Signaling Pathways Underlying Muscarinic Receptor-induced [Ca2+]iOscillations 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 μm) 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 μm) induced repetitive [Ca2+]ispikes or oscillations, the continuation of which was dependent on extracellular Ca2+. The entry of Ca2+with 100 μmcarbachol and with the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, was completely blocked by 1 μmGd3+, as well as 30–100 μmconcentrations of the membrane-permeant inositol 1,4,5-trisphosphate receptor inhibitor, 2-aminoethyoxydiphenyl borane (2-APB). Sensitivity to these inhibitors is indicative of capacitative calcium entry. Arachidonic acid, a candidate signal for Ca2+entry associated with [Ca2+]ioscillations in HEK293 cells, induced entry that was inhibited only by much higher concentrations of Gd3+and was unaffected by 100 μm2-APB. Like arachidonic acid-induced entry, the entry associated with [Ca2+]ioscillations was insensitive to inhibition by Gd3+but was completely blocked by 100 μm2-APB. These findings indicate that the signaling pathway responsible for the Ca2+entry driving [Ca2+]ioscillations in HEK293 cells is more complex than originally thought, and may involve neither capacitative calcium entry nor a role for PLA2and arachidonic acid.