MODULATION OF PHOSPHATIDYLSERINE SYNTHESIS BY A MUSCARINIC RECEPTOR OCCUPANCY HUMAN NEUROBLASTOMA CELL-LINE LA-N-1

The incorporation of [H-3]serine into lipids, water-soluble metabolite s 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 phosphati dylserine and proteins in a concentration-dependent manner, with the m aximal stimulation at 250 mu M. This activation was blunted by 100 mu M atropine. There were no detectable changes of the radioactivity in t he water-soluble metabolites. Acetylcholine, another muscarinic agonis t, slightly decreased the serine incorporation into lipids, but did no t affect the protein or water-soluble compartments. Several other musc arinic agonists, including 250 mu M pilocarpine, 100 mu M McN-A-343 an d 1 mM carbachol, did not effect these [H-3]serine incorporations. Pre incubation of cells with 1 mM oxotremorine M, or 1 mM carbachol, or 1 mM McN-A-343, for 4h prevented the oxotremorine-M-induced increase of serine incorporation. These observations are consistent with the oxotr emorine-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 mu M ) and AlF3, prevented the oxotremorine stimulation. The muscarinic ago nists, 250 mu M oxotremorine-M, 1 mM carbamoylcholine and 500 mu M ace tylcholine, triggered the accumulation of inositol mono- and di-phosph ates by cells that had been prelabelled with myo-[H-3]inositol, and th is phospholipase C activation was blunted by 100 mu M atropine. The pr otein kinase C inhibitor H7 prevented the oxotremorine-M stimulation o f serine incorporation. Overnight exposure of LA-N-1 cells to 100 nM p horbol 12-myristate 13-acetate resulted in a decrease of cytosolic pro tein kinase C activity, and prevented the oxotremorine-M stimulation o f serine incorporation. Neither oxotremorine-M nor acetylcholine cause d a redistribution of protein kinase C activity between the cytosol an d membrane compartments. In addition, oxotremorine-M did not activate phospholipase D of the LA-N-1 cells.