated cells. Using chemokine receptor activation as an example, we highlight the utility of this assay, demonstrating that only cells expressing a specific chemokine receptor are activated by cognate chemokine ligand. Furthermore, we describe a technique for simultaneously stimulating and monitoring calcium flux in vehicle and drug treated cells, demonstrating the effects of the Gαi inhibitor, pertussis toxin (PTX), on chemokine stimulated calcium flux. The described real time calcium flux assay provides a robust platform for characterizing cell activation within primary cells, and offers a more accurate technique for studying the effect of drug treatment on receptor activation in a heterogeneous population of primary cells.

Topics: Antigens, Surface; Benzofurans; Calcium; Flow Cytometry; Fluorescent Dyes; Humans; Imidazoles; Intracellular Space; Leukocytes, Mononuclear; T-Lymphocytes

Cytosolic free calcium ions represent important second-messengers in platelets. Therefore, quantitative measurement of intraplatelet calcium provides a popular and very sensitive tool to evaluate platelet activation and reactivity. Current protocols for determination of intracellular calcium concentrations in platelets have a number of limitations. Cuvette-based methods do not allow measurement of calcium flux in complex systems, such as whole blood, and therefore require isolation steps that potentially interfere with platelet activation. Flow cytometry has the potential to overcome this limitation, but to date the application of calibrated, quantitative readout of calcium kinetics has only been described for Indo-1. As excitation of Indo-1 requires a laser in the ultraviolet range, such measurements cannot be performed with a standard flow cytometer. Here, we describe a novel, rapid calibration method for ratiometric calcium measurement in platelets using both Ar(+)-laser excited fluorescence dyes Fluo-4 and Fura Red. We provide appropriate equations that allow rapid quantification of intraplatelet calcium fluxes by measurement of only two standardisation buffers. We demonstrate that this method allows quantitative calcium measurement in platelet rich plasma as well as in whole blood. Further, we show that this method prevents artefacts due to platelet aggregate formation and is therefore an ideal tool to determine basal and agonist induced calcium kinetics.

Topics: Adenosine Diphosphate; Aniline Compounds; Benzofurans; Biological Transport; Blood Platelets; Calcium; Calibration; Flow Cytometry; Humans; Imidazoles; Linear Models; Platelet Aggregation; Time Factors; Xanthenes

It has been shown that vitamin C (VC) is transported at synaptic boutons, but how this occurs has not been elucidated. This study investigates the role of the sodium-dependent vitamin C transporter-2 (SVCT2) in transporting VC at the cortical nerve terminal. Immunostaining of cultured mouse superior cervical ganglion cells showed the SVCT2 to be expressed in presynaptic boutons, colocalizing with the vesicular monoamine transporter-2 and the norepinephrine transporter. Immunoblotting of enriched cortical synaptosomes demonstrated that the SVCT2 was enriched in presynaptic fractions, confirming a predominantly presynaptic location. In crude synaptosomes, known inhibitors of SVCT2 inhibited uptake of VC. Furthermore, the kinetic features of VC uptake were consistent with SVCT2-mediated function. VC was also found to efflux from synaptosomes by a mechanism not involving the SVCT2. Indeed, VC efflux was substantially offset by reuptake of VC on the SVCT2. The presence and function of the SVCT2 at the presynaptic nerve terminal suggest that it is the transporter responsible for recovery of VC released into the synaptic cleft.

Topics: Analysis of Variance; Animals; Animals, Newborn; Ascorbic Acid; Benzofurans; Carbon Isotopes; Cells, Cultured; Cerebral Cortex; Disks Large Homolog 4 Protein; Guanylate Kinases; Imidazoles; Membrane Proteins; Mice; Mice, Inbred C57BL; Neurons; Norepinephrine Plasma Membrane Transport Proteins; Protein Transport; Sodium; Sodium-Coupled Vitamin C Transporters; Superior Cervical Ganglion; Synaptosomes; Vesicular Monoamine Transport Proteins

The activation of T cells is the fundamental on switch for the adaptive immune system. Ca(2+) signaling is essential for T cell activation and starts as initial, short-lived, localized Ca(2+) signals. The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) forms rapidly upon T cell activation and stimulates early Ca(2+) signaling. We developed a high-resolution imaging technique using multiple fluorescent Ca(2+) indicator dyes to characterize these early signaling events and investigate the channels involved in NAADP-dependent Ca(2+) signals. In the first seconds of activation of either primary murine T cells or human Jurkat cells with beads coated with an antibody against CD3, we detected Ca(2+) signals with diameters close to the limit of detection and that were close to the activation site at the plasma membrane. In Jurkat cells in which the ryanodine receptor (RyR) was knocked down or in primary T cells from RyR1(-/-) mice, either these early Ca(2+) signals were not detected or the number of signals was markedly reduced. Local Ca(2+) signals observed within 20 ms upon microinjection of Jurkat cells with NAADP were also sensitive to RyR knockdown. In contrast, TRPM2 (transient receptor potential channel, subtype melastatin 2), a potential NAADP target channel, was not required for the formation of initial Ca(2+) signals in primary T cells. Thus, through our high-resolution imaging method, we characterized early Ca(2+) release events in T cells and obtained evidence for the involvement of RyR and NAADP in such signals.

Topics: Aniline Compounds; Animals; Benzofurans; Calcium; Calcium Signaling; Cells, Cultured; Fluorometry; Humans; Imidazoles; Jurkat Cells; Lymphocyte Activation; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Fluorescence; Muromonab-CD3; NADP; Ryanodine Receptor Calcium Release Channel; T-Lymphocytes; Time Factors; TRPM Cation Channels; Xanthenes

We developed a conventional imaging method to measure Ca(2+) concentration in cytosol (using FuraRed as an indicator) and mitochondria (using Rhod-2 as an indicator), simultaneously, by alternative excitation with specific wave length. After confirming the availability of the method in Hela cells, we applied it to mouse whole-brain slice -preparation, which was exposed to oxygen- and glucose-deprived artificial cerebrospinal fluid (ischemic ACSF) for 12 min. The fluorescence (>570 nm) at the cerebral cortex and hippocampus due to FuraRed (excited by 480 ± 10 nm) decreased (indicating the increase in cytosolic Ca(2+)-concentration), while the fluorescence due to Rhod-2 (excited by 560 ± 10 nm) increased (indicating the increase in mitochondrial Ca(2+) concentration) during exposure to ischemic conditions. We found the characteristic protective effects of cyclosporine A (10(-6) M), a known blocker for mitochondrial permeability transition, and SEA0400 (10(-6) M), a blocker for Na(+)/Ca(2+) exchanger, on the abnormal Ca(2+) increase in cytosol. We confirmed that the present method will be useful for future pathological and pharmacological studies on ischemia-induced brain damage.

Topics: Aniline Compounds; Animals; Benzofurans; Calcium; Cytosol; Disease Models, Animal; Drug Evaluation, Preclinical; HeLa Cells; Heterocyclic Compounds, 3-Ring; Humans; Imidazoles; In Vitro Techniques; Ischemia; Mice; Mice, Inbred C57BL; Mitochondria; Neurons; Phenyl Ethers; Sodium-Calcium Exchanger; Time Factors

Calcium accumulation induces the breakdown of cytoskeleton and axonal fragmentation in the late stages of Wallerian degeneration. In the early stages there is no evidence for any long-lasting, extensive increase in intra-axonal calcium but there does appear to be some redistribution. We hypothesized that changes in calcium distribution could have an early regulatory role in axonal degeneration in addition to the late executionary role of calcium. Schmidt-Lanterman clefts (SLCs), which allow exchange of metabolites and ions between the periaxonal and extracellular space, are likely to have an increased role when axon segments are separated from the cell body, so we used the oxalate-pyroantimonate method to study calcium at SLCs in distal stumps of transected wild-type and slow Wallerian degeneration (Wld(S)) mutant sciatic nerves, in which Wallerian degeneration is greatly delayed. In wild-type nerves most SLCs show a step gradient of calcium distribution, which is lost at around 20% of SLCs within 3mm of the lesion site by 4-24h after nerve transection. To investigate further the association with Wallerian degeneration, we studied nerves from Wld(S) rats. The step gradient of calcium distribution in Wld(S) is absent in around 20% of the intact nerves beneath SLCs but 4-24h following injury, calcium distribution in transected axons remained similar to that in uninjured nerves. We then used calcium indicators to study influx and buffering of calcium in injured neurites in primary culture. Calcium penetration and the early calcium increase in this system were indistinguishable between Wld(S) and wild-type axons. However, a significant difference was observed during the following hours, when calcium increased in wild-type neurites but not in Wld(S) neurites. We conclude that there is little relationship between calcium distribution and the early stages of Wallerian degeneration at the time points studied in vivo or in vitro but that Wld(S) neurites fail to show a later calcium rise that could be a cause or consequence of the later stages of Wallerian degeneration.

Topics: Animals; Axons; Axotomy; Benzofurans; Calcium; Cells, Cultured; Ganglia, Spinal; Gene Expression Regulation; Imidazoles; Microscopy, Electron, Transmission; Mutation; Myelin Sheath; Nerve Tissue Proteins; Neurites; Neurons; Rats; Rats, Mutant Strains; Sciatic Neuropathy; Time Factors; Wallerian Degeneration

Trophoblasts are believed to play an important role in mitigating immunological responses against the fetus. To better understand the nature of trophoblast-leukocyte interactions, we have studied signal transduction during intercellular interactions.. Using a highly sensitive microfluorometric ratioing method and Ca²(+) -sensitive dyes, we measured Ca²(+) signals in trophoblast-like cell lines (JEG-3 and JAR) or in leukocytes (neutrophils and monocytes) during intercellular contact.. Trophoblast cell lines exhibit Ca²(+) signals during leukocyte contact. In contrast, leukocytes cannot elicit Ca²(+) signals in non-opsonized tumour cells, suggesting that Ca²(+) signaling is not a general feature of cell-cell encounters. Similarly, leukocytes demonstrate Ca²(+) signals during contact with trophoblast cell lines. Ca²(+) signals were confirmed using three dyes and with the Ca²(+) buffer BAPTA.. We suggest that leukocyte-to-trophoblast interactions lead to mutual Ca²(+) signaling events in both cell types, which may contribute to immunoregulation at the materno-fetal interface.

