Contribution of Na⁺-K⁺-Cl⁻cotransporter to high-[K⁺]_o- induced swelling and EAA release in astrocytes

Abstract

We hypothesized that high extracellular K(+) concentration ([K(+)](o))-mediated stimulation of Na(+)-K(+)-Cl(-) cotransporter isoform 1 (NKCC1) may result in a net gain 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 astrocytes. Under 75 mM [K(+)](o,) astrocytes swelled by 20.2 +/- 4.9%. This high-[K(+)](o)-mediated swelling was abolished by the NKCC1 inhibitor bumetanide (10 microM, 1.0 +/- 3.1%; P < 0.05). Intracellular (36)Cl(-) accumulation was increased from a control value of 0.39 +/- 0.06 to 0.68 +/- 0.05 micromol/mg protein in response to 75 mM [K(+)](o). This increase was significantly reduced by bumetanide (P < 0.05). Basal intracellular Na(+) concentration ([Na(+)](i)) was 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) on return to 5.8 mM [K(+)](o) was decreased by 40% in the presence of bumetanide (P < 0.05). Bumetanide inhibited high-[K(+)](o)-induced (14)C-labeled D-aspartate release by ~50% (P < 0.05). These results suggest that NKCC1 contributes to high-[K(+)](o)-induced astrocyte swelling and glutamate release.

Receptor-mediated glutamate release from volume sensitive channels in astrocytes

“…When K-gluconate replaced intracellular Cs-glutamate, a small outward current was recorded (5). The addition of 10 mM BAPTA in the pipette (same conditions as in 3) inhibited the inward current (6). When 100 M NPPB was added to the bath (same conditions as in 3), the inward current was inhibited other amino acids, such as leucine, phenylalanine, and tyrosine, were not released (Fig.…”

“…Astrocytes possess multiple mechanisms for several key functions. For example, the important task of K ϩ buffering is undertaken by several K ϩ channels expressed by astrocytes, including KIR4.1 and rSlo K(Ca) (5), but also by the K ϩ -Na ϩ -Cl Ϫ cotransporter (6).…”

Cytotoxic edema: mechanisms of pathological cell swelling

“…Genetic ablation of NKCC1, as well as its block by bumetanide,17 causes a decrease in intracellular Cl − in hypoxic neurons145 and blocks Na + -and Cl − -mediated cell swelling in astrocytes. 101,142,143 In vivo studies have shown that intracerebral bumetanide administered via a microdialysis probe, either prior to or during ischemia/hypoxia insult caused by temporary MCA occlusion, is neuroprotective, ameliorates brain damage, and reduces brain edema in rat focal ischemia,141,172,173 reinforcing the in vitro data. Focal cerebral ischemia in rats results in elevated NKCC1 protein levels in the ipsilateral cortex and striatum.…”

Novel Treatment Targets for Cerebral Edema

“…In a rat model of cerebral ischemia, NKCC1 transcripts and protein are significantly upregulated in cortical neurons, as well as in whole brain lysates [54,55]. These changes may occur in ischemic conditions secondary to elevated extracellular potassium and glutamate levels, substances known to stimulate NKCC1 activity in neurons and neuroglia [18,56,57]. Cytokines may also be involved in ischemiainduced upregulation of NKCC1, with interleukin-6 shown to activate NKCC1 in capillaries [58].…”