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 Ca²⁺ from intracellular stores and a sustained influx of external Ca²⁺ in PC12D cells, a subline of the widely studied cell line PC 12. Release of Ca²⁺ from intracellular stores and a sustained influx of Ca²⁺ are also observed following exposure to thapsigargin, a sesquiterpene lactone that depletes intracellular Ca²⁺ pools by irreversibly inhibiting the Ca²⁺ pump of the endoplasmic reticulum. In this study, we show that carbachol and thapsigargin empty the same intracellular Ca²⁺ stores, and that these stores are a subset of intracellular stores depleted by the Ca²⁺ ionophore ionomycin. Intracellular Ca²⁺ stores remain depleted during continuous stimulation of mAChR with carbachol in medium containing 2 mM extracellular Ca²⁺, but rapidly refill following inhibition of mAChRs with atropine. Addition of atropine to carbachol-stimulated cells causes intracellular Ca²⁺ levels to return to baseline levels in two steps: a rapid decrease that correlates with the reuptake of Ca²⁺ into internal stores and a delayed decrease that correlates with the inhibition of a Mn²⁺-permeable Ca²⁺ channel. Several lines of evidence suggest that carbachol and thapsigargin stimulate Ca²⁺ influx by a common mechanism: (i) pretreatment with thapsigargin occludes atropine-mediated inhibition of Ca²⁺ influx, (ii) carbachol and thapsigargin applied individually or together are equally efficient at stimulating the influx of Mn²⁺, and (iii) identical rates of Ca²⁺ influx are observed when Ca²⁺ is added to cells pretreated with carbachol, thapsigargin, or both agents in the absence of extracellular Ca²⁺. Taken together, these data suggest that the sustained influx of extracellular Ca²⁺ observed following activation of mAChRs in PC12D cells is mediated primarily by activation of a Mn²⁺-permeable, Ca²⁺ store-operated Ca²⁺ channel.