MUSCARINIC-RECEPTOR-MEDIATED CHANGES IN INTRACELLULAR CA-2+ AND INOSITOL 1,4,5-TRISPHOSPHATE MASS IN A HUMAN NEUROBLASTOMA CELL-LINE, SH-SY5Y

This study reports increased intracellular Ca2+ and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in response to muscarinic-cholinergic stimulation of human neuroblastoma (SH-SY5Y) cells. Carbachol stimulation leads to a rapid increase in intracellular Ca2+ and Ins(1,4,5)P3 mass, both reaching a peak at around 10 s and then declining to a new maintained phase significantly above basal. Dose-response analysis of peak and plateau phases of intracellular Ca2+ shows different agonist potencies for both phases, carbachol being more potent for the plateau phase. The plateau-phase intracellular Ca2+ was dependent on extracellular Ca2+, which is admitted to the cell through a non-voltage-sensitive Ni2+-blockable Ca2+ channel. Using a Mn2+ quench protocol, we have shown that Ca2+ entry occurs during the discharge of the internal stores. The plateau phase (Ca2+-channel opening) is dependent on the continued presence of agonist, since addition of atropine closes the Ca2+ channel and intracellular Ca2+ declines rapidly back to basal. We also failed to detect a refilling transient when we added back Ca2+ after intracellular Ca2+ had reached a peak and then declined in Ca2+-free conditions. These data strongly suggest that muscarinic stimulation of SH-SY5Y cells lead to a rapid release of Ca2+ from an Ins(1,4,5)P3-sensitive internal store and a parallel early entry of Ca2+ across the plasma membrane.