Topics: Aniline Compounds; Benzofurans; Calcium Signaling; Cell Line; Female; Fetus; Flow Cytometry; Humans; Imidazoles; Indoles; Leukocytes; Microscopy, Interference; Poloxamer; Pregnancy; Trophoblasts; Xanthenes

The present study outlines improved strategies for ratiometric imaging of cell calcium using a flash lamp-based excitation method and its application to neutrophil polarization. A brief (approximately 6 micros) and intense flash was used to excite the Fluo-4 and Fura Red calcium dye combination in morphologically polarized human neutrophils. These illumination conditions do not allow the dye or calcium ions to diffuse significant distances during the exposure period. Buffer conditions such as pH, pyruvate concentration, and glucose levels were adjusted to more faithfully replicate these parameters in sepsis patients. Fluorescence images at both dyes' emission wavelengths were simultaneously collected using a Dual-View apparatus and an ICCD camera. The ratiometric images, when viewed as single frames or averaged image stacks, clearly demonstrated high calcium probe ratios at the uropod and comparatively low ratios at the cell body that were not evident using conventional imaging methods with longer exposure times. Calcium signaling at the uropod is likely associated with cytoskeletal remodeling during cell motility.

Topics: Adenosine Triphosphatases; Benzofurans; Calcium; Calcium Signaling; Diffusion; Equipment Design; Humans; Hydrogen-Ion Concentration; Image Processing, Computer-Assisted; Imidazoles; Membrane Microdomains; Microscopy, Fluorescence; Microscopy, Video; Neutrophils; Protein Structure, Tertiary; Pyruvates

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.

Topics: Aniline Compounds; Benzofurans; Calcium; CD3 Complex; Flow Cytometry; Fluorescent Dyes; Humans; Imidazoles; Indoles; Jurkat Cells; Leukocytes, Mononuclear; Thapsigargin; Xanthenes

The stress of parturition in the dairy cow is associated with increased susceptibility to infectious disease. During the periparturient period the demands for calcium are increased; these increased demands for calcium can result in subclinical or clinical hypocalcemia. Periparturient cows also experience significant immune suppression. Because intracellular calcium signaling is a key early feature in immune cell activation, we have hypothesized that the increased demand for calcium in periparturient cows may adversely affect intracellular calcium stores of immune cells. This reduction in intracellular calcium stores in immune cells could blunt intracellular calcium release following an activating stimulus, contributing to the immune suppression seen in these animals. To test this hypothesis, peripheral mononuclear cells were obtained from 27 multiparous dairy cows spanning a period of 2 wk before and 2 wk after parturition. Following activation of these cells by anti-CD3 antibodies plus secondary antibodies, intracellular calcium release from intracellular stores was measured. The intracellular calcium released in response to the activation signal declined as calcium demand for lactation became more intense and recovered as plasma calcium normalized. Intracellular calcium stores in peripheral mononuclear cells, estimated by pretreating cells with pervanadate and ionomycin, significantly decreased at parturition and returned to normal levels as the cows' blood calcium returned to normal levels. Hypocalcemia, which is common in periparturient dairy cows, is associated with decreased intracellular calcium stores in peripheral mononuclear cells. Our data suggest that this is the cause of a blunted intracellular calcium release response to an immune cell activation signal. It is concluded that intracellular Ca stores decrease in peripheral blood mononuclear cells (PBMC) before parturition and development of hypocalcemia. This suggests that systemic calcium stress precedes measurable hypocalcemia, particularly in cows that will develop milk fever. Therefore, PBMC intracellular Ca stores are a more sensitive measure of calcium stresses in transition cow. This decrease in PBMC intracellular Ca stores before parturition and the development of hypocalcemia contributes to periparturient immune suppression.

Topics: Aniline Compounds; Animals; Benzofurans; Calcium; Cattle; Cattle Diseases; Endoplasmic Reticulum; Female; Flow Cytometry; Fluorescent Dyes; Hypocalcemia; Imidazoles; Immune Tolerance; Lactation; Leukocytes, Mononuclear; Neutrophils; Parturient Paresis; Parturition; Pregnancy; Signal Transduction; Xanthenes

The acidic food vacuole exerts several important functions during intraerythrocytic development of the human malarial parasite Plasmodium falciparum. Hemoglobin taken up from the host erythrocyte is degraded in the food vacuole, and the heme liberated during this process is crystallized to inert hemozoin. Several anti-malarial drugs target food vacuolar pathways, such as hemoglobin degradation and heme crystallization. Resistance and sensitization to some antimalarials is associated with mutations in food vacuolar membrane proteins. Other studies suggest a role of the food vacuole in ion homeostasis, and release of Ca2+ from the food vacuole may mediate adopted physiological responses. To investigate whether the food vacuole is an intracellular Ca2+ store, which in turn may affect other physiological functions in which this organelle partakes, we have investigated total and exchangeable Ca2+ within the parasite's food vacuole using x-ray microanalysis and quantitative confocal live cell Ca2+ imaging. Apparent free Ca2+ concentrations of approximately 90, approximately 350, and approximately 400 nM were found in the host erythrocyte cytosol, the parasite cytoplasm, and the food vacuole, respectively. In our efforts to determine free intracellular Ca2+ concentrations, we evaluated several Ca2+-sensitive fluorochromes in a live cell confocal setting. We found that the ratiometric Ca2+ indicator Fura-Red provides reliable determinations, whereas measurements using the frequently used Fluo-4 are compromised due to problems arising from phototoxicity, photobleaching, and the strong pH dependence of the dye. Our data suggest that the food vacuole contains only moderate amounts of Ca2+, disfavoring a role as a major intracellular Ca2+ store.

Topics: Aniline Compounds; Animals; Benzofurans; Calcium; Calibration; Coloring Agents; Cytoplasm; Cytosol; Erythrocytes; Fluorescent Dyes; Heme; Hemoglobins; Homeostasis; Humans; Hydrogen-Ion Concentration; Imidazoles; Ions; Light; Microscopy, Confocal; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Mutation; Plasmodium falciparum; Potassium; Time Factors; X-Rays; Xanthenes

The mechanism of collagen-induced human platelet activation was examined using Ca2+, Na+, and the pH-sensitive fluorescent dyes calcium green/fura red, sodium-binding benzofuran isophthalate, and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Administration of a moderate dose of collagen (10 microg/ml) to human platelets resulted in an increase in [Ca2+](i) and platelet aggregation. The majority of this increase in [Ca2+](i) resulted from the influx of calcium from the extracellular milieu via the Na+/Ca2+ exchanger (NCX) functioning in the reverse mode and was reduced in a dose-dependent manner by the NCX inhibitors 5-(4-chlorobenzyl)-2',4'-dimethylbenzamil (KD(50) = 4.7 +/- 1.1 microm) and KB-R7943 (KD(50) = 35.1 +/- 4.8 microm). Collagen-induced platelet aggregation was dependent on an increase in [Ca2+](i) and could be inhibited by chelation of intra- and extracellular calcium through the administration of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) and EGTA, respectively, or via the administration of BAPTA-AM to platelets suspended in no-Na+/HEPES buffer. Collagen induced an increase in [Ca2+](i) (23.2 +/- 7.6 mm) via the actions of thromboxane A(2) and, to a lesser extent, of the Na+/H+ exchanger. This study demonstrates that the collagen-induced increase in [Ca2+](i) is dependent on the concentration of Na+ in the extracellular milieu, indicating that the collagen-induced increase in [Ca2+](i) causes the reversal of the NCX, ultimately resulting in an increase in [Ca2+](i) and platelet aggregation.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Aspirin; Benzofurans; Blood Platelets; Calcium; Collagen; Dose-Response Relationship, Drug; Egtazic Acid; Ethers, Cyclic; Fluoresceins; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Imidazoles; Inositol 1,4,5-Trisphosphate; Models, Biological; Organic Chemicals; Platelet Activation; Platelet Aggregation; Sodium; Sodium-Calcium Exchanger; Specimen Handling; Temperature; Thromboxane A2; Time Factors; Vasoconstrictor Agents

Ca2+ signalling influences many processes in the adult and developing nervous system like exocytosis, synaptic plasticity, and growth cone motility. Optical techniques in combination with fluorescent Ca2+ indicators are the most frequently used methods to measure Ca2+ signalling in cells. In the present study, a new method for ratiometric confocal Ca2+ imaging was developed, and the usefulness of the system was tested with two different neuronal preparations. Developing Manduca sexta antennal lobe neurons were loaded with the Ca2+-sensitive dye Fura Red-AM, and the ratio of fluorescence excited at 457 and 488nm was measured with a confocal laser scanning microscope. During pupal stages 4-12, the antennal lobe neuropil is restructured which includes the ingrowth of olfactory receptor axons, dendritic outgrowth of antennal lobe neurons, and synaptogenesis. In antennal lobe neurons, application of the AChR agonist carbachol induced Ca2+ oscillations the amplitude and frequency of which changed during stages 4-9, while at the end of synaptogenesis, at stages 11 and 12, only single Ca2+ transients were elicited. The Ca2+ oscillations were blocked by D-tubocurarine and Cd2+, indicating that they were due to Ca2+ influx through voltage-gated Ca2+ channels, activated by nAChR-mediated membrane depolarization. To test whether single action potentials can induce Ca2+ transients detectable by Fura Red, individual leech Retzius neurons were injected iontophoretically with the Ca2+ indicator, and the membrane potential was recorded during Ca2+ imaging. Single action potentials induced transient increases in the Fura Red ratio measured in the axon, while trains of action potentials elicited Ca2+ transients that could also be recorded in the cell body and the nucleus. The results show that Fura Red can be used as a ratiometric Ca2+ indicator for confocal imaging.

