DIFFERENTIAL-EFFECTS OF CARBACHOL ON CALCIUM-ENTRY AND RELEASE IN CHOCELLS EXPRESSING THE M3-MUSCARINIC-RECEPTOR

Calcium signalling was examined in CHO-k1 cells that stably express th e 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 anlon transp orter. The addition of carbachol (CCh) to Fura-2 loaded cells in suspe nsion caused a rapid transient increase in intracellular calcium [Ca]( i) followed by a smaller sustained plateau phase. The transient rise i n [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 mu M CCh. This phase was accompanied by a similar dose-dep endent stimulation of total inositol phosphate production and was assu med to be generated by release from intracellular stores of the endopl asmic reticulum (ER). The sustained increase in [Ca](i) was generated by entry from the extracellular bath since it was blocked by pretreatm ent with La3+ (1 mu M) and was absent when bath calcium was chelated w ith EGTA. This phase was not dependent on CCh dose, and a stimulation of [Ca](i) of similar to 90 nM above baseline was observed with CCh co ncentrations between 50 nM and 10 mu M. With this dose range, the rate of Mn2+ quenching of Fura-2 at the Ca-insensitive excitation waveleng th of 360 nm was likewise maximally stimulated. At lower CCh concentra tions (10-50 nM), it was clear that the activation of Ca entry could n ot be dissociated from a threshold release of Ca from intracellular st ores. The phorbol ester PMA, which uncouples the muscarinic receptor f rom phospholipase C, reduced the transient rise in [Ca](i) by similar to 50% with little or no effect on Ca entry at higher CCh levels (grea ter than or equal to 1 mu M). At lower CCh concentrations (less than o r equal to 100 nM) however, pretreatment with PMA completely blocked a ll Ca mobilization and supports the contention that Ca entry is couple d to Ca release from stores or to store depletion. The emptying of ino sitol trisphosphate-sensitive stores with thapsigargin (10 nM) stimula ted Ca entry and also the rate of Mn2+ quenching. Store depletion by i ncubation in Ca-free media likewise stimulated Mn2+ uptake without a r ise in [Ca](i). Our data are therefore consistent with a 'capacitative ' coupling model, whereby the activation of the plasma membrane recept or leads to an lnsP(3)-induced change in the degree of filling of the ER Ca pool. This 'activated' form of the ER then transmits a signal wh ich increases the plasma membrane Ca permeability.