Artículos de revistas: "Fura-2"

1

Tran, N. N. P., P. Leroy, L. Bellucci, A. Robert, A. Nicolas, J. Atkinson y C. Capdeville-Atkinson. "Intracellular concentrations of Fura-2 and Fura-2/AM in vascular smooth muscle cells following perfusion loading of Fura-2/AM". Pharmacological Research 31 (enero de 1995): 206. http://dx.doi.org/10.1016/1043-6618(95)87082-2.

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2

Hurley, T. W., M. P. Ryan y R. W. Brinck. "Changes of cytosolic Ca2+ interfere with measurements of cytosolic Mg2+ using mag-fura-2". American Journal of Physiology-Cell Physiology 263, n.º 2 (1 de agosto de 1992): C300—C307. http://dx.doi.org/10.1152/ajpcell.1992.263.2.c300.

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In rat pancreatic and submandibular gland acini during exposure to carbachol, changes in the fluorescence emission intensity ratio (R) of acini loaded with mag-fura-2 resemble changes in cytosolic Ca2+ concentration (Ca2+i) in acini loaded with fura-2. Furthermore, changes of R depend on the presence of extracellular Ca2+ (Ca2+o) but are much less influenced by changes in extracellular Mg2+ (Mg2+o). To evaluate interference with measurement of cytosolic Mg2+ (Mg2+i) by changes in Ca2+i, we determined the dissociation constant (Kd) and Hill coefficient (NH) of the Ca(2+)-mag-fura-2 and Mg(2+)-mag-fura-2 complexes in standard solutions, in intact acini after loading with the acetoxymethyl ester of mag-fura-2 (mag-fura-2/AM), and in lysates derived from acini loaded with mag-fura-2/AM. The Kd of the Ca(2+)-mag-fura-2 complex in acini (determined with ionomycin) was 20.49 +/- 5.20 microM, and NH was 1.44 +/- 0.16 (n = 24). Kd of the Mg(2+)-mag-fura-2 complex in acini was 2.25 +/- 0.98 mM, and NH was 1.20 +/- 0.20 (n = 25). Mean Kd values were slightly lower in acinar lysates and in solutions of standard mag-fura-2. In acini from either gland, 1,2-bis(2-aminophenoxy)ethan-N,N,N',N'-tetraacetic acid (BAPTA) suppressed carbachol-induced Ca2+i transients. The R value in stimulated acini loaded with BAPTA and mag-fura-2 increased slightly when Mg2+o was increased from less than 10 nM to 1.2 mM, suggesting that Mg2+ influx contributes to the maintenance of Mg2+i during exposure to carbachol. Under these conditions, pancreatic acinar Mg2+i is 0.53 +/- 0.14 mM (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)

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3

Kass, G. E., D. L. Webb, S. C. Chow, J. Llopis y P. O. Berggren. "Receptor-mediated Mn2+ influx in rat hepatocytes: comparison of cells loaded with Fura-2 ester and cells microinjected with Fura-2 salt". Biochemical Journal 302, n.º 1 (15 de agosto de 1994): 5–9. http://dx.doi.org/10.1042/bj3020005.

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In single Fura-2 ester-loaded hepatocytes, stimulation by vasopressin, but not emptying of the agonist-sensitive Ca2+ store by 2,5-di-(t-butyl)hydroquinone, resulted in an increase in the rate of Fura-2 fluorescence-quenching by Mn2+. Similarly, in cells microinjected with Fura-2 salt, vasopressin stimulated Mn2+ entry while 2,5-di-(t-butyl)hydroquinone or thapsigargin did not. The pattern of Fura-2 quenching by Mn2+ only correlated with the movement of Mn2+ across the plasma membrane.

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4

Owen, C. S. "Spectra of intracellular Fura-2". Cell Calcium 12, n.º 6 (junio de 1991): 385–93. http://dx.doi.org/10.1016/0143-4160(91)90064-l.

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5

Becker, P. L. y F. S. Fay. "Photobleaching of fura-2 and its effect on determination of calcium concentrations". American Journal of Physiology-Cell Physiology 253, n.º 4 (1 de octubre de 1987): C613—C618. http://dx.doi.org/10.1152/ajpcell.1987.253.4.c613.

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This study was performed to determine the effect of photobleaching on the spectral properties of the calcium-sensitive fluorescent dye fura-2. Fura-2, whether in cells or in calibrating solutions, was found to be bleached when exposed to excitation light. In contrast to the widely held belief, photobleaching altered the spectral properties of the dye. Decomposition of the excitation spectra of partially bleached fura-2 solutions revealed an intermediate that is still fluorescent and is not sensitive to calcium over the same range as fura-2, but which can bind calcium in the millimolar range. The presence of this intermediate violates one of the assumptions on which the ratio method of calibration is based; that is, that the only fluorescent species present are the calcium-bound and the free anion forms of fura-2. Thus, if photobleaching occurs, the ratio method will not give accurate calcium concentration values. We calculate that as little as an 8% loss of total fluorescence intensity is sufficient to produce a large error. Photobleaching of fura-2-loaded cells and fura-2 containing calibrating solutions can be minimized by reducing the oxygen concentration and by reducing the excitation light intensity. Strategies are presented to help maintain a high signal-to-noise ratio in fura-2 fluorescence detection systems, despite a lower excitation intensity so that photobleaching, and the resulting inaccuracies in calculated [Ca2+], can be largely avoided.

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6

Jensen, P. E., M. J. Mulvany, C. Aalkjaer, H. Nilsson y H. Yamaguchi. "Free cytosolic Ca2+ measured with Ca(2+)-selective electrodes and fura 2 in rat mesenteric resistance arteries". American Journal of Physiology-Heart and Circulatory Physiology 265, n.º 2 (1 de agosto de 1993): H741—H746. http://dx.doi.org/10.1152/ajpheart.1993.265.2.h741.

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Free cytosolic Ca2+ was measured with sub-micrometer-tip, double-barrelled, Ca(2+)-selective electrodes and fura 2 in rat mesenteric resistance arteries. The purpose was to establish intracellular free Ca2+ concentration ([Ca2+]i) values in resting and stimulated vessels. Isolated vessels were mounted for isometric force measurements. Measured with electrodes, mean [Ca2+]i was 115 and 708 nM under resting and norepinephrine-activated conditions, respectively. Fura 2 was calibrated intracellularly including determination of the intracellular dissociation constant (Kd) of the fura 2:Ca2+ complex. The intracellular Kd was 342 nM. With this value of Kd, fura 2 measurements of mean [Ca2+]i were 129 and 537 nM under resting and norepinephrine-activated conditions, respectively. The values measured with the two techniques were thus in good accordance.

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7

Scaduto, Russell C. y Lee W. Grotyohann. "Hydrolysis of Ca2+-sensitive fluorescent probes by perfused rat heart". American Journal of Physiology-Heart and Circulatory Physiology 285, n.º 5 (noviembre de 2003): H2118—H2124. http://dx.doi.org/10.1152/ajpheart.00881.2001.

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Rat hearts were loaded with the fluorescent calcium indicators fura 2, indo 1, rhod 2, or fluo 3 to determine cytosolic calcium levels in the perfused rat heart. With fura 2, however, basal tissue fluorescence increased above anticipated levels, suggesting accumulation of intermediates of fura 2-AM deesterification. To examine this process, we separated the intermediates of the deesterification process using HPLC after incubation of fura 2-AM with tissue homogenates and after loading in the rat heart. Loading of hearts with fura 2-AM resulted in tissue levels of fura 2 free acid that were only 5% of the total heart dye content of all fura 2 species. The parent fura 2-AM form accumulated without accumulation of intermediate products. Similar results were obtained with indo 1-AM. Fluo 3 loaded very poorly in perfused hearts. Unlike other indictors, rhod 2 rapidly loaded in perfused hearts and was completely converted to the free acid form. To determine the subcellular localization of the free acid form of these indictors, mitochondria from indicator-loaded hearts were assayed for the free acid form. Approximately 75% of the total amount of rhod 2 in hearts could be recovered in isolated mitochondria. Subcellular localization of indo 1 and fura 2 was more evenly distributed between mitochondria and nonmitochondrial compartments. We conclude that measurement of calcium in the perfused rat heart using surface fluorescence with either indo 1 or fura 2 is complicated by an inconsistent accumulation of the parent ester and that the resulting signal cannot be easily calibrated using “in situ” methods using the free acid form. Rhod 2 does not display this shortcoming, but like other indicators, it also loads into the mitochondrial matrix.