Topics: Action Potentials; Algorithms; Animals; Benzofurans; Brain; Cadmium; Calcimycin; Calcium; Calcium Signaling; Calibration; Carbachol; Cell Nucleus; Cytoplasm; Data Interpretation, Statistical; Imidazoles; Ionomycin; Leeches; Manduca; Metamorphosis, Biological; Microscopy, Confocal; Microscopy, Fluorescence; Neurons; Photobleaching; Pupa; Tubocurarine

To elucidate the spatial and temporal relationships between phosphatidyl inositol metabolism and changes in intracellular calcium levels, we developed a simultaneous imaging system using green fluorescent protein fused with the pleckstrin homology domain, and the fluorescent calcium indicator, FuraRed. The redistribution of the fusion protein, which represents the phosphatidyl inositol metabolism process, was quantified by calculating the coefficient of variance of the fluorescence over the entire cytosolic region, excluding the nucleus. This calculation increased the reproducibility, compared to the normalized fluorescence changes in arbitrarily selected cytosolic regions used in conventional analysis. The application of this method to analyzing the response of PC12h cells to a number of pharmacological stimuli showed that the extent of the phosphatidyl inositol metabolism was related to the calcium level, but not induced by calcium alone.

Topics: Animals; Benzofurans; Calcium; Cell Nucleus; Cytosol; DNA; Green Fluorescent Proteins; Imidazoles; Luminescent Proteins; Microscopy, Confocal; PC12 Cells; Phosphatidylinositols; Protein Structure, Tertiary; Rats; Time Factors

Upon fertilisation by sperm, mammalian eggs are activated by a series of intracellular Ca(2+) oscillations that are essential for embryo development. The mechanism by which sperm induces this complex signalling phenomenon is unknown. One proposal is that the sperm introduces an exclusive cytosolic factor into the egg that elicits serial Ca(2+) release. The 'sperm factor' hypothesis has not been ratified because a sperm-specific protein that generates repetitive Ca(2+) transients and egg activation has not been found. We identify a novel, sperm-specific phospholipase C, PLC zeta, that triggers Ca(2+) oscillations in mouse eggs indistinguishable from those at fertilisation. PLC zeta removal from sperm extracts abolishes Ca(2+) release in eggs. Moreover, the PLC zeta content of a single sperm was sufficient to produce Ca(2+) oscillations as well as normal embryo development to blastocyst. Our results are consistent with sperm PLC zeta as the molecular trigger for development of a fertilised egg into an embryo.

Topics: Amino Acid Sequence; Animals; Benzofurans; Calcium Signaling; Cloning, Molecular; Embryonic and Fetal Development; Fertilization; Fluorescent Dyes; Imidazoles; Isoenzymes; Male; Mice; Microinjections; Molecular Sequence Data; Ovum; Phosphoinositide Phospholipase C; Phylogeny; Sequence Alignment; Spermatozoa; Tissue Distribution; Type C Phospholipases

Here we show that rabbit Müller cell differentiation from radial glial progenitor cells is accompanied by a decreasing capability to respond to specific stimuli (depolarization and extracellular adenosine 5'-triphosphate [ATP]) with an elevation of intracellular calcium. Intracellular free calcium was recorded in retinal wholemounts from young (postnatal days [P] 2 to 31) and adult rabbits. Images were taken from the nerve fiber/ganglion cell layers where the endfeet of radial glial/ Müller cells can be identified after selective uptake of calcium-sensitive dyes. The area of responding endfeet was determined as the percentage of the total area occupied by Müller cell endfeet, as an estimate of the percentage of responding cells. In response to depolarization (50 mM potassium), an increase of intracellular free calcium occurred in 19% of cells from young postnatal retinae (P2-31) but only in 2% from adults. This depolarization-induced calcium rise was caused both by a calcium influx from extracellular space and by an intracellular calcium release. The latter response was inhibited by the P2 receptor blocker pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS), indicating that extracellular calcium-independent ATP release into the extracellular space occurs during retinal depolarization. When extracellular ATP (200 microM) was applied, calcium responses were recorded in 83% of cells from young postnatal retinae (P2-6); in the course of further development, both the percentage of responding cells (7% in retinae from adult rabbits) and the amplitude of the calcium responses decreased. It is concluded that during the differentiation of immature radial glia into mature Müller cells, stimulus-evoked intracellular calcium signaling mechanisms change.

Topics: Adenosine; Adenosine Triphosphate; Aging; Animals; Benzofurans; Calcium; Calcium Signaling; Cell Count; Cell Differentiation; Cell Size; Fluorescent Dyes; Imidazoles; In Vitro Techniques; Magnesium; Neuroglia; Potassium; Pyridoxal Phosphate; Rabbits; Retina; Stem Cells

We have used the fluorescently labelled calmodulin TA-CaM to follow calmodulin activation during depolarization of adult rat sensory neurons. Calcium concentration was measured simultaneously using the low affinity indicator Oregon Green BAPTA 5N. TA-CaM fluorescence increased during a 200-ms depolarization but then continued to increase during the subsequent 500 ms, even though total cell calcium was falling at this time. In the next few seconds TA-CaM fluorescence fell, but to a new elevated level that was then maintained for several tens of seconds. During a train of depolarizations that evoked a series of largely independent calcium changes TA-CaM fluorescence was in contrast raised for the duration of the train and for many tens of seconds afterwards. The presence of a peptide corresponding to the calmodulin binding domain of myosin light chain kinase significantly increased the depolarization-induced TA-CaM fluorescence increase and slowed the subsequent fall of fluorescence. We interpret the slow recovery component of the TA-CaM signal as reflecting the slow dissociation of calcium--calmodulin--calmodulin binding protein complexes. Our results show that after brief electrical activity calmodulin's interaction with calmodulin binding proteins persists for approximately one minute.

Topics: Action Potentials; Animals; Benzofurans; Calcium; Calcium Signaling; Calmodulin; Female; Fluorescent Dyes; Ganglia, Spinal; Imidazoles; Intracellular Fluid; Male; Neurons, Afferent; Organic Chemicals; Peptide Fragments; Rats; Rats, Sprague-Dawley; Synaptic Transmission; Time Factors; Triazines

Purinergic receptors are important in the regulation of renal hemodynamics; therefore, this study sought to determine if such receptors influence macula densa cell function. Isolated glomeruli containing macula densa cells, with and without the cortical thick ascending limb, were loaded with the Ca(2+) sensitive indicators, Fura Red (confocal microscopy) or fura 2 (conventional video image analysis). Studies were performed on an inverted microscope in a chamber with a flow-through perfusion system. Changes in cytosolic calcium concentration ([Ca(2+)](i)) from exposed macula densa plaques were assessed upon addition of adenosine, ATP, UTP, ADP, or 2-methylthio-ATP (2- MeS-ATP) for 2 min added to the bathing solution. There was no change in [Ca(2+)](i) with addition of adenosine (10(-7) to 10(-3) M). UTP and ATP (10(-4) M) caused [Ca(2+)](i) to increase by 268 +/- 40 nM (n = 21) and 295 +/- 53 nM (n = 21), respectively, whereas in response to 2MesATP and ADP, [Ca(2+)](i) increased by only 67 +/- 13 nM (n = 8) and 93 +/- 36 nM (n = 14), respectively. Dose response curve for ATP (10(-7) to 10(-3) M) added in bath showed an EC(50) of 15 microM. No effect on macula densa [Ca(2+)](i) was seen when ATP was added from the lumen. ATP caused similar increases in macula densa [Ca(2+)](i) in the presence or absence of bath Ca(2+) and addition of 5 mM ethyleneglycotetraacetic acid (EGTA). Suramin (an antagonist of P2X and P2Y receptors) completely inhibited ATP-induced [Ca(2+)](i) dynamics. Also, ATP-Ca(2+) responsiveness was prevented by the phospholipase C inhibitor, U-73122, but not by its inactive analog, U-73343. These results suggest that macula densa cells possess P2Y(2) purinergic receptors on basolateral but not apical membranes and that activation of these receptors results in the mobilization of Ca(2+).

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Benzofurans; Calcium; Cytosol; Dose-Response Relationship, Drug; Estrenes; Female; Fura-2; Imidazoles; Kidney Glomerulus; Microscopy, Confocal; Pyrrolidinones; Rabbits; Receptors, Purinergic; Signal Transduction; Suramin; Thionucleotides; Uridine Triphosphate

Plug flow cytometry is a recently developed system for the automated delivery of multiple small boluses or "plugs" of cells or particles to the flow cytometer for analysis. Important system features are that sample plugs are of precisely defined volume and that the sample vessel need not be pressurized. We describe how these features enable direct cell concentration determinations and novel ways to integrate flow cytometers with other analytical instruments.. Adhesion assays employed human polymorphonuclear neutrophils (PMNs) loaded with Fura Red and Chinese hamster ovary (CHO) cells cotransfected with genes for green fluorescent protein (GFP) and human P-selectin. U937 cells expressing the human 7-transmembrane formyl peptide receptor were loaded with the fluorescent probe indo-1 for intracellular ionized calcium determinations. A computer-controlled syringe or peristaltic pump loaded the sample into a sample loop of the plug flow coupler, a reciprocating eight-port valve. When the valve position was switched, the plug of sample in the sample loop was transported to the flow cytometer by a pressure-driven fluid line.. In stirred mixtures of PMNs and CHO cells, we used plug flow cytometry to directly quantify changes in concentrations of nonadherent singlet PMNs. This approach enabled accurate quantification of adherent PMNs in multicell aggregates. We constructed a novel plug flow interface between the flow cytometer and a cone-plate viscometer to enable real-time flow cytometric analysis of cell-cell adhesion under conditions of uniform shear. The High Throughput Pharmacology System (HTPS) is an instrument used for automated programming of complex pharmacological cell treatment protocols. It was interfaced via the plug flow coupling device to enable rapid (< 5 min) flow cytometric characterization of the intracellular calcium dose-response profile of U937 cells to formyl peptide.. By facilitating the coupling of flow cytometers to other fluidics-based analytical instruments, plug flow cytometry has extended analytical capabilities in cell adhesion and pharmacological characterization of receptor-ligand interactions.

