Abstract

Agonist activation of thrombin and purinergic receptors endogenously expressed in human embryonic kidney (HEK) cells and of the stably expressed m3 muscarinic acetylcholine receptor (mAChR) induces phospholipase C (PLC) stimulation, with the most pronounced PLC stimulation observed on mAChR activation. These receptor responses were pertussis toxin (PTX) insensitive and nonadditive, suggesting that the receptors share common signaling pathways. Short term (2 min) pretreatment of HEK cells with carbachol (1 mM), but not ATP, followed by agonist washout, caused a long-lasting (> or = 90 min) sensitization of PLC responses. At 30 min after carbachol treatment and washout, mAChR-stimulated PLC activity, measured as formation of either total inositol phosphates or of inositol-1,4,5-trisphosphate, was enhanced by 1.5-2-fold. PLC stimulation by thrombin and purinergic receptors was increased by approximately 3-fold. Furthermore, carbachol pretreatment also enhanced, by approximately 2.5-fold, stimulation of PLC activity on direct activation of G proteins by AIF4- and guanosine-5'-O-(3-thio)-triphosphate in intact and permeabilized cells, respectively. In contrast, PLC activities, measured with exogenous phosphatidylinositol-4,5-bisphosphate [Ptdns(4,5)P2] in HEK cell lysates, were not altered, suggesting that carbachol pretreatment may enhance the cellular level of Ptdlns(4,5)P2. Indeed, the level of Ptdlns(4,5)P2 was found to be increased by approximately 50% in HEK cells 30 min after short term carbachol treatment, whereas the level of phosphatidylinositol was not altered and that of phosphatidylinositol-4-phosphate decreased (by 40-50%). Pretreatment of HEK cells with PTX prevented the m3 mAChR-induced PLC potentiation and reduced the elevation in Ptdlns(4,5)P2 level by approximately 50%. In conclusion, short term agonist activation of m3 mAChRs stably expressed in HEK cells can lead to a longlasting heterologous potentiation of PLC signaling, which processes apparently involve PTX-sensitive G proteins and an enhanced PLC substrate supply.