Ang II‐Mediated Regulation of AGT Protein Synthesis in Rat Brainstem Astrocytes is βarrestin1 Dependent

Abstract

ObjectiveStudy the role of βarrestins in Angiotensin (Ang) II‐mediated regulation of angiotensinogen (AGT) protein synthesis in astrocytes isolated from the brainstem of spontaneously hypertensive rats (SHRs) or normotensive rats.BackgroundThe βarrestins (βarrestin1 and‐2) terminate G protein signaling by G protein‐coupled receptors (GPCRs) and, at the same time, they initiate their own “second wave” of signaling from GPCRs, independently of G proteins. Ang II, through its cognate GPCR, Angiotensin II type1 receptor (AT1R), signals via both G proteins and βarrestins in various cell types. In our previous study, we reported that SII (an Ang II peptide analog which is a βarrestin‐biased agonist), similarly to Ang II, upregulates AGT synthesis in a time‐dependent manner. Here, we sought to delineate whether Ang II‐mediated regulation of AGT protein synthesis in brainstem‐derived astrocytes is G protein or βarrestin or both G protein and βarrestin dependent.MethodsPrimary cultures of brainstem astrocytes were isolated from the brains of 2–3 days old pups. Double‐stranded siRNAs (Dharmacon) targeting rat βarrestin1 or βarrestin2 were used to knockdown βarrestins. Non‐silencing siRNA (Dharmacon) was used as a control. RNiMAX (Invitrogen) was used as a transfection reagent. Ang II (100 nM) and 10 μM SII (the βarrestin‐biased agonist) were used to study AT1R‐mediated AGT protein expression. Protein analysis was done using Western blotting and mRNA measurement was done using quantitative real‐time PCR. The results in SHR astrocytes were compared to normotensive rat astrocytes.ResultsOur results indicate that βarrestin1 is the major βarrestin isoform in astrocytes isolated from both SHR and normotensive rat brainstem astrocytes. Ang II upregulated AGT protein synthesis in both SHR and normotensive rat astrocytes. This effect was higher in SHR as compared to normotensive rats. siRNA mediated knockdown of βarrestin1 abolished Ang II‐mediated AGT protein synthesis. However, βarrestin2 knockdown did not inhibit Ang II‐mediated AGT protein synthesis.ConclusionIn this study, we showed that βarrestin1 plays a major role in the Ang II‐AT1R‐mediated regulation of AGT protein synthesis in rat brainstem astrocytes in both SHR and normotensive rat models, which could be an important therapeutic target for the treatment of hypertension and other cardiovascular diseases.Support or Funding InformationFunding for this project was provided through a Nova Southeastern University President's Faculty Research and Development Grant (PFRDG)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.