George T P, Cook H W, Byers D M, Palmer F B, Spence M W
Department of Pediatrics, Dalhousie University, Halifax, Canada.
Biochim Biophys Acta. 1991 Jul 9;1084(2):185-93. doi: 10.1016/0005-2760(91)90219-8.
The major route of phosphatidylcholine (PtdCho) biosynthesis in mammalian cells is the sequence: choline (Cho)----phosphocholine (PCho)----cytidinediphosphate choline (CDP-Cho)----PtdCho. Recently, we have found that intermediates of this pathway are not freely diffusible in cultured rat glioma (C6) cells but are channeled towards PtdCho biosynthesis (George et al. (1989). Biochim. Biophys. Acta. 1004, 283-291). Channeling of intermediates in other mammalian systems is thought to be mediated through adsorption of enzymes to membranes and cytoskeletal elements to form multienzyme complexes. In this study, agents which perturb the structure and function of cytoskeletal elements were tested for effects on phospholipid metabolism in glioma cells. The filament-disrupting agent cytochalasin B (CB), but not other cytochalasins or the microtubule depolymerizer colchicine inhibited PtdCho and phosphatidylethanolamine (PtdEtn) biosynthesis as judged by dose-dependent reduction of labeling from [3H]Cho and [14C]ethanolamine (Etn). 32Pi pulse-labeling indicated that CB selectively decreased PtdCho and PtdEtn biosynthesis without affecting synthesis of other phospholipids. Synthesis of water-soluble intermediates of PtdCho metabolism was unaffected but the conversion of phosphoethanolamine to CDP-ethanolamine was reduced by CB. Effects of CB on phospholipid biosynthesis were not due to inhibition of glucose uptake as shown by experiments with 2-deoxyglucose, glucose-starved cells and other cytochalasins. Experiments with Ca(2+)-EGTA buffers and digitonin-permeabilized cells, and the Ca(2+)-channel blocker verapamil suggest that effects of CB on PtdCho and PtdEtn biosynthesis are due to alteration of intracellular Ca2+. Taken together, these results suggest that CB acts at sites distinct from glucose transport and cellular microfilaments to specifically inhibit PtdCho and PtdEtn biosynthesis by mechanisms dependent on intracellular Ca2+.
哺乳动物细胞中磷脂酰胆碱（PtdCho）生物合成的主要途径是：胆碱（Cho）→磷酸胆碱（PCho）→胞苷二磷酸胆碱（CDP-Cho）→PtdCho。最近，我们发现该途径的中间产物在培养的大鼠胶质瘤（C6）细胞中并非自由扩散，而是被导向PtdCho的生物合成（George等人，（1989年）。生物化学与生物物理学报。1004，283 - 291）。其他哺乳动物系统中中间产物的通道化被认为是通过酶吸附到膜和细胞骨架元件上以形成多酶复合物来介导的。在本研究中，测试了扰乱细胞骨架元件结构和功能的试剂对胶质瘤细胞磷脂代谢的影响。破坏细丝的试剂细胞松弛素B（CB），而非其他细胞松弛素或微管解聚剂秋水仙碱，抑制了PtdCho和磷脂酰乙醇胺（PtdEtn）的生物合成，这通过[3H]Cho和[14C]乙醇胺（Etn）标记的剂量依赖性降低来判断。32Pi脉冲标记表明CB选择性地降低了PtdCho和PtdEtn的生物合成，而不影响其他磷脂的合成。PtdCho代谢的水溶性中间产物的合成未受影响，但CB降低了磷酸乙醇胺向CDP - 乙醇胺的转化。如用2 - 脱氧葡萄糖、饥饿葡萄糖的细胞和其他细胞松弛素进行的实验所示，CB对磷脂生物合成的影响并非由于抑制葡萄糖摄取。用Ca(2 +)-EGTA缓冲液和洋地黄皂苷通透细胞进行的实验，以及Ca(2 +)通道阻滞剂维拉帕米表明，CB对PtdCho和PtdEtn生物合成的影响是由于细胞内Ca2 +的改变。综上所述，这些结果表明CB作用于与葡萄糖转运和细胞微丝不同的位点，通过依赖细胞内Ca2 +的机制特异性抑制PtdCho和PtdEtn的生物合成。
