Muscarinic acetylcholine receptor regulates phosphatidylcholine phospholipase D in canine brain.
The hydrolytic activity of phosphatidylcholine phospholipase D in the synaptosomes from canine brain was examined using a radiochemical assay with 1,2-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 2- to 3-fold stimulation of the basal activity (4.81 +/- 0.44 nmol choline released/mg protein/h) with guanosine 5'-(3-O-thiol)triphosphate (GTP gamma S), guanyl-5'-yl-(beta, gamma-methylene)diphosphonate, aluminum fluoride, or cholera toxin. The stimulation of phospholipase D hydrolytic activity by GTP gamma S was inhibited by 2 mM guanosine 5'-(2-O-thiol)diphosphate. GTP gamma S at the maximum stimulatory concentration (10 microM) 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, O-tetradecanoylphorbol 13-acetate, a protein kinase C activator, was able to stimulate the hydrolytic activity of phospholipase D more than 300% in the presence of 0.2 microM GTP gamma S. However, in the absence of GTP gamma S, stimulation was less than 60%. Our results not only indicate that the receptor-G 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-G protein-regulated phospholipase D is a novel signal transduction process coupling the neuronal muscarinic receptor to cellular responses.
Qian
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Department of Biochemistry, School of Medicine, University of Minnesota, Duluth 55812.
Drewes
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LR
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000378:metabolism*; D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002540:Cerebral Cortex / Q000201:enzymology*; D002772:Cholera Toxin / Q000494:pharmacology; D004285:Dogs; D019204:GTP-Binding Proteins / Q000378:metabolism; D006706:Homeostasis; D006868:Hydrolysis; D007700:Kinetics; D009116:Muscarine / Q000494:pharmacology; D010739:Phospholipase D / Q000378:metabolism*; D010740:Phospholipases / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology*; D013574:Synaptosomes / Q000201:enzymology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D011976:Receptors, Muscarinic; D015060:1,2-Dipalmitoylphosphatidylcholine; D009116:Muscarine; D002217:Carbachol; D002772:Cholera Toxin; D010740:Phospholipases; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D000109:Acetylcholine; D013755:Tetradecanoylphorbol Acetate