Quantal calcium release in electropermeabilized SH-SY5Y neuroblastoma cells perfused with myo-inositol 1,4,5-trisphosphate

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 (IP₃)-induced calcium (Ca²⁺) mobilisation (IICM). SH-SY5Y cells when stimulated with sub-optimal IP₃ exhibited a rapid concentration dependent activation of Ca²⁺ mobilization followed by a partial inactivation. Although this partial inactivation allowed net Ca²⁺ mobilized to be stringently returned to basal levels, a concentration-dependent depletion of the store was maintained while ever perfusion with the stimulating IP₃ concentration was sustained. This partial inactivation of IP₃-induced quantal Ca²⁺ release (QCR) was only compromised if cells, with replete Ca²⁺ stores, were perfused with supra-maximally effective concentrations of IP₃ (5-10 μM). Thus, at supra-optimal IP₃ concentrations, a reproducible plateau of Ca²⁺ release lying 50-150 nM above the basal Ca²⁺ concentration was observed. Feedback on IP₃R sensitivity by gross cytosolic Ca²⁺levels could be eliminated as the sustained and exclusive mediator of incremental activation/inactivation cycle of IICM in SH-SY5Y cells, since released Ca²⁺ was perfused away from the immobilized cells. Thus, while ever the cells were continuously perfused with IP₃, impressive incremental inactivation was apparent. Additionally, IP₃R partial agonists were found to exhibit lower intrinsic activity for both activation and inactivation of QCR, suggesting that ligand-induced inactivation of the IP₃R was more important than inactivation mechanisms reliant on either Ca²⁺ 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 IF₃ binding probably activates and then partially inactivates its receptor in a concentration-dependent fashion to produce the QCR phenomenon.