N2,O3-Diacylglucosamine 1-phosphate (
lipid X), a
monosaccharide precursor of Escherichia coli
lipid A, was used to stimulate RAW 264.7 macrophage
tumor cells, and the effects on macrophage
phospholipid metabolism were examined. The addition of E. coli
lipid X to the medium of cells that had been uniformly labeled with 32Pi resulted in a 4-8-fold increase in the level of
lysophosphatidylinositol. This effect was maximal at 5 microM
lipid X.
Lysophosphatidylinositol levels reached a maximum 45 min after stimulation, followed by a gradual decline to near normal levels within 2 h. The formation of
lysophosphatidylinositol was dependent upon extracellular
calcium and was almost completely inhibited when
cycloheximide was added at the time of stimulation. The addition of the
disaccharide lipid A precursor IVA, commercial
lipopolysaccharide (1 microgram/ml),
phorbol 12-myristate 13-acetate (10(-7) M), or
calcium ionophore A23187 (10(-6) M) to these cells resulted in a similar increase in
lysophosphatidylinositol levels, but
phosphatidic acid was inactive. The stimulation by IVA and
phorbol myristate acetate was blocked by
cycloheximide, but the stimulation by
lipopolysaccharide was only partially blocked. The stimulation by
A23187 was unaffected by
cycloheximide. The increase in
lysophosphatidylinositol levels might be related to the stimulation of arachidonate release and
prostaglandin synthesis that is also observed in cells treated with
lipid A precursors. The
disaccharide precursor, IVA, was at least 100 times more effective than
lipid X at stimulating
lysophosphatidylinositol formation and
prostaglandin release. The relative ability of
lipid X and IVA to stimulate these cells correlated well with their effects on other
lipopolysaccharide-responsive systems. Macrophage
tumor cells also had the ability to inactivate
lipid X by dephosphorylating it.