Subcellular distribution of agonist-stimulated phosphatidylinositol synthesis in 1321 N1 astrocytoma cells.
In an inositol-depleted 1321 N1 astrocytoma cell line, propranolol at 0.5 mM concentration and carbachol in the presence of Li+ induce a large increase (30-60-fold) in the amount of CMP-phosphatidate, the lipid substrate of PtdIns synthase. The actions of both agents on CMP-phosphatidate accumulation were reversed by co-incubation with 1 mM inositol. In cells grown in the presence of 40 microM inositol the propranolol- and carbachol-mediated CMP-phosphatidate accumulation was much smaller (2-4-fold). Propranolol- and carbachol-mediated increases in CMP-phosphatidate accumulation were at least additive in both inositol-replete and -depleted cells. The subcellular distribution of accumulated CMP-phosphatidate was investigated by sucrose-density-gradient centrifugation of a lysate of inositol-depleted cells. There were two coincident peaks of carbachol-stimulated [3H]CMP-phosphatidate and PtdIns synthase activity, respectively. The first peak of accumulated [3H]CMP-phosphatidate and PtdIns synthase activity is characteristic of a 'light vesicle' fraction, since it sediments at sucrose densities similar to that of endocytosed 125I-transferrin. The later peak, containing both carbachol-stimulated [3H]CMP-phosphatidate and PtdIns synthase activity, has a distribution in the gradient that is similar to NADPH-cytochrome c reductase activity, an endoplasmic-reticulum marker. By contrast, propranolol-stimulated [3H]CMP-phosphatidate accumulates in membranes which sediment as a single peak corresponding to the endoplasmic-reticulum marker. These observations suggest that agonist-stimulated PtdIns synthesis occurs in the endoplasmic reticulum and in at least one additional membrane compartment which is insensitive to propranolol, an inhibitor of endoplasmic-reticulum phosphatidate phosphohydrolase.
Sillence
D J
DJ
Department of Biochemistry, University of Dundee, U.K.
Downes
C P
CP
D001254:Astrocytoma; D002217:Carbachol / Q000494:pharmacology*; D006801:Humans; D007294:Inositol / Q000378:metabolism; D008094:Lithium / Q000378:metabolism; D010716:Phosphatidylinositols / Q000096:biosynthesis*; D011433:Propranolol / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D010716:Phosphatidylinositols; D007294:Inositol; D002217:Carbachol; D008094:Lithium; D011433:Propranolol