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8

Carroll, S. L., M. G. Klein y M. F. Schneider. "Calcium transients in intact rat skeletal muscle fibers in agarose gel". American Journal of Physiology-Cell Physiology 269, n.º 1 (1 de julio de 1995): C28—C34. http://dx.doi.org/10.1152/ajpcell.1995.269.1.c28.

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Intact single fibers enzymatically dissociated from rat flexor digitorum brevis muscle were suspended in 0.5% low-melting-temperature agarose gel to minimize fiber movement during action potentials or trains of action potentials. Resting Ca2+ concentration ([Ca2+]) and changes in [Ca2+] were monitored using the fluorescent calcium indicator fura 2. The time course and waveform of [Ca2+] transients during an action potential or trains of action potentials in fibers in agarose were calculated using kinetic parameters previously determined to correct for the calcium-fura 2 kinetic delay. Half times of the calculated calcium transients for single action potentials were 30-fold briefer than the original fura 2 signals. To confirm the time course and waveform of the calculated calcium transients, changes in [Ca2+] were monitored using the more rapidly equilibrating calcium indicator mag-fura 2. [Ca2+] transients for fibers containing fura 2 had very similar time courses and waveforms as mag-fura 2 signals from other fibers, indicating that the corrections for the calcium-fura 2 kinetic delay were accurate. The advantages of the agarose gel suspension are discussed.

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9

Steinberg, S. F., J. P. Bilezikian y Q. Al-Awqati. "Fura-2 fluorescence is localized to mitochondria in endothelial cells". American Journal of Physiology-Cell Physiology 253, n.º 5 (1 de noviembre de 1987): C744—C747. http://dx.doi.org/10.1152/ajpcell.1987.253.5.c744.

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The new, highly fluorescent, calcium-sensitive dye, fura-2, can be loaded nondisruptively into intact cells by means of its permeant ester and used to measure the free calcium ion concentration in individual cells. For fura-2 to signal cytosolic calcium, it must be distributed homogeneously and exclusively throughout the cytoplasmic space. However, microscopic examination of bovine aortic endothelial cells loaded with fura-2 by exposure to its permeant ester reveals fluorescence associated with discrete intracellular structures rather than the homogeneous distribution expected for a cytosolic stain. Simultaneous labeling of bovine aortic endothelial cells with fura-2 and rhodamine 123 (a mitochondrial fluorescent vital stain) identifies these structures as mitochondria. Subcellular dye localizations are not observed when the cells are loaded with other putative cytosolic stains that gain access to the cytosol by means of a membrane permeant ester. Both carboxyfluorescein and indo-1 (another member of the family of second generation calcium indicators) stain the cytoplasm diffusely. It is suggested that fura-2 fluorescence accumulates in certain cells in association with mitochondria. It is important to assess the intracellular distribution of fura-2 when this indicator is used to measure the free cytosolic calcium ion concentration.

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10

Tram, N. N. P., P. Leroy, L. Bellucci, A. Robert, A. Nicolas, J. Atkinson y C. Capdeville-Atkinson. "Intracellular concentrations of Fura-2 and Fura-2/AM in vascular smooth muscle cells following perfusion loading of Fura-2/AM in arterial segments". Cell Calcium 18, n.º 5 (noviembre de 1995): 420–28. http://dx.doi.org/10.1016/0143-4160(95)90057-8.

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11

Pape, P. C., D. S. Jong, W. K. Chandler y S. M. Baylor. "Effect of fura-2 on action potential-stimulated calcium release in cut twitch fibers from frog muscle." Journal of General Physiology 102, n.º 2 (1 de agosto de 1993): 295–332. http://dx.doi.org/10.1085/jgp.102.2.295.

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Cut fibers (striation spacing, 3.6-4.2 microns) were mounted in a double Vaseline-gap chamber and studied at 14-15 degrees C. One or both of the Ca indicators fura-2 and purpurate-3,3' diacetic acid (PDAA) were introduced into the optical recording site by diffusion from the end pools. Sarcoplasmic reticulum (SR) Ca release was elicited by action potential stimulation. With resting [fura-2] = 0 mM at the optical site, the [Ca] transient measured with PDAA was used to estimate SR Ca release (Baylor, S.M., W.K. Chandler, and M.W. Marshall. 1983. Journal of Physiology. 344:625-666). With resting [fura-2] > 0 mM, the contribution from Ca complexation by fura-2 was added to the estimate. When resting [fura-2] was increased from 0 to 0.5-2 mM, both the amount of SR Ca release and the maximal rate of release were increased by approximately 20%. These results are qualitatively similar to those obtained in intact fibers (Baylor, S.M., and S. Hollingworth. 1988. Journal of Physiology. 403:151-192; Hollingworth, S., A. B. Harkins, N. Kurebayashi, M. Konishi, and S. M. Baylor. 1992. Biophysical Journal. 63:224-234) and are consistent with a reduction of Ca inactivation of SR Ca release produced by 0.5-2 mM fura-2. With resting [fura-2] > or = 2 mM, the PDAA [Ca] transient was reduced to nearly zero and SR Ca release could be estimated from delta [Cafura-2] alone. When resting [fura-2] was increased from 2-4 to 5-6 mM, both the amount of SR Ca release and the maximal rate of release were decreased by approximately half, consistent with a possible reduction of Ca-induced Ca release (Jacquemond, V., L. Csernoch, M. G. Klein, and M. F. Schneider. 1991. Biophysical Journal. 60:867-873) or a possible pharmacological effect of fura-2.

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12

Tomsig, J. L. y J. B. Suszkiw. "Pb2(+)-induced secretion from bovine chromaffin cells: fura-2 as a probe for Pb2+". American Journal of Physiology-Cell Physiology 259, n.º 5 (1 de noviembre de 1990): C762—C768. http://dx.doi.org/10.1152/ajpcell.1990.259.5.c762.

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The effect of Pb2+ on catecholamine release was studied in isolated intact and permeabilized bovine chromaffin cells. Fura-2 was used to monitor intracellular Pb2+. A characterization of Pb2(+)-fura-2 interactions in solutions simulating intracellular ionic composition showed that Pb2+ forms a 1:1 Pb2(+)-fura-2 complex (apparent dissociation constant = 4.2 x 10(-12) M, pH 7.05) whose fluorescence resembles that of the Ca2(+)-fura-2 complex. Spectra recorded from fura-2-loaded cells indicate entry of Pb2+ into the cells. Intracellular Pb2+ concentrations were proportional to extracellular Pb2+ concentrations and were found to be 10(-11)-10(-12) M in cells exposed to micromolar Pb2+ concentrations. Pb2+ elicited the release of tritiated norepinephrine from fura-2-loaded cells, indicating the extraordinary effectiveness of Pb2+ as a releasing agent. Permeabilization of cells with digitonin showed that Pb2+ is able, in the absence of Ca2+, to produce exocytotic release at concentrations of 3.2 x 10(-10) M or higher (3 orders of magnitude lower than Ca2+). These results support the notion that Pb2+ can act as a potent Ca2+ surrogate in triggering secretion.

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13

Mazorow, D. L. y D. B. Millar. "Quin-2 and Fura-2 measure calcium differently". Analytical Biochemistry 186, n.º 1 (abril de 1990): 28–30. http://dx.doi.org/10.1016/0003-2697(90)90567-s.

