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(Received for publication, March 25, 1996, and in revised form, October 4, 1996)
From the Departments of Sphingosine, sphinganine, and other long-chain
(sphingoid) bases are highly bioactive intermediates of sphingolipid
metabolism that have diverse effects when added to cells, including the
inhibition of protein kinase C (PKC) as evaluated by both enzymatic
activity and [3H]phorbol dibutyrate
([3H]PDBu) binding. Nonetheless, changes in endogenous
sphingoid bases have not been proven to affect PKC or other signal
transduction pathways. We have discovered recently that changing
J774A.1 cells to new medium results in up to 10-fold increases in
sphingoid bases (Smith, E. R., and Merrill, A. H., Jr. (1995)
J. Biol. Chem. 270, 18749-18758); therefore, this
system was used to elevate sphingosine and sphinganine and determine if
PKC was affected. Incubation of J774A.1 cells in new medium for 30 min
increased the levels of these endogenous sphingoid bases to
approximately 0.5 nmol/mg of protein and decreased
[3H]PDBu binding by 40-60%. Addition of
NH4Cl, which suppresses the change in sphingosine, restored
[3H]PDBu binding. Elevation of endogenous sphinganine by
a second method (addition of fumonisin B1, an inhibitor of
ceramide synthase) also reduced [3H]PDBu binding;
therefore, elevations in sphingosine and sphinganine can both affect
PKC. The elevation in sphingoid bases was also associated with an
increase in the amount of PKC-
Volume 272, Number 9,
Issue of February 28, 1997
pp. 5640-5646
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
,
Biochemistry and
§ Microbiology and Immunology, Emory University School of
Medicine, Atlanta, Georgia 30322-3050
(the major PKC isozyme in J774A.1
cells) in the cytosol, as determined by activity assays and immunoblot
analyses. Changing the culture medium affected other PKC isozymes,
increased cellular levels of diacylglycerol, dihydroceramide, and
ceramide, and altered the expression of two genes (the expression of JE
was increased, and the induction of MnSOD by TNF-
was potentiated).
Thus, changing the culture medium has numerous effects on J774A.1
cells, including the modulation of PKC by endogenous sphingoid
bases.
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