Muscarinic Receptor Stimulation of d-Aspartate Uptake into Human SH-SY5Y Neuroblastoma Cells Is Attenuated by Hypoosmolarity

Abstract

In addition to its function as an excitatory neurotransmitter, glutamate plays a major role as an osmolyte within the central nervous system (CNS). Accordingly, mechanisms that regulate glutamate release and uptake are of physiological importance not only during conditions in which cell volume remains constant but also when cells are subjected to hypoosmotic stress. In the present study, the ability of muscarinic cholinergic receptors (mAChRs) to regulate the uptake of glutamate (monitored as D-aspartate) into human SH-SY5Y neuroblastoma cells under isotonic or hypotonic conditions has been examined. In isotonic media, agonist activation of mAChRs resulted in a significant increase (250 -300% of control) in the uptake of D-aspartate and, concurrently, a cellular redistribution of the excitatory amino acid transporter 3 (EAAT3) to the plasma membrane. mAChR-mediated increases in D-aspartate uptake were potently blocked by the EAAT3 inhibitor L-␤-threo-benzylaspartate. In hypotonic media, the ability of mAChR activation to facilitate D-aspartate uptake was significantly attenuated (40 -50%), and the cellular distribution of EAAT3 was disrupted. Reduction of mAChR-stimulated D-aspartate uptake under hypoosmotic conditions could be fully reversed upon re-exposure of the cells to isotonic media. Under both isotonic and hypotonic conditions, mAChR-mediated increases in D-aspartate uptake depended on cytoskeletal integrity, protein kinase C and phosphatidylinositol 3-kinase activities, and the availability of intracellular Ca 2ϩ . In contrast, dependence on extracellular Ca 2ϩ was observed only under isotonic conditions. The results suggest that, although the uptake of D-aspartate into SH-SY5Y cells is enhanced after mAChR activation, this process is markedly attenuated by hypoosmolarity.

Hypoosmotic and glutamate‐induced swelling of bipolar cells in the rat retina: comparison with swelling of Müller glial cells

“…In addition, a reduction in the glial and neuronal glutamate uptake may contribute to the hypoosmotic increase in extracellular glutamate. A reduction in the glutamate uptake under hypoosmotic conditions was recently described in neuroblastoma cells (Foster et al 2010).…”

“…A reduction in the glutamate uptake under hypoosmotic conditions was recently described in neuroblastoma cells (Foster et al . ).…”

The importance of the excitatory amino acid transporter 3 (EAAT3)

“…Foster and coworkers found that treatment with oxotremorine-M, a non-selective muscarinic acetylcholine receptor agonist that can activate PKC, increased the plasma membrane localization of EAAT3, an effect that was partially sensitive to PKC and PI3K inhibitors (Foster et al, 2010). PI3K-mediated EAAT3 increase in surface expression is stimulated by platelet-derived growth factor (PDGF) (Sims et al, 2000).…”

Receptor regulation of osmolyte homeostasis in neural cells

“…As observed for the taurine transporter, the osmosensitive attenuation of glutamate uptake reflects a reduction in the V max for transport without a change in K m and was accompanied by an altered trafficking pattern of the transporter (Fig. 3; Foster et al 2010). Endothelin‐1‐stimulated increases in glutamate uptake into C 6 glioma cells (mediated by EAAT3) were similarly attenuated by hyposmolarity indicating that receptor‐mediated regulation of glutamate transport may be a general, rather than cell‐specific, phenomenon.…”

“…To determine whether similar or distinct mechanisms exist for glutamate and taurine homeostasis, glutamate uptake into SH‐SY5Y cells (monitored as d ‐[ 3 H]aspartate influx) was measured as a function of osmolarity and receptor activation. Glutamate uptake was substantially increased under isotonic conditions by activation of mAChRs and to a lesser extent by endothelin or LPA receptors (Foster et al 2010). The mAChR‐mediated enhancement of glutamate uptake, which required the presence of Ca 2+ and activation of PKC and PI3K, was mediated by a cellular redistribution to the plasma membrane of the excitatory amino acid transporter 3 (EAAT3), a neuron‐specific transporter.…”