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14

Gunter, T. E., D. Restrepo y K. K. Gunter. "Conversion of esterified fura-2 and indo-1 to Ca2+-sensitive forms by mitochondria". American Journal of Physiology-Cell Physiology 255, n.º 3 (1 de septiembre de 1988): C304—C310. http://dx.doi.org/10.1152/ajpcell.1988.255.3.c304.

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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.

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15

Oakes, S. George, William J. Martin, Carol A. Lisek y Garth Powis. "Incomplete hydrolysis of the calcium indicator precursor fura-2 pentaacetoxymethyl ester (fura-2 AM) by cells". Analytical Biochemistry 169, n.º 1 (febrero de 1988): 159–66. http://dx.doi.org/10.1016/0003-2697(88)90267-9.

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16

Poenie, Martin, Akwasi Minta y Charles Vorndran. "A new family of fluorescent calcium indicators designed to resist leakage or for measuring calcium near membranes". Proceedings, annual meeting, Electron Microscopy Society of America 52 (1994): 168–69. http://dx.doi.org/10.1017/s0424820100168578.

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The use of fura-2 as an intracellular calcium indicator is complicated by problems of rapid dye leakage and intracellular compartmentalization which is due to a probenecid sensitive anion transporter. In addition there is increasing evidence for localized microdomains of high calcium signals which may not be faithfully reported by fura-2.We have developed a new family of fura-2 analogs aimed at addressing some of these problems. These new indicators are based on a modified bapta which can be readily derivatized to produce fura-2 analogs with a variety of new properties. The modifications do not affect the chromophore and have little impact on the spectral and metal binding properties of the indicator. One of these new derivatives known as FPE3 is a zwitterionic analog of fura-2 that can be loaded into cells as an acetoxymethyl ester and whose retention in cells is much improved. The improved retention of FPE3 is important for both cuvettebased measurements of cell suspensions and for calcium imaging.

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17

Jiang, Yandong y Fred J. Julian. "Pacing rate, halothane, and BDM affect fura 2 reporting of [Ca2+]iin intact rat trabeculae". American Journal of Physiology-Cell Physiology 273, n.º 6 (1 de diciembre de 1997): C2046—C2056. http://dx.doi.org/10.1152/ajpcell.1997.273.6.c2046.

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Experiments were done on intact trabeculae from rats. Fura 2 in the salt form was microinjected directly into the myoplasm. The experiments were conducted at 30°C, with 2 mM extracellular Ca2+ concentration and pacing at either 0.5 or 5 Hz. The aims were to establish a new method for in vivo calibration of fura 2 and to determine the effect of autofluorescence changes on intracellular Ca2+ concentration ([Ca2+]i) reported by fura 2. Autofluorescence was recorded under optimal conditions for fura 2 fluorescence (emission at 510 nm). By alteration of the oxidation-reduction state, it was shown that NADH is the main component of autofluorescence in heart. An increase in pacing frequency caused a decrease in autofluorescence. Both halothane and 2,3-butanedione monoxime (BDM) at 5-Hz pacing produced a substantial rise in autofluorescence, approaching the levels observed at 0.5-Hz pacing. The values for the dissociation constant (678 nM) and maximum fluorescence ratio of fura 2 for Ca2+ for the in vivo calibration are 3.4 times larger and 2.6 times smaller, respectively, than those found in vitro. Using the parameters obtained in vivo, we found that the diastolic and systolic [Ca2+]iof a twitch at 30°C were 0.2 and 2.4 μM, respectively. Proper correction of the autofluorescence change unmasks the [Ca2+]ielevation caused by 5-Hz pacing. It was concluded that autofluorescence is not constant and that interventions affecting autofluorescence need correction if fura 2 is used to report [Ca2+]i.

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18

Seymour, M. T., M. L. Slevin, D. J. Kerr, D. Cunningham, R. D. James, J. A. Ledermann, T. J. Perren et al. "Randomized trial assessing the addition of interferon alpha-2a to fluorouracil and leucovorin in advanced colorectal cancer. Colorectal Cancer Working Party of the United Kingdom Medical Research Council." Journal of Clinical Oncology 14, n.º 8 (agosto de 1996): 2280–88. http://dx.doi.org/10.1200/jco.1996.14.8.2280.

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PURPOSE To determine the effects of interferon alpha-2a (IFN alpha) on the efficacy and toxicity of fluorouracil (FUra) and leucovorin (LV) in patients with advanced colorectal cancer. PATIENTS AND METHODS Two hundred sixty chemotherapy-naive patients were randomized to FUra/LV alone or FUra/LV plus IFN alpha. All patients received: LV 200 mg/m2 intravenous (IV) infusion over 2 hours, then FUra 400 mg/m2 i.v. bolus plus 400 mg/m2 i.v. infusion over 22 hours, all repeated on day 2. Treatment was every 2 weeks for up to 12 cycles. Patients randomized to IFN alpha received 6 x 10(6) IU subcutaneously every 48 hours throughout. Objective response (OR) and toxicity were assessed conventionally; in addition, palliative benefit and adverse effects were assessed using quality-of-life (QoL) questionnaires. RESULTS There were no differences in OR rate, progression-free survival, or overall survival. OR rates in assessable patients were as follows: FUra/LV alone (n = 104), complete or partial response (OR) = 27%, no change (NC) = 34%; FUra/LV/IFN alpha (n = 101), OR = 28%, NC = 30%. Median survival was 10 months in both arms. Dose-limiting FUra toxicities were not significantly increased by co-administration of IFN alpha, and the delivered FUra dose-intensity was not significantly reduced. However, QoL was adversely affected: patients on IFN alpha were less likely to report improvement in pretreatment physical and psychologic symptoms, and more likely to report new or worsening symptoms. CONCLUSION IFN alpha, at a dose that impaired QoL, did not improve the efficacy of FUra/LV. The power of this trial is sufficient to exclude with 95% confidence a benefit of 15% in OR or 10 weeks in median survival. Accordingly, we cannot recommend the use of IFN alpha as a clinical modulator of FUra/LV in the treatment of advanced colorectal cancer.

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19

Futsaether, C. M. y A. Johnsson. "Using fura-2 to measure intracellular free calcium in Propionibacterium acnes". Canadian Journal of Microbiology 40, n.º 6 (1 de junio de 1994): 439–45. http://dx.doi.org/10.1139/m94-072.

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The fluorescent probe fura-2 was used to measure the intracellular free calcium concentration [Ca2+]i in the Gram-positive skin bacterium Propionibacterium acnes. Three methods for loading the probe into P. acnes were used. Two consisted of using the membrane permeable acetoxymethyl ester of fura-2, fura-2/AM, whereas in the third method, fura-2 was loaded directly into the bacteria by means of an acid shock procedure. One method using fura-2/AM was found to be satisfactory. In this case, a statistical experimental design [Formula: see text] was used to devise a suitable loading procedure. [Ca2+]i of P. acnes was dependent upon the external calcium concentration. At low external concentrations (0.2 < [Ca2+]o < 5 μM), [Ca2+]i was 135 ± 13 nM (n = 20). An increase in [Ca2+]o to 1 mM resulted in an increase in [Ca2+]i to approximately 280 ± 40 nM whereas in a Ca-free solution ([Ca2+]o < 5 nM), [Ca2+]i decreased to a lower resting value of 70 ± 7 nM. The time constants for calcium regulation upon step changes in the external concentration were of the order of 10 min. Intracellular alkalinization induced by changes in the external pH (pHo) resulted in an increase in [Ca2+]i when [Ca2+]o was in the range 0.2–5 μM. [Ca2+]i was approximately constant in the range pHo 5.5–7.7 but increased when pHo > 8.0. This increase was partially reversed when pHo was again lowered to below 8.0. In a Ca-free solution, little or no increase in [Ca2+]i was observed when pHo > 8.0.Key words: Propionibacterium acnes, fura-2, calcium.

