Quantal calcium release in electropermeabilized SH-SY5Y neuroblastoma cells perfused with myo-inositol 14,5-trisphosphate

Abstract

Continuous perfusion of immobilized electropermeabilized SH-SY5Y neuroblastoma cells was utilised as a novel approach to the assessment of incremental activation and inactivation of myo-inositol 1,4,5-trisphosphate (IP 3)-induced calcium (Ca 2+) mobilisation (IICM). SH-SY5Y cells when stimulated with sub-optimal IP 3 exhibited a rapid concentration dependent activation of Ca 2+ mobilization followed by a partial inactivation. Although this partial inactivation allowed net Ca 2+ mobilized to be stringently returned to basal levels, a concentration-dependent depletion of the store was maintained while ever perfusion with the stimulating IP 3 concentration was sustained. This partial inactivation of IP 3-induced quantal Ca 2+ release (QCR) was only compromised if cells, with replete Ca 2+ stores, were perfused with supra-maximally effective concentrations of IP 3 (5–10 μM). Thus, at supra-optimal IP 3 concentrations, a reproducible plateau of Ca 2+ release lying 50–150 nM above the basal Ca 2+ concentration was observed. Feedback on IP 3R sensitivity by gross cytosolic Ca 2+ levels could be eliminated as the sustained and exclusive mediator of incremental activation/inactivation cycle of IICM in SH-SY5Y cells, since released Ca 2+ was perfused away from the immobilized cells. Thus, while ever the cells were continuously perfused with IP 3, impressive incremental inactivation was apparent. Additionally, IP 3R partial agonists were found to exhibit lower intrinsic activity for both activation and inactivation of QCR, suggesting that ligand-induced inactivation of the IP 3R was more important than inactivation mechanisms reliant on either Ca 2+ flux through the channel and/or calcium store depletion. Therefore, we suggest that, in perfused SH-SY5Y cells, the most parsimonious explanation of our data is that IP 3 binding probably activates and then partially inactivates its receptor in a concentration-dependent fashion to produce the QCR phenomenon.