The effects of glutamate and adenosine receptor ligands on neuronal and bone cell cultures

Effect of glutamate and adenosine A2A receptor agonists and antagonists on neuronal cell cultures. In vivo data have shown that both adenosine A2A receptor agonists and antagonists are protective against neuronal damage produced by ischaemia. This project aimed to elucidate the direct role of the A2A receptor in neuronal viability using cortical neuronal cultures so that specific experimental questions in the whole animal can be formulated. Cell death was assessed by trypan blue uptake. In cortical neuronal cultures, N-methyl-D-aspartate ((NMDA) 0, 10, 30 & 100 muM)) induced concentration-dependent cell death. This project sought to examine if adenosine A2A receptors could modulate this foral of cell death in MC3T3-E1 cells, an osteoblastic cell line. Cells were characterised by assaying for alkaline phosphatase to ensure they differentiated in accordance with the literature. MC3T3-E1 cells cultured in media containing ascorbate and glycerol phosphate showed an almost 2-fold higher alkaline phosphatase activity than cells cultured in media without these supplements (p<0.01). Cell viability was assessed by Alamar Blue reduction. Exposure of MC3T3-E1 cells to 100 muM and 300 muM NMD A caused a time- and dose-dependent increase in their viability (p<0.05, n=3). However, a prolonged exposure (72 hr) of MC3T3-E1 cells to NMDA did not induce an increase in their viability. NMDA (100, 300 & 1000 muM) increased alkaline phosphatase activity in MC3T3-E1 cells, an effect which AP5 tended to reduce. Quinolinic acid (QA 300, 1000 & 3000 muM) induced a time and dose dependent increase in MC3T3-E1 cell viability (p<0.01, n=3). However, a prolonged exposure (72 hr) of MC3T3-E1 cells to 3000 muM QA did not induce an increase in their viability. QA (300 & 1000) increased alkaline phosphatase activity in MC3T3-E1 cells, an effect which APS tended to reduce. However, 3000 muM QA did not increase alkaline phosphatase activity. CGS 21680 alone elicited an increase in MC3T3-E1 cell viability. ZM 241385 alone elicited an increase in MC3T3-E1 cell viability (p<0.05, n=6). CGS 21680 induced a greater increase in cell viability when it was combined with ZM 241385 than it did alone. CGS 21680 increased alkaline phosphatase activity in MC3T3-E1 cells, an effect which ZM 241385 trended to reduce. Hydrogen peroxide (1000 muM) dramatically reduced cell viability in MC3T3-E1 cells (p<0.001, n=6). However, 10 and 100 muM hydrogen peroxide were without effect. NMDA, QA, CGS 21680 and ZM 241385 had no effect on the response to hydrogen peroxide. However, 100 muM H2O2 significantly reduced the proliferative effect of the CGS 21680 and ZM 241385 (p<0.05, n=6). Serum withdrawal increased the number of MC3T3-E1 cells positively immunostaining for caspase-3 (p