David Saffen

In this paper we report that stimulation of mAChRs in PC12D cells activates Ca²⁺ channels that are regulated independently of intracellular Ca²⁺ stores. In nominally Ca²⁺-free medium, exposure of PC12D cells to carbachol stimulates a robust influx of Ba²⁺, a Ca²⁺ substitute. This influx is blocked by atropine, but not by inhibitors of the nicotinic acetylcholine receptor or L-, N-, or T-type voltage-regulated Ca²⁺ channels. By contrast, depletion of intracellular Ca²⁺ stores with thapsigargin only weakly stimulates Ba²⁺ influx. Unlike store-operated Ca²⁺ channels (SOCCs), which close only after intracellular Ca²⁺ stores refill, channels mediating carbachol-stimulated Ba²⁺ influx rapidly close following the inactivation of mAChRs with atropine. Ba²⁺ influx is inhibited by extracellular Ca²⁺, by the Ca²⁺ channel blocker SKF-96365, and by activation of protein kinase C (PKC). Exogenous expression of antisense RNA encoding the rat canonical-transient receptor potential Ca²⁺ channel subtype 6 (TRPC6) or the N-terminal domain of TRPC6 blocks carbachol-stimulated Ba²⁺ influx in PC12D cells. Expression of TRPC6 antisense RNA or the TRPC6 N-terminal domain also blocks Ba²⁺ influx stimulated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a diacylglycerol analog previously shown to activate exogenously expressed TRPC6 channels. These data show that mAChRs in PC12D cells activate endogenous Ca²⁺ channels that are regulated independently of Ca²⁺ stores and require the expression of TRPC6.

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 PC12. 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.

An insertion/deletion polymorphism in the SERT linked promoter region (SERTLPR), previously reported to regulate mRNA expression in vitro, has been associated with mental disorders and response to psychotropic drugs. Contradictory evidence, however, has raised questions about the role of SERTLPR in regulating mRNA expression in vivo. We have used analysis of allelic expression imbalance (AEI) of SERT mRNA to assess quantitatively the contribution of SERTLPR to mRNA expression in human post-mortem pons tissue sections containing serotonergic neurons of the dorsal and median raphe nuclei. Any difference in the expression of one allele over the other indicates the presence of cis-acting elements that differentially affect transcription and/or mRNA processing and turnover. Using a marker SNP in the 3' untranslated region of SERT mRNA, statistically significant differences in allelic mRNA levels were detected in nine out of 29 samples heterozygous for the marker SNP. While the allelic expression differences were relatively small (15–25%), they could nevertheless be physiologically relevant. Although previous results had suggested that the long form of SERTLPR yields higher mRNA levels than the short form, we did not observe a correlation between SERTLPR and allelic expression ratios. Also in contrast to previous results, we found no correlation between SERTLPR and allelic expression ratios or SERT mRNA levels in B-lymphocytes. This study demonstrates that regulation of SERT mRNA is independent of SERTLPR, but could be associated with polymorphisms in partial linkage disequilibrium with SERTLPR.