Topics: Animals; Benzofurans; Cell Adhesion; CHO Cells; Cricetinae; Equipment Design; Flow Cytometry; Fluorescent Dyes; Green Fluorescent Proteins; Humans; Imidazoles; In Vitro Techniques; Luminescent Proteins; Neutrophils; P-Selectin; Reproducibility of Results; Stress, Mechanical; Transfection; U937 Cells; Viscosity

The human colon carcinoma cell line HT29 c1.19A was studied for organic anion transporter activity by determining intracellular fluo-3 and fura-red accumulation and by measuring fluo-3 efflux. Modulators of organic anion transport systems were used to identify the transporters that are involved in dye extrusion. Addition of probenecid to the dye-loading medium, containing 10 microM fluo-3/AM and fura-red/AM, resulted in a dose-dependent increase in fluo-3 and fura-red accumulation in the cells. The increase in fluo-3 accumulation in the cells in the presence of probenecid was explained by the inhibitory effect of this compound on fluo-3 efflux. Fluo-3 efflux from the cells was also inhibited by sulfinpyrazone, another inhibitor of organic anion transport. Substrates of renal probenecid-sensitive organic anion exchange mechanisms as well as modulators of multidrug resistance associated protein (MRP) activity did not influence fluo-3 extrusion rates. However, reducing intracellular ATP contents completely blocked fluo-3 extrusion. Moreover, MK571, an inhibitor of MRP, significantly stimulated dye accumulation, whereas inhibitors of the multidrug resistance gene (MDR1) product Pglycoprotein, cyclosporin A and verapamil, did not. As probenecid inhibits fluo-3 efflux across the apical membrane of cells grown on permeable supports, we conclude that a probenecid-sensitive organic anion transporter is present in the apical membrane of HT29 c1.19A cells. This organic anion transport system differs from MDRI and MRP2.

Topics: Adenosine Triphosphate; Aniline Compounds; Anions; ATP Binding Cassette Transporter, Subfamily B, Member 1; Benzofurans; Biological Transport; Carrier Proteins; Electric Impedance; Fluorescent Dyes; HT29 Cells; Humans; Imidazoles; Membrane Potentials; Microscopy, Fluorescence; Probenecid; Xanthenes

Using the low-affinity fluorescent Ca(2+) indicators, Mag-Fura-2 and Mag-Fura Red, we studied light- and InsP(3)-induced Ca(2+) release in permeabilized microvillar photoreceptors of the medicinal leech, Hirudo medicinalis. Two major components of the phosphoinositide signaling pathway, phospholipase-C and the InsP(3) receptor, were characterized immunologically and appropriately localized in photoreceptors. Whereas phospholipase-C was abudantly expressed in photoreceptive microvilli, InsP(3) receptors were found mostly in submicrovillar endoplasmic reticulum (SER). Permeabilization of the peripheral plasma membrane with saponin allowed direct measurements of luminal free Ca(2+) concentration (Ca(L)) changes. Confocal Ca(2+) imaging using Mag-Fura Red demonstrated that Ins(1,4,5)P(3) mobilizes Ca(2+) from SER. As detected with Mag-Fura-2, a brief 50ms light flash activated rapid Ca(2+) depletion of SER, followed by an effective refilling within 1min of dark adaptation after the light flash. Sensitivity to Ins(1,4,5)P(3) of the Ca(2+) release from SER in leech photoreceptors was accompanied by irreversible uncoupling of phototransduction from Ca(2+) release. Depletion of Ca(2+) stores was induced by Ins(1,4,5)P(3)(EC(50)= 4.75 microM) and the hyper-potent agonist adenophostin A (EC(50)/40nM) while the stereoisomer L-myo Ins(1,4,5)P(3) was totally inactive. Ins(1,4,5)P(3)- or adenophostin A-induced Ca(2+) release was inhibited by 0.1-1mg/ml heparin. The Ca(2+) pump inhibitors, cyclopiazonic acid and thapsigargin, in the presence of Ins(1,4,5)P(3), completely depleted Ca(2+) stores in leech photoreceptors.

Topics: Animals; Benzofurans; Calcium; Calcium Channels; Endoplasmic Reticulum; Fluorescent Dyes; Fura-2; Imidazoles; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Leeches; Phosphatidylinositol Diacylglycerol-Lyase; Photoreceptor Cells, Invertebrate; Receptors, Cytoplasmic and Nuclear; Type C Phospholipases; Vision, Ocular

We have studied the degree to which fluorescent Ca(2+) indicator dyes, and green fluorescent protein and its variants, can be used together. We find that the most commonly used fluorescent protein, enhanced green fluorescent protein (EGFP), seriously contaminates fura 2 signals. We suggest two alternative combinations for which there is no detectable contamination of the Ca(2+) indicator signal by the fluorescent protein. Blue fluorescent protein can be used with the Ca(2+) indicator Fura Red; EGFP can be used with the Ca(2+) indicator X-Rhod 1. The use of these combinations will permit the accurate measurement of Ca(2+) signals in cells transfected with fluorescent proteins.

Topics: Animals; Benzofurans; Calcium; Calcium Signaling; Fluorescent Dyes; Fura-2; Genetic Variation; Green Fluorescent Proteins; Imidazoles; Luminescent Proteins; Rats; Spectrometry, Fluorescence; Transfection; Tumor Cells, Cultured

The regulation of cytosolic-free calcium concentration of smooth-muscle and endothelial cells was mainly studied on cultured cells where the cross talk between these two coupled cell types is lost. In the present study, the cytosolic-free calcium concentration in the endothelial and the smooth-muscle cells was examined in an intact arterial wall in vitro. Strips of the main branch of rat mesenteric artery were used. Cytosolic-free calcium concentration [Ca2+]i was estimated by determining the fluorescence ratio of the two calcium probes, Fluo-4 and Fura red. The emitted fluorescence of both probes was measured with a confocal microscope. We showed that potassium and phenylephrine, which increase the cytosolic -free calcium concentration of the smooth-muscle cells, also indirectly influence the calcium concentration in the endothelial cells. By simultaneously determining [Ca2+]i in the endothelial and the smooth-muscle cells of an arterial strip, we observed that when calcium increases in the endothelial cells in response to acetylcholine, it slightly decreases in the smooth-muscle cells. We conclude that the regulation of [Ca2+]i in the arterial endothelial cell, depends according to the stimuli either upon the endothelial cells themselves, or upon the coupled smooth-muscle cells.

Topics: Acetylcholine; Adrenergic alpha-Agonists; Aniline Compounds; Animals; Benzofurans; Calcium; Cytosol; Endothelium, Vascular; Fluorescent Dyes; Imidazoles; In Vitro Techniques; Male; Mesenteric Arteries; Muscle, Smooth; Phenylephrine; Potassium; Rats; Rats, Sprague-Dawley; Vasodilator Agents; Xanthenes

Flow cytometry offers numerous advantages over traditional techniques for measuring intracellular Ca(2+) in lymphoid and nonlymphoid cells. In particular, the heterogeneity of cell responses can be defined by flow cytometry, and multiparameter analyses permit the determination of intracellular Ca(2+) in surface-marker-defined target cells as well as correlation of changes in Ca(2+) with other biochemical markers, including ligand binding. This article presents several established methods for measuring intracellular Ca(2+) by flow cytometry in lymphoid and nonlymphoid cells. Examples are provided for determination of Ca(2+) in human peripheral blood leukocytes and two human epithelial cell lines grown in monolayer. In addition, applications are reviewed or presented for correlating changes in intracellular Ca(2+) with other cell parameters, including cell cycle analysis, changes in cell membrane integrity, and the induction of apoptosis markers. Finally, a number of novel sample handling capabilities useful for performing kinetic analyses of Ca(2+) changes by flow cytometry are now available and one application is presented which is finding utility in pharmacologic studies.

Topics: Aniline Compounds; Benzofurans; Calcium; Calibration; Cell Adhesion; Cell Line; Cell Membrane; Cells, Cultured; Chelating Agents; Epithelial Cells; Flow Cytometry; Fluorescent Dyes; Humans; Imidazoles; Intracellular Fluid; Leukocytes, Mononuclear; Propidium; Receptors, Formyl Peptide; Receptors, Immunologic; Receptors, Peptide; Spectrometry, Fluorescence; Transfection; Xanthenes

A method for visualisation of cytosolic [Ca(2+)] distribution was applied to living plant tissue. A mixture of the fluorescent probes Fluo-3 and Fura Red was used. The emitted fluorescence was scanned simultaneously in two channels with a laser-scanning confocal microscope and rationing was performed. The homogeneity of the Fluo-3/Fura Red concentration ratio throughout the tissue after AM-ester loading was proven. In vitro calibration permitted conversion of Fluo-3/Fura Red fluorescence ratios to [Ca(2+)] values. Apparent K(D)of 286 nM, R(min)of 0.43 and R(max)of 18 were calculated. The in vivo determination of extreme ratio values was performed by permeabilizing the plasmalemma for Ca(2+)with a ionophore and manipulating the extracellular [Ca(2+)]. The resultant R(minv)of 1.33 and R(maxv)of 2.69 for vegetative apices, and R(mini)of 1.26 and R(maxi)of 3.45 for apices induced to flowering, suggested incomplete equalization of extra- and intracellular Ca(2+)levels in these experiments. In Chenopodium rubrum, the cytosolic [Ca(2+)] patterns of apical tissue obtained using Fluo-3 and Fura Red were significantly different between vegetative apices and apices after photoperiodic flower induction. This methodological approach may also be helpful for studying cytosolic [Ca(2+)] distribution in other living plant tissues.

