Fluorescent Ca2+ indicators

Hasan, M. T., Friedrich, R. W., Euler, T., Larkum, M. E., Giese, G., Both, M., Duebel, J., Waters, J., Bujard, H. Griesbeck, O. el al. (2004). Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control. PLoS Biol. 2, el63. 

The development of fluorescent Ca2+ indicators that can be introduced easily into almost any vertebrate cell revolutionized calcium signaling research in the 1980s (Fig. 22-1) [1]. The majority of these compounds are... 

Furthermore, in more recent studies high [Ca2+]mt signals are seen in only a few mitochondria within a given cell, and reports claiming very high [Ca2+]mt under physiological conditions are based on cells isolated by enzymatic dispersion. This, coupled with the uncertainty of calibration of luminescent and fluorescent Ca2+ indicators within the mitochondrial matrix, (for nuclei see Perez-Terzic et al 1997) should raise serious questions about the correct values of [Ca2+]mt. It is unfortunate that, with rare exceptions, very few available studies compare free with total mitochondrial Ca in the same cell type observed under the same condition. 

Recently, Tsubosaka et al. (2010a) reported that halichlorine was also revealed to inhibit L-type Ca2+ channels, which leads to inhibit smooth muscle contraction. In their report, the direct effect of halichlorine on vascular contractility was investigated. Then, halichlorine was found to inhibit both high concentration of K+- and phenylephrine-induced contractions in rat aorta dose dependently. The effect of halichlorine on high K+-induced contraction was shown to be stronger than that on phenylephrine-induced contraction. Because known L-type Ca2+ channel blockers, verapamil and nifedipine, were observed to show the similar effect by them, it was suggested that halichlorine selectively inhibits L-type Ca2+ channels. Then, the effect of halichlorine on intracellular Ca2+ concentration in vascular smooth muscle tissue was examined using a fluorescent Ca2+ indicator, Fura-2. 

G Grynkiewioz, M Poenie, RY Tsien. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260 3440-3450. 

FIG. 3. Confocal images showing the location of the SR in live myocytes within an intact, small diameter (< 250 nm passive diameter), pressurized (70 mmHg) artery from the rat mesenteric artery arcade. The artery was loaded with Fluo-4 as the membrane-permeant acetoxymethyl ester. Some of this high-affinity, Ca2+ indicator dye is often sequestered in the SR (cf. Goldman et al 1990). The SR can then be readily visualized, especially when [Ca2+]CYx is low (as in the panels at 0 and 6.8 s), because the intra-SR dye is saturated with Ca2+, and fluoresces brightly. This artery was treated with 1.0 fim phenylephrine (PE), which caused the [Ca2+]CYT level to oscillate asynchronously in the cells seen in the centre of the panel. The cell outlines are clearly visible when [Ca2+]CYT tiscs, as in the panels at 3.4 and 10.2 s. Note that nearly all of the SR (the very bright areas, especially in the 0 and 3.4 s panels) lies parallel to, and immediately beneath the PL (from Miriel at al 1999, with permission). 

Most of the fluorescent calcium indicators and their cell-permeant acetoxymethyl (AM) esters are variations of the nonfluorescent calcium chelator BAPTA and have been proposed by Tsien/134-1365 Among them Fura-2 and Indo-1 (Figure 5.22) are particularly used formeasuring Ca2+in single cells by imaging or flow cytometry/65... 

Fig. 8. R/Platelet in individual platelets adhering to polymer surfaces. HSB data were statistically confirmed to be different from PSt (P < 0.5), HSR (P < 0.5) and PHEMA (P < 0.5) after 40 s R/Platelet (an index of cytoplasmic free calcium concentration) is the ratio of fluorescence emission intensitie of a Ca2 + indicator dye (Fura 2) loaded in platelets when they are excited at 340 nm and 380 nm. (Reproduced from J Biomed Mater Res [Ref 84 Prevention of changes in platelet cytoplasmic free calcium levels by interaction with 2-hydroxyethyl methacrylate/styrene block copolymer surfaces] through the courtesy of John Wiley Sons, Inc.)...

Calcium oscillations observed with six cultured pancreatic p cells after a single infusion of 0.2 mM carbamoylcholine. Tire fluorescence intensity of the Ca2+ indicator dye fura 2, with excitation at 380 nm, was recorded versus time. From Pretki et al.ss... 

For example, the oscillatory change in intracellular [Ca2+] shown above was observed in pancreatic insulin-secreting P cells responding to stimulation by the agonist carbamoylcholine. The free [Ca2+] was evaluated from fluorescence measurements using the Ca2+ indicator dye fura 2 (From Prentki et alss). Oscillations in [Ca2+] have been observed... 

Fig. 9. Kinetics of Ca2+ dissociation from the cPLA, C2 domain in the absence and presence of phosphatidylcholine vesicles, (a) Stopped-flow measurement of the Ca2t off rate using the fluorescent calcium indicator Quin-2, (b) Conformational changes triggered by Ca- removal from the C2 domain monitored using the intrinsic fluorescence of Trp71. (c) Membrane release of the C2 domain followed by protein-to-membrane FRET. (From Nalefski et al. 1997 with permission.)...

A powerful new approach is the direct measurement of released Ca + with fluorescent Ca + indicators such as fura-2 or fluo-3, whereby the permeabilized smooth muscle has to be placed into microcuvettes (lino, 1991). Since force is no longer required as an indicator, Ca + release may be measured in the absence of MgATP, which allowed the study of adenine nucleotides on the IP3-induced Ca + release without the interference by Ca + uptake (lino, 1991 Hirose and lino, 1994). Inclusion of ryanodine allows a separate study of the IP3- and caffeine-sensitive stores (Hirose et al., 1993). Experimental protocols have been designed that allow the use of high concentrations of EGTA, thereby preventing Ca -mediated feedback regulation of the Ca2+ release (Hirose and lino, 1994). Direct measurement of Ca + release with indicators becomes even more powerful in combination with... 

Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M. and Miyawaki, A. (2004). Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc. Natl. Acad. Sci. USA 101, 10554-9. 

Romoser, V. A., Hinkle, P. M. and Persechini, A. (1997). Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators. J. Biol. Chem. 272, 13270-4. 

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