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 accumulatio...
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Veröffentlicht in: | Biochemical journal 1993-03, Vol.290 (2), p.381-387 |
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Zusammenfassung: | 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. |
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ISSN: | 0264-6021 1470-8728 |
DOI: | 10.1042/bj2900381 |