Effects of Ischaemic Conditions on Uptake of Glutamate, Aspartate, and Noradrenaline by Cell Lines Derived from the Human Nervous System

Abstract

The effect of hypoglycaemic, hypoxic, and ischaemic conditions on high‐affinity neurotransmitter transport was studied in the human astrocytoma clone D384 and the human neuroblastoma clone SH‐SY5Y. Both cell lines expressed a sodium‐dependent glutamate/aspartate transporter. Km values for d‐[3H]aspartate uptake were 6.1 ± 0.9 µM for D384 cells and 5.3 ± 0.3 µM for SH‐SY5Y cells (mean ± SEM of three experiments). In addition, SH‐SY5Y, but not D384, expressed a sodium‐dependent noradrenaline transporter with Km = 0.6 ± 0.1 µM (mean ± SEM of three experiments). Up to 3 h of hypoglycaemic conditions had no effect on neurotransmitter uptake or on ATP levels of each cell line. In sharp contrast, during hypoxic conditions, the uptake of d‐[3H]aspartate and [3H]noradrenaline declined by 43–56% within 5 min. These reduced rates of neurotransmitter uptake were maintained over 30 min of hypoxic conditions. Five minutes of ischaemic conditions caused similar reductions in neurotransmitter uptake rates. A correlation between reductions in rates of neurotransmitter uptake and in ATP levels was observed for each cell line. Results are discussed in relation to other brain preparations, which are used as models of the nervous system to study the effects of ischaemic conditions on neurotransmitter and energy metabolism.

Glucose Regulates Glutamate‐Evoked Arachidonic Acid Release from Cultured Striatal Neurons

“…It is generally proposed that neuronal injury involves calciummediated events such as the activation of proteases or lipases, resulting in the degradation of the plasma membrane and subsequent cell lysis or apoptosis . The precise mechanism leading to the elevation of extracellular glutamate during ischemia is not known, but it has been suggested, by experiments performed on human astrocytoma and neuroblastoma cell lines, that the reuptake of glutamate is reduced by hypoxia (but not under glucose deprivation) (O'Neill et al, 1994), producing a predicted reinforcement of the glutamate effect. However, no information is available concerning the modification of the efficacy of glutamate on neurons during ischemia .…”

Overexpression of the myristoylated alanine‐rich C kinase substrate decreases uptake and K+‐evoked release of noradrenaline in the human neuroblastoma SH‐SY5Y

“…The uptake of [ 3 H]NA by SH‐SY5Y was measured essentially as described previously (O'Neill et al ., 1994). Cells were grown in 24‐well plates and assayed for NA uptake after 4 days.…”

Hypoxia Enhances [3H]Noradrenaline Release Evoked by Nicotinic Receptor Activation from the Human Neuroblastoma SH‐SY5Y

“…One possible mechanism to explain the effects of hypoxia on nAChRs in SH-SY5Y is that hypoxia might alter the phosphorylation state of these receptors. Similar hypoxic conditions have previously been shown to reduce intracellular ATP levels in SH-SY5Y by~50% (O'Neill et al, 1994). The nAChR is well known to be phosphorylated by protein kinase A, PKC, and tyrosine kinase, and such phosphorylation can alter receptor!…”