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Hiroshi Kitazato - One of the best experts on this subject based on the ideXlab platform.
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Real‐time visualization of calcium ion activity in shallow benthic foraminiferal cells using the Fluorescent indicator Fluo‐3 AM
Geochemistry Geophysics Geosystems, 2008Co-Authors: Takashi Toyofuku, Lennart Jan De Nooijer, Hiroyuki Yamamoto, Hiroshi KitazatoAbstract:[1] Calcium ion storage and movements within foraminiferal cells were observed using the Fluorescent cell-permeant calcium indicator Fluo-3 AM. Living specimens of the shallow-water benthic foraminifer Ammonia beccarii were incubated with 8 μM Fluo-3 AM in both natural seawater and calcium-free artificial seawater. No Fluorescence was observed in specimens that were incubated in Fluo-3 AM/calcium-free seawater, while Fluorescent emission was identified in all foraminifera that were incubated in Fluo-3 AM/natural seawater. In another series of experiments, juvenile specimens were incubated with Fluo-3 AM to trace calcium ion activity during the process of chamber formation. The method described here is a potential tool to observe the flow of calcium ions within living foraminiferal cells and may yield valuable information on the process of calcite precipitation.
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real time visualization of calcium ion activity in shallow benthic foraminiferal cells using the Fluorescent indicator Fluo 3 am
Geochemistry Geophysics Geosystems, 2008Co-Authors: Takashi Toyofuku, Lennart Jan De Nooijer, Hiroyuki Yamamoto, Hiroshi KitazatoAbstract:[1] Calcium ion storage and movements within foraminiferal cells were observed using the Fluorescent cell-permeant calcium indicator Fluo-3 AM. Living specimens of the shallow-water benthic foraminifer Ammonia beccarii were incubated with 8 μM Fluo-3 AM in both natural seawater and calcium-free artificial seawater. No Fluorescence was observed in specimens that were incubated in Fluo-3 AM/calcium-free seawater, while Fluorescent emission was identified in all foraminifera that were incubated in Fluo-3 AM/natural seawater. In another series of experiments, juvenile specimens were incubated with Fluo-3 AM to trace calcium ion activity during the process of chamber formation. The method described here is a potential tool to observe the flow of calcium ions within living foraminiferal cells and may yield valuable information on the process of calcite precipitation.
M J Croucher - One of the best experts on this subject based on the ideXlab platform.
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comparison of human recombinant adenosine a2b receptor function assessed by Fluo 3 am Fluorometry and microphysiometry
British Journal of Pharmacology, 2003Co-Authors: H Patel, R H P Porter, A M Palmer, M J CroucherAbstract:The aim of this study was to establish the utility of a Fluorometric imaging plate reader (FLIPR) assay to assess human adenosine A2B receptor function by characterizing its receptor pharmacology and comparing this profile to that obtained using a microphysiometer. FLIPR was used, in conjunction with a Ca2+-sensitive dye (Fluo-3-AM), to measure rapid rises in intracellular calcium in a Chinese Hamster Ovary (CHO-K1) cell line stably transfected with both the human A2B receptor and a promiscuous Gα16 protein. Microphysiometry was used to measure rapid changes in the rate of extracellular acidification in a Human Embryonic Kidney (HEK-293) cell line also stably transfected with human A2B receptor. Activation of A2B receptors by various ligands caused a concentration-dependent increase in both the intracellular calcium concentration and the extracellular acidification rate in the cells tested, with a similar rank order of potency for agonists: NECA>N6-Benzyl NECA>adenosine⩾R-PIA>CPA>S-PIA>CHA>CGS 21680. No comparable effects were observed in the non-transfected control cell lines. The rank order of potency of the agonists examined was the same in all studies, whereas absolute potency and efficacy varied. Thus, all compounds exhibited greater potency in FLIPR than the microphysiometer and the efficacies obtained with CHO-K1+Gα16+A2B cell line and FLIPR were greater than those obtained with HEK-293+A2B cell line in the microphysiometer. ZM-241385 was the most potent of a range of adenosine antagonists tested with a pA2 of 8.0 in both the FLIPR and microphysiometer assays. In conclusion, the