SPHINGOLIPIDS AS MEDIATORS OF EFFECTS OF PLATELET-DERIVED GROWTH-FACTOR IN VASCULAR SMOOTH-MUSCLE CELLS

The role of sphingolipids in mediating the action of platelet-derived growth factor (PDGF) has been investigated in the vascular smooth muscle-derived A7r5 cell line. L-Cycloserine (2 mM), an inhibitor of sphingolipid synthesis, caused time-dependent inhibition of [H-3]serine incorporation into [H-3]sphingomyelin in A7r5 cells. PDGF-AB (10 ng/ml), PDGF-BB (10 ng/ml), or sphingosine (10 muM) independently stimulated [H-3]thymidine incorporation into DNA in A7r5 cells. L-Cycloserine (2 mM) inhibited stimulation of DNA synthesis by both PDGF-AB and PDGF-BB. L-Cycloserine (2 mM, 16 h) did not affect the ability of PDGF or sphingosine to increase intracellular free calcium ([Ca2+]i) in A7r5 cells loaded with the fluorescent indicator fura 2. Measurement of adenine nucleotide levels in A7r5 cell extracts by reverse-phase high-performance liquid chromatography indicated that treatment with L-cycloserine did not adversely affect cellular metabolism. To determine directly whether PDGF activates sphingolipid metabolism, A7r5 cells were labeled with [H-3] serine for 48 h and then treated with PDGF-AB (10 ng/ml) for 1 h. Sphingolipids were separated by thin-layer chromatography and quantified by liquid scintillation counting. PDGF-AB stimulated an increase in [H-3]sphingosine from 25.5 +/- 3.0 to 37.5 +/- 4.1 counts . min-I (cpm) . mug protein-I and a concomitant decrease in [H-3]ceramide from 24.3 +/- 3.2 to 18.5 +/- 2.9 cpm/mug protein. These data suggest that the PDGF-stimulated increase in [Ca2+], is not sufficient for induction of DNA synthesis and that mitogenic effects of PDGF in vascular smooth muscle cells are mediated by sphingolipid metabolism.