Abstract 14628: Angiotensin II Type 1 Receptor Autoantibody Prolonged Angiotensin II Type 1 Receptor Activation Through Attenuating Receptor Internalization in Cultured Vascular Smooth Muscle Cells
Introduction: Angiotensin II type 1 receptor autoantibody (AT1-AA) can persistently activate angiotensin II type 1 receptor (AT1R), thereby has pathogenetic relevance to hypertension, but the exact mechanisms remain obscure. Current study aimed to identify whether AT1-AA can attenuate AT1R internalization and its possible role in vasoconstriction.
Methods and Results: AT1-AA induced sustained vasoconstriction via AT1R was confirmed by using isolated vascular rings method. Vascular smooth muscle cells (VSMC) were isolated from rat aorta and identified by immunostaining of α-SMA and calponin. Immunoprecipitation verified that AT1-AA could bind to AT1R in VSMC. The AT1R downstream PKC and ERK1/2 phosphorylation were upregulated due to AT1-AA stimulation. After incubated VSMC with atto488 labeled AT1-AA at 37°C for 30 minutes, AT1-AA could translocate from cell surface into cytoplasm, which indicated AT1-AA could induce AT1R internalization. To confirm this, RFP-tagged AT1R overexpressed cells were incubated with atto488 labeled AT1-AA at 37°C. Confocal images showed that AT1-AA and AT1R colocalized on cell surface at first and then translocated into cytoplasm together. To compare the degree of AT1R internalization caused by AT1-AA and angiotensin II (Ang II), cells were stimulated by the two reagents in same concentration and harvested at each time point, respectively. Subcellular protein fractionation combined western blot showed that Ang II could induce large number of AT1Rs to be internalized into cytoplasm which characterized by AT1R decreased on membrane and increased in cytoplasm. Surprisingly, compared with Ang II, internalization of AT1R was not detected obviously under AT1-AA stimulate condition. Furthermore, cell surface protein biotinylation indicated that although AT1-AA could induce AT1R internalization, its effect was much lower than Ang II.
Conclusions: Our data suggested that AT1-AA could activate AT1R and simultaneously induce weak AT1R internalization in VSMC. Non-internalization of AT1-AA activated AT1R may contribute to prolonged AT1R activation and sustained vasoconstriction. Whether the internalized AT1-AA can activate intracellular AT1R thereby induce sustained vasoconstriction remains to be determined.
Author Disclosures: J. Lei: None. S. Zhang: None. J. Bian: None. X. Yin: None. P. Wang: None. P. Wang: None. Y. Wu: None. H. Liu: None.
- © 2016 by American Heart Association, Inc.