profile of the responses to both A2B receptor agonists and antagonists in FLIPR were similar to those obtained by the microphysiometer, although both potency and efficacy values were higher in the FLIPR assay. With this caveat in mind, this study shows that FLIPR coupled with a cell line transfected with both the human A2B receptor and a promiscuous Gα16 protein provides a useful, high throughput method for the assessment of A2B receptor function. Keywords: Adenosine, receptors, A2B, microphysiometry, Fluorescence, receptor coupling Introduction The endogenous nucleoside adenosine mediates its action on cells through at least four G-protein-coupled receptor subtypes, which have been classified as A1, A2A, A2B and A3 (Linden & Jacobson, 1998; Ralevic & Burnstock, 1998). Adenosine A2B receptors have been implicated in neurosecretion, intestinal function, asthma and inflammation (Feoktistov et al., 1998b). Elucidation of the pharmacology of A2B receptors has lagged behind that of the A1 and A2A subtypes, largely because of the absence of A2B receptor-specific ligands. The assessment of A2B receptor pharmacology has also relied largely on the measurement of cyclic AMP accumulation or adenylate cyclase activity (Martin, 1992; Brackett & Daly, 1994; Peakman & Hill, 1994; Klotz et al., 1998; Feoktistov & Biaggioni, 1997; 1998). FLIPR is increasingly being used to assess the functional response of G protein-coupled receptors (Porter et al., 1999; Wood et al., 1999; Jerman et al., 2000), particularly when the receptor of interest is co-expressed with the promiscuous G-protein Gα16 to couple the receptor responses to phospholipase C (Milligan et al., 1996). The present study represents the first assessment of human A2B receptor function using a calcium-sensitive dye (Fluo-3-AM) coupled with a Fluorometric imaging plate reader (FLIPR). The pharmacological characteristics of the A2B receptor have been defined and compared with those obtained by microphysiometry, a method that uses small alterations in pH as a signal of changes in cellular metabolism induced by a variety of ligand-receptor interactions, including G-protein linked receptors (Owicki et al., 1990; McConnell et al., 1992; Coldwell et al., 1999). A preliminary report of this work has previously been published (Randall et al., 2000).
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Comparison of human recombinant adenosine A2B receptor function assessed by Fluo‐3‐AM Fluorometry and microphysiometry
British journal of pharmacology, 2003Co-Authors: H Patel, R H P Porter, A M Palmer, M J CroucherAbstract:The aim of this study was to establish the utility of a Fluorometric imaging plate reader (FLIPR) assay to assess human adenosine A2B receptor function by characterizing its receptor pharmacology and comparing this profile to that obtained using a microphysiometer. FLIPR was used, in conjunction with a Ca2+-sensitive dye (Fluo-3-AM), to measure rapid rises in intracellular calcium in a Chinese Hamster Ovary (CHO-K1) cell line stably transfected with both the human A2B receptor and a promiscuous Gα16 protein. Microphysiometry was used to measure rapid changes in the rate of extracellular acidification in a Human Embryonic Kidney (HEK-293) cell line also stably transfected with human A2B receptor. Activation of A2B receptors by various ligands caused a concentration-dependent increase in both the intracellular calcium concentration and the extracellular acidification rate in the cells tested, with a similar rank order of potency for agonists: NECA>N6-Benzyl NECA>adenosine⩾R-PIA>CPA>S-PIA>CHA>CGS 21680. No comparable effects were observed in the non-transfected control cell lines. The rank order of potency of the agonists examined was the same in all studies, whereas absolute potency and efficacy varied. Thus, all compounds exhibited greater potency in FLIPR than the microphysiometer and the efficacies obtained with CHO-K1+Gα16+A2B cell line and FLIPR were greater than those obtained with HEK-293+A2B cell line in the microphysiometer. ZM-241385 was the most potent of a range of adenosine antagonists tested with a pA2 of 8.0 in both the FLIPR and microphysiometer assays. In conclusion, the profile of the responses to both A2B receptor agonists and antagonists in FLIPR were similar to those obtained by the microphysiometer, although both potency and efficacy values were higher in the FLIPR assay. With this caveat in mind, this study shows that FLIPR coupled with a cell line