PMID: 2112545Jun 15, 1990Paper

Analysis by base exchange of thyrotropin-releasing hormone responsive and unresponsive inositol lipid pools in rat pituitary tumor cells.

The Journal of Biological Chemistry
A B CubittM C Gershengorn

Abstract

We have shown that there is an inositol (Ins) lipid pool in cloned rat pituitary tumor (GH3) cells that is hydrolyzed in response to thyrotropin-releasing hormone (TRH) and an unresponsive pool. Because others have suggested that incorporation of [3H]Ins by base exchange may not occur uniformly into Ins lipids in other cell types, we established conditions using permeabilized cells under which labeling occurs by Ins-phosphatidylinositol (PI) exchange in the absence of de novo PI synthesis to further characterize these pools in GH3 cells. In permeabilized cells incubated in buffer containing 10 mM Mg2+ and 0.1 mM CMP, [3H]Ins incorporation into lipids occurred by base exchange only. This was so because: 1) [3H]Ins incorporation into lipids displayed properties similar to that for release of 3H-labeled Ins by unlabeled Ins from PI in cells prelabeled in situ prior to permeabilization; and 2) there was no change in PI mass under these conditions. In permeabilized cells incubated in buffer with 0.1 mM [3H]Ins for 60 min, incorporation was 0.61 +/- 0.05 nmol of [3H]Ins/10(6) permeabilized cells, which amounted to 35% of PI, while the level of PI, measured as nonradioactive phosphorus, was 94 +/- 8.0% of control. Permeabilized GH3 ce...Continue Reading

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