Retraction Note: Functional kinomics establishes a critical node of volume-sensitive cation-Cl- cotransporter regulation in the mammalian brain.
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Journal Article↗
Biomedical subjects
Dandan Sun
Publications and source records attributed to Dandan Sun.
Na-K-Cl cotransporter contributes to glutamate-mediated excitotoxicity.
We hypothesized that cation-dependent Cl- transport protein Na-K-Cl cotransporter isoform 1 (NKCC1) plays a role in the disruption of ion homeostasis in cerebral ischemia. In the current study, a role for NKCC1 in neuronal death was elucidated in neurotoxicity induced by glutamate and oxygen and glucose deprivation (OGD). Incubation of cortical neurons cultured for 14-15 d in vitro (DIV) with 100 microm glutamate for 24 hr resulted in 50% cell death. Three hours of OGD followed by 21 hr of reoxygenation led to 70% cell death. Inhibition of NMDA receptors with dizocilpine hydrogen maleate (1 microm) prevented both OGD- and glutamate-mediated cell death. Moreover, blocking of NKCC1 activity with bumetanide (5-10 microm) abolished glutamate- or OGD-induced neurotoxicity. Bumetanide was ineffective if added after 10-120 min of glutamate incubation or 3-6 hr of OGD treatment. Accumulation of intracellular Na+ and 36Cl content after NMDA receptor activation was inhibited by bumetanide. Blockage of NKCC1 significantly attenuated cell swelling after OGD or NMDA receptor activation. This neuroprotection was age dependent. Inhibition of NKCC1 did not protect DIV 7-8 neurons against OGD-mediated cell death. In contrast, cell death in DIV 7-8 neurons was prevented by the protein-synthesis inhibitor, cycloheximide. Taken together, the results suggest that NKCC1 activity is involved in the acute excitotoxicity as a result of excessive Na+ and Cl- entry and disruption of ion homeostasis.
Animals↗
Inhibition of Na(+)-K(+)-Cl(-) cotransporter during focal cerebral ischemia decreases edema and neuronal damage.
Our previous study demonstrated that pharmacological inhibition of the Na(+)-K(+)-Cl(-) cotransporter isoform 1 (NKCC1) during ischemia and reperfusion attenuated neuronal damage and edema. In this study, we further investigated whether NKCC1 activity contributes to ischemic damage during either ischemia or reperfusion. Immunoblotting revealed that expression of NKCC1 protein was increased following 2-h focal ischemia in cerebral cortex. A sustained up-regulation of NKCC1 in cortex was detected at 4, 8, 12, and 24 h of reperfusion. An increase in the phosphorylated NKCC1 (NKCC1-p) was found at 4 and 8 h of reperfusion. In striatum, a significant increase in NKCC1 expression occurred between 4 and 24 h of reperfusion and no elevation of NKCC1-p signal was observed. Artificial cerebral spinal fluid (aCSF) or 100 microM bumetanide in aCSF were continuously microdialyzed into left cortices either 1 h prior to ischemia plus 2-h ischemia, or only during 24-h reperfusion. Infarction volume was significantly decreased in the pre-ischemic bumetanide-treated group (P<0.05) but not in the post-ischemic treatment group (P>0.05). In addition, pre-ischemic bumetanide treatment reduced the ipsilateral water content increase by 70% (P<0.05). Inhibition of NKCC1 did not attenuate poly (ADP-ribose) polymerase cleavage or the number of TUNEL-labeled apoptotic cells in ischemic brains. These results suggest that inhibition of NKCC1 attenuates cytotoxic edema and necrotic neuronal death during focal ischemia. Activation of NKCC1 activity plays a role in the early stage of ischemic damage.
Animals↗
Adult olfactory ensheathing cell transplantation for acute spinal cord injury.
Cellular transplantation strategies have been explored for the treatment of spinal cord injury. In particular, olfactory nerve ensheathing cell (OEC) transplantation has been reported to improve functional outcome following injury. We investigated the effect of OEC transplantation using cells derived from adult animals on the restoration of function following a contusion injury to the spinal cord. The NYU impactor was used to create a moderate to severe spinal cord injury in 17 rats. Hoescht stained cultured OECs derived from adult rats (n = 7) or culture medium alone (n = 10) were injected into the injury site immediately following injury. Histological and functional outcomes were measured using immunohistochemistry and the Basso, Beattie, and Bresnahan (BBB) scale. All animals transplanted with OECs were found to have surviving Hoescht positive cells within the spinal cord when sacrificed 6 weeks following injury. Immunohistochemical staining of the explanted cords revealed that the surviving cells stained positively for nerve growth factor receptor. Functional outcomes were not different between the transplanted and control groups. OECs transplanted immediately following a contusion injury to the spinal cord survive during the first 6 weeks following injury. These cells do not appear to influence functional outcome during the first 6 weeks following injury. Additional studies are required in order to definitively determine the utility of this type of cellular transplantation for spinal cord injury.
Animals↗
Cross talk between the GABA(A) receptor and the Na-K-Cl cotransporter is mediated by intracellular Cl-.
