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J. Biol. Chem., Vol. 265, Issue 14, 7959-7966, May, 1990
MS Pessin, JJ Baldassare and DM Raben
Recent studies have implicated the hydrolysis of phosphoinositides and
phosphatidylcholine in agonist-stimulated events. The potent mitogen,
alpha-thrombin, stimulates the generation of diglycerides in a biphasic and
sustained manner in IIC9 fibroblasts (Wright, T. M., Rangan, L. A., Shin,
H. S., and Raben, D. M. (1988) J. Biol. Chem. 263, 9374-9380). Using
measurements of radiolabeled headgroup release and molecular species
analysis, we previously determined that alpha-thrombin generates
diglycerides through the hydrolysis of both the phosphoinositides and
phosphatidylcholine at early times (15 s), and at later times (greater than
or equal to 5 min) through the hydrolysis of primarily, if not exclusively,
phosphatidylcholine (Pessin, M. S., and Raben, D. M. (1989) J. Biol. Chem.
264, 8729-8738). In contrast, IIC9 fibroblasts respond to the mitogenic
treatments of (a) alpha-thrombin following chymotrypsin pretreatment or (b)
epidermal growth factor by increasing their levels of diglycerides in a
monophasic and sustained manner (Wright, T. M., Rangan, L. A., Shin, H. S.,
and Raben, D. M. (1988) J. Biol. Chem. 263, 9374-9380). In this report, we
have analyzed the molecular species of the diglycerides generated by these
two different treatments and have also examined the lipid response of IIC9
fibroblasts to platelet-derived growth factor. Based on both the molecular
species analyses and the release of radiolabeled head-groups, all three of
these different mitogenic treatments generate diglycerides primarily
through the stimulation of phosphatidylcholine hydrolysis. However, while
similar, the molecular species profiles of the diglycerides generated by
these three treatments are not identical to the molecular species profile
of total cellular phosphatidylcholine. In addition, the molecular species
profiles of the diglycerides generated by these three mitogenic treatments
greatly resemble each other, with significant differences between any two
profiles occurring in at most one molecular species. This finding differs
from that seen with alpha- thrombin stimulation alone, where the molecular
species profile of the diglycerides generated following 5 min of
alpha-thrombin stimulation is nearly identical to the molecular species
profile of total cellular phosphatidylcholine. These data support the
possibility of hormone- sensitive phosphatidylcholine pools or selective
diglyceride metabolism.
Molecular species analysis of mitogen-stimulated 1,2-diglycerides in fibroblasts. Comparison of alpha-thrombin, epidermal growth factor, and platelet-derived growth factor [published erratum appears in J Biol Chem 1990 Sep 5;265(25):15347]
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
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