J. Biol. Chem., Vol. 265, Issue 17, 9707-9714, Jun, 1990
Analysis by base exchange of thyrotropin-releasing hormone responsive and unresponsive inositol lipid pools in rat pituitary tumor cells
AB Cubitt, B Zhang and MC Gershengorn
Department of Medicine, Cornell University Medical College, New York, New York.
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 cells were responsive to TRH. In cells
prelabeled in situ and then permeabilized, TRH stimulated an increase in
3H-labeled Ins phosphates (IPs) in 20 min which was 10% of 3H radioactivity
initially present in lipids. This increase in 3H-labeled IPs was 6.3 times
the 3H radioactivity present in phosphatidylinositol 4,5-bisphosphate prior
to stimulation. When prelabeled cells were exchanged with unlabeled Ins
after permeabilization there was only a 10- 16% decrease in 3H-labeled IP
accumulation stimulated by TRH even though 3H-labeled lipids decreased to
52% of control. TRH did not affect labeling by [3H]Ins-PI exchange. In
cells labeled by base exchange after permeabilization TRH stimulated a very
small increase in 3H-labeled IPs of only 0.21 +/- 0.02% of 3H-labeled
lipids in 20 min or only 7% of the 3H radioactivity in phosphatidylinositol
4,5- bisphosphate. These data show that in permeabilized GH3 cells base
exchange can occur in the absence of de novo PI synthesis and that lipids
that are preferentially labeled by base exchange comprise a pool that is
less responsive to TRH than total Ins lipids.
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