Muscarinic acetylcholine receptors stimulate Ca2+ influx in PC12D cells predominantly via activation of Ca2+ store-operated channels

Activation of muscarinic acetylcholine receptors (mAChRs) causes the rapid release of Ca(2+) from intracellular stores and a sustained influx of external Ca(2+) in PC12D cells, a subline of the widely studied cell line PC12. Release of Ca(2+) from intracellular stores and a sustained influx of Ca(2+) are also observed following exposure to thapsigargin, a sesquiterpene lactone that depletes intracellular Ca(2+) pools by irreversibly inhibiting the Ca(2+) pump of the endoplasmic reticulum. In this study, we show that carbachol and thapsigargin empty the same intracellular Ca(2+) stores, and that these stores are a subset of intracellular stores depleted by the Ca(2+) ionophore ionomycin. Intracellular Ca(2+) stores remain depleted during continuous stimulation of mAChR with carbachol in medium containing 2 mM extracellular Ca(2+), but rapidly refill following inhibition of mAChRs with atropine. Addition of atropine to carbachol-stimulated cells causes intracellular Ca(2+) levels to return to baseline levels in two steps: a rapid decrease that correlates with the reuptake of Ca(2+) into internal stores and a delayed decrease that correlates with the inhibition of a Mn(2+)-permeable Ca(2+) channel. Several lines of evidence suggest that carbachol and thapsigargin stimulate Ca(2+) influx by a common mechanism: (i) pretreatment with thapsigargin occludes atropine-mediated inhibition of Ca(2+) influx, (ii) carbachol and thapsigargin applied individually or together are equally efficient at stimulating the influx of Mn(2+), and (iii) identical rates of Ca(2+) influx are observed when Ca(2+) is added to cells pretreated with carbachol, thapsigargin, or both agents in the absence of extracellular Ca(2+). Taken together, these data suggest that the sustained influx of extracellular Ca(2+) observed following activation of mAChRs in PC12D cells is mediated primarily by activation of a Mn(2+)-permeable, Ca(2+) store-operated Ca(2+) channel.