Sphingosine inhibits phosphatidate phosphohydrolase in human neutrophils by a protein kinase C-independent mechanism.
Human neutrophils have been labeled in 1-O-alkyl-phosphatidylcholine with 3H in both the alkyl chain and the choline moiety. Upon stimulation of these labeled cells with formyl-Met-Leu-Phe, C5a, or phorbol 12-myristate 13-acetate, phospholipase D is activated to produce 1-O-[3H]alkylphosphatidic acid ([3H]alkyl-PA) and [3H]choline. The [3H]alkyl-PA is then dephosphorylated by phosphatidate phosphohydrolase (PPH) to produce 1-O-[3H]alkyldiglyceride ([3H]alkyl-DG). Sphingosine, a sphingoid base known to inhibit protein kinase C (PKC), causes a dose-dependent inhibition of [3H]alkyl-DG formation. This inhibition is accompanied by increased accumulation of [3H]alkyl-PA without alterations in [3H]choline formation. Studies using various other sphingoid bases demonstrate that a long hydrocarbon chain and an amino group are required for the inhibition of DG formation. These results suggest that sphingoid bases inhibit PPH activity without altering phospholipase D activation and that they exhibit a similar structure-activity relationship for both PPH and PKC. K252a, a PKC inhibitor which acts by competing for ATP binding sites, does not inhibit the formation of [3H]alkyl-DG, [3H]alkyl-PA, or [3H]choline at a concentration (3 microM) that completely blocks phorbol 12-myristate 13-acetate-induced protein phosphorylation. Moreover, in neutrophil homogenates, sphingosine but not octylamine, inhibits PPH activity in a dose-dependent manner. Thus sphingosine inhibits PPH activity by a PKC-independent mechanism, raising the possibility that sphingoid bases may play a role in regulating PPH-mediated lipid metabolism in stimulated cells.
Mullmann
T J
TJ
Department of Allergy and Immunology, Schering-Plough Research, Bloomfield, New Jersey 07003.
Siegel
M I
MI
Egan
R W
RW
Billah
M M
MM
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D009504:Neutrophils / Q000201:enzymology*; D010711:Phosphatidate Phosphatase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013110:Sphingosine / Q000494:pharmacology*; D013329:Structure-Activity Relationship
D004075:Diglycerides; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D010711:Phosphatidate Phosphatase; D010739:Phospholipase D; D013110:Sphingosine