Ca2* indicators

Blinks, J. R. (1989). Use of calcium-regulated photoproteins as intracellular Ca2+ indicators. Method. Enzymol. 172 164-203. 

Shimomura, O., and Shimomura, A. (1985). Halistaurin, phialidin and modified forms of aequorin as Ca2+ indicator in biological systems. Biochem. J. 228 745-749. 

A typically prolonged transient increase in intracellular Ca2+ concentration, which is detected by a Ca2+ indicator. The increased Ca2+ levels are due to Ca2+... 

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

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. 

Obelin is a Ca2+-activated bioluminescent photoprotein that has been isolated from the marine polyp Obelia longissima. Binding of calcium ions determines a luminescent emission. The protein consists of 195 amino acid residues [264] and is composed of apoobelin, coelenterazine, and oxygen. As aequorin, it contains three EF-hand Ca2+-binding sites and the luminescent reaction may be the result of coelenterazine oxidation by way of an intramolecular reaction that produces coelenteramide, C02, and blue light. As for aequorin, the luminescent reaction of obelin is sensitive to calcium and the protein was used in the past as an intracellular Ca2+ indicator. More recently, the cloning of cDNA for apoobelin led to the use of recombinant obelin as a label in different analytical systems. 

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... 

Wier We don t find an effect of APB on high K+ contraction if phentolamine is present. With respect to your mechanism of tonic contraction, which you indicated was dependent on Ca2+ influx, in the mesenteric small artery that we study, which develops a tonic contraction, this isn t accompanied by tonic elevation of Ca2+, but rather by these asynchronous Ca2+ waves that we have already seen. Nevertheless, when we add up all these Ca2+ indicator signals we get something that looks like a steady elevated level of Ca2+. We would say that in that tissue the dependence on Ca2+ influx is to keep the SR going, to keep generating these waves. Is this relevant to your tissue ... 

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). 

We carefully dissected rat tail arteries and loaded them with a Ca2+ indicator, Fluo-3. After a rectangular glass capillary was inserted into the lumen of the excised arteries, [Ca2+] in smooth muscle cells within the arterial wall was visualized using a confocal microscope. Brief electrical shocks were delivered at 5 Hz to the preparations to stimulate the sympathetic nerve network present in the adventitia. We found Ca2+ signals with diverse spatiotemporal patterns, Ca2+ waves and oscillations in individual smooth muscle cells during the sympathetic nerve stimulation (lino et al 1994). 

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. 

Increases in the levels of Ins 1,4,5-P3 can be measured in several ways (e.g. by radioimmunoassay), whilst intracellular levels of Ca2+ can be monitored by the inclusion into the neutrophil cytoplasm of suitable Ca2+ indicators. Some elegant work using the Ca2+-sensitive photoprotein obelin (obtained from the luminous jellyfish Obelia) has shown that increases in intracellular Ca2+ can be detected within seconds of activation of neutro-... 

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.)...

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. 

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... 

Pologruto, T.A., R. Yasuda, and K. Svoboda. 2004. Monitoring neural activity and Ca2+ with genetically encoded Ca2+ indicators. J. Neurosci. 24 9572-9579. 

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