(for review see Refs. 6, 11). A change in the balance between influx and efflux will alter i and the gradient, thereby modulating ,-coupled processes and cellular activity. For example, alterations of Lli influence the cytosolic Ca2+ concentration ([Caâ+]& via -Ca2+ exchange, thereby affecting the intracellular storage of Ca2+ and cell reactivity (5). Direct measurements of [ ] i previously required the laborious application of ion-selective microelectrodes (e.g., see Ref. 36). Recently, Minta and Tsien (25) developed a reliable -sensitive fluorescent indicator, sodium-binding benzofuran isophthalate (SBFI), which enables convenient direct monitoring of i at the single-cell level. SBFI, like fura-2, is a ratiometric dye; the ratio of fluorescent emission excited by 340 and 380 nm illumination is proportional to concentration. The sensitivity of SBFI is sufficient to detect concentration changes of ~1 mM (1, 7, 19, 27). SBFI fluorescence reflects accurately the predicted changes in i that result from lowering extracellular ([ ],), inhibiting the pump, or applying cell agonists (1,7,8,19,27,29). Use of SBFI has revealed some differences between intracellular free and total cell content. For example, i measured with SBFI is always lower than total cell content (7, 19, 27); this may indicate that some is bound
AJP - Cell Physiology – The American Physiological Society
Published: Jun 1, 1993
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