Analyzing Angiotensin II Receptor Type 1 Clustering in PC12 Cells in Response to Hypoxia Using Direct Stochastic Optical Reconstruction Microscopy (dSTORM)

Abstract

Angiotensin II (Ang II) is a hormone that plays a major role in maintaining homeostasis. The Ang II receptor type 1 (AT₁R) is expressed in acute O₂ sensitive cells, including carotid body (CB) type I cells and pheochromocytoma 12 (PC12) cells, and Ang II increases cell activity. While a functional role for Ang II and AT₁Rs in increasing the activity of O₂ sensitive cells has been established, the nanoscale distribution of AT₁Rs has not. Furthermore, it is not known how exposure to hypoxia may alter the single-molecule arrangement and clustering of AT₁Rs. In this study, the AT₁R nanoscale distribution under control normoxic conditions in PC12 cells was determined using direct stochastic optical reconstruction microscopy (dSTORM). AT₁Rs were arranged in distinct clusters with measurable parameters. Across the entire cell surface there averaged approximately 3 AT₁R clusters/μm² of cell membrane. Cluster area varied in size ranging from 1.1 × 10^-4 to 3.9 × 10^-2 μm². Twenty-four hours of exposure to hypoxia (1% O₂) altered clustering of AT₁Rs, with notable increases in the maximum cluster area, suggestive of an increase in supercluster formation. These observations could aid in understanding mechanisms underlying augmented Ang II sensitivity in O₂ sensitive cells in response to sustained hypoxia.

Keywords: Angiotensin II; Angiotensin II receptor type 1; Chronic hypoxia; Direct stochastic optical reconstruction microscopy (dSTORM); PC12 cells; Single-molecule localization.