transfected with both the human A2B receptor and a promiscuous Gα16 protein provides a useful, high throughput method for the assessment of A2B receptor function. Keywords: Adenosine, receptors, A2B, microphysiometry, Fluorescence, receptor coupling Introduction The endogenous nucleoside adenosine mediates its action on cells through at least four G-protein-coupled receptor subtypes, which have been classified as A1, A2A, A2B and A3 (Linden & Jacobson, 1998; Ralevic & Burnstock, 1998). Adenosine A2B receptors have been implicated in neurosecretion, intestinal function, asthma and inflammation (Feoktistov et al., 1998b). Elucidation of the pharmacology of A2B receptors has lagged behind that of the A1 and A2A subtypes, largely because of the absence of A2B receptor-specific ligands. The assessment of A2B receptor pharmacology has also relied largely on the measurement of cyclic AMP accumulation or adenylate cyclase activity (Martin, 1992; Brackett & Daly, 1994; Peakman & Hill, 1994; Klotz et al., 1998; Feoktistov & Biaggioni, 1997; 1998). FLIPR is increasingly being used to assess the functional response of G protein-coupled receptors (Porter et al., 1999; Wood et al., 1999; Jerman et al., 2000), particularly when the receptor of interest is co-expressed with the promiscuous G-protein Gα16 to couple the receptor responses to phospholipase C (Milligan et al., 1996). The present study represents the first assessment of human A2B receptor function using a calcium-sensitive dye (Fluo-3-AM) coupled with a Fluorometric imaging plate reader (FLIPR). The pharmacological characteristics of the A2B receptor have been defined and compared with those obtained by microphysiometry, a method that uses small alterations in pH as a signal of changes in cellular metabolism induced by a variety of ligand-receptor interactions, including G-protein linked receptors (Owicki et al., 1990; McConnell et al., 1992; Coldwell et al., 1999). A preliminary report of this work has previously been published (Randall et al., 2000).
Takashi Toyofuku - One of the best experts on this subject based on the ideXlab platform.
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Real‐time visualization of calcium ion activity in shallow benthic foraminiferal cells using the Fluorescent indicator Fluo‐3 AM
Geochemistry Geophysics Geosystems, 2008Co-Authors: Takashi Toyofuku, Lennart Jan De Nooijer, Hiroyuki Yamamoto, Hiroshi KitazatoAbstract:[1] Calcium ion storage and movements within foraminiferal cells were observed using the Fluorescent cell-permeant calcium indicator Fluo-3 AM. Living specimens of the shallow-water benthic foraminifer Ammonia beccarii were incubated with 8 μM Fluo-3 AM in both natural seawater and calcium-free artificial seawater. No Fluorescence was observed in specimens that were incubated in Fluo-3 AM/calcium-free seawater, while Fluorescent emission was identified in all foraminifera that were incubated in Fluo-3 AM/natural seawater. In another series of experiments, juvenile specimens were incubated with Fluo-3 AM to trace calcium ion activity during the process of chamber formation. The method described here is a potential tool to observe the flow of calcium ions within living foraminiferal cells and may yield valuable information on the process of calcite precipitation.
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real time visualization of calcium ion activity in shallow benthic foraminiferal cells using the Fluorescent indicator Fluo 3 am
Geochemistry Geophysics Geosystems, 2008Co-Authors: Takashi Toyofuku, Lennart Jan De Nooijer, Hiroyuki Yamamoto, Hiroshi KitazatoAbstract:[1] Calcium ion storage and movements within foraminiferal cells were observed using the Fluorescent cell-permeant calcium indicator Fluo-3 AM. Living specimens of the shallow-water benthic foraminifer Ammonia beccarii were incubated with 8 μM Fluo-3 AM in both natural seawater and calcium-free artificial seawater. No Fluorescence was observed in specimens that were incubated in Fluo-3 AM/calcium-free seawater, while Fluorescent emission was identified in all foraminifera that were incubated in Fluo-3 AM/natural seawater. In another series of experiments, juvenile specimens were incubated with Fluo-3 AM to trace calcium ion activity during the process of chamber formation. The method described here is a potential tool to observe the flow of calcium ions within living foraminiferal cells and may yield valuable information on the process of calcite precipitation.
