Modulation of angiotensin-converting enzyme 2 (ACE2) expression, subcellular localization, and enzymatic activity by angiotensin II: Implication in neurogenic hypertension.

摘要

Angiotensin-converting enzyme 2 (ACE2) is a transmembrane carboxypeptidase that counter-balances the vasoconstrictive axis of the renin-angiotensin system (RAS) by hydrolyzing angiotensin-II (ang-II) to form the vasodilator peptide angiotensin-(1-7)[ang-(1-7)]. Since its discovery in 2000, extensive research has focused on the protective effects of ACE2 expression and activity in the cardiovascular system. However, little is known about the regulation of ACE2 expression and subcellular localization during pathological conditions associated with the development and maintenance of hypertension. In models of ang-II-mediated hypertension, ACE2 protein expression and enzymatic activity are down-regulated in brain regions associated with regulation of cardiovascular function. Therefore, the aim of this study was to investigate the regulation of ACE2 expression, subcellular localization, and enzymatic activity in presence of elevated extracellular ang-II in vitro. To this end, we used a GFP-tagged human ACE2 plasmid construct (ACE2-GFP) in transfected cell lines. Our results demonstrate that after ang-II treatment, ACE2 expression and activity were reduced at the plasma membrane in Neuro2A (N2A) cell line, which express endogenous AT1R. Further, after 4 hours ang-II exposure, ACE2 co-localized with the lysosomal marker Rab7, suggesting that the enzyme undergoes degradation. These observations were confirmed by experiments demonstrating a decrease in total cellular ACE2 protein levels following 18 hour ang-II treatment. Ang-II-mediated reduction of ACE2 activity was blocked by pre-treatment with the angiotensin-II type 1 receptor (AT1R) antagonist losartan and lysosomal inhibitor leupeptin. In HEK293T cells, which lack endogenous AT1R expression, ang-II stimulated internalization of ACE2 only after co-transfection of AT1R. In addition, ACE2 co-localized with AT1R at the plasma membrane in co-transfected HEK293T cells. This interaction was decreased by ang-II treatment in a time-dependent manner, which was paralleled by increased ubiquitination of ACE2. Taken together, these data demonstrate for the first time that elevated ang-II decreases ACE2 expression and activity through promoting internalization followed by lysosomal degradation in an AT1R-dependent manner. These results may contribute to expand therapeutic strategies for the treatment of neurogenic hypertension.