Jacobs L S, Kester M
Department of Physiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Am J Physiol. 1993 Sep;265(3 Pt 1):C740-7. doi: 10.1152/ajpcell.1993.265.3.C740.
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.
在血管平滑肌来源的A7r5细胞系中，研究了鞘脂在介导血小板衍生生长因子（PDGF）作用中的角色。L-环丝氨酸（2 mM）是一种鞘脂合成抑制剂，可导致A7r5细胞中[3H]丝氨酸掺入[3H]鞘磷脂的时间依赖性抑制。PDGF-AB（10 ng/ml）、PDGF-BB（10 ng/ml）或鞘氨醇（10 microM）可分别刺激A7r5细胞中[3H]胸腺嘧啶掺入DNA。L-环丝氨酸（2 mM）抑制了PDGF-AB和PDGF-BB对DNA合成的刺激。L-环丝氨酸（2 mM，16小时）不影响PDGF或鞘氨醇增加负载荧光指示剂fura 2的A7r5细胞内游离钙（[Ca2+]i）的能力。通过反相高效液相色谱法测量A7r5细胞提取物中的腺嘌呤核苷酸水平表明，用L-环丝氨酸处理不会对细胞代谢产生不利影响。为了直接确定PDGF是否激活鞘脂代谢，将A7r5细胞用[3H]丝氨酸标记48小时，然后用PDGF-AB（10 ng/ml）处理1小时。通过薄层层析分离鞘脂，并通过液体闪烁计数进行定量。PDGF-AB刺激[3H]鞘氨醇从25.5±3.0增加到37.5±4.1计数·分钟-1（cpm）·微克蛋白-1，并伴随[3H]神经酰胺从24.3±3.2减少到18.5±2.9 cpm/微克蛋白。这些数据表明，PDGF刺激的[Ca2+]i增加不足以诱导DNA合成，并且PDGF在血管平滑肌细胞中的促有丝分裂作用是由鞘脂代谢介导的。
