Cellular responses to stimulation of the M5 muscarinic acetylcholine receptor as seen in murine L cells

The membrane signaling properties of the neuronal type-5 muscarinic acetylcholine receptor (M5 AChR) as expressed in murine L cells were studied. Recipient Ltk- cells responded to ATP acting through a P2-purinergic receptor by increasing phosphoinositide hydrolysis 2-fold but were unresponsive to 17 receptor agonists that are stimulatory in other cells. L cells expressing the M5 AChR responded to carbachol (CCh) with an approximately 20-fold increase in phospholipase C activity, mobilization of Ca2+ from endogenous stores, causing a transient peak increase in the intracellular concentration of Ca2+ (Ca2+]i), influx of extracellular Ca2+, causing a sustained increase in Ca2+]i dependent on extracellular Ca2+, and release of 3H]arachidonic acid from prelabeled cells, without altering resting or prostaglandin E1-elevated intracellular cAMP levels. None of the effects of the M5 AChR were inhibited by pertussis toxin. The regulation of L cell Ca2+]i was studied further. ATP had the same effects as CCh and the two agonists acted on a shared intracellular pool of Ca2+. The peak and sustained Ca2+]i increases were reduced by cholera toxin and forskolin, neither of which altered significantly phosphoinositide hydrolysis. This is consistent with interference with the action of inositol 1,4,5-trisphosphate (IP3) through cAMP-mediated phosphorylation and suggests a continued involvement of IP3 during the sustained phase of Ca+]i increases. The temporal pattern of the sustained Ca2+]i increase differed whether elicited by CCh or ATP, and was enhanced in pertussis toxin-treated cells. This is consistent with existence of a kinetic control of the sustained Ca2+]i change by a receptor-G protein-dependent mechanism independent of the IP3 effector site(s) (e.g. pulsatile activation of phospholipase C and/or pulsatile activation of a receptor/G protein-operated plasma membrane Ca2+ channel). Thus, the non-excitable L cell may be a good model for studying Ca2+]i regulations, as may occur in other nonexcitable cells of which established cell lines do not exist, and for studying of receptors that as yet cannot be studied in their natural environment.