Ca2+-sensitive dyes

Since the first report by lino and co-workers in 1994 (lino Tsukioka 1994), asynchronous wave-like [Ca2+]j oscillations have emerged as an universal mode of Ca2+ signalling in in situ VSMCs (Table 1). Confocal microscopy of intracellular Ca2+-sensitive dyes in intact blood vessels, reveals recurrent intracellular Ca2+ waves travelling through the longitudinal axis of the ribbonshaped VSMCs. These waves, which are usually but not always initiated by agonists, do not propagate between cells. 

With cell retention accomplished by 3D flow control, calcium mobilization in a single yeast cell has been studied over a long period of time, and in the manner of multiple stimuli and multiple responses. Loading of the Ca2+- sensitive dye Fluo-4 in the cell was accomplished either (1) after cell wall removal or (2)... 

Since Ca2+ waves are rises of intracellular free Ca2+, Ca2+ imaging is required to visualize them. Generally fast, high-affinity Ca2+-sensitive fluorescent dyes such... 

FIG. 2. Imaging of Ca2+ within the SR stores using the low-affinity Ca2+-sensitive fluorescent dye Fluo-3-FF. This cell is a myocyte freshly dispersed from term pregnant myometrium, but similar results are seen with cultured cells. Scale bar = 10 /im (reprinted from Morgan et al 1996). 

Sensitization of honeybees by sucrose stimulation led to an overall increase of Ca2+ signals in PNs (Weidert et al., 2001). The PNs were selectively labeled with a calcium-sensitive dye (see below) and the response in activated glomeruli was significantly stronger than in control animals. Hence, neural correlates of both associative and non-associative processes are formed already at the level of the AL. A sensitized animal will be in a state more ready to pay attention to environmental stimuli, e.g. foodsearch. 

Earlier techniques for measuring cytosolic free Ca2+ (1-2) such as the luminescent photoprotein aequorin, the absorbance dye arsenazo III and Ca2+-sensitive microelectrodes, all required microinjection or impalements, and were therefore applied mainly to giant cells. Later, photoproteins have been loaded with various reversible permeabilization procedures (3), but the largest expansion in the range of cell types in which Ca2+ signals can be quantified has come from the development of new fluorescent indicators that can be loaded using hydrolyzable esters. Currently four fluorescent indicators are used frequently quin-2, fura-2, indo-1 and fluo-3. 

Several ions are involved in the regulation of gap junctional conductance including Ca2+, Mg2+, H+ and Na+. Very early Ca2+-induced reduction of junctional permeability has been described in Chironomus salivary glands [Rose and Loewenstein, 1975] and heart [De Mello, 1975]. Using the calcium-sensitive fluorescent dye, aequorin, Rose and Loewenstein [1975, 1976] demonstrated parallel changes in pCa and uncoupling, concluding that the actual pCaj is... 

The free dye (M) has an absorption maximum at 520 nm while Ca M absorbs maximally at 480 nm. The concentration of Ca2 + can be calculated from a standard curve in which A480 is plotted as a function of [Ca2+] or preferably from a standard curve of (A520 — A480) as a function of [Ca2+] which is less curved and more sensitive (Figure 5.9). Using this method, the [Ca2+] in milk was found to be 2.53-3.4 mM and appears to be 0.8 mM higher than that determined by the other methods. 

Annexins isolated from chondroblast matrix vesicles may be reconstituted with phospholipids to form calcium ion channels in the complete absence of Ca2+ ions. Indeed, annexin V has domains that directly bind calcium ions glutamate and aspartate residues provide the ion binding site (EF-hand domains). Figure 9.6b illustrates putative annexin V channels that mediate an influx of Ca2+ ions into artificial bilayers and liposomes (detectable with a calcium-sensitive fluorescent dye). These in vitro annexin Ca2+ channels, and also the Ca2+ influx into matrix vesicles in cell culture and in vivo, are blocked by Zn2+ ions, or a derivative of 1,4-benzothiazepine (inhibitor K201). 

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