1. The role of Na+-Ca2+ exchange in the regulation of the cytosolic free Ca2+ concentration ([Ca2+](i)) was studied in primary cultured rat brain capillary endothelial cells. [Ca2+](i) measured by digital fluorescence imaging in cells loaded with fura-2. 2. ATP (100 μM) applied for a short period of time (6 s) caused a rise in [Ca2+](i) from 127 ± 3 (n = 290) to 797 ± 25 nM, which then declined to the resting level, with a t 1/2 (time required for [Ca2+](i) to decline to half of peak [Ca2+](i)) of 5.4 ± 0.09 s. This effect was independent of external Ca2+ and could be abolished by previously discharging the Ca2+ pool of the endoplasmic reticulum with thapsigargin (1 μM). 3. Application of thapsigargin (1 μM) or cyclopiazonic acid (10 μM) to inhibit the Ca2+-ATPase of the endoplasmic reticulum 6 s prior to ATP application did not influence the peak [Ca2+](i) but greatly reduced the rate of decline of [Ca2+](i), with t 1/2 values of 15 ± 1.6 and 23 ± 3 s, respectively 4. In the absence of external Na+ (Na+ replaced by Li+ or N-methylglucamine) the basal [Ca2+](i) was slightly elevated (152 ± 6 nM) and the restoration of [Ca2+](i) after the ATP stimulation was significantly slower (t 1/2 , 7.3 ± 0.46 s in Li+ medium, 8.12 ± 0.4 s in N-methylglucamine medium). 5. The external Na+-dependent component of the [Ca2+](i) sequestration was also demonstrated in cells stimulated by ATP subsequent to addition of cyclopiazonic acid; in a Na+-free medium [Ca2+](i) remained at the peak level in 88% of the cells after stimulation with ATP. 6. Addition of monensin (10 μM) in the presence of external Na+ increased the resting [Ca2+](i) to 222 ± 9 nM over ~ 1 min and subsequent removal of extracellular sodium resulted in a further increase in [Ca2+](i) to a peak of 328 ± 11 nM, which was entirely dependent on external Ca2+. 7. These findings indicate that a functional Na+-Ca2+ exchanger is present at the blood-brain barrier, which plays a significant role in shaping the stimulation-evoked [Ca2+](i) signal and is able to work in reverse mode under pharmacological conditions.
ASJC Scopus subject areas