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Am J Physiol Cell Physiol (December 12, 2001). doi:10.1152/ajpcell.00478.2001
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Articles in PresS, published online ahead of print December 12, 2001
Am J Physiol Cell Physiol, 10.1152/ajpcell.00478.2001
Submitted on October 9, 2001
Accepted on December 7, 2001

Contribution of Na+-K+-Cl- cotransporter to high [K+]o-induced swelling and excitatory amino acid release in rat astrocytes

Gui Su1, Douglas B Kintner2, and Dandan Sun1*

1 Neurosurgery, University of Wisconsin Medical School, Madison, Wisconsin, USA; Physiology, University of Wisconsin Medical School, Madison, Wisconsin, USA
2 Neurosurgery, University of Wisconsin Medical School, Madison, Wisconsin, USA

* To whom correspondence should be addressed. E-mail: sun{at}neurosurg.wisc.edu.

We previously found that Na+-K+-Cl- cotransporter activity in rat astrocytes was significantly stimulated in 75mM [K+]o. We hypothesized that this stimulation may result in an net influx of K+ and Cl- and thus lead to high [K+]o-induced swelling and glutamate release. In the current study, relative cell volume changes were determined in a single astrocyte using video-enhanced differential interference contrast (DIC) microscopy. In 75mM [K+]o, astrocytes swelled by 120.2 ± 4.9 %. This high [K+]o-mediated swelling was abolished by the NKCC inhibitor bumetanide (10µM, 98.5 ± 3.1 %, p < 0.05). Intracellular 36Cl accumulation was increased from a control value of 0.39 ± 0.06 to 0.68 ± 0.05µmol/mg protein in response to 75mM [K+]o. The increase of intracellular 36Cl level was significantly reduced by bumetanide (p < 0.05). [Na+]i was determined using the ratiometric dye SBFI. Basal levels of [Na+]i were reduced from 19.1 ± 0.8 to 16.8 ± 1.9 mM by bumetanide (p<0.05). [Na+]i decreased to 8.4 ± 1.0 mM under 75 mM [K+]o and was further reduced to 5.2 ± 1.7 mM by bumetanide. In addition, the recovery rate of [Na+]i upon returning to 5.8mM [K+]o was decreased by 40% in the presence of bumetanide (p < 0.05). We then examined if inhibition of the Na+-K+-Cl- activity could block the release of pre-loaded [14C]-D-aspartate (Asp). Exposing cells to 75 mM [K+]o induced a release of the pre-loaded [14C]-D-Asp by 1.52 ± 0.17%. [14C]-D-Asp release was reduced to 0.68 ± 0.13% by bumetanide (p < 0.05). Taken together, the results suggest that the Na+-K+-Cl- cotransporter contributes to high [K+]o-induced astrocyte swelling and glutamate release.




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