AJP - Cell Physiology, Vol 265, Issue 3 C740-C747, Copyright © 1993 by American Physiological Society

ARTICLES

Sphingolipids as mediators of effects of platelet-derived growth factor in vascular smooth muscle cells

L. S. Jacobs and M. Kester
Department of Physiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.

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.

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