Calcium transient measurements

Calcium transient measurements The detection of Ca transients is widely used in high-throughput screening assays in cells preloaded with a fluorescent... 

Practical problems with hemi-equilibria can be avoided by allowing sufficient time for equilibrium to occur. However, there are some situations where this may not be possible. One is where the functional system desensitizes during the span of time required for equilibrium to be attained. Another is where the actual type of response being measured is transitory and where the only measurement of calcium transients where a spike of effect is the only response observed in the experimental system. 

Figure 4.7 Changes in intraceiiuiar calcium in cultured rat ventricular myocytes exposed to oxidant stress. Calcium was measured using the fluorescent probe Fura>2. The ratio of the Fura-2 fluorescence measured at 340 and 380 nm excitation is shown and this is proportional to the intracellular calcium concentration. The fast-speed traces shown (note the 3.5 s time-scale) were recorded after various durations of oxidant stress. Myocytes under control conditions (before t = 0) show spontaneous calcium transients. These transients decreased in frequency with oxidant stress until cells failed to show spontaneous activity but continued to maintain a low intracellular calcium.

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). 

Kobayashi, S., Kanaide, H., and Nakamura, M. (1985). Cytosolic free calcium transients in cultured vascular smooth muscle cells Microfluormetric measurements. Science 229, 533-556. 

Konishi, M. Berlin, J. R. Calcium transients in cardiac myocytes measured with a low affinity fluorescent indicator, Furaptra. Biophys. J. 1993, 64, 1331-1343. 

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. 

A. Azzi B. Chance (1969) Biochim. Biophys. Acta 189, 141. Selected entries from Methods in Enzymology [vol, page(s)] Calibration of calcium binding, 260, 425-427 luminescence measurement, 260, 424-425 mitochondria-targeted hybrid protein [calcium quantitation, 260, 418, 422, 424-428 stable expression, 260, 418-421 transient expression, 260, 420 intracellular localization, 260, 421-425 reconstitution with coelenterazine, 260, 422-422] structure, 260, 418. 

Hence, surface films composed mostly of CaCl2 cover calcium electrodes in T. As is usual for SEI-type electrodes [32], the films grow and reach a steady thickness during storage. It is assumed that the major ionic conductor in these films is Cl-, as they are totally blocking for Ca deposition [32], Their resistivity was measured by transient techniques and impedance spectroscopy [28], and is around 109 Q cm for a fresh Ca electrode and 1010 Q cm for an aged Ca electrode (500-1000 h in a TC-Ca(AlCl4)2 solution) [28], The thickness of the surface films is estimated as several tens of A [32],... 

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. 

Thermal ionization has been used to determine isotopic abundance of virtually all the elements We have recently extnded our own capability in this direction by adapting the silica gel/phosphoric acid filament coating technique (5) to our system Five 1 of a fine silica gel suspension is placed on a filament Five l of the analyte ion solution is coated, dried then coated with 2 pi of a 0 7N phosphoric acid solution and heated until dry again The analysis is performed in a similar manner as before, except that the signal is more transient and somewhat less intense than the calcium analysis With this approach, however, we have made natural abundance isotope ratio measurements on zinc, copper, and magnesium Table II shows our measurements compared to the accepted values, shown in parenthesis, for these elements The isotope used as reference... 

When environmental chemists measure the amounts of chemical substances, for example in a water sample, they are usually measuring the concentration of that substance, for example the concentration of calcium (Ca) in the water. It is very easy to assume that the analysis has measured all of the free Ca2+ ions in the sample, but in fact it will almost certainly have measured all of the calciumbearing dissolved species, called ion pairs, as well. Ions in solution are often sufficiently close to one another for electrostatic interactions to occur between oppositely charged species. These interactions reduce the availability of the free ion to participate in reactions, thereby reducing the effective concentration of the free ion. Collisions between oppositely charged ions also allow the transient formation of ion pairs, for example ... 

Strontium stimulates bone formation and decreases bone resorption. In a preliminary study of postmenopausal women with severe osteoporosis, strontium ranelate 1 g twice daily or 2 g once daily reduced new vertebral fractures by 41%, and increased lumbar spine BMD by 14% and femoral neck BMD by 8% compared with placebo. Nonvertebral fracture rates were similar. Diarrhea was more common in the strontium group. Small decreases in serum calcium and PTH, small increases in serum phosphate, and transient increases in creatine kinase were measured. 

If physiological approaches are to be used to resolve issues about the relationship between calcium and cytokinins, then a system is needed in which one can add hormone and rapidly measure local Ca" " transients or waves. Experimental conditions need to be found to avoid mechanical disturbances known to induce strong calcium responses as measured by aequorine luminescence [70]. So far, our attempts to measure Ca transients using aequorine luminescence in Nicotiana plumbaginifolia cells and seedlings have been unsuccessful (Faure and Howell, unpublished data). 

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