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20

DeFeo, T. T., G. M. Briggs y K. G. Morgan. "Ca2+ signals obtained with multiple indicators in mammalian vascular muscle cells". American Journal of Physiology-Heart and Circulatory Physiology 253, n.º 6 (1 de diciembre de 1987): H1456—H1461. http://dx.doi.org/10.1152/ajpheart.1987.253.6.h1456.

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Enzymatically isolated single cells from ferret portal vein were loaded with the fluorescent dyes fura-2 and chlortetracycline. Ferret portal vein intact strips were loaded with the luminescent indicator aequorin. At short loading times, fura-2 loading resulted in relatively homogeneous images of labeled cells. At longer loading times, extremely heterogeneous images were obtained that were similar to those produced by chlortetracycline, an indicator recognized to enter calcium-storage organelles. A significant effect of fura-2 on contractile function was detected at the long but not at the short loading time. Caffeine, which is known to deplete calcium from sarcoplasmic reticulum, decreased the fura-2 fluorescent intensity when cells were incubated for a long loading time but caused no statistically significant change at the short loading time. Caffeine caused no drop in the aequorin signal but did cause a drop in the chlortetracycline fluorescence. These results are consistent with the idea that aequorin reports cytoplasmic intracellular calcium concentration ([Ca2+]i), chlortetracycline reports stored calcium, and fura-2 reports a mixed signal from the cytoplasm and calcium-storage organelles depending on incubation time.

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21

Black, B. L. y J. O. Rogers. "Development of Ca2+ homeostasis in epithelial cells from embryonic and neonatal intestine". American Journal of Physiology-Gastrointestinal and Liver Physiology 263, n.º 3 (1 de septiembre de 1992): G371—G379. http://dx.doi.org/10.1152/ajpgi.1992.263.3.g371.

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The fluorescent probe fura-2 was used to assay Ca2+ levels in epithelial cell suspensions from embryonic and neonatal chick duodenum. Cell preparations maintained high viability, completely hydrolyzed fura-2/AM to fura-2, retained 92% of cellular fura-2 within the cytoplasmic compartment, and gave low autofluorescence values during assay. Fura-2 leakage from loaded cells occurred at all ages, but could be corrected for in subsequent calculations of cellular Ca2+. Cytoplasmic Ca2+ concentration was 76-80 nM in cells from 14- to 16-day embryonic intestine, rose significantly to 92-98 nM at 17-20 days, and reached 209 nM at 1-day post-hatch when assayed in buffers containing 1.3 mM Ca2+. The developmental rise in cytoplasmic Ca2+ was accompanied by an enhanced ability of cells to maintain a constant Ca2+ concentration at increased levels of extracellular Ca2+ and by a highly correlated rise in alkaline phosphatase (ALP) activity. Epithelial Ca2+ subsequently decreased to the "adult" value of 133-142 nM and was constant along the crypt-villus axis of neonatal intestine. These results verify that fura-2 can be used to compare baseline cytoplasmic Ca2+ values of epithelial cells from developing intestine, reveal that significant changes in Ca2+ homeostasis occur during ontogeny, and suggest that epithelial Ca2+ may modulate ALP activity during the differentiation of embryonic enterocytes.

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22

Di Virgilio, F., C. Fasolato y T. H. Steinberg. "Inhibitors of membrane transport system for organic anions block fura-2 excretion from PC12 and N2A cells". Biochemical Journal 256, n.º 3 (15 de diciembre de 1988): 959–63. http://dx.doi.org/10.1042/bj2560959.

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The neuroblastoma-like cell line N2A and the pheochromocytoma-like cell line PC12 excrete about 20-25% of the intracellular fluorescent Ca2+ indicator fura-2 during 10 min of incubation at 37 degrees C. The drug probenecid, known to inhibit membrane systems for the transport of organic anions [Cunningham, Israili & Dayton (1981) Clin. Pharmacol. 6, 135-151], inhibited fura-2 excretion in both cell types. However, probenecid also had untoward effects on intracellular Ca2+ homeostasis in N2A and PC12 cells. We therefore tested the drug sulphinpyrazone, another known inhibitor of organic-anion transport systems. Sulphinpyrazone fully inhibited excretion of fura-2 at 250 microM, a concentration one order of magnitude lower than that of probenecid. At this concentration and for incubation times up to 20 min, sulphinpyrazone had no untoward effects on cell viability and metabolic functions. Fura-2 was also loaded into the cytoplasm of N2A cells by permeabilization of the plasma membrane with extracellular ATP. In this case as well, the dye was rapidly released from the cells and the efflux was blocked by sulphinpyrazone. These findings suggest that N2A and PC12 cells possess a membrane system for the transport of the free-acid form of fura-2. This transport system is probably responsible for the excretion of fura-2 from these cells. Incubation of N2A and PC12 cells with sulphinpyrazone may help overcome problems arising in the investigation of [Ca2+]i homeostasis in these cell types.

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23

Kargacin, M. E., C. R. Scheid y T. W. Honeyman. "Continuous monitoring of Ca2+ uptake in membrane vesicles with fura-2". American Journal of Physiology-Cell Physiology 255, n.º 5 (1 de noviembre de 1988): C694—C698. http://dx.doi.org/10.1152/ajpcell.1988.255.5.c694.

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The Ca2+-selective, fluorescent dye, fura-2, was used to monitor ATP-dependent Ca2+ uptake by membrane vesicles isolated from rabbit skeletal muscle. Micromolar fura-2 concentrations, added outside the vesicles, served as a high-affinity, low-capacity Ca2+ buffer. Changes in fura-2 fluorescence resulted from the decline in free Ca2+ concentration [( Ca2+]free) owing to active Ca2+ accumulation by the vesicles. Ca2+ uptake (delta[Ca2+]total) was calculated from changes in [Ca2+]free and from the Kd value for the fura-2-Ca2+ complex. The velocity of Ca2+ uptake determined in this manner had an apparent [Ca2+]0.5 of approximately 200 nM. The Hill coefficient for dependence of uptake velocity on [Ca2+]free was congruent to 2. Changes in [Ca2+]free and Ca2+ uptake expected for Ca2+ transport by skeletal muscle sarcoplasmic reticulum were determined theoretically from known kinetic parameters and found to be similar to experimental values. This method of directly monitoring Ca2+ uptake can be used to determine the kinetic parameters for Ca2+ transport with small amounts of vesicles and with greater precision than possible with radiometric techniques.

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24

Jacob, R., E. Murphy y M. Lieberman. "Free calcium in isolated chick embryo heart cells measured using quin2 and fura-2". American Journal of Physiology-Cell Physiology 253, n.º 2 (1 de agosto de 1987): C337—C342. http://dx.doi.org/10.1152/ajpcell.1987.253.2.c337.

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Cytosolic free calcium (Cai) was measured using quin2 and fura-2 in isolated chick embryo heart cells. Account was taken of extracellular quin2 and fura-2 (which could not be entirely washed away) by adding Mn. Shortly after loading with quin2, Cai was 49 nM (n = 7) but then rose continuously at a rate varying between 13 and 88%/h. By varying the time between cell isolation and quin2 loading, it was ascertained that the loading was causing the rise in Cai. In one set of experiments, Cai was stable in time and the apparent Cai increased steadily from 55 to 179 nM as dye loading (quin2 or fura-2) was decreased from 1 mM to 5 microM. We conclude that although quin2 and fura-2 are useful for comparing Cai levels and determining whether Cai changes as a result of certain maneuvers, they do not provide an absolute measure of Cai in isolated embryonic heart cells.

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25

Wetzel, Petra, Tanja Kleinke, Simon Papadopoulos y Gerolf Gros. "Inhibition of muscle carbonic anhydrase slows the Ca2+ transient in rat skeletal muscle fibers". American Journal of Physiology-Cell Physiology 283, n.º 4 (1 de octubre de 2002): C1242—C1253. http://dx.doi.org/10.1152/ajpcell.00106.2002.