Topics: Aniline Compounds; Benzofurans; Calcimycin; Calcium; Chenopodiaceae; Cytosol; Fluorescent Dyes; Imidazoles; Ionophores; Meristem; Microscopy, Confocal; Photoperiod; Xanthenes

Quantifying the magnitude of Ca2+ signals from changes in the emission of fluorescent indicators relies on assumptions about the indicator behaviour in situ. Factors such as osmolarity, pH, ionic strength and protein environment can affect indicator properties making it advantageous to calibrate indicators within the required cellular or subcellular environment. Selecting Ca2+ indicators appropriate for a particular application depends upon several considerations including Ca2+ binding affinity, dynamic range and ease of loading. These factors are usually best determined empirically. This study describes the in-situ calibration of a number of frequently used fluorescent Ca2+ indicators (Fluo-3, Fluo-4, Calcium Green-1, Calcium Orange, Oregon Green 488 BAPTA-1 and Fura-Red) and their use in reporting low- and high-amplitude Ca2+ signals in HeLa cells. All Ca2+ indicators exhibited lower in-situ Ca2+ binding affinities than suggested by previously published in-vitro determinations. Furthermore, for some of the indicators, there were significant differences in the apparent Ca2+ binding affinities between nuclear and cytoplasmic compartments. Variation between indicators was also found in their dynamic ranges, compartmentalization, leakage and photostability. Overall, Fluo-3 proved to be the generally most applicable Ca2+ indicator, since it displayed a large dynamic range, low compartmentalization and an appropriate apparent Ca2+ binding affinity. However, it was more susceptible to photobleaching than many of the other Ca2+ indicators.

Topics: Aniline Compounds; Benzofurans; Calcium; Calcium Signaling; Calibration; Cell Compartmentation; Cell Nucleus; Cytosol; Fluorescent Dyes; HeLa Cells; Humans; Imidazoles; Organic Chemicals; Xanthenes

The intracellular magnesium and calcium concentrations in cultured dorsal root ganglion neurons were measured using a fluorescent Mg2+ indicator, Mag-Fura-2 and a Ca2+ indicator, Fura-2, respectively. The magnesium concentration in the cytoplasm was higher than that in the nuclei at rest; 0.68+/-0.10 mM (mean+/-S.E.M., n=7) in the cytoplasm and 0.11+/-0.05 mM in the nucleus. When depolarized by a 60 mM KCl solution, the magnesium concentration increased remarkably in the cytoplasm; 1.52+/-0.26 mM (n=7) in the cytoplasm and 0.25+/-0. 12 mM in the nucleus. This is in contrast to a Ca2+ increase due to depolarization in which the increase was remarkable also in the nucleus. The Mg2+ response displayed a rapid spontaneous recovery even in the presence of the high K+ solution. The Ca2+ response, on the other hand, accompanied a slow recovery 'plateau'. Simultaneous measurements of Mg2+ and Ca2+ by a double-labeling experiment revealed that the Ca2+ concentration started to rise 0.46+/-0.05 s (n=32) earlier, and it reached its peak 1.38+/-0.12 s (n=32) earlier than Mg2+. These results support the scheme of 'calcium induced magnesium release', that the depolarization-induced elevation of the Ca2+ concentration causes an increase in the Mg2+ concentration in the cytoplasm.

Topics: Animals; Benzofurans; Calcium; Cells, Cultured; Fluorescent Dyes; Fura-2; Ganglia, Spinal; Image Processing, Computer-Assisted; Imidazoles; Magnesium; Male; Membrane Potentials; Microscopy, Confocal; Neurons; Rats; Rats, Sprague-Dawley

An increase in intracellular calcium during cerebral ischemia has been proposed as a common final pathway underlying the events leading to neuronal death. Intracellular calcium has been measured with ion selective electrodes during energy deprivation (ED) in hippocampal slices and with fluorescent techniques in neuronal cultures. In the present study, we describe a novel method to visualize and quantify changes in intracellular calcium in brain slices using Confocal Laser Scanning Microscopy (CLSM). CA 1 pyramidal neurons in hippocampal slices were filled by intracellular injection with a 1:2 mixture of the fluorescent dyes Fluo 3 and Fura Red. The neurons were then visualized using CLSM, and the ratio of the fluorescence from each probe used to quantify intracellular calcium concentrations before and during ED. The free intracellular calcium concentration was 60 nM prior to ED and increased to 24 microM during ED. These results demonstrates that CLSM and fluorescent probes can be used in functional neuronal networks in addition to cell cultures as previously described.

Topics: Aniline Compounds; Animals; Benzofurans; Brain Ischemia; Calcium; Calibration; Energy Metabolism; Fluorescent Dyes; Hippocampus; Imidazoles; In Vitro Techniques; Kinetics; Microscopy, Confocal; Neurons; Pyramidal Cells; Rats; Rats, Wistar; Xanthenes

Cytoplasmatic calcium concentrations are elevated two to three fold during cerebral ischemia. In order to determine the role of calcium-release from intracellular stores vs. calcium entry from the extracellular space, intracellular stores were depleted by the use of thapsigargin and calcium was removed from the incubation fluid prior to energy deprivation (ED). CA 1 pyramidal neurons in hippocampal rat slices were filled with a 1:2 mixture of Fluo-3 and Fura Red by intracellular injection. The neurons were visualized in a Confocal Laser Scanning Microscope (CLSM) and the fluorescence ratio from the probe mixture was used to quantify the calcium concentration. Intracellular calcium concentration was monitored before and during ED. The intracellular calcium concentration was 55 nM prior to ED and increased to 25 microM during ED. The resting levels were the same in the experimental groups, but the increase during ED was significantly lower in the intervention groups. The increase in the calcium free group was to 1 microM and in the thapsigargin group to 5 microM. In the last experimental group, thapsigargin treatment and removal of extracellular calcium, the intracellular calcium increased to 630 nM. These results demonstrate that the increased intracellular calcium seen during ED originates from several sources. Calcium-release from intracellular stores may be of major importance in calcium-related neuronal injury during cerebral ischemia.

Topics: Aniline Compounds; Animals; Benzofurans; Calcium; Calcium Channel Blockers; Energy Metabolism; Extracellular Space; Fluorescent Dyes; Hippocampus; Imidazoles; In Vitro Techniques; Intracellular Membranes; Microscopy, Confocal; Osmolar Concentration; Pyramidal Cells; Rats; Rats, Wistar; Thapsigargin; Xanthenes

The spatial characteristics of inositol 1,4,5-trisphosphate (IP3)-induced quantal Ca2+ release were examined by imaging Ca2+ concentrations within Ca2+ stores ([Ca2+]L) in permeabilized HSY cells. The image of mag-fura-2 fluorescent ratio with dual excitation (344 nm/360 nm) demonstrated that a sequential application of different concentrations of IP3 (0.1, 0.3, 10 microM) resulted in a stepwise decrease in the ratio at all regions of the cytoplasm. This change in the ratio suggests that the stepwise decrease in [Ca2+]L is associated with the quantal Ca2+ release. To monitor the change in [Ca2+]L within a single organelle, IP3-dependent changes in the mag-fura-red fluorescence of permeabilized cells were studied by confocal microscopy. Applications of increasing concentrations of IP3 caused a stepwise increase in fluorescence within ER-like reticulum structures of the cytoplasm. This finding suggests that the [Ca2+]L in a single Ca2+ store was not depleted by submaximal concentrations of IP3, and supports the steady-state model of quantal Ca2+ release.

Topics: Adenocarcinoma; Benzofurans; Calcium; Cell Membrane Permeability; Diagnostic Imaging; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Fluorescent Dyes; Fura-2; Humans; Imidazoles; Inositol 1,4,5-Trisphosphate; Microscopy, Confocal; Organelles; Salivary Gland Neoplasms; Saponins; Tumor Cells, Cultured

Calcium distribution and mobilization during mechanical stimulation in outer hair cells of the guinea pig were monitored using laser scanning confocal microscopy and co-loaded fluo-3 and fura-red fluorescent probes. Spatial calcium gradients were revealed among various subcellular areas. The ratios of the fluorescence intensity of fluo-3 and fura-red were 1.71 +/- 0.85, 1.61 +/- 0.75, 1.47 +/- 0.65 and 1.39 +/- 0.66 for the cytoplasm, the cytoplasmic membrane, the cuticular plate and the nucleus respectively, indicating that free calcium ion concentrations are the highest in the cytoplasm and the lowest in the nucleus. While the calcium concentration remained relatively constant under resting conditions, it increased during mechanical stimulation. The results show that confocal ratio imaging of fluo-3 and fura-red enables us to determine more accurately the subcellular calcium distribution and that the calcium ions make a contribution to the mechanic-electrical transduction in hair cells.

