Sphingolipids as mediators of effects of platelet-derived growth factor in vascular smooth muscle cells
Jacobs, L.S.; Kester, M.
American Journal of Physiology 265(3 Pt 1): C740-C747
1993
ISSN/ISBN: 0002-9513 PMID: 8214030 Document Number: 412326
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 (3H)serine incorporation into (3H)sphingomyelin in A7r5 cells. PDGF-AB (10 ng/ml), PDGF-BB (10 ng/ml), or sphingosine (10 mu-M) independently stimulated (3H)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 ((Ca-2+)-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 (3H) 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 (3H)sphingosine from 25.5 +- 3.0 to 37.5 +- 4.1 counts cntdot min-1 (cpm) cntdot mu-g protein-1 and a concomitant decrease in (3H)ceramide from 24.3 +- 3.2 to 18.5 +- 2.9 cpm/mu-g protein. These data suggest that the PDGF-stimulated increase in (Ca-2+)-i is not sufficient for induction of DNA synthesis and that mitogenic effects of PDGF in vascular smooth muscle cells are mediated by sphingolipid metabolism.