benzofurans and indo-1

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

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

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

We studied the effect of ionotropic glutamate receptor agonists on the release of endogenous glutamate or of [3H]D-aspartate from reaggregate cultures (retinospheroids) or from monolayer cultures of chick retinal cells, respectively. Kainate increased the fluorescence ratio of the Na+ indicator SBFI and stimulated a dose-dependent release of glutamate in low (0.1 mM) Ca2+ medium, as measured using a fluorometric assay. Under the same experimental conditions, the release evoked by N-methyl-D-aspartate (NMDA; 400 microM) was about half of that evoked by the same kainate concentration; alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid (AMPA; 400 microM) did not trigger a significant response. In the presence of 1 mM CaCl2, all of the agonists increased the [Ca2+]i, as determined with the fluorescence dye Indo-1, but the glutamate release evoked by NMDA and kainate was significantly lower than that measured in 0.1 mM CaCl2 medium. Inhibition by Ca2+ of the kainate-stimulated release of glutamate was partially reversed by the phospholipase A2 inhibitor oleiloxyethyl phosphorylcholine (OPC), suggesting that the effect was mediated by the release of arachidonic acid, which inhibits the glutamate carrier. Accordingly, kainate, NMDA, and AMPA stimulated a Ca(2+)-dependent release of [3H]arachidonic acid, and the direct addition of the exogenous fatty acid to the medium decreased the release of glutamate evoked by kainate in low (0.1 mM) CaCl2 medium. In monolayer cultures, we showed that NMDA, kainate, and AMPA also stimulated the release of [3H]D-aspartate, but in this case release in the presence of 1 mM CaCl2 was significantly higher than that evoked in media with no added Ca2+. The ranking order of efficacy for stimulation of Ca(2+)-dependent release of [3H]D-aspartate was NMDA > > kainate > AMPA.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Arachidonic Acid; Benzofurans; Calcium Chloride; Cells, Cultured; Chick Embryo; Ethers, Cyclic; Excitatory Amino Acid Agonists; Fluorescent Dyes; Glutamic Acid; Indoles; Kainic Acid; Kinetics; N-Methylaspartate; Receptors, Glutamate; Retina; Sodium; Spectrometry, Fluorescence

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

1. In skeletal muscle there is generally a slowing of relaxation with increasing tetanus duration and it has been suggested that this is due to Ca2+ loading of parvalbumin (PA). To study this we have produced prolonged tetani in intact, single fibres from a mouse foot muscle which contain a high concentration of PA. We measured the rate of tension relaxation and also various aspects of Ca2+ handling. 2. During 'interrupted' tetani (15 repeated cycles of 100 ms with stimulation and 50 ms without) we observed a marked slowing of the relaxation both under control conditions and in acidosis (obtained by increasing the bath CO2 content). This slowing was not accompanied by any reduction of the initial rate of decline of the free myoplasmic Ca2+ concentration ([Ca2+]i), which was measured with indo-1. 3. The functioning of the sarcoplasmic reticulum (SR) pump after tetani of various durations was analysed by plotting d[Ca2+]i/dt vs. [Ca2+]i during the final slow decline of [Ca2+]i after tetani. This analysis showed that the rate of SR Ca2+ pumping after a 1 s tetanus is less than half of that after a 100 ms tetanus. 4. The amplitude of the tail of [Ca2+]i 250 ms into relaxation was measured after tetani of various durations. This amplitude increased with tetanus duration and could be fitted to the sum of one exponential and one linear function. The exponential component increased with a time constant of 0.17 s and probably reflects Ca2+ loading of PA. 5. Ca2+ binding to PA will displace Mg2+ and hence the free myoplasmic concentration of Mg2+ ([Mg2+]i) will increase. To study this we used the fluorescent Mg2+ indicator furaptra. The results showed an increase of [Mg2+]i during prolonged tetani which, after removing the Ca2+ component of the fluorescent signal, amounted to about 0.5 mM. 6. A model of Ca2+ movements and tension production in skeletal muscle was used. The model showed that the increase of the amplitude of [Ca2+]i tails after tetani of various durations can be explained by the combined effect of Ca2+ loading of PA and slowed SR Ca2+ pumping. In contrast to the experimental data, the model predicted a slight reduction of the initial rate of [Ca2+]i decline with increased tetanus duration. 7. In conclusion, we observed a marked slowing of relaxation during prolonged tetanic stimulation. Altered Ca2+ handling, including Ca2+ loading of PA, seems not to be important for this slowing. Thus, the slowing appears to be due to altered cross-bridge kinetics.