Topics: Aniline Compounds; Animals; Benzofurans; Calcium; Cell Nucleus; Cytoplasm; Fluorescent Dyes; Guinea Pigs; Hair Cells, Auditory, Outer; Imidazoles; Microscopy, Confocal; Xanthenes

Fusion of sperm and egg plasma membranes is an early and essential event at fertilization but it is not known if it plays a part in the signal transduction mechanism that leads to the oscillations in the cytoplasmic free Ca2+ concentration ([Ca2+]i) that accompany mammalian egg activation. We have used two independent fluorescence methods and confocal microscopy to show that cytoplasmic continuity of egg and sperm precedes the onset of the first [Ca2+]i increase in mouse eggs. The Ca2+ indicator dye Ca2+-green dextran was microinjected and its transfer from egg to sperm was monitored. We found that it occurred before, and without a requirement for, any detectable [Ca2+]i increase in the egg. In separate experiments [Ca2+]i changes were recorded in populations of eggs, using fura red, and the eggs fixed at various times after some of the eggs had shown a [Ca2+]i transient. Fusion of the sperm and egg was then assessed by Hoechst dye transfer. All eggs that showed a [Ca2+]i increase had a fused sperm but more than half of the eggs contained a sperm but had not undergone a [Ca2+]i increase. These data indicate that sperm-egg fusion precedes [Ca2+]i changes and we estimate that the elapsed time between sperm-egg fusion and the onset of the [Ca2+li oscillations is 1-3 minutes. Finally, sperm-egg fusion was prevented by using low pH medium which reversibly prevented [Ca2+]i oscillations in eggs that had been inseminated. This was not due to disruption of signalling mechanisms, since [Ca2+]i changes still occurred if low pH was applied after the onset of oscillations at fertilization. [Ca2+]i changes also occurred in eggs in low pH in response to the muscarinic agonist carbachol. These data are consistent with the idea that the [Ca2+]i signals that occur in mammalian eggs at fertilization are initiated by events that are closely coupled to the fusion of the sperm and egg membranes.

Topics: Acrosome; Animals; Benzimidazoles; Benzofurans; Calcium; Cell Fusion; Cell Membrane; Cytoplasm; Female; Fluorescent Dyes; Imidazoles; Kinetics; Male; Membrane Fusion; Mice; Mice, Inbred Strains; Microscopy, Confocal; Oscillometry; Ovum; Sperm-Ovum Interactions; Spermatozoa; Time Factors

Receptors for neuroligands in the paranodal Schwann cell region of a myelinated nerve fiber could have important functions. We have used confocal laser scanning microscopy in combination with Ca(2+)-sensitive fluorescent dyes to study the possible effects of purinergic agonists on the free intracellular calcium concentration ([Ca2+]i) in paranodes of isolated rat spinal roots. Application of ATP in concentrations of 100 and 300 microM resulted in a transient rise in [Ca2+]i in about 57% of the paranodal Schwann cell regions studied. UTP was equipotent to ATP whereas adenosine, beta,gamma-methylene ATP, and elevation of the extracellular K+ concentration by 10 mM had no effect on [Ca2+]i. These data indicate the presence of the P2Y2 (previously termed P2U) subtype of P2 receptors in the paranodal Schwann cell membrane of rat spinal root myelinated nerve fibers.

Topics: Adenosine Triphosphate; Animals; Axons; Benzofurans; Calcium; Fluorescent Dyes; Imidazoles; Intracellular Membranes; Male; Microscopy, Confocal; Nerve Fibers, Myelinated; Organic Chemicals; Rats; Rats, Wistar; Receptors, Purinergic; Schwann Cells; Spinal Nerve Roots; Uridine Triphosphate

In Paramecium tetraurelia cells analysis of transient changes in Ca2+ concentration, [Ca2+]i, during aminoethyldextran (AED) stimulated synchronous (<1 second) trichocyst exocytosis has been hampered by various technical problems which we now have overcome. While Fura Red was found appropriate for quantitative double wavelength recordings, Fluo-3 allowed to follow, semi-quantitatively but with high time resolution, [Ca2+]i changes by rapid confocal laser scanning microscopy (CLSM). Resting values are between 50 and 70 nM in the strains analysed (7S wild type, as well as a non-discharge and a trichocyst-free mutant, nd9-28 degrees C and tl). In all strains [Ca2+]i first increases at the site of AED application, up to 10-fold above basal values, followed by a spillover into deeper cell regions. This might: (i) allow a vigorous Ca2+ flush during activation, and subsequently (ii) facilitate re-establishment of Ca2+ homeostasis within > or =20 seconds. Because of cell dislocation during vigorous trichocyst exocytosis, 7S cells could be reasonably analysed only by CLSM after Fluo-3 injection. In 7S cells cortical [Ca2+]i transients are strictly parallelled by trichocyst exocytosis, i.e. in the subsecond time range and precisely at the site of AED application. Injection of Ca2+ is a much less efficient trigger for exocytosis. Ca2+-buffer injections suggest a requirement of [Ca2+]i >1 to 10 microM for exocytosis to occur in response to AED. In conclusion, our data indicate: (i) correlation of cortical [Ca2+]i transients with exocytosis, as well as (ii) occurrence of a similar signal transduction mechanism in mutant cells where target structures may be defective or absent.

Topics: Animals; Benzofurans; Calcium; Dextrans; Fluorescent Dyes; Imidazoles; Ion Transport; Paramecium

The localizations of Ca2+ extrusion sites in mouse pancreatic acinar cells during elevation of the intracellular free calcium concentration ([Ca2+]i) have been studied. During an agonist stimulated calcium elevation as well as when intracellular calcium is released from a 'caged compound', Ca2+ is primarily extruded from the apical secretory pole of the cells in spite of different spatial patterns of [Ca2+]i different sources of Ca2+, and the presence or absence of agonist. This is most likely due to a relatively high density of calcium pumps in the secretory granule region, although it could be explained by calcium pumps in this part of the cell having different characteristics from those in the basal membrane. The intensity of Ca2+ extrusion in the apical secretory pole is such that substantial (several millimoles per litre) changes of the free calcium concentration in the lumen of the acinus can occur during agonist stimulation.

Topics: Acetylcholine; Animals; Benzofurans; Calcium; Cell Membrane; Dextrans; Egtazic Acid; Fluorescent Dyes; Imidazoles; Kinetics; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Organic Chemicals; Pancreas

To investigate the calcium homeostasis in single fiber cells isolated from rat ocular lens cortex and to quantify the changes in the concentration of free intracellular calcium [Ca2+]i during the process of disintegrative globulization.. Individual fiber cells from the cortex of the adult rat lens were isolated by treatment with trypsin in ion-free buffered sucrose. The isolated fiber cells were loaded with the acetoxymethyl esters of Fluo-3 or Calcium Green-2, or with Fluo-3 and Fura Red, and changes in [Ca2+]i of single cortical fibers were measured using a microfluorometer. The time course of increase of [Ca2+]i in fiber cells exposed to Ringer's solution was measured, and the effects on the increase of [Ca2+]i of calcium channel blocker, verapamil, Na-Ca exchange inhibitors Ni2+ and Zn2+, and protease inhibitor, leupeptin, Na+-free and K+-free media and Ca2+-containing isotonic sucrose solution, were investigated.. In Hepes sucrose solution (containing approximately 1.5 microM Ca2+), the isolated fiber cells maintained stable values of [Ca2+]i at 99.6+/-10 nM (n = 32). Exposure of the isolated fibers to Ringer's solution (containing 2 mM Ca2+) led to a monoexponential increase of [Ca2+]i at a rate of 0.12 min(-1). This increase in [Ca2+]i was accompanied by disintegration of the isolated fibers into discrete but resealed globules. Changes in [Ca2+]i, monitored by using a two-dye ratiometric method using Fura Red and fluo-3, showed a progressive increase in [Ca2+]i in fibers exposed to Ringer's solution, preceding globulization. The [Ca2+]i in the globules in Ringer's solution, determined using Calcium Green-2, was 3.6+/-0.7 microM (n = 23). Compared with that in fibers in Ringer's solution, the rate of increase of [Ca2+]i in fibers was much slower in the presence of 50 microM verapamil (0.047 min[-1]), in Na+-free (0.086 min[-1]) and in K+-free (0.062 min[-1]) Ringer's solution, or when the fibers were suspended in Hepes-sucrose solution, containing 2 mM Ca2+ (0.046 min[-1]). After 30 minutes, the [Ca2+]i of fiber cells exposed to Ringer's solution, containing 2 mM Ni2+ (574.7+/-29 nM; n = 7) or Zn2+ (402.6+/-77 nM; n = 7) was significantly lower (P < 0.001) compared with that in fiber cells exposed to Ringer's solution alone (1995+/-461 nM, n = 10). In Ringer's solution, leupeptin delayed globulization without significantly affecting the increase in [Ca2+]i. The [Ca2+]i of fiber cells isolated from outer and inner cortex and suspended in Hepes-sucrose was comparable; however, after 15 minutes of exposure to Ringer's solution, [Ca2+]i in fibers from the outer cortex was approximately three times higher than [Ca2+]i in those from the inner cortex.. Exposure to high (millimolar) concentrations of calcium in the external medium leads to an increase in [Ca2+]i of isolated individual fiber cells, which precedes disintegrative globulization. The protective effects of Na+-free and K+-free solutions on globulization appear to be due to a lower rate of increase of [Ca2+]i. Part of the calcium influx may be mediated by L-type calcium channels and by Na-Ca exchange, operating in reverse. Proteolytic inhibitors do not affect the increase in [Ca2+]i but delay globulization by inhibiting calcium-mediated proteolysis. The isolated fiber cells and the disintegrated globules maintain a 100- to 300-fold gradient of calcium across their plasma membranes.

