Abstract
Astrocytes are plastic cells that play key roles in brain physiology and pathology, including via their glutamate transporters, excitatory amino acid transporter (EAAT)1 and EAAT2, maintaining low extracellular glutamate concentrations and protecting against excitotoxic neuronal injury. Alterations in cell surface expression of EAATs and astrocytic cytoskeleton are important for regulating transporter activity. This study employed the actions of rottlerin, to interrogate the regulation of EAAT activity, expression and localization, and interfaces with Na+/K+-ATPase and astrocytic morphology. EAAT activity and expression were determined in primary cultures of mouse astrocytes in the presence of and after rottlerin removal, with or without trafficking inhibitors, using uptake ([3H]d-aspartate, 86Rb+) and molecular analyses. Astrocytic morphology and EAAT localization were investigated using Western blotting and immunocytochemistry, in concert with image analysis of glial fibrillary acidic protein, F-actin and EAAT1/2. Rottlerin induced a time-dependent inhibition of glutamate transport (Vmax). Rapid changes in cytoskeletal arrangement were observed and immunoblotting revealed increases in EAAT2 total and cell surface expression, despite reduced EAAT activity. Rottlerin-induced inhibition was reversible and its rate was increased by monensin co-treatment. Rottlerin inhibited, while monensin stimulated Na+/K+-ATPase. Removal of rottlerin rapidly elevated Na+/K+-ATPase activity beyond control levels, while co-treatment with monensin failed to stimulate the Na+/K+-ATPase. These data reveal inhibition of EAAT activity by rottlerin is not associated with loss of EAATs at the cell surface, but rather linked to cytoskeletal rearrangement, and inhibition of the Na+/K+-ATPase. Rapid recovery of Na+/K+-ATPase activity, and subsequent restoration of glutamate uptake indicates that astrocytic morphology and EAAT activity are co-regulated by a tightly coupled, homeostatic relationship between l-glutamate uptake, the electrochemical gradient and the activity of the Na+/K+-ATPase.
| Original language | English |
|---|---|
| Pages (from-to) | 335-346 |
| Number of pages | 12 |
| Journal | Neuroscience |
| Volume | 254 |
| DOIs | |
| Publication status | Published - 19 Dec 2013 |
| Externally published | Yes |
Keywords
- Astrocytes
- Cytoskeleton
- Glutamate transporters
- Na/K-ATPase
- Rottlerin
- Trafficking
Cite this
}
Links between l-glutamate transporters, Na+/K+-ATPase and cytoskeleton in astrocytes : Evidence following inhibition with rottlerin. / Sheean, R. K.; Lau, C. L.; Shin, Y. S.; O'Shea, R. D.; Beart, P. M.
In: Neuroscience, Vol. 254, 19.12.2013, p. 335-346.Research output: Contribution to journal › Article › Research › peer-review
TY - JOUR
T1 - Links between l-glutamate transporters, Na+/K+-ATPase and cytoskeleton in astrocytes
T2 - Evidence following inhibition with rottlerin
AU - Sheean, R. K.
AU - Lau, C. L.
AU - Shin, Y. S.
AU - O'Shea, R. D.
AU - Beart, P. M.
PY - 2013/12/19
Y1 - 2013/12/19
N2 - Astrocytes are plastic cells that play key roles in brain physiology and pathology, including via their glutamate transporters, excitatory amino acid transporter (EAAT)1 and EAAT2, maintaining low extracellular glutamate concentrations and protecting against excitotoxic neuronal injury. Alterations in cell surface expression of EAATs and astrocytic cytoskeleton are important for regulating transporter activity. This study employed the actions of rottlerin, to interrogate the regulation of EAAT activity, expression and localization, and interfaces with Na+/K+-ATPase and astrocytic morphology. EAAT activity and expression were determined in primary cultures of mouse astrocytes in the presence of and after rottlerin removal, with or without trafficking inhibitors, using uptake ([3H]d-aspartate, 86Rb+) and molecular analyses. Astrocytic morphology and EAAT localization were investigated using Western blotting and immunocytochemistry, in concert with image analysis of glial fibrillary acidic protein, F-actin and EAAT1/2. Rottlerin induced a time-dependent inhibition of glutamate transport (Vmax). Rapid changes in cytoskeletal arrangement were observed and immunoblotting revealed increases in EAAT2 total and cell surface expression, despite reduced EAAT activity. Rottlerin-induced inhibition was reversible and its rate was increased by monensin co-treatment. Rottlerin inhibited, while monensin stimulated Na+/K+-ATPase. Removal of rottlerin rapidly elevated Na+/K+-ATPase activity beyond control levels, while co-treatment with monensin failed to stimulate the Na+/K+-ATPase. These data reveal inhibition of EAAT activity by rottlerin is not associated with loss of EAATs at the cell surface, but rather linked to cytoskeletal rearrangement, and inhibition of the Na+/K+-ATPase. Rapid recovery of Na+/K+-ATPase activity, and subsequent restoration of glutamate uptake indicates that astrocytic morphology and EAAT activity are co-regulated by a tightly coupled, homeostatic relationship between l-glutamate uptake, the electrochemical gradient and the activity of the Na+/K+-ATPase.
AB - Astrocytes are plastic cells that play key roles in brain physiology and pathology, including via their glutamate transporters, excitatory amino acid transporter (EAAT)1 and EAAT2, maintaining low extracellular glutamate concentrations and protecting against excitotoxic neuronal injury. Alterations in cell surface expression of EAATs and astrocytic cytoskeleton are important for regulating transporter activity. This study employed the actions of rottlerin, to interrogate the regulation of EAAT activity, expression and localization, and interfaces with Na+/K+-ATPase and astrocytic morphology. EAAT activity and expression were determined in primary cultures of mouse astrocytes in the presence of and after rottlerin removal, with or without trafficking inhibitors, using uptake ([3H]d-aspartate, 86Rb+) and molecular analyses. Astrocytic morphology and EAAT localization were investigated using Western blotting and immunocytochemistry, in concert with image analysis of glial fibrillary acidic protein, F-actin and EAAT1/2. Rottlerin induced a time-dependent inhibition of glutamate transport (Vmax). Rapid changes in cytoskeletal arrangement were observed and immunoblotting revealed increases in EAAT2 total and cell surface expression, despite reduced EAAT activity. Rottlerin-induced inhibition was reversible and its rate was increased by monensin co-treatment. Rottlerin inhibited, while monensin stimulated Na+/K+-ATPase. Removal of rottlerin rapidly elevated Na+/K+-ATPase activity beyond control levels, while co-treatment with monensin failed to stimulate the Na+/K+-ATPase. These data reveal inhibition of EAAT activity by rottlerin is not associated with loss of EAATs at the cell surface, but rather linked to cytoskeletal rearrangement, and inhibition of the Na+/K+-ATPase. Rapid recovery of Na+/K+-ATPase activity, and subsequent restoration of glutamate uptake indicates that astrocytic morphology and EAAT activity are co-regulated by a tightly coupled, homeostatic relationship between l-glutamate uptake, the electrochemical gradient and the activity of the Na+/K+-ATPase.
KW - Astrocytes
KW - Cytoskeleton
KW - Glutamate transporters
KW - Na/K-ATPase
KW - Rottlerin
KW - Trafficking
UR - http://www.scopus.com/inward/record.url?scp=84886681672&partnerID=8YFLogxK
U2 - 10.1016/j.neuroscience.2013.09.043
DO - 10.1016/j.neuroscience.2013.09.043
M3 - Article
VL - 254
SP - 335
EP - 346
JO - Neuroscience
JF - Neuroscience
SN - 0306-4522
ER -