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Angiotensin II AT 1 receptor internalization, translocation and de novo synthesis modulate cytosolic and nuclear calcium in human vascular smooth muscle cells

2003; NRC Research Press; Volume: 81; Issue: 3 Linguagem: Inglês

10.1139/y03-007

1205-7541

Ghassan Bkaily, Sama F. Sleiman, Joseph S. Stephan, Claude Asselin, Sanaa Choufani, Maud T Kamal, Danielle Jacques, Fernand Gobeil, Pedro DʼOrléans-Juste,

Protein Kinase Regulation and GTPase Signaling

The present study was designed to verify if human (h) Angiotensin II (Ang II) type-1 receptor (hAT 1 R) undergoes internalization, nuclear translocation, and de novo synthesis in primary culture of human aortic vascular smooth muscle cells (hVSMCs) and if overexpression of this receptor modulates sustained free cytosolic ([Ca] c ) and nuclear ([Ca] n ) calcium. 3-dimensional (3-D) confocal microscopy was used to monitor free intracellular Ca 2+ and hAT 1 R-green fluorescence protein (GFP) fusion protein in cultured hVSMCs. Immunofluorescence studies showed the presence of hAT 1 R and the absence of hAT 2 R in normal hVSMCs. Using 3-D imaging technique, hAT 1 receptors were localized at the sarcolemma and in the cytosolic and nuclear compartments. In native as well as in normal hAT 1 R or hAT 1 R –GFP overexpressing hVSMCs, Ang II (10 –9 and 10 –4 M) induced internalization and nuclear translocation of this type of receptor. The internalization of hAT 1 Rs is mediated via clathrin-coated pits and vesicles pathway. This phenomenon of trancellular trafficking of receptors was associated with an increase of hAT 1 R. The Ang II induced increase of hAT 1 R density was prevented by the protein synthesis inhibitor cycloheximide. Overexpression of hAT 1 R and hAT 1 R–GFP decreased both basal cytosolic and nuclear Ca 2+ . In normal hVSMCs and low hAT 1 R–GFP overexpressing hVSMCs, Ang II (10 –15 to 10 –4 M) induced a dose-dependent sustained increase of [Ca] c and [Ca] n with an EC 50 near 5 × 10 –11 and 5 × 10 –9 M, respectively. Our results suggest that hAT 1 Rs are the predominant type of Ang II receptors in aortic hVSMCs and are present in the sarcolemma, the cytosolic and the nuclear compartments. Ang II rapidly induces hAT 1 R internalization, nuclear translocation, as well as nuclear de novo synthesis of this receptor. The hAT 1 R overexpression in hVSMCs modulates sustained [Ca] c and [Ca] n .Key words: angiotensin, calcium, protein synthesis, nucleus, AT 1 receptor, nuclear de novo synthesis.