Edelman_1994_Cell.Calcium_16_181

Calcium signalling was examined in CHO-k1 cells that stably express the m3 subtype of the muscarinic receptor. The calcium indicator Fura-2 was retained in these cells only in the presence of probenecid (1 mM), suggesting that Fura-2 efflux was mediated by an organic anion transporter. The addition of carbachol (CCh) to Fura-2 loaded cells in suspension caused a rapid transient increase in intracellular calcium [Ca]i followed by a smaller sustained plateau phase. The transient rise in [Ca]i was dose-dependent with a threshold response of 89 +/- 18 nM above baseline with 10 nM CCh and a maximum stimulation of 734 +/- 46 nM with 10 microM CCh. This phase was accompanied by a similar dose-dependent stimulation of total inositol phosphate production and was assumed to be generated by release from intracellular stores of the endoplasmic reticulum (ER). The sustained increase in [Ca]i was generated by entry from the extracellular bath since it was blocked by pretreatment with La3+ (1 microM) and was absent when bath calcium was chelated with EGTA. This phase was not dependent on CCh dose, and a stimulation of [Ca]i of approximately 90 nM above baseline was observed with CCh concentrations between 50 nM and 10 microM. With this dose range, the rate of Mn2+ quenching of Fura-2 at the Ca-insensitive excitation wavelength of 360 nm was likewise maximally stimulated. At lower CCh concentrations (10-50 nM), it was clear that the activation of Ca entry could not be dissociated from a threshold release of Ca from intracellular stores. The phorbol ester PMA, which uncouples the muscarinic receptor from phospholipase C, reduced the transient rise in [Ca]i by approximately 50% with little or no effect on Ca entry at higher CCh levels (> or = 1 microM). At lower CCh concentrations (< or = 100 nM) however, pretreatment with PMA completely blocked all Ca mobilization and supports the contention that Ca entry is coupled to Ca release from stores or to store depletion. The emptying of inositol trisphosphate-sensitive stores with thapsigargin (10 nM) stimulated Ca entry and also the rate of Mn2+ quenching. Store depletion by incubation in Ca-free media likewise stimulated Mn2+ uptake without a rise in [Ca]i. Our data are therefore consistent with a 'capacitative' coupling model, whereby the activation of the plasma membrane receptor leads to an InsP3-induced change in the degree of filling of the ER Ca pool.(ABSTRACT TRUNCATED AT 400 WORDS)