A Ca2+-insensitive form of fura-2 associated with polymorphonuclear leukocytes. Assessment and accurate Ca2+ measurement.

Scanlon, Mary; Williams, David A.; Fay, Fredric S.

doi:10.1016/s0021-9258(18)45570-6

Cited by 308 publications

(17 citation statements)

References 12 publications

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“…Additional problems with the indicator dyes result from photobleaching (Becker and Fay, 1987), the presence of uncleaved ester (Scanlon et al, 1987), viscosity (Konishi et al, 1988;Tsien and Poenie, 1986), protein binding (Konishi et al, 1988), and light/dye toxicity (Callaham, D. A., and P. Hepler, unpublished observations). Some of these difficulties such as protein binding are not restricted to the fluorescence indicators since studies with arsenazo-III indicate that as much as 80-90% of the dye in muscle cells may be complexed with protein (Beeler et al, 1980).…”

Section: Interpretation Of the Resultsmentioning

confidence: 99%

Calcium transients during mitosis: observations in flux.

Hepler

1989

The Journal of Cell Biology

105

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Section: Interpretation Of the Resultsmentioning

confidence: 99%

Calcium transients during mitosis: observations in flux.

Hepler

1989

The Journal of Cell Biology

105

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“…The [Ca2+]i was then calculated as the mean of the individual pixel ratios for the entire cell as well as for a 20-Am' window of the cell immediately adjacent to the bound bead using the following expression originally described by Grynkiewicz et al . (9) and adapted by others (6,19) .…”

Section: Measurement Of[ca2+] During Binding and Phagocytosis Oflatex Beads By Single Cellsmentioning

confidence: 99%

Calcium transients during Fc receptor-mediated and nonspecific phagocytosis by murine peritoneal macrophages.

Hishikawa

Cheung

Yelamarty

et al. 1991

The Journal of Cell Biology

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Abstract. Studies with populations of macrophages have produced conflicting results concerning the possibility that the concentration of intracellular ionized calcium ([Ca2+]i) may act as an important mediator for phagocytosis. Since asynchronous changes in [Ca"] i in individual cells undergoing phagocytosis may be averaged to undetectability in population studies, we studied single adhering murine macrophages using fura-2 and our previously described digital imaging system . The proportion of macrophages phagocytosing IgG-coated latex beads was greater than for uncoated beads (percent phagocytosing cells: 71 f 7 vs. 27 t 7, P < 0.01) . Phagocytosis of IgG-coated and uncoated beads was always associated with a calcium transient that preceded the initiation of phagocytosis . No calcium transients were detected in cells that bound but did not phagocytose beads . Four major differences between Fc receptor-mediated and nonspecific phagocytosis were detected : (a) the duration of calcium transients was longer for nonspecific phagocytosis compared with Fc B NDING and endocytosis of immune complexes by macrophages are mainly mediated by membrane receptors for the Fc portion of IgG (Fc receptors) (20) . Transmembrane signaling for the initiation of phagocytosis of bound immune complexes is not currently understood. Since calcium regulates the assembly and disassembly of cytoskeletal elements (7,18,22), it has long been proposed that a transient increase in the concentration of intracellular ionized calcium ([Ca2+]i) acts as a second messenger for phagocytosis . However, available studies with populations of macrophages have shown conflicting results concerning this possibility (8,14,26) . Studies of cell populations in which no calcium transients were observed may have been hampered by the fact that phagocytosis is an asynchronous event in different cells (14). Thus, calcium transients occurring in different cells at different times might have been averaged to undetectability as previously postulated by Kruskal and Maxfield (12). To overcome these difficulties, we used our previously described digital video imaging system (5,15,25) receptor-mediated phagocytosis (69.9 f 10.2 vs. 48.7 f 4 .7 s, P < 0.05) and the magnitude of calcium transients was less for nonspecific phagocytosis (178 f 43 vs. 349 t 53 nM, P < 0.05); (b) removal of extracellular calcium abolished the calcium transients associated with nonspecific phagocytosis but had no effect on those associated with receptor-mediated phagocytosis; (c) in the absence of extracellular calcium, buffering intracellular calcium with a chelator reduced Fc receptor-mediated phagocytosis but had no additive inhibitory effect on nonspecific phagocytosis ; and (d) inhibition of protein kinase C (PKC) with staurosporine inhibited nonspecific phagocytosis but had no effect on receptor-mediated phagocytosis. Our observations suggest that despite both types of phagocytosis being associated with intracellular calcium transients, the role played by intracellular calcium in the sign...

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“…Mn ~+ binds to fura-2 with a higher affinity than Ca 2+ (48-fold) and its entry is also facilitated by ionomycin. Mn ~+ quenches the fura-2 fluorescence, hut does not affect the cell autofluorescence, the fluorescence of the acetoxymethylester (Luckhoff, 1986) or of the partial hydrolysis products of fura-2AM (Scanlon et al, 1987). Therefore, the value at each individual wavelength represents the proportion that has to be subtracted from each signal before making the ratio signal.…”

Section: Fura-2 Measurementsmentioning

confidence: 99%

Cytoplasmic free calcium, myosin light chain phosphorylation, and force in phasic and tonic smooth muscle.

Himpens

Matthijs

Somlyo

et al. 1988

The Journal of General Physiology

143

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The time course of [Ca~+]i, tension, and myosin light chain phosphorylation were determined during prolonged depolarization with high K + in intact tonic (rabbit pulmonary artery) and phasic (longitudinal layer of guinea pig ileum) smooth muscles. [CaZ+]i was monitored with the 340 nm/380 nm signal ratio of the fluorescent indicator fura-2. The fluorescence ratio had a similar time course in both muscle types during depolarization with 109 mM [K+]o; after a transient peak, there was a decline to 70% of its peak value in tonic smooth muscle, and to 60% in phasic smooth muscle. Tension, however, continued to increase in the pulmonary artery, while in the ileum it declined in parallel with the [CaZ+] i. On changing [K+]o from 109 to 20 raM, tension and [CaZ+]i either remained unchanged or declined in parallel in the pulmonary artery. Phosphorylation of the 20-kD myosin light chain, measured during stimulation of muscle strips with 109 mM [K+]o in another set of experiments, increased from 3% to a peak of 50% in the intact pulmonary artery, and then declined to a steady state value of 23%. In the intact ileum, a very rapid, early transient phosphorylation (up to 50%) at 2-3 s was seen. This transient declined by 30 s to a value that was close to the resting level (7%), while tension remained at 55% of its peak force. A quick release during maintained stimulation induced no detectable change in the [CaZ+]i in either type of smooth muscle. We discuss the possibility that the slowly rising tonic tension in pulmonary artery could be due to cooperativity between phosphorylated and nonphosphorylated crossbridges.

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A Ca2+-insensitive form of fura-2 associated with polymorphonuclear leukocytes. Assessment and accurate Ca2+ measurement.

Cited by 308 publications

References 12 publications

Calcium transients during mitosis: observations in flux.

Calcium transients during mitosis: observations in flux.

Calcium transients during Fc receptor-mediated and nonspecific phagocytosis by murine peritoneal macrophages.

Cytoplasmic free calcium, myosin light chain phosphorylation, and force in phasic and tonic smooth muscle.

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