H Patel - One of the best experts on this subject based on the ideXlab platform.
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comparison of human recombinant adenosine a2b receptor function assessed by Fluo 3 am Fluorometry and microphysiometry
British Journal of Pharmacology, 2003Co-Authors: H Patel, R H P Porter, A M Palmer, M J CroucherAbstract:The aim of this study was to establish the utility of a Fluorometric imaging plate reader (FLIPR) assay to assess human adenosine A2B receptor function by characterizing its receptor pharmacology and comparing this profile to that obtained using a microphysiometer. FLIPR was used, in conjunction with a Ca2+-sensitive dye (Fluo-3-AM), to measure rapid rises in intracellular calcium in a Chinese Hamster Ovary (CHO-K1) cell line stably transfected with both the human A2B receptor and a promiscuous Gα16 protein. Microphysiometry was used to measure rapid changes in the rate of extracellular acidification in a Human Embryonic Kidney (HEK-293) cell line also stably transfected with human A2B receptor. Activation of A2B receptors by various ligands caused a concentration-dependent increase in both the intracellular calcium concentration and the extracellular acidification rate in the cells tested, with a similar rank order of potency for agonists: NECA>N6-Benzyl NECA>adenosine⩾R-PIA>CPA>S-PIA>CHA>CGS 21680. No comparable effects were observed in the non-transfected control cell lines. The rank order of potency of the agonists examined was the same in all studies, whereas absolute potency and efficacy varied. Thus, all compounds exhibited greater potency in FLIPR than the microphysiometer and the efficacies obtained with CHO-K1+Gα16+A2B cell line and FLIPR were greater than those obtained with HEK-293+A2B cell line in the microphysiometer. ZM-241385 was the most potent of a range of adenosine antagonists tested with a pA2 of 8.0 in both the FLIPR and microphysiometer assays. In conclusion, the profile of the responses to both A2B receptor agonists and antagonists in FLIPR were similar to those obtained by the microphysiometer, although both potency and efficacy values were higher in the FLIPR assay. With this caveat in mind, this study shows that FLIPR coupled with a cell line transfected with both the human A2B receptor and a promiscuous Gα16 protein provides a useful, high throughput method for the assessment of A2B receptor function. Keywords: Adenosine, receptors, A2B, microphysiometry, Fluorescence, receptor coupling Introduction The endogenous nucleoside adenosine mediates its action on cells through at least four G-protein-coupled receptor subtypes, which have been classified as A1, A2A, A2B and A3 (Linden & Jacobson, 1998; Ralevic & Burnstock, 1998). Adenosine A2B receptors have been implicated in neurosecretion, intestinal function, asthma and inflammation (Feoktistov et al., 1998b). Elucidation of the pharmacology of A2B receptors has lagged behind that of the A1 and A2A subtypes, largely because of the absence of A2B receptor-specific ligands. The assessment of A2B receptor pharmacology has also relied largely on the measurement of cyclic AMP accumulation or adenylate cyclase activity (Martin, 1992; Brackett & Daly, 1994; Peakman & Hill, 1994; Klotz et al., 1998; Feoktistov & Biaggioni, 1997; 1998). FLIPR is increasingly being used to assess the functional response of G protein-coupled receptors (Porter et al., 1999; Wood et al., 1999; Jerman et al., 2000), particularly when the receptor of interest is co-expressed with the promiscuous G-protein Gα16 to couple the receptor responses to phospholipase C (Milligan et al., 1996). The present study represents the first assessment of human A2B receptor function using a calcium-sensitive dye (Fluo-3-AM) coupled with a Fluorometric imaging plate reader (FLIPR). The pharmacological characteristics of the A2B receptor have been defined and compared with those obtained by microphysiometry, a method that uses small alterations in pH as a signal of changes in cellular metabolism induced by a variety of ligand-receptor interactions, including G-protein linked receptors (Owicki et al., 1990; McConnell et al., 1992; Coldwell et al., 1999). A preliminary report of this work has previously been published (Randall et al., 2000).
