Angiotensin II type 1 receptor (AT1R) internalization and translocation modulate cytosolic and nuclear calcium in human vascular smooth muscle cells
Sleiman, Sama Fawwak
The Angiotensin II (Ang II) type-1 receptor (ATIR) has been reported to accumulate in the nuclei of cells upon Ang II stimulation of the plasma membrane receptors. This extracellular Ang II induced nuclear accumulation of the AT1R was also linked to cell function.The objective of this work was to verify whether the human (h) AT1R undergoes internalization and nuclear translocation and whether the overexpression of this receptor modulates free cytosolic ([Ca] c ) and nuclear ([Ca]n ) calcium in human aortic vascular smooth muscle cells (hVSMCs). Immunofluorescence studies showed the presence of hAT1R and the absence of hAT2R in normal hVSMCs. Using 3-dimensional (3-D) imaging technique, hATIR were localized at the sarcolemma and in the cytosolic and nuclear compartments. In addition, 3-D confocal microscopy was also used to monitor cellular hAT1R-green fluorescence protein (GFP) fusion protein in cultured hVSMCs. In normal hVSMCs and in hAT1R-GFP expressing hVSMCs, extracellular Ang II (10 -4 M) induced the mobilization and the nuclear accumulation of both the native hAT1R and hAT1R-GFP. Overexpression of hAT1R-GFP decreased both cytosolic and nuclear free Ca2+ . This effect was blocked by the AT1R antagonist Losartan (10-5 M). In normal hVSMCs and low hAT1R-GFP overexpressing hVSMCs, extracellular Ang II (10-15 to 10-4 M) induced a dose-dependent increase of [Ca]c and [Ca]n with an EC50 near 5 × 10-11 M and 5 × 10-9 M, respectively. In conclusion, the hAT1Rs are the predominant type of Ang II receptors in aortic hVSMCs and are present in the sarcolemma, the cytosolic and nuclear compartments.The hATIR undergoes internalization and nuclear translocation in response to its ligand Ang II. Finally, its overexpression in hVSMCs modulates both [Ca] c and [Ca]n .