Links between l-glutamate transporters, Na+/K+-ATPase and cytoskeleton in astrocytes: Evidence following inhibition with rottlerin

- Rottlerin induces cytoskeletal changes consistent with astrocyte activation and stellation.
- Rottlerin inhibits glutamate uptake via a decreased rate of transport, not a loss in cell surface glutamate transporters.
- Loss of glutamate uptake was reversible and enhanced by co-treatment with monensin.
- Na+/K+-ATPase activity and ATP levels are also reduced with exposure to rottlerin.
- Recovery of Na+/K+-ATPase activity preceded Glu uptake rescue supporting partial uncoupling of these processes.

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.