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A countertransport of H+ is coupled to Ca2+ transport across the sarcoplasmic reticulum (SR) membrane. We propose that SR carbonic anhydrase (CA) accelerates the CO2-HCO[Formula: see text]reaction so that H+ ions, which are exchanged for Ca2+ ions, are produced or buffered in the SR at sufficient rates. Inhibition of this SR-CA is expected to reduce the rate of H+ fluxes, which then will retard the kinetics of Ca2+ transport. Fura 2 signals and isometric force were simultaneously recorded in fiber bundles of the soleus (SOL) and extensor digitorum longus (EDL) from rats in the absence and presence of the lipophilic CA inhibitors L-645151, chlorzolamide (CLZ), and ethoxzolamide (ETZ), as well as the hydrophilic inhibitor acetazolamide (ACTZ). Fura 2 and force signals were analyzed for time to peak (TTP), 50% decay time ( t 50), and their amplitudes. L-645151, CLZ, and ETZ significantly increased TTP of fura 2 by 10–25 ms in SOL and by 5–7 ms in EDL and TTP of force by 6–30 ms in both muscles. L-645151 and ETZ significantly prolonged t 50 of fura 2 and force by 20–55 and 40–160 ms, respectively, in SOL and EDL. L-645151, CLZ, and ETZ also increased peak force of single twitches and amplitudes of fura fluorescence ratio (R340/380) at an excitation wavelength of 340 to 380 nm. All effects of CA inhibitors on fura 2 and force signals could be reversed. ACTZ did not affect TTP, t 50, and amplitudes of fura 2 signals or force. L-645151, CLZ, and ETZ had no effects on myosin-, Ca2+-, and Na+-K+-ATPase activities, nor did they affect the amplitude and half-width of action potentials. We conclude that inhibition of SR-CA by impairing H+ countertransport is responsible for deceleration of intracellular Ca2+transients and contraction times.

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26

Koorts, A. M., M. Viljoen y M. C. Kruger. "Die bepaling van intrasellulere vry kalsium in die neutrofiel met die fluoressente kalsiumindikators, fura-2 en fura-PE3". Suid-Afrikaanse Tydskrif vir Natuurwetenskap en Tegnologie 19, n.º 3/4 (15 de julio de 2000): 96–102. http://dx.doi.org/10.4102/satnt.v19i3/4.750.

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A large number of fluorescent calcium indicators are available for the determination of intracellular free calcium concentrations. The best known of these is fura-2. However, the employment of fura-2, amongst others, for the determination of intracellular free calcium is not problem-free.

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27

Alonso, M. "Fura-2 antagonises calcium-induced calcium release". Cell Calcium 33, n.º 1 (enero de 2003): 27–35. http://dx.doi.org/10.1016/s0143-4160(02)00179-3.

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28

TATSUMI, Hitoshi y Yoshifumi KATAYAMA. "Measurement of intracellular calcium ion concentration by using fura-2; estimation of the chelating action of fura-2." Seibutsu Butsuri 32, n.º 4 (1992): 226–28. http://dx.doi.org/10.2142/biophys.32.226.

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29

Sobrero, A. F., C. Aschele y J. R. Bertino. "Fluorouracil in colorectal cancer--a tale of two drugs: implications for biochemical modulation." Journal of Clinical Oncology 15, n.º 1 (enero de 1997): 368–81. http://dx.doi.org/10.1200/jco.1997.15.1.368.

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PURPOSE To determine if fluorouracil (FUra) has different mechanisms of action as a function of the dose schedule used. DESIGN The preclinical and clinical literature relating toxicity and antitumor effects of FUra as a function of its dose schedule, with and without modulating agents, was reviewed. RESULTS The data support the hypothesis that FUra may be considered to be two different drugs, depending on its dose schedule (bolus v continuous infusion [CI]). CONCLUSION These results suggest that additional therapeutic benefit may be obtained from FUra regimens by (1) appropriate schedule-dependent modulation, (2) the sequential or alternating use of cycles of bolus followed by cycles of CI FUra appropriately modulated, or (3) hybrid regimens, ie, those that contain both pulse and CI schedules.

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30

Merritt, J. E., S. A. McCarthy, M. P. A. Davies y K. E. Moores. "Use of fluo-3 to measure cytosolic Ca2+ in platelets and neutrophils. Loading cells with the dye, calibration of traces, measurements in the presence of plasma, and buffering of cytosolic Ca2+". Biochemical Journal 269, n.º 2 (15 de julio de 1990): 513–19. http://dx.doi.org/10.1042/bj2690513.

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A description is given of the methodology, and problems encountered, for the use of a new fluorescent Ca2(+)-indicator dye, fluo-3, in neutrophils and platelets. The higher Kd and longer excitation wavelength of fluo-3 can have significant advantages over fura-2. Although neutrophils and platelets are used as examples, these observations will be applicable to other cell types. The Kd of fluo-3 for binding Ca2+ at 37 degrees C was measured and found to be 864 nM; the previously published value was 400 nM at 22 degrees C. The Kd of fluo-3, like that of fura-2, is therefore very temperature-dependent. Protocols for loading cells, and preventing leakage of fluo-3, are described; probenecid, known to inhibit fura-2 leakage from cells, was found to be essential to get good fluo-3 signals from platelets. Calibration of fluo-3 fluorescence signals to [Ca2+] and methods for obtaining maximum and minimum fluorescence signals are described; these methods differ from those used with fura-2. Agonist-stimulated responses of fluo-3-loaded neutrophils and platelets are shown, and the calculated cytosolic [Ca2+] is comparable with that previously obtained with fura-2. Responses of cells in the presence of plasma are also shown; such measurements, unobtainable with quin2, fura-2 or indo-1, are possible with fluo-3, owing to its longer excitation wavelengths. Co-loading of cells with bis-(o-aminophenoxy)ethane-NNN′N′-tetra-acetic acid and fluo-3 is included as an example of how cytosolic [Ca2+] can be buffered and manipulated. Many of these observations will be of value when using fluo-3 (or other Ca2(+)-indicator dyes) in most cell types.

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31

Vonderlage, M. y V. Schreiner. "[Ca2+]i and contraction of ear artery in response to rapid stimulation by NE: measurements with quin2 and fura-2". American Journal of Physiology-Heart and Circulatory Physiology 257, n.º 2 (1 de agosto de 1989): H649—H657. http://dx.doi.org/10.1152/ajpheart.1989.257.2.h649.

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The relationship between the quin2 and fura-2 fluorescence and isovolumetric contraction of smooth muscle cells of everted segments of the rabbit ear artery in response to rapid stimulation by norepinephrine (NE) was investigated. The resting level measured with quin2 (91 +/- 10 nM, n = 4) and with fura-2 (87 +/- 9 nM, n = 5) did not significantly differ. After addition of NE, an initial slow increase (ISI) in emission could be measured with quin2; however, Ca2+ buffering by this indicator slowed the increase of intracellular Ca2+ concentration ([Ca2+]i) and of contraction. When using fura-2, no ISI could be measured, but this indicator did not significantly interfere with the normal time course of contraction. Maximal response in [Ca2+]i to 1 microM NE was 424 +/- 30 nM (with quin2) and 337 +/- 46 nM (with fura-2). Mechanical latency depended not only on the onset but also on the initial rate of the increase in [Ca2+]i. Contractile response was quickest around 0.1 s after the onset of the fura-2 signal. During its rising phase, the rate of pressure development was linearly correlated with the -log molar Ca2+ concentration (pCa) of the cytoplasm (pCai), whereas during slow relaxation, pressure was linearly related to the pCai. These results suggest that contraction may depend not only on [Ca2+]i but also depend on its rate of change.

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32

Danhauser, L. L., J. H. Freimann, T. L. Gilchrist, J. U. Gutterman, C. Y. Hunter, A. C. Yeomans y A. B. Markowitz. "Phase I and plasma pharmacokinetic study of infusional fluorouracil combined with recombinant interferon alfa-2b in patients with advanced cancer." Journal of Clinical Oncology 11, n.º 4 (abril de 1993): 751–61. http://dx.doi.org/10.1200/jco.1993.11.4.751.

