Na⁺–Ca²⁺ exchange and its implications for calcium homeostasis in primary cultured rat brain microvascular endothelial cells

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 was measured by digital fluorescence imaging in cells loaded with fura-2. 2. ATP (100 microM) 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 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 microM). 3. Application of thapsigargin (1 microM) or cyclopiazonic acid (10 microM) 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 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 , 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 microM) in the presence of external Na+ increased the resting [Ca2+]i to 222 +/- 9 nM over approximately 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.