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

Angiotensin II (Ang II) is a hormone that plays a major role in maintaining homeostasis. The Ang II receptor type 1 (AT(1)R) is expressed in acute O-2 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 AT(1)Rs in increasing the activity of O-2 sensitive cells has been established, the nanoscale distribution of AT(1)Rs has not. Furthermore, it is not known how exposure to hypoxia may alter the single-molecule arrangement and clustering of AT(1)Rs. In this study, the AT(1)R nanoscale distribution under control normoxic conditions in PC12 cells was determined using direct stochastic optical reconstruction microscopy (dSTORM). AT(1)Rs were arranged in distinct clusters with measurable parameters. Across the entire cell surface there averaged approximately 3 AT(1)R clusters/mu m(2) of cell membrane. Cluster area varied in size ranging from 1.1 x 10(-4) to 3.9 x 10(-2) mu m(2). Twenty-four hours of exposure to hypoxia (1% O-2) altered clustering of AT(1)Rs, 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 O-2 sensitive cells in response to sustained hypoxia.