Recruitment of RGS5 Protein to Mechanically Activated AT1R in Arteriolar VSMC

Abstract

Ligand‐independent activation of G protein‐coupled receptors (GPCR) contributes to pressure‐induced vasoconstriction. Thus, stretch/mechanical activation of angiotensin II (Ang II) type 1 receptors (AT1R) likely mobilizes second messengers (e.g. Ca2+ and diacylglycerol) leading to constriction through ion channel activation and/or PKC‐dependent Ca2+ sensitization or actin polymerization. As enhanced pressure‐induced constriction may contribute to vascular disorders, mechanoactivation of AT1R is likely regulated by negative feedback mechanisms. This study explored whether regulators of G‐protein signaling (RGS) proteins, key molecules for the negative regulation of GPCR signaling, are co‐localized with mechanically activated AT1R in vascular smooth muscle cells (VSMC). An in situ proximity ligation assay (PLA) was performed with or without candesartan, an AT1R blocker, in cremaster arteriolar VSMC. VSMC were treated with Ang II or hypotonic solution to determine if ligand dependent‐ or independent‐activation of AT1R leads to translocation of RGS proteins. qPCR data indicated that RGS5 protein was abundantly expressed in cremaster arterioles (n=6). In PLA using antibodies specific for AT1R and RGS5 protein there were significantly higher fluorescent labeling, indicative of co‐localization, per VSMC detected in the presence of Ang II (1 µM, 24.25 ± 2.19) or hypotonic solution (150 mOsm/L, 22.86 ± 2.83) than at baseline (7.23 ± 0.89). Labeling produced by hypotonic solution was significantly reduced by candesartan (10 µM) (24.00 ± 5.00 VS 11.46 ± 1.30). These findings indicate that mechanoactivation of AT1R causes recruitment of RGS5 protein, which may prevent exaggerated AT1R‐mediated myogenic vasoconstriction by terminating GPCR signaling.