Topics: Acidosis; Animals; Benzofurans; Calcium; Electric Stimulation; Fluorescent Dyes; Fura-2; In Vitro Techniques; Indoles; Magnesium; Male; Mice; Muscle Fibers, Skeletal; Muscle Relaxation; Muscle, Skeletal; Oxazoles; Parvalbumins; Sarcoplasmic Reticulum; Troponin

Na(+)-Ca2+ exchange has been shown to contribute to reperfusion- and reoxygenation-induced cellular Ca2+ loading and damage in the heart. Despite the fact that both [Na+]i and [Ca2+]i have been documented to rise during ischemia and hypoxia, it remains unclear whether the rise in [Ca2+]i occurring during hypoxia is linked to the rise in [Na+]i via Na(+)-Ca2+ exchange before reoxygenation and how this relates to cellular injury. Single electrically stimulated (0.2 Hz) adult rat cardiac myocytes loaded with Na(+)-sensitive benzofuran isophthalate (SBFI), the new fluorescent probe, were exposed to glucose-free hypoxia (PO2 less than 0.02 mm Hg), and SBFI fluorescence was monitored to index changes in [Na+]i. Parallel experiments were performed with indo-1-loaded cells to index [Ca2+]i. The SBFI fluorescence ratio (excitation, 350/380 nm) rose significantly during hypoxia after the onset of ATP-depletion contracture, consistent with a rise in [Na+]i. At reoxygenation, the ratio fell rapidly toward baseline levels. The indo-1 fluorescence ratio (emission, 410/490 nm) also rose only after the onset of rigor contracture and then often showed a secondary rise early after reoxygenation at a time when [Na+]i fell. The increase in both [Na+]i and [Ca2+]i, seen during hypoxia, could be markedly reduced by performing experiments in Na(+)-free buffer. These experiments suggested that hypoxic Ca2+ loading is linked to a rise in Na+i via Na(+)-Ca2+ exchange. To show that Na(+)-Ca2+ exchange activity was not fully inhibited by profound intracellular ATP depletion, cells were exposed to cyanide, and then buffer Na+ was abruptly removed after contracture occurred. The sudden removal of buffer Na+ would be expected to stimulate cell Ca2+ entry via Na(+)-Ca2+ exchange. A large rapid rise in the indo-1 fluorescence ratio ensued, which was consistent with abrupt cell Ca2+ loading via the exchanger. The effect of reducing hypoxic buffer [Na+] on cell morphology after reoxygenation was examined. Ninety-five percent of cells studied in a normal Na(+)-containing buffer (144 mM NaCl, n = 38) and reoxygenated 30 minutes after the onset of hypoxic rigor underwent hypercontracture. Only 12% of cells studied in Na(+)-free buffer (144 mM choline chloride, n = 17) hypercontracted at reoxygenation (p less than 0.05). Myocytes were also exposed to hypoxia in the presence of R 56865, a compound that blocks noninactivating components of the Na+ current. R 56865 blunted the rise in [Na+]i typi

Topics: Animals; Benzofurans; Benzothiazoles; Calcium; Cell Hypoxia; Ethers, Cyclic; Hydrogen-Ion Concentration; Indoles; Ischemia; Myocardium; Piperidines; Rats; Sodium; Sodium Channels; Sodium-Potassium-Exchanging ATPase; Thiazoles

Fluorescent calcium indicators fluo-3, fura-2 and indo-1, and fluorescent magnesium indicators mag-fura-2 (FURAPTRA) and mag-indo-1 were evaluated for the effects of pH on their association and dissociation rates, ion selectivity and thermodynamic properties. Calcium indicator affinities for Ca and Mg were reduced and the discrimination between Ca and Mg decreased in fura-2 and indo-1 at acidic pH. Alterations in apparent dissociation constants were caused primarily by reduced association rates. Magnesium indicators did not show these changes. The enthalphies of the calcium indicators' Ca complex were 1-3 kcal/mole and magnesium indicators' Mg complex were 7-9 kcal/mole. The potential effects of a biexponential dissociation rate of fluo-3 and of Ca interactions with magnesium indicators were examined.

Topics: Aniline Compounds; Benzofurans; Calcium; Fluorescent Dyes; Fura-2; Hydrogen-Ion Concentration; Indoles; Magnesium; Oxazoles; Spectrometry, Fluorescence; Thermodynamics; Xanthenes

A novel method of determining the apparent dissociation constants of fluorescent calcium indicators is described which utilizes Chelex-100 ion exchange resin and 45Ca. The affinity for calcium of indicators fluo-3, fura-2 and indo-1 measured at either 22 degrees or 37 degrees C decreases as pH is decreased from 7.4 to 5.5. These measurements agree with determinations made using EDTA-calcium buffers. The 1:1 calcium:indicator complex is maintained under all conditions. The necessity to correct dissociation constants during intracellular acidification to properly interpret fluorescence measurements is illustrated by indo-1 measurements in the ischemic rat heart.

