Calcium intracellular, measurement

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.

A calcium ion indicator dye (based on the structure of the chelator EGTA) that exhibits a strong fluorescence at 385 nm and can be used to measure changes in intracellular Ca concentration. The approximate dissociation constant for the Ca -Fura-2 complex is 0.1 juM, depending on cellular ion composition and pH. An esteri-fied derivative of Fura-2 readily crosses the peripheral membrane of many cells and, after hydrolysis, the release of Fura-2 permits calcium ion measurements within cells. See Calcium Ion Indicator Dyes Metal Ion Complex-ation... 

Collet, C., Allard, B., Tourneur, Y., and Jacquemond, V., 1999, Intracellular calcium signals measured with indo-1 in isolated skeletal muscle fibres from control and mdx mice, J Physiol, 520 Pt 2, pp 417—429. 

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). 

All nicotinic receptors are somewhat calcium permeable the most permeable are neuronal homomeric receptors (a7, a9) and the least permeable, embryonic muscle receptors. It must be noted that the measurement of relative calcium permeability by the simplest technique (reversal potential shift induced by changes in extracellular calcium concentrations) is error-prone for neuronal nicotinic receptors because their extreme inward rectification makes it difficult to measure reversal potentials accurately. A further technical difficulty (for recombinant receptors) arises from the presence in Xenopus oocytes of a calcium-dependent chloride conductance that has to be suppressed or minimized by either intracellular calcium chelation or chloride depletion. Some of these problems can be overcome by expressing the receptors in mammalian cell lines and using ratiometric measurements of intracellular calcium and coupled with wholecell recording, to obtain a measure of what proportion of the nicotinic current is carried by calcium (a measure that also has the advantage of being physiologically more relevant). 

Calcium Sensing. Measurements of intracellular concentrations of Ca are of wide interest in cell physiology and biology. There is a vast literature concerning the measurements of the intracellular concentrations of Ca using fluorescent indicators and the journal Cell Calcium is devoted to this topic 11,101,103). 

White, J. R., Ishizaka, T., Ishizaka, K., and Sha afi, R. I., 1984, Direct demonstration of increased intracellular concentration of free calcium as measured by quin-2 in stimulated rat peritoneal mast cell, Proc. Natl. Acad. Sci. USA 81 3978-3982. 

Boltz, R. C., Jr. A high capacity screen for immunoregulants using intracellular calcium concentration measurement. PCT hit. Appl. WO 9610090, 1996 Chem. Abstr. 1996,125, 76326. 

Brown, H. M., Pemberton, J. P., Owen, J. D., Calcium-sensitive microelectrode suitable for intracellular measurement of calcium(II) activity. Anal Chim Acta 1976, 85(2), 261-276. 

A single 337-nm flash (<1 ns) released sufficient bradykinin to excite the BK2 receptors on single rat sensory neurons, which dramatically increased the intracellular calcium concentration measured with Indo-1, a Ca +-chelating fluorescent indicator. The quantum efficiency of bradykinin appearance was idependently determined to be 0.22, as shown in Table 69.12. 

Sheu, Y. A., Kricka, L. J., and Pritchett, D. B. (1993). Measurement of intracellular calcium using bioluminescent aequorin expressed in human cells. Anal. Biochem. 209 343-347. 

Figure 1. Original records of tension and intracellular free calcium concentration Caf ) obtained from a single mouse muscle fiber during a fatigue run (modified from Westerblad and Allen, 1991). A continuous tension record in which each vertical line represents a tetanus. B (Ca ] (measured with fura-2) and tension records obtained from the individual tetani (a, b, and c) indicated above the record in A. Three major features are illustrated 1.) the initial tension decline is accompanied by an increase in tetanic ICa li, 2.) late in fatigue the tetanic [Ca li is reduced, and 3.) the resting [Ca li increases during fatiguing stimulation (dashed line indicates resting [Ca ] in control). Stimulation periods are shown below tension records in B. From Westerblad et al., 1991, with permission from the Amer. Physiol. Society.

Another popular assay for GPCR activation is to measure the increase in intracellular Ca2+ that occurs upon activation. GPCRs on the cell surface produce inositol triphosphate (IP3) via the action of Phospholipase C (PLC). IP3 stimulates calcium channels called IP3 receptors on the endoplasmic reticulum, which raise... 

Patients with acute hyperkalemia usually require other therapies to manage hyperkalemia until dialysis can be initiated. Patients who present with cardiac abnormalities caused by hyperkalemia should receive calcium gluconate or chloride (1 g intravenously) to reverse the cardiac effects. Temporary measures can be employed to shift extracellular potassium into the intracellular compartment to stabilize cellular membrane effects of excessive serum potassium levels. Such measures include the use of regular insulin (5 to 10 units intravenously) and dextrose (5% to 50% intravenously), or nebulized albuterol (10 to 20 mg). Sodium bicarbonate should not be used to shift extracellular potassium intracellularly in patients with CKD unless severe metabolic acidosis (pH less than 7.2) is present. These measures will decrease serum potassium levels within 30 to 60 minutes after treatment, but potassium must still be removed from the body. Shifting potassium to the intracellular compartment, however, decreases potassium removal by dialysis. Often, multiple dialysis sessions are required to remove potassium that is redistributed from the intracellular space back into the serum. 

As light emission from aequorin is dependent on Ca2+, the protein has been widely employed for determination of this ion. In particular, the protein was used in the past in the highly sensitive measurement of intracellular calcium concentration in several kind of cells [16]. More recently immobilized aequorin was used to develop an optical biosensor for measurement of calcium ions in complex... 

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