It has been suggested that the GABA(A) receptor-mediated depolarization in immature neurons depends on a high intracellular Cl(-) concentration maintained by Na-K-Cl cotransporter isoform1 (NKCC1). We previously found that activation of the GABA(A) receptor led to stimulation of NKCC1. This stimulation could be a result of GABA(A) receptor-mediated Cl(-) efflux. However, a loss of intracellular Cl(-) is associated with cell shrinkage, membrane depolarization, as well as a rise of intracellular Ca(2+) concentration ([Ca(2+)](i)). To determine which cellular mechanism is underlying NKCC1 stimulation, we investigated changes of intracellular Cl(-) content, [Ca(2+)](i), cell volume, and NKCC1 activity following GABA(A) receptor activation. The basal levels of intracellular (36)Cl were 0.70 +/- 0.04 micromol/mg protein. The intracellular (36)Cl content decreased to 0.53 +/- 0.03 micromol/mg protein in response to 30 microM muscimol (P < 0.05). The loss of intracellular (36)Cl was blocked by 10 microM bicuculline. Muscimol triggered a rise in [Ca(2+)](i), but did not cause cell shrinkage. In contrast, 10-50 mM [Cl(-)](o) or hypertonic HEPES-MEM resulted in reversible cell shrinkage (P < 0.05). Moreover, the GABA-mediated stimulation of NKCC1 activity was not abolished either by removal of extracellular Ca(2+) or BAPTA-AM. An increase in phosphorylation of NKCC1 was detected under both 10 mM [Cl(-)](o) and muscimol conditions. These results suggest that a GABA-mediated loss of intracellular Cl(-), but not a subsequent rise in [Ca(2+)](i) or shrinkage, leads to stimulation of NKCC1. This stimulation may be an important positive feedback mechanism to maintain intracellular Cl(-) level and GABA function in immature neurons.
Animals↗
GABA-mediated trophic effect on oligodendrocytes requires Na-K-2Cl cotransport activity.
The Na-K-2Cl cotransporter isoform1 (NKCC1) is present in many animal cells where it plays prominent roles in regulating cell volume and maintaining intracellular Cl(-) concentration ([Cl(-)]i) above electrochemical equilibrium. We show here that NKCC1 is present and active in cultured oligodendrocytes. Expression of NKCC1 in the rat spinal cord increased during development from postnatal day 6 through 21 in parallel with that of myelin basic protein. In cultured oligodendrocytes, 39% of the total K+ (86Rb+) influx represented NKCC1 activity. Activation of GABA(A) receptors with muscimol produced a reduction in intracellular Cl(-) content, cell shrinkage, and a stimulation of NKCC1 activity. Muscimol also triggered an increase in intracellular Ca2+, which depended on NKCC1 activity. Survival of oligodendrocytes following withdrawal of growth factors was enhanced by muscimol and this effect also required NKCC1 activity. Our results suggest that NKCC1 functions in oligodendrocytes to maintain [Cl(-)]i above electrochemical equilibrium and that NKCC1 is required for GABAergic trophic effects.
Animals↗
Contribution of Na(+)-K(+)-Cl(-) cotransporter to high-[K(+)](o)- induced swelling and EAA release in astrocytes.
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.
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Astrocytes from Na(+)-K(+)-Cl(-) cotransporter-null mice exhibit absence of swelling and decrease in EAA release.
We reported previously that inhibition of Na(+)-K(+)-Cl(-) cotransporter isoform 1 (NKCC1) by bumetanide abolishes high extracellular K(+) concentration ([K(+)](o))-induced swelling and intracellular Cl(-) accumulation in rat cortical astrocytes. In this report, we extended our study by using cortical astrocytes from NKCC1-deficient (NKCC1(-/-)) mice. NKCC1 protein and activity were absent in NKCC1(-/-) astrocytes. [K(+)](o) of 75 mM increased NKCC1 activity approximately fourfold in NKCC1(+/+) cells (P < 0.05) but had no effect in NKCC1(-/-) astrocytes. Intracellular Cl(-) was increased by 70% in NKCC1(+/+) astrocytes under 75 mM [K(+)](o) (P < 0.05) but remained unchanged in NKCC1(-/-) astrocytes. Baseline intracellular Na(+) concentration ([Na(+)](i)) in NKCC1(+/+) astrocytes was 19.0 +/- 0.5 mM, compared with 16.9 +/- 0.3 mM [Na(+)](i) in NKCC1(-/-) astrocytes (P < 0.05). Relative cell volume of NKCC1(+/+) astrocytes increased by 13 +/- 2% in 75 mM [K(+)](o), compared with a value of 1.0 +/- 0.5% in NKCC1(-/-) astrocytes (P < 0.05). Regulatory volume increase after hypertonic shrinkage was completely impaired in NKCC1(-/-) astrocytes. High-[K(+)](o)-induced (14)C-labeled D-aspartate release was reduced by approximately 30% in NKCC1(-/-) astrocytes. Our study suggests that stimulation of NKCC1 is required for high-[K(+)](o)-induced swelling, which contributes to glutamate release from astrocytes under high [K(+)](o).
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