Topics: Aniline Compounds; Benzofurans; Calcium; Chelating Agents; Edetic Acid; Fura-2; Hydrogen-Ion Concentration; Indoles; Resins, Synthetic; Temperature; Xanthenes

The effects of different concentrations of the fluorometric Ca2+ probes, fura-2 and indo-1, on Ca2+ transients in cultured rat aortic smooth muscle cells were examined. When stimulated with the agonists, angiotensin II and arginine vasopressin, cells incubated with low concentrations of fura-2 or indo-1 (less than 1 microM) produced Ca2+ transients characterized by a small increase followed by a dramatic decrease in fluorescence below the original baseline. This effect of agonists was concentration-dependent, reversible, and blocked by receptor antagonists. In contrast to the agonists, stimulation of Ca2+ transients with depolarizing concentrations of K+ or with caffeine did not produce decreases in fluorescence and Ca2+ levels at any loading concentration of probe. The decrease in Ca2+ observed with agonists was dependent on the presence of extracellular Na+. These data suggest that under certain loading conditions, fluorescent Ca2+ indicators measure agonist-stimulated Ca2+ efflux mediated by a Na+/Ca2+ exchange mechanism.

Topics: Animals; Aorta; Benzofurans; Calcium; Fluorescent Dyes; Fluorometry; Fura-2; Indoles; Muscle, Smooth, Vascular; Rats; Sodium

The highly fluorescent probes Indo-1 and Fura-2 were employed to detect intracellular calcium responses in murine splenic lymphocytes following cross-linking of cell surface Ig. Inhibition by phorbol ester (12-O-tetradecanoylphorbol 13-acetate) was rapid and showed a strong preference for the very transient phase of the response which has been identified as a mobilization of intracellular calcium. 12-O-Tetradecanoylphorbol-13-acetate had significantly less effect on the longer lasting increase in intracellular free calcium which involved an influx of extracellular calcium. Whole spectra were used as a check on transients, which were monitored at a single wavelength, in order to eliminate changes that were not calcium-dependent. It was found that such changes could arise from the association of Indo-1, or its acetoxymethyl ester, with phospholipid bilayers since this affected their fluorescence spectra. In addition, the loading of resting cells with dye esters was shown to be greatly enhanced by the inclusion of a small amount of the detergent Pluronic F-127 in the incubation medium. A spectral analysis of labeled cells showed that the extent of hydrolysis of intracellular dye was improved as well as the rate of uptake by cells.

Topics: Algorithms; Animals; Antibodies, Anti-Idiotypic; Benzofurans; Calcium; Female; Fura-2; Hydrolysis; Immunoglobulins; Indoles; Lymphocytes; Mice; Mice, Inbred BALB C; Poloxalene; Spectrophotometry; Tetradecanoylphorbol Acetate

Using ratio-imaging fluorescence microscopy, we have investigated the changes in intracellular Ca2+ [( Ca2+]i) that occurred in cytotoxic T lymphocytes (CTL) upon target-cell binding. This process resulted in a rapid increase in [Ca2+]i, which was localized in the region of the CTL in contact with the target cell. This increase was mediated both by influx from the external medium as well as by release from intracellular stores. Although the magnitude of the initial increase in [Ca2+]i was not dependent upon the presence of extracellular Ca2+, influx was necessary for sustained elevation of [Ca2+]i. Inasmuch as target-cell lysis by the CTL clone used is dependent on extracellular Ca2+, this suggests that a prolonged elevation of [Ca2+]i is necessary for lytic function. It was also shown that the increase in [Ca2+]i and its subsequent decay show several pulsations. The mechanism by which these variations are generated and their possible function is not known. Finally, a role for K+ efflux in the control of the antigen-induced increase in [Ca2+]i was demonstrated. Thus it is becoming clear that signal transduction in CTL is remarkably complex, involving several ionic species and second messengers.

Topics: Animals; Benzofurans; Calcium; Cell Line; Cytotoxicity, Immunologic; Fluorescent Dyes; Fura-2; Indoles; Kinetics; Mice; Microscopy, Fluorescence; Signal Transduction; Spectrometry, Fluorescence; T-Lymphocytes, Cytotoxic

Rat liver mitochondria are shown to convert the acetoxymethyl ester forms of fura-2 and indo-1 into Ca2+-dependent forms of these indicators. The excitation spectrum of the Ca2+-dependent conversion product of fura-2 acetoxymethyl ester is shown to be similar to that of the pentacarboxylic acid form of fura-2. A systematic investigation of the ionic strength and pH dependences of the fluorescence of the pentacarboxylic acid forms of these indicators shows small changes within the ranges thought to obtain within the mitochondrial matrix after Ca2+ uptake. Intramitochondrial free Ca2+ levels are studied both before and after Ca2+ sequestration by mitochondria, and a rough estimate is made of the mitochondrial contribution to the Ca2+-dependent fura-2 fluorescence of a hepatocyte suspension.