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PURPOSE Enhanced fluorouracil (FUra) cytotoxicity caused by recombinant interferon alfa-2b (rIFN-a) has been reported, but the mechanism, optimal dose, and schedule remain unknown. Therefore, a phase I and pharmacokinetic study of FUra with escalating doses of rIFN-a was initiated. PATIENTS AND METHODS FUra (750 mg/m2/d) was given by continuous intravenous (IV) infusion for 5 days. rIFN-a (0.1 to 15 x 10(6) U/m2/d) was given subcutaneously (SC) daily for 5 days concurrent with FUra. Courses were repeated every 14 to 21 days. Forty-four patients were enrolled; 39 received at least two courses. During the first course of therapy, FUra levels before and after administration of rIFN-a were quantitated in 26 patients by high-pressure liquid chromatography. RESULTS The maximum-tolerated dose of rIFN-a was 10 x 10(6) U/m2/d. Stomatitis was dose-limiting. Three partial and five minor responses occurred. Interpatient pharmacokinetics showed that rIFN-a did not alter steady-state plasma concentration (Css; range, 0.77 +/- 0.35 mumol/L to 1.85 +/- 0.48 mumol/L), elimination half-life (t1/2; mean, 9.7 +/- 4.3 minutes), area under the concentration-versus-time curve (AUC; range, 93 to 224 mumol/L x hours), total-body clearance (CI; range, 1,172 to 3,236 mL/min), or volume of distribution (range, 11.9 to 49.2 L) of FUra. Intrapatient data evaluation revealed a dose-independent effect of rIFN-a. The mean FUra Css after rIFN-a administration (1.31 mumol/L) was greater than that before rIFN-a administration (1.02 mumol/L, P < .0001). FUra Cl after rIFN-a administration was reduced by 20% to 35% compared with use of FUra alone (P < .0001). Patients with a greater than 20% decrease in FUra Cl had a fourfold greater incidence of diarrhea. CONCLUSION rIFN-a reduces FUra Cl and, consequently, increases FUra-associated toxicity. Phase II studies of FUra and rIFN-a seem to be warranted.

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33

Restrepo, D. y J. H. Teeter. "Olfactory neurons exhibit heterogeneity in depolarization-induced calcium changes". American Journal of Physiology-Cell Physiology 258, n.º 6 (1 de junio de 1990): C1051—C1061. http://dx.doi.org/10.1152/ajpcell.1990.258.6.c1051.

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Olfactory neurons from the channel catfish, Ictalurus punctatus, were isolated by a brief (15 min) treatment with papain. After incubation with fura-2 acetoxymethyl ester (fura-2/AM) for 1 h, the isolated olfactory receptor cells are found to hydrolyze fura-2/AM to fura-2 free acid without detectable traces of intermediate products of hydrolysis. Intracellular calcium measured with fura-2 in single cells covers a wide range (from less than 2 to 100 nM with a median of 17.6 nM, n = 140 cells). Twenty-one percent of the cells respond to potassium-induced depolarization with an increase in intracellular calcium mediated by influx of extracellular calcium. The L-type calcium channel antagonist nimodipine inhibits the increase in intracellular calcium triggered by membrane depolarization and blocks small unitary barium currents displaying the characteristics of L-type calcium currents (unitary conductance of 29 +/- 5 pS in 55 mM BaCl2 and high selectivity for Ba2+ over Na+ and K+) recorded from azolectin bilayers at the tip of patch pipettes into which isolated olfactory cilia membrane vesicles had been incorporated. Olfactory neurons are found to be functionally heterogeneous in their response to membrane depolarization and can be separated into three groups: one in which the increase in intracellular calcium is rapid and transient, another in which calcium increases slowly, and a third group of cells in which depolarization causes no change in intracellular calcium.

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34

Qian, Li, Ruth A. Altschuld y Bradford T. Stokes. "Quantitation of intracellular free calcium in single adult cardiomyocytes by fura-2 fluorescence microscopy: Calibration of fura-2 ratios". Biochemical and Biophysical Research Communications 147, n.º 1 (agosto de 1987): 120–26. http://dx.doi.org/10.1016/s0006-291x(87)80095-5.

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35

Hyrc, K. L., J. M. Bownik y M. P. Goldberg. "Ionic selectivity of low-affinity ratiometric calcium indicators: mag-Fura-2, Fura-2FF and BTC". Cell Calcium 27, n.º 2 (febrero de 2000): 75–86. http://dx.doi.org/10.1054/ceca.1999.0092.

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36

Backx, P. H. y H. E. Ter Keurs. "Fluorescent properties of rat cardiac trabeculae microinjected with fura-2 salt". American Journal of Physiology-Heart and Circulatory Physiology 264, n.º 4 (1 de abril de 1993): H1098—H1110. http://dx.doi.org/10.1152/ajpheart.1993.264.4.h1098.

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We have measured force, sarcomere length, and Ca2+ during twitches in rat cardiac trabeculae. To avoid the difficulties associated with fura-2/acetoxymethyl ester (AM), fura-2 salt was iontophoretically microinjected into the preparation using a single impalement site; this is possible because fura-2 diffuses through the gap junctions between cells. By use of this method, the estimated peak of the [Ca2+] transient during a twitch was not statistically different at different sarcomere lengths: 875 +/- 92 nM at a sarcomere length of 2.15 microns vs. 905 +/- 67 nM at a sarcomere length of 1.65 microns (means +/- SD, n = 10). When trabeculae were loaded using fura-2/AM, the estimated peak of the [Ca2+] transient at a sarcomere length of 2.15 microns was 540 +/- 180 nM (means +/- SD, n = 5). The time course of the Ca2+ transients at different sarcomere lengths is qualitatively similar, but small systematic differences were observed during the relaxation period. On the other hand, the duration of twitch force increases dramatically as the muscle length is increased. As a result, when the trabeculae were held at short muscle lengths, the temporal relationship between force and the Ca2+ transient resembled the relationship between cell shortening and the Ca2+ transient measured in isolated myocytes. At longer lengths the temporal relationship between force and the Ca2+ transient more closely resembles that obtained in papillary muscles using aequorin.

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37

Mázala, Davi A. G., Robert W. Grange y Eva R. Chin. "The role of proteases in excitation-contraction coupling failure in muscular dystrophy". American Journal of Physiology-Cell Physiology 308, n.º 1 (1 de enero de 2015): C33—C40. http://dx.doi.org/10.1152/ajpcell.00267.2013.

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Duchenne muscular dystrophy (DMD) is one of the most frequent types of muscular dystrophy. Alterations in intracellular calcium (Ca2+) handling are thought to contribute to the disease severity in DMD, possibly due to the activation of Ca2+-activated proteases. The purpose of this study was twofold: 1) to determine whether prolonged excitation-contraction (E-C) coupling disruption following repeated contractions is greater in animals lacking both dystrophin and utrophin ( mdx/Utr−/−) compared with mice lacking only dystrophin ( mdx); and 2) to assess whether protease inhibition can prevent E-C coupling failure following repeated tetani in these dystrophic mouse models. Excitation-contraction coupling was assessed using Fura-2 ratio, as an index of intracellular free Ca2+ concentration, in response to electrical stimulation of single muscle fibers from the flexor digitorum brevis muscle. Resting Fura-2 ratio was higher in dystrophic compared with control (Con) fibers, but peak Fura-2 ratios during stimulation were similar in dystrophic and Con fibers. One hour after a series of repeated tetani, peak Fura-2 ratios were reduced by 30 ± 5.6%, 23 ± 2%, and 36 ± 3.1% in mdx, mdx/Utr+/−, and mdx/Utr−/−, respectively, with the greatest reduction in mdx/Utr−/− fibers ( P < 0.05). Protease inhibition attenuated this decrease in peak Fura-2 ratio. These data indicate that E-C coupling impairment after repeated contractions is greatest in fibers lacking both dystrophin and utrophin and that prevention of protease activation can mitigate the prolonged E-C coupling impairment. These data further suggest that acute protease inhibition may be useful in reducing muscle weakness in DMD.

