Muscarinic Acetylcholine Receptor Regulates Phosphatidylcholine Phospholipase

The hydrolytic activity of phosphatidylcholine phospholipase D in the synaptosomes from canine brain was examined using a radiochemical assay with 1,Z-dipalmitoyl-sn-glycerol-3-phosphoryl[3H]choline as the exogenous substrate. The involvement of G protein(s) in regulation of this enzyme was demonstrated by a 2to %fold stimulation of the basal activity (4.81 2 0.44 nmol choline released/mg protein/h) with guanosine 5’(3-0-thio1)triphosphate (GTPyS), guanyl-5’-yl-(Byymethylene)diphosphonate, aluminum fluoride, or cholera toxin. The stimulation of phospholipase D hydrolytic activity by GTP-yS was inhibited by 2 mM guanosine 5’-(2-O-thiol)diphosphate. GTPyS at the maximum stimulatory concentration (10 WM) had an additive effect on the maximum cholera toxin stimulation of phospholipase D activity. However, the reverse was not true, thus indicating the possibility that more than one G protein may be involved. Furthermore, cholinergic agonists, including acetylcholine, carbachol, and muscarine, were able to increase the phospholipase D hydrolytic activity at low but not maximally stimulatory concentrations of guanine nucleotide. These cholinergic stimulations were antagonized by atropine, a muscarinic blocker. In addition, 0-tetradecanoylphorbol 13-acetateY a protein kinase C activator, was able to stimulate the hydrolytic activity of phospholipase D more than 300% in the presence of 0.2 I.LM GTPyS. However, in the absence of GTPyS, stimulation was less than 60%. Our results not only indicate that the receptor-(= protein-regulated phospholipase D may be directly responsible for the rapid accumulation of choline and phosphatidic acid in the central nervous system but also reveal that muscarinic acetylcholine receptor-(; protein-regulated phospholipase D is a novel signal transduction process coupling the neuronal muscarinic receptor to cellular responses.