Signaling pathways underlying muscarinic receptor-induced [Ca2+]i oscillations in HEK293 cells

We have investigated the signaling pathways underlying muscarinic receptor-induced calcium oscillations in human embryonic kidney (HEK293) cells. Activation of muscarinic receptors with a maximal concentration of carbachol (100 muM) induced a biphasic rise in cytoplasmic calcium ([Ca2+](i)) comprised of release of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular space. A lower concentration of carbachol (5 muM) induced repetitive [Ca2+](i) spikes or oscillations, the continuation of which was dependent on extracellular Ca2+. The entry of Ca2+ with 100 muM carbachol and with the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, was completely blocked by 1 muM Gd3+, as well as 30-100 muM concentrations of the membrane-permeant inositol 1,4,5-trisphosphate receptor inhibitor, 2-aminoethyoxydiphenyl borane (8-APE). Sensitivity to these inhibitors is indicative of capacitative calcium entry. Arachidonic acid, a candidate signal for Ca2+ entry associated with [Ca2+](i) oscillations in HEK293 cells, induced entry that was inhibited only by much higher concentrations of Gd3+ and was unaffected by 100 muM 2-APB. Like arachidonic acid-induced entry, the entry associated with [Ca2+](i) oscillations was insensitive to inhibition by Gd3+ but was completely blocked by 100 muM 8-APE. These findings indicate that the signaling pathway responsible for the Ca2+ entry driving [Ca2+](i) oscillations in HEK293 cells is more complex than originally thought, and may involve neither capacitative calcium entry nor a role for PLA(2) and arachidonic acid.