Rapid and Persistent Desensitization of m3 Muscarinic Acetylcholine Receptor-stimulated Phospholipase D

Abstract

Activation of muscarinic acetylcholine receptors (mAChR) in human embryonic kidney (HEK) cells stably expressing the human m3 subtype leads to stimulation of both phospholipase C (PLC) and D (PLD). mAChR-stimulated PLD was turned off after 2 min of receptor activation with either the full (carbachol) or partial agonist (pilocarpine) and remained completely suppressed for at least 4 h. Partial recovery was observed 24 h after agonist removal. This rapid arrest of PLD response was not due to a loss of cell surface receptors and was also not caused by negative feedback due to concomitant activation of protein kinase C, tyrosine phosphorylation, increase in cytosolic calcium, or activation of Gi proteins. Furthermore, PLD stimulation by directly activated protein kinase C and GTP-binding proteins was unaltered in carbachol-pretreated cells. Finally, neither prevention of PLD stimulation during carbachol pretreatment by genistein nor inhibition of protein synthesis by cycloheximide, added before or after carbachol challenge, resulted in recovery of mAChR-stimulated PLD. The short term carbachol pretreatment nearly completely abolished agonist-induced binding of guanosine 5'-O-(3-thiotriphosphate) to membranes or permeabilized adherent cells. Full recovery of this response was achieved after 4 h. Similar to transfected m3 mAChR, PLD stimulation by endogenously expressed purinergic receptors was also fully blunted after 2 min of agonist (ATP) treatment. Preexposure of HEK cells to either receptor agonist partially, but not completely, reduced PLD stimulation by the other agonist. In contrast to desensitization of PLD stimulation, 2 min of carbachol treatment led to a sensitization, by up to 2-fold, of mAChR-stimulated inositol phosphate formation. This supersensitivity was also observed with pilocarpine, which acted as a full agonist on PLC. On the basis of these results, we conclude that the m3 mAChR stimulates PLD and PLC in HEK cells with distinct efficiencies and with very distinct durations of each response. The rapid and long lasting desensitization of the PLD response is apparently not due to a loss of cell surface receptors or PLD activation by GTP-binding proteins, but it may involve, at least initially, an uncoupling of receptors from GTP-binding proteins and most likely a loss of an as yet undefined essential transducing component.

Regulation of Peripheral Cannabinoid Receptor CB2Phosphorylation by the Inverse Agonist SR 144528

“…On the other hand, it was found that the recovery period for the thrombin receptor is 1 h in endothelial cells, but 16 -18 h in megacaryoblastic cell lines (45). The m3-cholinergic receptor transfected in HEK 293 cells remained fully desensitized at 4 h and only partially desensitized 24 h after a short carbachol treatment (2 min) (46).…”

Muscarinic Receptor Activation of Arachidonate-mediated Ca2+ Entry in HEK293 Cells Is Independent of Phospholipase C

“…This desensitization is apparently not a result of the loss of cell-surface receptors, but reflects a rapid and sustained uncoupling of the receptors from the activation of PLD. Second, it has been shown that addition of the phorbol ester PMA induces a pronounced stimulation of PLD activity in m3-HEK cells (40,41) as well as in many other mammalian cells (42,43). Examination of the published data indicate that 0.1 M PMA increased PLD activity to levels ϳ4-fold higher than those seen with a maximal concentration of carbachol (40).…”

“…However, several characteristics of this response in the m3-mAChR-transfected HEK293 cells indicate that this is unlikely to be the basis for the observed carbachol-induced increase in arachidonic acid generation. First, the activation of PLD by the m3-mAChR stably transfected into HEK293 cells has been shown to completely desensitize within 2 min of stimulation, even at the low concentrations of agonist used in this study (40). This desensitization is apparently not a result of the loss of cell-surface receptors, but reflects a rapid and sustained uncoupling of the receptors from the activation of PLD.…”

“…This, in turn, is mainly derived from the hydrolysis of phosphatidylcholine as a result of receptor-induced increases in either PLD activity (via phosphatidic acid) (35,36) or PLC (37,38). In this context, it has previously been reported that the m3-mAChR stably transfected into HEK293 cells is capable of coupling to a PLD (39,40). However, several characteristics of this response in the m3-mAChR-transfected HEK293 cells indicate that this is unlikely to be the basis for the observed carbachol-induced increase in arachidonic acid generation.…”

Implication of Ca2+‐Dependent Protein Tyrosine Phosphorylation in Carbachol‐Induced Phospholipase D Activation in Rat Pheochromocytoma PC12 Cells

“…In human embryonal kidney cells, muscarinic activation of PLD ceased after 2 mm due to desensitization (Schmidt et al, 1995). Thus, it was conceivable that CCh-induced PLD activation was desensitized rapidly in PC12 cells.…”