LAUSR

AIMS Activation of the angiotensin (Ang) (1-7)/Mas receptor (R) axis protects from sympathetic overactivity. Endocytic trafficking is an essential process that regulates receptor (R) function and its ultimate cellular responses. We investigated whether the blunted responses to Ang-(1-7) in hypertensive rats are associated to an alteration in MasR trafficking. METHODS AND RESULTS Brainstem neurons from Wistar-Kyoto (WKY) or spontaneously hypertensive rats (SHRs) were investigated for (1) Ang-(1-7) levels and binding and MasR expression, (2) Ang-(1-7) responses (arachidonic acid and nitric oxide release and Akt and ERK1/2 phosphorylation) and (3) MasR trafficking. Ang-(1-7) were determined by radioimmunoassay. MasR expression and functionality were evaluated by Western blot and binding assays. MasR trafficking was evaluated by immunofluorescence. Ang-(1-7) treatment induced an increase in nitric oxide and arachidonic acid release and ERK1/2 and Akt phosphorylation in WKY neurons but did not have an effect in SHR neurons. Although SHR neurons showed greater MasR expression, Ang-(1-7)-elicited responses were substantially diminished presumably due to decreased Ang-(1-7) endogenous levels concomitant with impaired binding to its receptor. Through immunocolocalization studies, we evidenced that upon Ang-(1-7) stimulation MasRs were internalized through clathrin-coated pits and caveolae into early endosomes and slowly recycled back to the plasma membrane. However, the fraction of internalized MasRs into early endosomes was larger and the fraction of MasRs recycled back to the plasma membrane was smaller in SHR than in WKY neurons. Surprisingly, in SHR neurons but not in WKY neurons, Ang-(1-7) induced MasR translocation to the nucleus. Nuclear MasR expression and Ang-(1-7) levels were significantly greater in the nuclei of Ang-(1-7)-stimulated SHR neurons, indicating that the MasR is translocated with its ligand bound to it. CONCLUSIONS MasRs display differential trafficking in brainstem neurons from SHRs, which may contribute to the impaired responses to Ang-(1-7). TRANSLATIONAL PERSPECTIVE R trafficking is not a universal mechanism for every R and it critically determines the ultimate cellular response. Alterations in R trafficking have been associated with several diseases, hypertension and neurodegenerative diseases among others. We provide evidence, for the first time, about MasR trafficking in brainstem neurons from SHRs. The present work opens the way in comprehending the mechanisms underlying MasR regulation in a pathological situation. The differential MasR trafficking in neurons from SHRs may contribute to the impaired responses of Ang-(1-7) and to hypertension development.