Wang D J, Huang N N, Heppel L A
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
J Cell Physiol. 1992 Nov;153(2):221-33. doi: 10.1002/jcp.1041530202.
The mitogenic effect of extracellular ATP on porcine aortic smooth muscle cells (SMC) was examined. Stimulation of [3H]thymidine incorporation by ATP was dose-dependent; the maximal effect was obtained at 100 microM. ATP acted synergistically with insulin, IGF-1, EGF, PDGF, and various other mitogens. Incorporation of [3H]thymidine was correlated with the fraction of [3H]thymidine-labeled nuclei and changes in cell counts. The stimulation of proliferation was also determined by measurement of cellular DNA using bisbenzamide and by following the increase of mitochondrial dehydrogenase protein. The effect of ATP was not due to hydrolysis to adenosine, which shows synergism with ATP. ATP acted as a competence factor. The mitogenic effect of ATP, but not adenosine, was further increased by lysophosphatidate, phosphatidic acid, or norepinephrine. The inhibitor of adenosine deaminase, EHNA, stimulated the effect of adenosine but not ATP. The adenosine receptor antagonist theophylline depressed adenosine-induced mitogenesis. ADP and the non-hydrolyzable analogue adenosine 5'-[beta, gamma-imido]triphosphate (AMP-PNP) were equally mitogenic. Thus extracellular ATP stimulated mitogenesis of SMC via P2Y purinoceptors. The mechanism of ATP acting as a mitogen in SMC was further explored. Extracellular ATP stimulated the release of [3H]arachidonic acid (AA) and prostaglandin E2 (PGE2) into the medium, and enhanced cAMP accumulation in a dose-dependent fashion similar to ATP-induced [3H]thymidine incorporation. Inhibitors of the arachidonic acid metabolism pathway, quinacrine and indomethacin, partially inhibited the mitogenic effect of ATP but not of adenosine. Pertussis toxin inhibited ATP-stimulated DNA synthesis, AA release, PGE2 formation, and cAMP accumulation. Down-regulation of protein kinase C (PKC) by long-term exposure to phorbol dibutyrate (PDBu) partially prevented stimulation of DNA synthesis and activation of the AA pathway by ATP. The PKC inhibitor, staurosporine, antagonized mitogenesis stimulated by ATP. No synergistic effect was found when PDBu and ATP were added together. Therefore, a dual mechanism, including both arachidonic acid metabolism and PKC, is involved in ATP-mediated mitogenesis in SMC. In addition, ATP acted synergistically with angiotensin II, phospholipase C, serotonin, or carbachol to stimulate DNA synthesis. Finally, the possible physiological significance of ATP as a mitogen in SMC was further studied. The effect of endothelin and heparin, which are released from endothelial cells, on ATP-dependent mitogenesis was investigated. Extracellular ATP acted synergistically with endothelin to stimulate a greater extent of [3H]thymidine incorporation than was seen with PDGF plus endothelin. Heparin, believed to have a regulatory role, partially inhibited the stimulation of DNA synthesis caused both by ATP and PDGF.(ABSTRACT TRUNCATED AT 400 WORDS)
研究了细胞外ATP对猪主动脉平滑肌细胞（SMC）的促有丝分裂作用。ATP对[³H]胸腺嘧啶核苷掺入的刺激呈剂量依赖性；在100微摩尔时获得最大效应。ATP与胰岛素、胰岛素样生长因子-1、表皮生长因子、血小板衍生生长因子及其他多种促有丝分裂原协同作用。[³H]胸腺嘧啶核苷的掺入与[³H]胸腺嘧啶核苷标记的细胞核比例及细胞计数变化相关。增殖刺激也通过使用双苯甲酰胺测量细胞DNA以及跟踪线粒体脱氢酶蛋白的增加来确定。ATP的作用并非由于水解为腺苷，腺苷与ATP表现出协同作用。ATP作为一种感受态因子发挥作用。溶血磷脂酸、磷脂酸或去甲肾上腺素进一步增强了ATP而非腺苷的促有丝分裂作用。腺苷脱氨酶抑制剂EHNA刺激了腺苷的作用，但不影响ATP。腺苷受体拮抗剂茶碱抑制腺苷诱导的有丝分裂。ADP和不可水解类似物腺苷5'-[β,γ-亚氨基]三磷酸（AMP-PNP）具有同等的促有丝分裂作用。因此，细胞外ATP通过P2Y嘌呤受体刺激SMC的有丝分裂。进一步探讨了ATP在SMC中作为促有丝分裂原的作用机制。细胞外ATP刺激[³H]花生四烯酸（AA）和前列腺素E2（PGE2）释放到培养基中，并以类似于ATP诱导的[³H]胸腺嘧啶核苷掺入的剂量依赖性方式增强cAMP积累。花生四烯酸代谢途径抑制剂奎纳克林和吲哚美辛部分抑制ATP的促有丝分裂作用，但不影响腺苷。百日咳毒素抑制ATP刺激的DNA合成、AA释放、PGE2形成和cAMP积累。长期暴露于佛波醇二丁酸酯（PDBu）导致蛋白激酶C（PKC）下调，部分阻止了ATP对DNA合成的刺激和AA途径的激活。PKC抑制剂星形孢菌素拮抗ATP刺激的有丝分裂。当PDBu和ATP一起添加时未发现协同效应。因此，包括花生四烯酸代谢和PKC的双重机制参与了ATP介导的SMC有丝分裂。此外，ATP与血管紧张素II、磷脂酶C、5-羟色胺或卡巴胆碱协同作用以刺激DNA合成。最后，进一步研究了ATP作为SMC促有丝分裂原的可能生理意义。研究了从内皮细胞释放的内皮素和肝素对ATP依赖性有丝分裂的影响。细胞外ATP与内皮素协同作用，比血小板衍生生长因子加内皮素更能刺激[³H]胸腺嘧啶核苷掺入。据信具有调节作用的肝素部分抑制了ATP和血小板衍生生长因子引起的DNA合成刺激。（摘要截短于400字）