Topics: Aniline Compounds; Animals; Benzofurans; Calcium; Calcium Channel Blockers; Fluorescent Dyes; Homeostasis; Imidazoles; Lens Cortex, Crystalline; Leupeptins; Nickel; Organic Chemicals; Rats; Rats, Sprague-Dawley; Spectrometry, Fluorescence; Verapamil; Xanthenes; Zinc

Ischemia produces striking electrophysiological abnormalities in blood-perfused hearts that may be caused, in part, by effects of ischemia on intracellular calcium. To test this hypothesis, intracellular Ca2+ concentration ([Ca2+]i) transients were recorded from the epicardial surface of blood- and saline-perfused rabbit hearts using the long-wavelength indicator Fura Red. Calcium transients were much larger than the movement artifact, representing up to 29% of the total signal. Switching the perfusate from saline to blood did not affect the time course of the transients or the apparent level of [Ca2+]i. Compartmentation of Fura Red fluorescence was estimated by exposure to Mn2+. The results were cytosol 60 +/- 3%, organelles 12 +/- 2%, and autofluorescence plus partly deesterified Fura Red 29 +/- 4%. [Ca2+]i transients were calibrated in situ by perfusion of the extracellular space with high-Ca2+ and Ca(2+)-free EGTA solutions. Peak systolic [Ca2+]i was 663 +/- 74 nM, and end-diastolic [Ca2+]i was 279 +/- 59 nm. Ischemia was produced by interruption of aortic perfusion for 2.5 min during pacing (150 beats/min). Ischemia produced broadening of the [Ca2+]i transient, along with beat-to-beat alternations in the peak systolic and end-diastolic level of [Ca2+]i (calcium transient alternans). [Ca2+]i transient alternans occurred in 82% of blood-perfused hearts vs. 43% of saline-perfused hearts. The discrepancy between large and small transients (mean alternans ratio) was larger in the blood-perfused hearts (0.23 +/- 0.04 vs. 0.07 +/- 0.03, P = 0.005). These observations are important because of the apparent relationship of [Ca2+]i transient alternans to electrical alternans and arrhythmias during ischemia.

Topics: Animals; Benzofurans; Calcium; Calibration; Female; Fluorescent Dyes; Heart; Imidazoles; Ionomycin; Kinetics; Male; Manganese; Myocardial Ischemia; Myocardium; Rabbits; Spectrometry, Fluorescence; Subcellular Fractions; Time Factors

Here we describe the subcellularly uniform control of the intracellular Ca2+ concentration ([Ca2+]i) by flash photolysis of caged Ca2+ or a caged Ca2+ buffer. A mixture of the two Ca2+ indicators Fluo-3 and Fura-red was used together with a laser-scanning confocal microscope to reveal spatial aspects of intracellular Ca2+ signals. The patch clamp technique in the whole-cell variant was applied to load the cells with the indicator mixture together with either DM-nitrophen or diazo-2 and to measure changes in the membrane current. An in vivo calibration was performed to convert the Fluo-3/Fura-red fluorescence ratios to [Ca2+] values. The resulting calibration curve suggested an apparent KD of 1.6 microM, Rmax of 2.15, Rmin of 0.08 and a Hill-coefficient of 0.75 for the indicator mixture. Controlled rupture of the cell membrane revealed a large fraction of immobile intracellular Fura-red fluorescence that may account for the reduced in vivo Rmax value when compared to the in vitro value of 3.1. In cardiac myocytes, flash photolytic release of Ca2+ from DM-nitrophen generated inwardly directed Na+/Ca2+ exchange currents and Ca2+ signals that were graded with the discharged flash-energy. Rapid line-scans revealed subcellularly homogeneous [Ca2+] jumps regardless of the discharged flash energy. Ca2+ signals evoked by L-type Ca2+ currents (ICa) could be terminated rapidly in a spatially homogeneous manner by UV flash photolysis of diazo-2. No side-effects of the photolytic products of DM-nitrophen or diazo-2 with the mixture of Fluo-3/Fura-red were detectable in our experiments. The combination of UV flash photolysis and laser scanning confocal microscopy enabled us to control [Ca2+]i homogeneously on the subcellular level. This approach may improve our understanding of the subcellular properties of cardiac Ca2+ signalling. The technique can also be applied in other cell types and with other signalling systems for which caged compounds are available.

Topics: Acetates; Aniline Compounds; Animals; Benzofurans; Calcium; Cells, Cultured; Chelating Agents; Diazonium Compounds; Ethylenediamines; Female; Fluorescent Dyes; Guinea Pigs; Image Processing, Computer-Assisted; Imidazoles; Male; Microscopy, Confocal; Myocardium; Phenoxyacetates; Photolysis; Rats; Rats, Wistar; Ultraviolet Rays; Xanthenes

Bradykinin and caffeine were used as two different agonists to study inositol 1,4,5-trisphosphate (IP3)-sensitive and caffeine/ryanodine-sensitive intracellular Ca2+ release in the outgrowing neurites of nerve-growth-factor (NGF)-treated rat phaeochromocytoma cells (PC12). Changes in neuritic intracellular free Ca2+ ([Ca2+]i) in single cells were measured after loading with a 1:1 mixture of the acetoxymethyl (AM) ester of the Ca2+-sensitive dyes Fura-red and Fluo-3, in combination with confocal microscopy. Bradykinin-induced Ca2+ release was blocked by U73211, a specific phospholipase C inhibitor. Caffeine-induced Ca2+ release was very low in neurites at rest. It increased after the cells were preloaded with Ca2+. The Ca2+ signal evoked at high concentrations of bradykinin (>500 nM) arose from a trigger zone in the proximal part of the neurite, as a bi-directional wave towards the growth cone and cell body. The speed of neuritic Ca2+ waves was reduced in cells loaded with the Ca2+ chelator 1, 2-bis(2-aminophenoxy)ethane-tetraacetic acid/AM. Preloading of Ca2+ stores led to increased bradykinin-induced Ca2+ release, as seen for caffeine, and faster Ca2+ wave speeds. Caffeine evoked a simultaneous [Ca2+]i rise along the neurites of Ca2+ preloaded cells. Higher Ca2+ signal amplitudes and faster Ca2+ wave speeds, but no longer-lasting IP3-induced [Ca2+]i signals, correlated with increased caffeine-induced Ca2+ release in the neurites. At low concentrations of bradykinin (<1.0 nM), the Ca2+ signals ceased to propagate as complete Ca2+ waves. Instead, repetitive stochastic Ca2+ release events (neuritic Ca2+ puffs) were observed. Neuritic Ca2+ puffs spread across only a few microns, at a slower speed than neuritic Ca2+ waves. These Ca2+ puffs represent elementary Ca2+ release units, whereby the released Ca2+ ions form these elementary events into the shape of a Ca2+ wave.

Topics: Aniline Compounds; Animals; Benzofurans; Bradykinin; Caffeine; Calcium; Cell Line; Egtazic Acid; Evoked Potentials; Imidazoles; Inositol 1,4,5-Trisphosphate; Microscopy, Confocal; Nerve Growth Factors; Neurites; Neurons; PC12 Cells; Rats; Ryanodine; Xanthenes

Recently a number of lower-affinity fluorescent Ca2+ indicators have become available with principal absorbance bands at visible wavelengths. This article evaluates these indicators, as well as two shorter wavelength indicators, mag-fura-5 and mag-indo-1, for their suitability as rapid Ca2+ indicators in frog skeletal muscle fibers. With three lower-affinity tricarboxylate indicators (mag-fura-5, mag-indo-1, and magnesium orange), the change in fluorescence in response to an action potential (delta F) appeared to track the myoplasmic Ca2+ transient (delta[Ca2+]) without delay. With three lower-affinity tetracarboxylate indicators (BTC, calcium-orange-5N, and calcium-green-5N) and one tricarboxylate indicator (magnesium green), delta F responded to delta[Ca2+] with a small delay. Unfortunately, with the tetracarboxylate indicators, other problems were detected that appear to limit their usefulness as reliable Ca2+ indicators. Surprisingly, delta F from mag-fura-red, another tricarboxylate indicator, was biphasic (with 480 nm excitation), a feature that also greatly limits its usefulness. With several of the indicators, estimates were obtained for the myoplasmic value of KD, Ca (the indicator's dissociation constant for Ca2+) and found to be elevated severalfold in comparison with the value measured in a simple salt solution. These and other problems related to the quantitative use of Ca2+ indicators in the intracellular environment are evaluated and discussed.

Topics: Action Potentials; Animals; Benzofurans; Biophysical Phenomena; Biophysics; Calcium; Fluorescent Dyes; Fura-2; Imidazoles; In Vitro Techniques; Indicators and Reagents; Indoles; Kinetics; Muscle, Skeletal; Organic Chemicals; Rana temporaria; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet

Laser scanning confocal microscopy in combination with the fluorescent calcium indicators Fluo-3 and Fura-Red was employed to estimate the intracellular concentration of free calcium ions in individual olfactory receptor neurons and to monitor temporal and spatial changes in the Ca(2+)-level upon stimulation. The chemosensory cells responded to odorants with a significant increase in the calcium concentration, preferentially in the dendritic knob. Applying various stimulation paradigma, it was found that in a population of isolated cells, subsets of receptor neurons display distinct patterns of responsiveness.

Topics: Aniline Compounds; Animals; Benzofurans; Biological Transport; Calcium; Dose-Response Relationship, Drug; Fluorescent Dyes; Imidazoles; In Vitro Techniques; Lasers; Microscopy, Confocal; Microscopy, Fluorescence; Odorants; Olfactory Receptor Neurons; Rats; Rats, Sprague-Dawley; Signal Transduction; Xanthenes

A continuous, spectrophotometric assay to simultaneously measure Ca uptake and ATP hydrolysis has been developed, in order to assess the function of the Ca-ATPase in skeletal and cardiac sarcoplasmic reticulum (SR) vesicles. The absorbance of Fura Red was measured continuously at 490 nm, in EGTA-buffered solutions containing initial free ionized calcium concentrations of 300 nM, 500 nM, 790 nM, and 2 microM, during assays of oxalate-facilitated or phosphate-facilitated active calcium uptake in skeletal SR. Simultaneous measurement of ATP hydrolysis during the measurement of phosphate-facilitated Ca uptake was accomplished by measuring the disappearance of NADH at 340 nm, coupled to the hydrolysis of ATP by an enzyme-linked, continuous ATPase assay. This new method, unlike the standard 45Ca-filtration assay, measures calcium uptake in real time and eliminates the need for radioactivity. Moreover, the rates of calcium uptake and ATP hydrolysis are measured simultaneously, allowing the direct quantitative comparison of the two parameters. This assay will facilitate the characterization of Ca-ATPase function and malfunction in skeletal and cardiac SR and advances the methodology for comparison of normal and physically, chemically, or biologically altered Ca-ATPase.

