Li Xiao C, Carretero Oscar A, Navar L Gabriel, Zhuo Jia L
Laboratory of Receptor and Signal Transduction, Division of Hypertension and Vascular Research, Henry Ford Hospital, Detroit 48202, USA.
Am J Physiol Renal Physiol. 2006 Aug;291(2):F375-83. doi: 10.1152/ajprenal.00405.2005. Epub 2006 Feb 14.
Long-term angiotensin II (ANG II) administration is associated with increased ANG II accumulation in the kidney, but intrarenal compartment(s) involved in this response remains to be determined. We tested the hypothesis that 1) extracellular ANG II is taken up by proximal tubule cells (PTCs) through AT(1) receptor-mediated endocytosis, 2) this process is regulated by cytoskeleton microtubule- and tyrosine phosphatase-dependent mechanisms, and 3) AT(1) receptor-mediated endocytosis of ANG II has a functional relevance by modulating intracellular cAMP signaling. In cultured PTCs, [(125)I]Tyr-labeled ANG II and fluorescein labeled-ANG II were internalized in a time-dependent manner and colocalized with the endosome marker Alexa Fluor 594-transferrin. Endocytosis of extracellular ANG II was inhibited by the AT(1) receptor blocker losartan (16.5 +/- 4.6%, P < 0.01 vs. ANG II, 78.3 +/- 6.2%) and by the tyrosine phosphatase inhibitor phenylarsine oxide (PAO; 30.0 +/- 3.5%, P < 0.05 vs. ANG II). Intracellular ANG II levels were increased by approximately 58% (basal, 229.8 +/- 11.4 vs. ANG II, 361.3 +/- 11.8 pg ANG II/mg protein, P < 0.01), and the responses were blocked by losartan (P < 0.01), the cytoskeleton microtubule inhibitor colchicine (P < 0.05), and PAO (P < 0.01), whereas depletion of clathrin-coated pits with hyperosmotic sucrose had no effect (356.1 +/- 25.5 pg ANG II/mg protein, not significant). ANG II accumulation was associated with significant inhibition of both basal (control, 15.5 +/- 2.8 vs. ANG II, 9.1 +/- 2.4 pmol/mg protein, P < 0.05) and forskolin-stimulated cAMP signaling (forskolin, 68.7 +/- 8.6 vs. forskolin + ANG II, 42.8 +/- 13.8 pmol/mg protein, P < 0.01). These effects were blocked by losartan and PAO. We conclude that extracellular ANG II is internalized in PTCs through AT(1) receptor-mediated endocytosis and that internalized ANG II may play a functional role in proximal tubule cells by inhibiting intracellular cAMP signaling.
长期给予血管紧张素II（ANG II）与肾脏中ANG II蓄积增加有关，但参与该反应的肾内区间仍有待确定。我们验证了以下假设：1）细胞外ANG II通过AT(1)受体介导的内吞作用被近端小管细胞（PTCs）摄取；2）该过程受细胞骨架微管和酪氨酸磷酸酶依赖性机制调节；3）ANG II的AT(1)受体介导的内吞作用通过调节细胞内cAMP信号传导具有功能相关性。在培养的PTCs中，[(125)I]酪氨酸标记的ANG II和荧光素标记的ANG II以时间依赖性方式内化，并与内体标记物Alexa Fluor 594 -转铁蛋白共定位。细胞外ANG II的内吞作用受到AT(1)受体阻滞剂氯沙坦（16.5±4.6%，与ANG II相比P<0.01，ANG II为78.3±6.2%）和酪氨酸磷酸酶抑制剂苯砷酸氧化物（PAO；30.0±3.5%，与ANG II相比P<0.05）的抑制。细胞内ANG II水平增加了约58%（基础水平，229.8±11.4对ANG II，361.3±11.8 pg ANG II/mg蛋白，P<0.01），氯沙坦（P<0.01）、细胞骨架微管抑制剂秋水仙碱（P<0.05）和PAO（P<0.01）可阻断该反应，而用高渗蔗糖耗尽网格蛋白包被小窝则无影响（356.1±25.5 pg ANG II/mg蛋白，无显著性差异）。ANG II蓄积与基础（对照，15.5±2.8对ANG II，9.1±2.4 pmol/mg蛋白，P<0.05）和福斯高林刺激的cAMP信号传导（福斯高林，68.7±8.6对福斯高林+ANG II，42.8±13.8 pmol/mg蛋白，P<0.01）的显著抑制有关。这些效应被氯沙坦和PAO阻断。我们得出结论，细胞外ANG II通过AT(1)受体介导的内吞作用在PTCs中内化，并且内化的ANG II可能通过抑制细胞内cAMP信号传导在近端小管细胞中发挥功能作用。
