E. Domotor et al., Na+-Ca2+ exchange and its implications for calcium homeostasis in primary cultured rat brain microvascular endothelial cells, J PHYSL LON, 515(1), 1999, pp. 147-155
1. The role of Na+-Ca2+ exchange in the regulation of the cytosolic free Ca
2+ concentration ([Ca2+](i) was studied in primary cultured rat brain capil
lary endothelial cells. [Ca2+]i was measured by digital fluorescence imagin
g in cells loaded with fura-2.
2. ATP (100 mu M) 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 t
o 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 o
f external Ca2+ and could be abolished by previously discharging the Ca2+ p
ool of the endoplasmic reticulum with thatpsigargin (1 mu M).
3. Application of thapsigargin (1 mu M) or cyclopiazonic acid (10 mu M) to
inhibit the Ca2+-ATPase of the endoplasmic reticulum 6 s prior to ATP appli
cation did not influence the peak [Ca2+], but greatly reduced the rate of d
ecline of [Ca2+](i) with t(1/2) values of 15 +/- 1.6 and 23 +/- 3 s, respec
tively.
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 restorat
ion 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 cycl
opiazonic acid; in a Na+-free medium [Ca2+](i) remained at the peak level i
n 88% of the cells after stimulation with ATP.
6. Addition of monensin (10 mu M) in the presence of external Na+ increased
the resting [Ca2+](i) to 222 +/- 9 nM over similar to 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 s
timulation-evoked [Ca2+](i) signal and is able to work in reverse mode unde
r pharmacological conditions.