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 [3H]serine incorporation into [3H]sphingomyelin in A7r5 cells. PDGF-AB (10 ng/ml), PDGF-BB (10 ng/ml), or sphingosine (10 microM) 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 ([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 [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.min-1 (cpm).micrograms protein-1 and a concomitant decrease in [3H]ceramide from 24.3 +/- 3.2 to 18.5 +/- 2.9 cpm/micrograms protein. These data suggest that the PDGF-stimulated increase in [Ca2+]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.
Jacobs
L S
LS
Department of Physiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Kester
M
M
D000227:Adenine Nucleotides / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002518:Ceramides / Q000378:metabolism; D003523:Cycloserine / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000502:physiology*; D013107:Sphingolipids / Q000378:metabolism*; D013110:Sphingosine / Q000378:metabolism
D000227:Adenine Nucleotides; D002518:Ceramides; D010982:Platelet-Derived Growth Factor; D013107:Sphingolipids; D004247:DNA; D003523:Cycloserine; D013110:Sphingosine; D002118:Calcium