Cytosolic calcium measurement

Another major second messenger in cells is calcium ion. Virtually any mammalian cell line can be used to measure transient calcium currents in fluorescence assays when cells are preloaded with an indicator dye that allows monitoring of changes in cytosolic calcium concentration. These responses can be observed in real time, but a characteristic of these responses is that they are transient. This may lead to problems with hemi-equilibria in antagonist studies whereby the maximal responses to agonists may be depressed in the presence of antagonists. These effects are discussed more fully in Chapter 6. 

Snowdowne, K. W., Ertel, R. J., and Borle, A. B. (1985). Measurement of cytosolic calcium with aequorin in dispersed rat ventricular cells. J. Mol. Cell. Cardiol. 17 233-241. 

J.C. Metcalfe, T.R. Hesketh, G.A. Smith, Free cytosolic Ca measurements with fluorine labelled indicators using F NMR, Cell Calcium 6 (1985) 183-195. 

Once mobilized, a large proportion of the cytosolic calcium load is extruded from the cell across the plasma membrane. This extrusion can be demonstrated by measuring the efflux of radiocalcium from previously labeled cells. All stimulation of adrenal, hepatic and vascular smooth muscle cells preloaded with radioactive calcium induces a marked increase in efflux of the radiolabel. This increased efflux is transient, peaking between 4 and 5 minutes after All addition and is observed in the absence of extracellular calcium [39]. Furthermore, under conditions of zero calcium, treatment of cells with dantrolene prior to hormonal stimulation abolishes the All-induced calcium efflux [40], confirming that the radiocalcium lost from the cell is mobilized from a component of the ER. 

Figure 1.8. Influence of extracellular Ca + on 30 gM gamblerol-Induced response. This effect was measured In a Ca +-free and Ca +-contalnlng solution. Intracellular calcium was monitored In fura-loaded neuroblastoma cells. Gambierol produced no Increment In cytosolic calcium when It was added In the Ca +-free solution.

Connor, j- A-, Cornwall, M. C., and Williams, G. H. (1987). Spatially resolved cytosolic calcium response to angiotensin II and potassium in rat glomemlosa cells measured by digital imaging techniques. J. Bioi. Chem. 262, 2919-2927. 

In the cell suspension culture treated with the processed water, CLA chemiluminescence (Fig. 2) and aequorin luminescence (Fig. 3) reflecting the generation of superoxide and increase in cytosolic calcium ion concentration ([Ca2+]c), respectively, were measured. We observed the spikes of CLA chemiluminescence in the cell suspension culture after addition of photo-catalytically processed waters, suggesting that photo-catalytic process generated or conditioned the waters enabling the stimulation of plant cells with oxidative stress. 

The isolated renal vessel technique has been modified so that vessels can be isolated from rats instead of rabbits [256]. The preparation has also been modified for fluorescence measurements of cytosolic calcium in the smooth muscle cell layer [257]. Measurements of changes in smooth muscle calcium can be carried out simultaneously with determinations of changes in lumen diameters. The effects of various agonists and pressure stimulation in the presence and absence of pharmacologic agents can be determined. Angiotensin... 

Cytosolic calcium overload is generally associated with cell injury and energy deprivation increases intracellular calcium. Kondo et al. measured 2 mM... 

That nicotine directly interacts with receptors on the afferent nerves is supported by studies showing that nodose neurons express nicotinic receptors (Mao et al. 2006). Furthermore, in functional studies that assess increases in cytosolic calcium as a measure of neuronal activation, nicotine and other nicotinic receptor agonists cause activation of a subset of capsaicin-sensitive neurons retrogradely labeled from the lungs. Nicotine also activated a subset of capsaicin-insensitive lung afferent nodose neurons (perhaps RAR neurons) (Xu et al. 2007). 

Braun, F.-J. and Hegemann, P. (1999) Direct measurement of cytosolic calcium and pH in living Chlamydomonas reinhardtii cells. Eur. J. CeUBiol. 78, 199-208. 

In Vivo Measurements of Cytosolic Calcium in Dictyostelium discoideum... 

Hurley, T. W. Ryan, M. R Brinck, R. W. Changes of cytosolic calcium interfere with measurements of cytosolic magnesium using Mag-Fura-2. Am. J. Physiol. 1992, 263, C300-C307. 

Tsien, R. Y., Rink, T. J. and Poenie, M. Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths. Cell Calcium 6 145-157,1985. 

Read ND, Allan WTG, Knight H, Knight MR, Malho R, Russell A, Shacklock PS, Trewavas AJ. Imaging and measurement of cytosolic free calcium in plant and fungal cells. JMicrosc 1992 166 57-86. 

Vomdran, C. et al. (1995) New fluorescent calcium indicators designed for cytosolic retention or measuring calcium near membranes, Biophys. J. 69, 2112-2124. 

The mobilization of calcium results not only in the observed transient rise in intracellular free calcium and enhanced cellular efflux, but also in a net loss of calcium from the cell (Fig. 1). Thus, total cell calcium declines with All stimulation of adrenal and vascular smooth muscle cells [44]. Furthermore, total cell calcium remains low throughout the duration of exposure to All, suggesting that the continued formation of small amounts of 1,4,5-IP3 prevents refilling of the ER pool. Upon the removal of All and the immediate reduction in IP3 concentration, total cell calcium rapidly recovers to prestimulation levels without a detectable change in cytosolic free calcium, as measured by calcium-sensitive dyes. This observation has been taken as evidence that the IP3-releasable ER pool is in direct communication with the plasma membrane and that extracellular calcium refills the pool without entering the bulk cytosol (see Ref. 45). The location of this pool within the cell (cytosolic vs. adjacent to the plasma membrane) remains a matter of controversy (see Rasmussen arid Barrett, Chapter 4). 

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