Topics: Adenosine Triphosphate; Animals; Benzofurans; Calcium; Calcium-Transporting ATPases; Fluorescent Dyes; Hydrolysis; Imidazoles; Muscle, Skeletal; Myocardium; Rabbits; Sarcoplasmic Reticulum; Spectrophotometry

We have investigated, at the single cell level, intracellular Ca2+ ([Ca2+]i) modulations triggered by the high affinity receptor for IgG, Fc gamma RI, in the monocytic cell line, U937. Cells were co-loaded with the Ca(2+)-sensitive dyes, Fluo-3 and Fura-Red, by incubation with their acetoxymethyl (AM) esters and confocal ratio imaging was used to monitor the [Ca2+]i changes induced by antibody cross-linking of IgG-loaded Fc gamma RI. A single Ca2+ spike was observed in 81% of untreated cells whereas dibutyryl cAMP-induced differentiation into a more macrophage cell type resulted in a sub-population of cells (44%) responding to receptor cross-linking with calcium oscillations. This change in calcium signalling may explain the difference in functional responses triggered by Fc gamma RI in monocytes and macrophages. Analysis of the Fluo-3 and Fura-Red fluorescence, after AM-ester loading, showed that both dyes have similar photobleach rates and intracellular localization allowing compensation for shifts in focal plane, dye photobleaching and non-uniformity of dye loading. In addition, because the binding kinetics of both dyes are equivalent, accurate temporal information can be gained about [Ca2+] changes. There are, however, two major problems with this dual indicator technique. Firstly, loading from AM esters results in considerable variation between cells in the intracellular concentration ratio of the two dyes, making calibration difficult. Secondly, the fluorescence ratio, Fluo-3/Fura-Red, behaves non-linearly at Ca2+ concentrations less than approximately 500 nM and comparison with Fura-2-loaded single cell photometry studies suggests there is considerable amplitude distortion of the signal when the ratios are displayed on a linear scale. These problems may considerably limit the application of Fluo-3/Fura-Red ratiometric measurements.

Topics: Adenosine Triphosphate; Aniline Compounds; Benzofurans; Calcium; Cell Differentiation; Fluorescent Dyes; Humans; Imidazoles; Immunoglobulin G; Microscopy, Confocal; Receptors, Fc; Tumor Cells, Cultured; Xanthenes

Measurement of changes in intracellular ionized calcium concentrations ([Ca2+]i) has proved to be of wide use in the study of cellular responses to activating stimuli. The fluorescent dye Indo-1 has successfully been used in flow cytometry for this purpose, and when used as a ratiometric indicator it provides optimum sensitivity and accuracy. Unfortunately, this dye requires ultraviolet (UV) excitation which is often not available. We show here that similar results can be obtained using a ratio of green to red fluorescence from the simultaneous loading of the dyes Fura Red and fluo-3. Both Fura Red and fluo-3 are excited using the commonly available blue 488 nm laser line. With appropriate concentrations of the two dyes, the magnitude of response with the fluo-3/Fura Red ratio is greater than that achieved with indo-1, while the intercellular variation in measurement is similar to that seen with indo-1. Analyses can be simultaneously combined with immunofluorescent detection of PE-labeled antibodies to enable [Ca2+]i measurement within cell subsets.

Topics: Aniline Compounds; Benzofurans; Calcium; Flow Cytometry; Fluorescent Dyes; Humans; Imidazoles; In Vitro Techniques; Indoles; Intracellular Fluid; Lymphocytes; Sensitivity and Specificity; Xanthenes

The intracellular calcium concentration [Ca2+]i in olfactory receptor neurones of Xenopus laevis was imaged with high spatial and temporal resolution. A new method using a mixture of the calcium indicator dyes Fluo-3 and Fura-Red was employed. The fluorescence patterns in two wavelength bands were measured on the emission side of a confocal laser scanning microscope, and the ratio R of the fluorescence intensities was taken as an estimate of [Ca2+]i. When the neurones were depolarized by elevating the extracellular potassium concentration [K+]o they showed one of three types of responses: a fast increase in [Ca2+]i, a slow increase in [Ca2+]i, or no change in [Ca2+]i. The fast increase in [Ca2+]i took place in the soma compartment. For at least 4 s after the onset of depolarization the calcium distribution in the dendrite remained essentially unchanged. To study the fast increase with high time resolution, line scan images were taken. The neurones were depolarized for brief periods applying a solution containing high [K+] onto the soma from an application pipette. The fast increase in [Ca2+]i began with a delay of about 200 ms and went from the resting concentration to about 110 nM above resting concentration. Following the depolarization, recovery from elevated [Ca2+]i to resting levels had a time constant of about 15 s. The slow response seemed to depend on the removal of [Na+] from the bath rather than on the elevated [K+] in the bath. The response was also observed with Cd2+, Ni2+, and Co2+ (1.5 mM each) in the bath.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aniline Compounds; Animals; Benzofurans; Calcium Channels; Imidazoles; In Vitro Techniques; Microscopy, Fluorescence; Olfactory Receptor Neurons; Potassium; Xanthenes; Xenopus laevis

The regenerative Ca(2+)-induced Ca2+ release mechanism is an important amplifier of signal transduction in diverse cells. In heart muscle cells, this mechanism contributes to the Ca2+ transient activating the mechanical contraction, but it is also believed to drive Ca2+ waves propagating within the cytosol. We investigated the subcellular Ca2+ distribution in heart muscle cells during spontaneous Ca2+ release using laser scanning confocal microscopy with a ratiometric fluorescent indicator technique. Besides planar Ca2+ waves with linear propagation, sequences of confocal optical sections also revealed spiral Ca2+ waves spinning around a subcellular core at approximately 1 Hz. Although the Ca2+ spirals were continuous processes they frequently exhibited an apparently oscillatory output function into the elongated cell body. These oscillatory waves emanating from the spiral at regular intervals were formally considered to be short outer segments of the spiral but could not be distinguished from planar Ca2+ waves propagating along the longitudinal cell axis. The complex spatiotemporal pattern of spiral Ca2+ waves implies the participation of an active process exhibiting a large degree of positive feedback, most likely the Ca(2+)-induced Ca2+ release mechanism.

Topics: Aniline Compounds; Animals; Benzofurans; Calcium; Cells, Cultured; Feedback; Fluorescent Dyes; Guinea Pigs; Heart; Imidazoles; Microscopy, Fluorescence; Models, Cardiovascular; Signal Transduction; Subcellular Fractions; Xanthenes

Topics: Benzofurans; Calcium; Fluorescent Dyes; Fura-2; Imidazoles; Muscles; Sarcoplasmic Reticulum; Spectrometry, Fluorescence

Fura red, a fluorescent Ca2+ indicator with absorbance bands at visible wavelengths, was injected into intact single muscle fibers that had been stretched to a long sarcomere length (approximately 3.8 microns) and bathed in a 'high-Ca2+' Ringer ([Ca2+] = 11.8 mM). From fura red's slow diffusion coefficient in myoplasm, 0.16 (+/- 0.01, SEM) x 10(-6) cm2 s-1 (N = 5; 16 degrees C), it is estimated that approximately 85% of the indicator molecules are bound to muscle constituents of large molecular weight. Binding appears to elevate, by 3- to 4-fold, the indicator's apparent dissociation constant for Ca2+ (KD), which is estimated to be 1.1-1.6 microM in myoplasm. Fura red's myoplasmic absorbance spectrum was used to estimate fr, the fraction of fura red molecules in the Ca2+-bound form at rest. In 3 fibers thought to be minimally damaged by the micro-injection, fr was estimated to be 0.15 (+/- 0.01). Thus, resting myoplasmic free [Ca2+] ([Ca2+]r) is estimated to be 0.19-0.28 microM. For fibers in normal Ringer solution ([Ca2+] = 1.8 mM), at shorter sarcomere length (approximately 2.7 microns), and containing a nonperturbing concentration of indicator (< or = 0.2 mM), [Ca2+]r is estimated to be 0.18-0.27 microM. This range is higher than estimated previously in frog fibers with other techniques. In 6 fibers, R, the indicator's fluorescence ratio signal (equal to the emission intensity measured with 420 nm excitation divided by that measured with 480 nm excitation), was measured at rest and following electrical stimulation and compared with absorbance measurements made from the same fiber region. The analysis implies that RMIN and RMAX (the values of R that would be measured if all indicator molecules were in the Ca(2+)-free and Ca(2+)-bound states, respectively) were substantially smaller in myoplasm than in calibration solutions lacking muscle proteins. Several methods for estimation of [Ca2+]r from R are analyzed and discussed.

Topics: Animals; Benzofurans; Calcium; Cytoplasm; Fluorescent Dyes; Fructose-Bisphosphate Aldolase; Hydrogen-Ion Concentration; Imidazoles; In Vitro Techniques; Kinetics; Magnesium; Muscles; Rabbits; Rana catesbeiana; Rana temporaria; Sarcomeres; Spectrometry, Fluorescence; Spectrophotometry