QUANTAL CALCIUM-RELEASE IN ELECTROPERMEABILIZED SH-SY5Y NEUROBLASTOMA-CELLS PERFUSED WITH MYOINOSITOL 1,4,5-TRISPHOSPHATE

Continuous perfusion of immobilized electropermeabilized SH-SY5Y neuro blastoma cells was utilised as a novel approach to the assessment of i ncremental activation and inactivation of myo-inositol 1,4,5-trisphosp hate (IP3)-induced calcium (Ca2+) mobilisation (IICM). SH-SY5Y cells w hen stimulated with sub-optimal IP3 exhibited a rapid concentration de pendent activation of Ca2+ mobilization followed by a partial inactiva tion. Although this partial inactivation allowed net Ca2+ mobilized to be stringently returned to basal levels, a concentration-dependent de pletion of the store was maintained while ever perfusion with the stim ulating 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 mu M). Thus, at supra-optimal IP3 concent rations, a reproducible plateau of Ca2+ release lying 50-150 nM above the basal Ca2+ concentration was observed. Feedback on IP(3)R sensitiv ity by gross cytosolic Ca2+ levels could be eliminated as the sustaine d and exclusive mediator of incremental activation/inactivation cycle of IICM in SH-SY5Y cells, since released Ca2+ was perfused away from t he immobilized cells. Thus, while ever the cells were continuously per fused with IP3, impressive incremental inactivation was apparent. Addi tionally, IP(3)R partial agonists were found to exhibit lower intrinsi c activity for both activation and inactivation of QCR, suggesting tha t ligand-induced inactivation of the IP(3)R was more important than in activation mechanisms reliant on either Ca2+ flux through the channel and/or calcium store depletion. Therefore, we suggest that, in perfuse d SH-SY5Y cells, the most parsimonious explanation of our data is that IP3 binding probably activates and then partially inactivates its rec eptor in a concentration-dependent fashion to produce the QCR phenomen on.