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38

Hopf, F. W., P. R. Turner, W. F. Denetclaw, P. Reddy y R. A. Steinhardt. "A critical evaluation of resting intracellular free calcium regulation in dystrophic mdx muscle". American Journal of Physiology-Cell Physiology 271, n.º 4 (1 de octubre de 1996): C1325—C1339. http://dx.doi.org/10.1152/ajpcell.1996.271.4.c1325.

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There are conflicting reports regarding whether resting free calcium levels ([Ca2+]i) are elevated in dystrophic mouse (mdx) myotubes and adult myofibers. We reinvestigated this question and found several lines of evidence supporting the hypothesis that increased calcium influx via leak channels leads to increases in resting [Ca2+]i. 1) Step calibration of fura 2/free acid in myofibers with use of microinjected Ca(2+)-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffers revealed greater [Ca2+]i in dystrophic cells. Careful calibration of fura PE3-AM, a compartmentalization-resistant derivative of fura 2, also showed elevated [Ca2+]i in mdx myotubes. 2) Chronic, but not acute, application of tetrodotoxin reduced resting [Ca2+]i in dystrophic myotubes, suggesting that elevated resting [Ca2+]i is a consequence of previous long-term contractile activity. 3) Rates of manganese quenching of fura 2 fluorescence, an indirect indicator of calcium influx, were significantly higher in mdx myotubes and were increased by nifedipine, a calcium leak channel agonist. 4) Calcium leak channel activity, measured using patch clamping, was greater in the sarcolemma of adult non-enzyme-treated mdx myofibers.

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39

Malaiyandi, Latha M., Harsh Sharthiya, Ameir N. Barakat, Joshua R. Edwards y Kirk E. Dineley. "Using FluoZin-3 and fura-2 to monitor acute accumulation of free intracellular Cd2+ in a pancreatic beta cell line". BioMetals 32, n.º 6 (21 de noviembre de 2019): 951–64. http://dx.doi.org/10.1007/s10534-019-00226-z.

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AbstractThe understanding of cellular Cd2+ accumulation and toxicity is hampered by a lack of fluorescent indicators selective for intracellular free Cd2+ ([Cd2+]i). In this study, we used depolarized MIN6 mouse pancreatic beta cells as a model for evaluating [Cd2+]i detection with commercially available fluorescent probes, most of which have been traditionally used to visualize [Ca2+]i and [Zn2+]i. We trialed a panel of 12 probes including fura-2, FluoZin-3, Leadmium Green, Rhod-5N, indo-1, Fluo-5N, and others. We found that the [Zn2+]i probe FluoZin-3 and the traditional [Ca2+]i probe fura-2 responded most consistently and robustly to [Cd2+]i accumulation mediated by voltage-gated calcium channels. While selective detection of [Cd2+]i by fura-2 required the omission of Ca2+ from extracellular buffers, FluoZin-3 responded to [Cd2+]i similarly in the presence or absence of extracellular Ca2+. Furthermore, we showed that FluoZin-3 and fura-2 can be used together for simultaneous monitoring of [Ca2+]i and [Cd2+]i in the same cells. None of the other fluorophores tested were effective [Cd2+]i detectors in this model.

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40

Palmer, Bradley M., Anne M. Thayer, Steven M. Snyder y Russell L. Moore. "Shortening and [Ca2+] dynamics of left ventricular myocytes isolated from exercise-trained rats". Journal of Applied Physiology 85, n.º 6 (1 de diciembre de 1998): 2159–68. http://dx.doi.org/10.1152/jappl.1998.85.6.2159.

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The effects of run endurance training and fura 2 loading on the contractile function and Ca2+ regulation of rat left ventricular myocytes were examined. In myocytes not loaded with fura 2, the maximal extent of myocyte shortening was reduced with training under our pacing conditions [0.5 Hz at 2.0 and 0.75 mM external Ca2+ concentration ([Ca2+]o)], although training had no effect on the temporal characteristics. The “light” loading of myocytes with fura 2 markedly suppressed (∼50%) maximal shortening in the sedentary and trained groups, although the temporal characteristics of myocyte shortening were significantly prolonged in the trained group. No discernible differences in the dynamic characteristics of the intracellular Ca2+ concentration ([Ca2+]) transient were detected at 2.0 mM [Ca2+]o, although peak [Ca2+] and rate of [Ca2+] rise during caffeine contracture were greater in the trained state at 0.75 mM [Ca2+]o. We conclude that training induced a diminished myocyte contractile function under the conditions studied here and a more effective coupling of inward Ca2+ current to sarcoplasmic reticulum Ca2+ release at low [Ca2+]o, and that fura 2 and its loading vehicle DMSO significantly alter the intrinsic characteristics of myocyte contractile function and Ca2+ regulation.

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41

Zheng, Xian‐Fu, Ying‐Xia Zhou, Hong‐Yun Zhang, Yun‐Yin Niu, Xiao‐Qing Shen, Cao‐Yuan Niu y Ben‐Lai Wu. "Synthesis, Crystal Structure and Properties of a Novel Cu(II)Complex with α‐Furan Carboxylate, [Cu(fura)2(bpy)(H2O)], (fura=α‐Furan Carboxylate, bpy=2,2′‐bispyridine)". Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry 37, n.º 4 (1 de mayo de 2007): 235–39. http://dx.doi.org/10.1080/15533170701316437.

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42

Zheng, Xian‐Fu, Ying‐Xia Zhou, Hong‐Yun Zhang, Xiao‐Qing Shen, Yun‐Yin Niu, Cao‐Yuan Niu y Ben‐Lai Wu. "Synthesis, Crystal Structure and Properties of a Novel Cu(II) Complex with α‐Furan Carboxylate, [Cu(fura)2(bpy)(H2O)], (fura=α‐Furan Carboxylate, bpy=2,2′‐bispyridine)". Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry 37, n.º 7 (1 de septiembre de 2007): 541–45. http://dx.doi.org/10.1080/15533170701563749.

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43

Van den Bergh, V., N. Boens, F. C. De Schryver, M. Ameloot, P. Steels, J. Gallay, M. Vincent y A. Kowalczyk. "Photophysics of the fluorescent Ca2+ indicator Fura-2". Biophysical Journal 68, n.º 3 (marzo de 1995): 1110–19. http://dx.doi.org/10.1016/s0006-3495(95)80285-7.

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44

Abe, Asaki, Minori Mitsui y Hideaki Karaki. "Leakage of fura-2 from smooth muscle tissue." Japanese Journal of Pharmacology 55 (1991): 321. http://dx.doi.org/10.1016/s0021-5198(19)39351-5.

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45

Kopp, Richard F., Colin A. Leech y Michael W. Roe. "Resveratrol Interferes with Fura-2 Intracellular Calcium Measurements". Journal of Fluorescence 24, n.º 2 (23 de octubre de 2013): 279–84. http://dx.doi.org/10.1007/s10895-013-1312-9.

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46

Abdulmalek, S., D. P. Montoya y C. M. McCarthy. "Incorporation of 5-fluorouracil into RNA by Mycobacterium avium complex strain LM1". Canadian Journal of Microbiology 39, n.º 6 (1 de junio de 1993): 616–22. http://dx.doi.org/10.1139/m93-089.

