| Regional Biophysics Meeting 2005, March 16-20, Zreče, Slovenia | [CellBiophys] |
Caffeine can effectively modulate intracellular calcium signalling and cellular functional responses via activation of Ca2+-induced Ca2+-release (CICR) from endoplasmic reticulum (ER). Total cellular Ca2+ content was measured by arsenazo III dye. The fluorescent high-affinity and low-affinity Ca2+ indicators fura 2/AM and mag-fura 2/AM were used to measure of the cytosolic ([Ca2+]i) and intrareticular Ca2+ concentration ([Ca2+]L). β-escin (40 mg/ml) was used to cell permeabilization. Changes in [Ca2+]L are reflected by the mag-fura 360/380 nm fluorescence ratio, which is directly proportional to the [Ca2+]L. Following permeabilization the cells were perfused with intracellular like solution containing 3 mM ATP ([Ca2+]i = 300 nM) to reload ER with Ca2+. In experiments with intact cells we did not observed significant changes in total cellular Ca2+ content upon 10 min incubation of salivary cells with caffeine (10, 20, 30 and 40 mM) and any detectable changes in [Ca2+]I in fura 2/AM-loaded intact salivary cells. On the other hand, when caffeine (0.2, 0.5, 1 mM) was added to the incubation medium (for 10 min) of permeabilized acinar cells, the total cellular Ca2+ content significantly decreased on 13±3%, 33±9%, 47±9% respectively (n=8; p<0,05). In mag-fura 2/AM-loaded permeabilized acinar cells brief application of caffeine triggered fall in 360/390 ratio. The mean ratio decrease was 15±0.2%, 32±2% (n=19, p<0,001), 46±5% (n=14, p<0,001) and 56±8% (n=9, p<0,001) upon application of 1, 10, 20 and 30 mM caffeine, respectively. This decrease in the 360/390 ratio upon caffeine application was blocked by 10 mM ryanodine. We suppose that caffeine-induced changes of Ca2+ homeostasis could be induced by the CICR following the activation of RyRs in the ER membrane of salivary cells.
Email: kopach_o@yahoo.com
Address: Ivan Franko Lviv State University, Department of Human and, Animal Physiology,, 4, Grushevsky Str., 79005, Lviv, Ukraine,