Topics: Animals; Benzofurans; Biotransformation; Calcium; Esters; Fluorescent Dyes; Fura-2; Indoles; Kinetics; Mitochondria, Liver; Rats; Spectrometry, Fluorescence

The kinetics of Ca2+ dissociation from fura-2 and indo-1 were measured using a stopped-flow spectrofluorimeter. The dissociation rate constants were 84 s-1 and 130 s-1, respectively, in 0.1 M KC1 at 20 degrees C. The rate constants were insensitive to pH over the range 7.0 to 8.0. The second order association rate constants were estimated indirectly to be in the region of 5 X 10(8) M-1 X s-1 and thus approach the diffusion-controlled limit. The results demonstrate that these new generation indicators are well-suited to measure rapid changes in concentration of intracellular Ca2+.

Topics: Benzofurans; Calcium; Edetic Acid; Egtazic Acid; Fura-2; Indoles; Kinetics; Magnesium; Spectrometry, Fluorescence

The role of changes in intracellular [Ca2+]i as a second messenger in response to either adrenergic or cholinergic agonists was determined in isolated bovine corneal epithelial cells. [Ca2+]i was measured in suspensions of cells loaded with either of the fluorescent indicators quin2 or indo-1, as well as in single cells loaded with fura-2. Fluorescence from the cell suspensions was measured in a spectrofluorometer while single cell fluorescence was measured using a modified fluorescence microscope with a photon counting photometer. Cells were loaded with these dyes by incubation in Ringer's (pH 8.1) containing 2-50 microM of the acetoxymethyl ester of the indicator. Fluorescence was measured before and after exposure to either, one of the adrenergic agonists isoproterenol, phenylephrine or epinephrine, or the cholinergic agonist carbachol. The resting [Ca2+]i level from the quin2 experiments was 115 nM +/- 41 nM (SEM) (n = 23) whereas with fura-2 it was 71 +/- 10 nM (n = 30). In no case did we see any change in [Ca2+]i within 15 min after addition of any agonist but we were able to observe increased calcium when 0.5 microM ionomycin was added to either the same or untreated cells. The disparity in the resting levels determined by the two methods may result from various calibration problems. Our results indicate that changes in [Ca2+]i have no second messenger role in response to these agonists.

Topics: Aminoquinolines; Animals; Benzofurans; Calcium; Cattle; Cornea; Epithelial Cells; Epithelium; Fura-2; Indoles; Parasympathomimetics; Sympathomimetics

Previous attempts to measure cytoplasmic Ca2+ in plant cells using the new generation of fluorescent probes, indo-1 and fura-2, have been unsuccessful. We investigated the use of indo-1 and fura-2 to measure cytoplasmic Ca2+ in barley aleurone protoplasts and found that indo-1 could be successfully used when it was loaded into protoplasts in the Ca2+-sensitive form. The acetoxymethyl esters of both dyes accumulated in aleurone protoplasts, but fura-2 was sequestered in the vacuole and indo-1 was not adequately hydrolyzed. We developed a non-disruptive method for loading the Ca2+-sensitive form of indo-1 into aleurone protoplasts in mildly acidic solutions. Using this approach, protoplasts accumulate indo-1 in a pH-dependent manner. The accumulated dye is Ca2+-sensitive, it is not sequestered in vacuoles or the endomembrane system, and it is not rapidly secreted. Fluorescence from indo-1 in individual cells was quenched by Mn2+ in the presence of digitonin. We estimate the cytoplasmic Ca2+ concentration in aleurone protoplasts to be approximately 250 nM. The Ca2+ ionophore, ionomycin does not induce changes in the fluorescence of protoplasts loaded with indo-1, but fluorescence changes could be induced by changes in extracellular Ca2+ in the presence of digitonin. We conclude that the strategy of loading indo-1 at acidic pH provides a useful means of measuring cytoplasmic Ca2+ in the barley aleurone that may also be applicable to other types of plant cells.

Topics: Benzofurans; Calcium; Cytoplasm; Fluorescent Dyes; Fluorometry; Fura-2; Hordeum; Hydrogen-Ion Concentration; Indoles; Protoplasts