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The incorporation of 5-fluorouracil (FUra) into RNA of Mycobacterium avium complex strain LM1 was evaluated. Cells were labeled with either [14C]FUra or [3H]uracil and the ribonucleosides were analyzed by high-performance liquid chromatography. The identification of the ribonucleosides was facilitated by the use of an isocratic system that provided unambiguous identification of the RNA pyrimidine components. Uracil was incorporated into RNA as uridine, but an equal amount was converted to cytidine. [14C]FUra was incorporated directly into RNA as 5-fluorouridine and there was no evidence of its conversion to other pyrimidines. The ratio of 5-fluorouridine:uridine was 2.8-fold greater for cells grown in 100 μg FUra/mL than for cells grown in 20 μg FUra/mL. Analysis of the RNA nucleotides was performed and deoxyribonucleotides were present; DNA contamination was estimated to range from about 2 to 8% of the RNA preparations.Key words: 5-fluorouracil, RNA, Mycobacterium avium complex, HPLC, ribonucleoside.

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47

Cheung, J. Y., D. L. Tillotson, R. V. Yelamarty y R. C. Scaduto. "Cytosolic free calcium concentration in individual cardiac myocytes in primary culture". American Journal of Physiology-Cell Physiology 256, n.º 6 (1 de junio de 1989): C1120—C1130. http://dx.doi.org/10.1152/ajpcell.1989.256.6.c1120.

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Cytosolic free Ca2+ concentration, [Ca2+]i, of single isolated Ca2+-tolerant rat ventricular myocytes in primary culture was determined by digital video imaging of intracellular fura-2 fluorescence. In deenergized myocytes in which contractile elements were uncoupled by 2,3-butanedione monoxime, the maximum and minimum fluorescence intensity ratio values of fura-2 in the cell were similar when compared with those of fura-2 solutions observed in the microscope. Through the use of in vitro calibration, [Ca2+]i in quiescent, rod-shaped myocytes was 90 +/- 6 nM. There was no detectable spatial heterogeneity in [Ca2+]i in resting myocytes. Localized regions of [Ca2+]i elevation were observed in cells undergoing spontaneous rhythmic contractions or when subjected to mild depolarization by KCl. Additions of gramicidin or veratridine resulted in massive increases in [Ca2+]i (greater than 1 microM) and immediate cell hypercontracture. Ruthenium red elicited a modest increase in [Ca2+]i but extracellular ATP or epinephrine had no effect. We conclude the following: 1) digital video imaging of resting cardiac cells did not reveal any subcellular Ca2+ gradients; 2) the fluorescence properties of intracellular fura-2 were similar to that in free solution; and 3) subcellular heterogeneity of [Ca2+]i in isolated myocytes was observed in cells undergoing spontaneous rhythmic contraction.

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48

Simpson, A. W. M., A. Stampfl y C. C. Ashley. "Evidence for receptor-mediated bivalent-cation entry in A10 vascular smooth-muscle cells". Biochemical Journal 267, n.º 1 (1 de abril de 1990): 277–80. http://dx.doi.org/10.1042/bj2670277.

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In fura-2-loaded A10 vascular smooth-muscle cells, 1 nM-vasopressin and 200 nM-endothelin evoked a rapid transient rise in intracellular free Ca2+ concentration [(Ca2+]i), which was then followed by a maintained elevation of [Ca2+]i. The maintained elevation of [Ca2+]i was only partially inhibited by 5 microM-nifedipine, but completely abolished in the presence of 1 mM-EGTA. When extracellular Ca2+ was replaced with 1 mM-Mn2+ (Mn2+ quenches fura-2 fluorescence), both endothelin and vasopressin evoked an Mn2+ quench of the fluorescence from the intracellularly trapped fura-2, even in the presence of 5 microM-nifedipine. These data suggest that both vasopressin and endothelin promote a bivalent-cation influx and provide further evidence for receptor-mediated Ca2+ entry in vascular smooth muscle.

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49

Tyagi, Suresh C. y Sanford R. Simon. "Interaction of neutrophil elastase with hydrophobic polyanionic chelators". Biochemistry and Cell Biology 69, n.º 9 (1 de septiembre de 1991): 624–29. http://dx.doi.org/10.1139/o91-092.

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The polyanionic calcium chelators, ethylenediamine-tetraacetic acid (EDTA), ethylene-bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA), [bis-(O-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA), 1-[2-amino-5-(6-carboxy-indol-2-yl)phenoxyl]-2-(2′-amino-5′-methylphenoxy)ethane- N,N,N′,N′-tetraacetic acid (INDO-1), 1-[2-(5-carboxyoxazol-2yl)-6-phenoxyl]-2-(2′-amino-5′-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid (FURA-2), and 2-{[2-bis-(carboxymethyl)-amino-5-methylphenoxy]-methyl}-6-methyl-8-bis-(bis-(carboxymethyl)-aminoquinoline (QUIN-2), are all inhibitors of amidolytic activity of human neutrophil elastase (HNE). With MeOSuc-Ala-Ala-Pro-Val-pNA as substrate, these chelators all display mixed partial competitive and partial noncompetitive inhibition, but with the smaller substrate, pGlu-Pro-Val-pNA, only the noncompetitive component persists. The most effective inhibitor is FURA-2, with an apparent Ki of 0.5–0.7 mM. QUIN-2 is somewhat less effective, with a Ki of 2 mM, while EDTA is much less effective, with a Ki of 7 mM. In general, the more hydrophobic chelators are the best inhibitors, although INDO-1, which is about the same size as FURA-2, is surprisingly ineffective as an inhibitor. The chelators no longer function as effective inhibitors if their carboxyl groups are blocked by esterification with acetoxymethyl groups or by complexation with calcium ions, indicating that their binding to HNE is mediated in part through electrostatic interactions with a center of positive charge on the protein. The excitation spectrum of the complex of FURA-2 with HNE differs from that of the chelator with calcium ions, indicating that the structure of the enzyme-inhibitor complex is not like that of the coordination complex of the chelator with the metal ion. The inhibitory capacity of FURA-2 apparently arises from binding to a site that is in the vicinity of the S4 and S5 subsites of the extended substrate binding domain on HNE through a combination of hydrophobic and electrostatic interactions with the enzyme.Key words: elastase, protease inhibitors, chelators, poly anions, calcium.

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50

Terada, Shin, Isao Muraoka y Izumi Tabata. "Changes in [Ca2+]i induced by several glucose transport-enhancing stimuli in rat epitrochlearis muscle". Journal of Applied Physiology 94, n.º 5 (1 de mayo de 2003): 1813–20. http://dx.doi.org/10.1152/japplphysiol.00780.2002.

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The purpose of the present investigation was to establish a method for estimating intracellular Ca2+ concentrations ([Ca2+]i) in isolated rat epitrochlearis muscles. Epitrochlearis muscles excised from 4-wk-old male Sprague-Dawley rats were loaded with a fluorescent Ca2+indicator, fura 2-AM, for 60–90 min at 35°C in oxygenated Krebs-Henseleit buffer. After fura 2 loading and subsequent 20-min incubation, the intensities of 500-nm fluorescence, induced by 340- and 380-nm excitation lights (Ftotal340 and Ftotal380), were measured. The fluorescences specific to fura-2 (Ffura 2340 and Ffura 2380) were calculated by subtracting the non-fura 2-specific component from Ftotal340 and Ftotal380, respectively. The ratio of Ffura 2340 to Ffura 2380 was calculated as R, and the change in the ratio from the baseline value (ΔR) was used as an index of the change in [Ca2+]i. In resting muscle, ΔR was stable for 60 min. Incubation for 20 min with caffeine (3–10 mM) significantly increased ΔR in a concentration-dependent manner. Incubation with hypoxic Krebs-Henseleit buffer for 10–60 min significantly elevated ΔR, depending on the duration of the incubation. Incubation with 50 μM N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide for 20 min significantly elevated ΔR ( P < 0.05). No significant increases in ΔR were observed during incubation for 20 min with 2 mM 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside or with 2 mU/ml insulin. These results demonstrated that, by using the fura 2-AM fluorescence method, the changes in [Ca2+]i can be monitored in the rat epitrochlearis muscle and suggest that the method can be utilized to observe quantitative information regarding [Ca2+]i that may be involved in contraction- and hypoxia-stimulated glucose transport activity in skeletal muscle.

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