Role of sphingolipids in regulation of vascular smooth muscle-derived A7r5 cell proliferation
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 uM) independently stimulated (^3H) thymidine incorporation into DNA of 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 cpm/ug protein and a concomitant decrease in (^3H) ceramide from 24.3 ± 3.2 to 18.5 ± 2.9 cpm/ug protein. Exogenous sphingosine (10 uM, 6 min) stimulated an increase in (^32P) lysophosphatidic acid but had no effect on (^32P) lysophosphatidylcholine, suggesting specificity of sphingosine action. In A7r5 cells labeled with 1-O- (^3H) alkyl-2-lyso-sn-glycero-3-phosphocholine, exogenous sphingosine stimulated a dose-dependent increase in (^3H) phosphatidic acid. Exogenous sphingosine produced a dose-dependent increase in (^32P) sphingosine-1-phosphate, indicating that A7r5 cells have an active sphingosine kinase. 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 in part by sphingolipid metabolism.
School:Case Western Reserve University
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:sphingolipids regulation vascular smooth muscle derived a7r5 cell proliferation
Date of Publication:01/01/1993