Modulation of phosphatidylserine synthesis by a muscarinic receptor occupancy in human neuroblastoma cell line LA-N-1.
The incorporation of [3H]serine into lipids, water-soluble metabolites and proteins by the human neuroblastoma cell line LA-N-1 exposed to oxotremorine-M, a muscarinic agonist, was investigated. Oxotremorine-M increased the incorporation of this labelled precursor into phosphatidylserine and proteins in a concentration-dependent manner, with the maximal stimulation at 250 microM. This activation was blunted by 100 microM atropine. There were no detectable changes of the radioactivity in the water-soluble metabolites. Acetylcholine, another muscarinic agonist, slightly decreased the serine incorporation into lipids, but did not affect the protein or water-soluble compartments. Several other muscarinic agonists, including 250 microM pilocarpine, 100 microM McN-A-343 and 1 mM carbachol, did not effect these [3H]serine incorporations. Preincubation of cells with 1 mM oxotremorine M, or 1 mM carbachol, or 1 mM McN-A-343, for 4 h prevented the oxotremorine-M-induced increase of serine incorporation. These observations are consistent with the oxotremorine-M action being mediated by muscarinic-receptor occupancy. The G-protein inhibitor guanosine 5'-[beta-thio]diphosphate (1 mM) and the G-protein activators, guanosine 5'-[gamma-thio]triphosphate (100 microM) and A1F3, prevented the oxotremorine stimulation. The muscarinic agonists, 250 microM oxotremorine-M, 1 mM carbamoylcholine and 500 microM acetylcholine, triggered the accumulation of inositol mono- and di-phosphates by cells that had been prelabelled with myo-[3H]inositol, and this phospholipase C activation was blunted by 100 microM atropine. The protein kinase C inhibitor H7 prevented the oxotremorine-M stimulation of serine incorporation. Over-night exposure of LA-N-1 cells to 100 nM phorbol 12-myristate 13-acetate resulted in a decrease of cytosolic protein kinase C activity, and prevented the oxotremorine-M stimulation of serine incorporation. Neither oxotremorine-M nor acetylcholine caused a redistribution of protein kinase C activity between the cytosol and membrane compartments. In addition, oxotremorine-M did not activate phospholipase D of the LA-N-1 cells.
Mikhaevitch
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IS
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
Singh
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IN
Sorrentino
G
G
Massarelli
R
R
Kanfer
J N
JN
D000109:Acetylcholine / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001285:Atropine / Q000494:pharmacology*; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007294:Inositol / Q000378:metabolism; D007700:Kinetics; D009447:Neuroblastoma; D010095:Oxotremorine / Q000494:pharmacology; D010277:Parasympathomimetics / Q000494:pharmacology*; D010716:Phosphatidylinositols / Q000096:biosynthesis; D010718:Phosphatidylserines / Q000096:biosynthesis*; D011493:Protein Kinase C / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology*; D012694:Serine / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D010277:Parasympathomimetics; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; D011976:Receptors, Muscarinic; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012694:Serine; D007294:Inositol; D010095:Oxotremorine; C023427:guanosine 5'-O-(2-thiodiphosphate); D001285:Atropine; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D000109:Acetylcholine