Clathrin-dependent internalization of the angiotensin II AT1A receptor links receptor internalization to COX-2 protein expression in rat aortic vascular smooth muscle cells

The major effects of Angiutensin II (AngII) in vascular tissue are mediated by AngII AT(1A) receptor activation. Certain effects initiated by AT(1A) receptor activation require receptor internalization. In rat aortic vascular smooth muscle cells (RASMC). AngII stimulates cyclooxygenase 2 protein expression. We have previously shown this is mediated by beta-arrestin-dependent receptor internalization and NF-kappa B activation. In this study, a specific inhibitor of clathrin-mediated endocytosis (CME), pitstop-2, was used to test the hypothesis that clathrin-dependent internalization or activated AT(1A) receptor mediates NF-kappa B activation and subsequent cyclooxygenase 2 expression. Radioligand binding assays, real time qt-PCR and immunoblotting were used to document the effects of pitstop-2 on AngII binding and signaling in RASMC. Laser scanning confocal microscopy (LSCM) was used to image pitstop-2's effects on AT(1) receptor/GFP internalization in HEK-293 cells and p65 NF-kappa B nuclear localization in RASMC. Pitstop-2 significantly inhibited internalization of AT(1A) receptor (44.7% +/- 3.1% Control vs. 13.2% perpendicular to 8.3% Pitstop-2; n=3) as determined by radioligand binding studies in RASMC. Studies utilizing AT(1A) receptor/GFP expressed in HEK 293 cells and LSCM confirmed these findings. Pitstop-2 significantly inhibited AngII-induced p65 NE-kappa B phosphorylation and nuclear localization, COX-2 message and protein expression in RASMC without altering activation of p42/44 ERK or TNF alpha signaling. Pitstop-2, a specific inhibitor of clathrin-mediated endocytosis, confirms that internalization of activated AT(1A) receptor mediates AngII activation of cyclooxygenase 2 expression in RASMC. These data provide support for additional intracellular signaling pathways activated through beta-arrestin mediated internalization of G protein-coupled receptors, such as AT(1A) receptors. (C) 2014 Elsevier By. All rights reserved,