Cellular calcium levels are one of a cells most responsive elements in many aspects of cellular regulation. In the astrocytes, which form the subject of this ongoing study, waves of calcium, initiated by mechanical stimuli or agonist, correlate with glutamate secretion. It is not known whether this release is vesicular in nature nor what calcium regulatory mechanisms are involved in the aftermath of the wave itself. Wave propagation is an IP3 dependent phenomenon and it is speculated that the rapid release of calcium from the endoplasmic reticulum is replenished by capacitative coupling through a calcium release activated current (ICRAC). The objective of this study was to investigate the interactions of the different calcium regulatory mechanisms and capitalize on the high sensitivity, microdomain analysis, of the self-referencing calcium probe. This technique was coupled to near-field optical fibres enabling cell specific release of caged calcium. An imaging system enabled the imaging of glutamate release via L-glutamate dehydrogenase and NADH fluorescence Still in progress the results of this study remain inconclusive. We have recorded weak steady-state calcium efflux signals from cultured confluent and single astrocytes. The signal, however, is not responsive to norepinephrine or cage calcium release, although both stimuli increase cellular free-calcium. Interestingly the efflux is eliminated in cells injected with either the caged calcium or BAPTA, suggesting that it is derived from transmembrane transport. Data indicate a response to mechanical stimuli but cellular variability still requires further analysis. Further experiments are planned to pursue the question of ICRAC contribution to the glutamate secretory response and its response to reticular calcium depletion. A different cellular model will be sought.
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