Zhang Jing, Lauf Peter K, Adragna Norma C
Department of Pharmacology and Toxicology; and Physiology and Biophysics, Wright State University, School of Medicine, Dayton, Ohio 45435, USA.
Am J Physiol Cell Physiol. 2003 Mar;284(3):C674-80. doi: 10.1152/ajpcell.00312.2002.
Platelet-derived growth factor (PDGF), a potent serum mitogen for vascular smooth muscle cells (VSMCs), plays an important role in membrane transport regulation and in atherosclerosis. K-Cl cotransport (K-Cl COT/KCC), the coupled-movement of K and Cl, is involved in ion homeostasis. VSMCs possess K-Cl COT activity and the KCC1 and KCC3 isoforms. Here, we report on the effect of PDGF on K-Cl COT activity and mRNA expression in primary cultures of rat VSMCs. K-Cl COT was determined as the Cl-dependent Rb influx and mRNA expression by semiquantitative RT-PCR. Twenty four-hour serum deprivation inhibited basal K-Cl COT activity. Addition of PDGF increased total protein content and K-Cl COT activity in a time-dependent manner. PDGF activated K-Cl COT in a dose-dependent manner, both acutely (10 min) and chronically (12 h). AG-1296, a selective inhibitor of the PDGF receptor tyrosine kinase, abolished these effects. Actinomycin D and cycloheximide had no effect on the acute PDGF activation of K-Cl COT, suggesting posttranslational regulation by the drug. Furthermore, PDGF increased KCC1 and decreased KCC3 mRNA expression in a time-dependent manner. These results indicate that chronic activation of K-Cl COT activity by PDGF may involve regulation of the two KCC mRNA isoforms, with KCC1 playing a dominant role in the mechanism of PDGF-mediated activation.
血小板衍生生长因子（PDGF）是一种对血管平滑肌细胞（VSMC）有强大作用的血清促有丝分裂原，在膜转运调节和动脉粥样硬化中发挥重要作用。钾氯协同转运体（K-Cl COT/KCC），即钾和氯的偶联转运，参与离子稳态。VSMC具有K-Cl COT活性以及KCC1和KCC3亚型。在此，我们报告PDGF对大鼠VSMC原代培养物中K-Cl COT活性和mRNA表达的影响。K-Cl COT通过依赖氯的铷流入量来测定，mRNA表达通过半定量逆转录聚合酶链反应（RT-PCR）测定。24小时血清剥夺抑制基础K-Cl COT活性。添加PDGF以时间依赖性方式增加总蛋白含量和K-Cl COT活性。PDGF在急性（10分钟）和慢性（12小时）时均以剂量依赖性方式激活K-Cl COT。AG-1296，一种PDGF受体酪氨酸激酶的选择性抑制剂，消除了这些作用。放线菌素D和环己酰亚胺对PDGF对K-Cl COT的急性激活没有影响，提示该药物的翻译后调节作用。此外，PDGF以时间依赖性方式增加KCC1 mRNA表达并降低KCC3 mRNA表达。这些结果表明，PDGF对K-Cl COT活性的慢性激活可能涉及对两种KCC mRNA亚型的调节，其中KCC1在PDGF介导的激活机制中起主导作用。
