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

摘要

class="enumerated" style="list-style-type:decimal">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.