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Comparison of human recombinant adenosine A2B receptor function assessed by Fluo‐3‐AM Fluorometry and microphysiometry
British journal of pharmacology, 2003Co-Authors: H Patel, R H P Porter, A M Palmer, M J CroucherAbstract:The aim of this study was to establish the utility of a Fluorometric imaging plate reader (FLIPR) assay to assess human adenosine A2B receptor function by characterizing its receptor pharmacology and comparing this profile to that obtained using a microphysiometer. FLIPR was used, in conjunction with a Ca2+-sensitive dye (Fluo-3-AM), to measure rapid rises in intracellular calcium in a Chinese Hamster Ovary (CHO-K1) cell line stably transfected with both the human A2B receptor and a promiscuous Gα16 protein. Microphysiometry was used to measure rapid changes in the rate of extracellular acidification in a Human Embryonic Kidney (HEK-293) cell line also stably transfected with human A2B receptor. Activation of A2B receptors by various ligands caused a concentration-dependent increase in both the intracellular calcium concentration and the extracellular acidification rate in the cells tested, with a similar rank order of potency for agonists: NECA>N6-Benzyl NECA>adenosine⩾R-PIA>CPA>S-PIA>CHA>CGS 21680. No comparable effects were observed in the non-transfected control cell lines. The rank order of potency of the agonists examined was the same in all studies, whereas absolute potency and efficacy varied. Thus, all compounds exhibited greater potency in FLIPR than the microphysiometer and the efficacies obtained with CHO-K1+Gα16+A2B cell line and FLIPR were greater than those obtained with HEK-293+A2B cell line in the microphysiometer. ZM-241385 was the most potent of a range of adenosine antagonists tested with a pA2 of 8.0 in both the FLIPR and microphysiometer assays. In conclusion, the profile of the responses to both A2B receptor agonists and antagonists in FLIPR were similar to those obtained by the microphysiometer, although both potency and efficacy values were higher in the FLIPR assay. With this caveat in mind, this study shows that FLIPR coupled with a cell line transfected with both the human A2B receptor and a promiscuous Gα16 protein provides a useful, high throughput method for the assessment of A2B receptor function. Keywords: Adenosine, receptors, A2B, microphysiometry, Fluorescence, receptor coupling Introduction The endogenous nucleoside adenosine mediates its action on cells through at least four G-protein-coupled receptor subtypes, which have been classified as A1, A2A, A2B and A3 (Linden & Jacobson, 1998; Ralevic & Burnstock, 1998). Adenosine A2B receptors have been implicated in neurosecretion, intestinal function, asthma and inflammation (Feoktistov et al., 1998b). Elucidation of the pharmacology of A2B receptors has lagged behind that of the A1 and A2A subtypes, largely because of the absence of A2B receptor-specific ligands. The assessment of A2B receptor pharmacology has also relied largely on the measurement of cyclic AMP accumulation or adenylate cyclase activity (Martin, 1992; Brackett & Daly, 1994; Peakman & Hill, 1994; Klotz et al., 1998; Feoktistov & Biaggioni, 1997; 1998). FLIPR is increasingly being used to assess the functional response of G protein-coupled receptors (Porter et al., 1999; Wood et al., 1999; Jerman et al., 2000), particularly when the receptor of interest is co-expressed with the promiscuous G-protein Gα16 to couple the receptor responses to phospholipase C (Milligan et al., 1996). The present study represents the first assessment of human A2B receptor function using a calcium-sensitive dye (Fluo-3-AM) coupled with a Fluorometric imaging plate reader (FLIPR). The pharmacological characteristics of the A2B receptor have been defined and compared with those obtained by microphysiometry, a method that uses small alterations in pH as a signal of changes in cellular metabolism induced by a variety of ligand-receptor interactions, including G-protein linked receptors (Owicki et al., 1990; McConnell et al., 1992; Coldwell et al., 1999). A preliminary report of this work has previously been published (Randall et al., 2000).
Stephen M Baylor - One of the best experts on this subject based on the ideXlab platform.
