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

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Abstract
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. 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 t½ (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). 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 t½ values of 15 ± 1.6 and 23 ± 3 s, respectively. 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 (t½, 7.3 ± 0.46 s in Li+ medium, 8.12 ± 0.4 s in N-methylglucamine medium). 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. 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+. 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.

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