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Comparison of Simulated and Measured Calcium Sparks in Intact Skeletal Muscle Fibers of the Frog
The Journal of general physiology, 2002Co-Authors: Stephen M Baylor, Stephen Hollingworth, W. K. ChandlerAbstract:Calcium sparks in frog intact skeletal muscle fibers were modeled as stereotypical events that arise from a constant efflux of Ca2+ from a point source for a fixed period of time (e.g., 2.5 pA of Ca2+ current for 4.6 ms; 18°C). The model calculates the local changes in the concentrations of free Ca2+ and of Ca2+ bound to the major intrinsic myoplasmic Ca2+ buffers (troponin, ATP, parvalbumin, and the SR Ca2+ pump) and to the Ca2+ indicator (Fluo-3). A distinctive feature of the model is the inclusion of a binding reaction between Fluo-3 and myoplasmic proteins, a process that strongly affects Fluo-3′s Ca2+-reaction kinetics, its apparent diffusion constant, and hence the morphology of sparks. ΔF/F (the change in Fluo-3′s Fluorescence divided by its resting Fluorescence) was estimated from the calculated changes in Fluo-3 convolved with the microscope point-spread function. To facilitate comparisons with measured sparks, noise and other sources of variability were included in a random repetitive fashion to generate a large number of simulated sparks that could be analyzed in the same way as the measured sparks. In the initial simulations, the binding of Ca2+ to the two regulatory sites on troponin was assumed to follow identical and independent binding reactions. These simulations failed to accurately predict the falling phase of the measured sparks. A second set of simulations, which incorporated the idea of positive cooperativity in the binding of Ca2+ to troponin, produced reasonable agreement with the measurements. Under the assumption that the single channel Ca2+ current of a ryanodine receptor (RYR) is 0.5–2 pA, the results suggest that 1–5 active RYRs generate an average Ca2+ spark in a frog intact muscle fiber.
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resting myoplasmic free calcium in frog skeletal muscle fibers estimated with Fluo 3
Biophysical Journal, 1993Co-Authors: A B Harkins, N Kurebayashi, Stephen M BaylorAbstract:Fluo-3 is an unusual tetracarboxylate Ca2+ indicator. For recent lots supplied by Molecular Probes Inc. (Eugene, OR), FMAX, the Fluorescence intensity of the indicator in its Ca(2+)-bound form, is approximately 200 times that of FMIN, the Fluorescence intensity of the indicator in its Ca(2+)-free form. (For earlier lots, impurities may account for the smaller reported values of FMAX/FMIN, 36–40). We have injected Fluo-3 from a high-purity lot into intact single fibers from frog muscle and measured the indicator's absorbance and Fluorescence signals at rest (A and F, respectively) and changes in absorbance and Fluorescence following action potential stimulation (delta A and delta F signals substantially lagged behind that of the myoplasmic free Ca2+ transient. Our analysis of Fluo-3's signals from myoplasm therefore focused on information about the level of resting myoplasmic free [Ca2+] ([Ca2+]r). From A, delta A, and in vitro estimates of Fluo-3's molar extinction coefficients, the change in the fraction of Fluo-3 in the Ca(2+)-bound form during activity (delta f) was estimated. From delta f, delta F, and F, the fraction of the indicator in the Ca(2+)-bound form in the resting fiber (fr) was estimated by fr = (delta f x F/delta F) + (1-FMAX/FMIN)-1. Since FMAX/FMIN is large, the contribution of the second term to the estimate of fr is small. At 16 degrees C, the mean value (mean +/- S.E.) of fr was 0.086 +/- 0.004 (N = 15). From two estimates of the apparent dissociation constant of Fluo-3 for Ca2+ in the myoplasm, 1.09 and 2.57 microM, the average value of [Ca2+]r is calculated to be 0.10 and 0.24 microM, respectively. The smaller of these estimates lies near the upper end of the range of values for [Ca2+]r in frog fibers (0.02–0.12 microM) estimated by others with aequorin and Ca(2+)-selective electrodes. The larger of the estimates lies within the range of values (0.2–0.3 microM) previously estimated in this laboratory with fura red. We conclude that [Ca2+]r in frog fibers is at least 0.1 microM and possibly as large as 0.3 microM.