Angiotensin-(1-7) downregulates the angiotensin II type 1 receptor in vascular smooth muscle cells

Abstract

Angiotensin (Ang)-(1-7) is a biologically active peptide of the renin-angiotensin system that has both vasodilatory and antiproliferative activities that are opposite the constrictive and proliferative effects of angiotensin II (Ang II). We studied the actions of Ang-(1-7) on the Ang II type 1 (AT1) receptor in cultured rat aortic vascular smooth muscle cells to determine whether the effects of Ang-(1-7) are due to its regulation of the AT1 receptor. Ang-(1-7) competed poorly for [125I]Ang II binding to the AT1 receptor on vascular smooth muscle cells, with an IC50 of 2.0 μmol/L compared with 1.9 nmol/L for Ang II. The pretreatment of vascular smooth muscle cells with Ang-(1-7) followed by treatment with acidic glycine to remove surface-bound peptide resulted in a significant decrease in [125I]Ang II binding; however, reduced Ang II binding was observed only at micromolar concentrations of Ang-(1-7). Scatchard analysis of vascular smooth muscle cells pretreated with 1 μmol/L Ang-(1-7) showed that the reduction in Ang II binding resulted from a loss of the total number of binding sites [Bmax 437.7±261.5 fmol/mg protein in Ang-(1-7)-pretreated cells compared with 607.5±301.2 fmol/mg protein in untreated cells, n=5, P<0.05] with no significant effect on the affinity of Ang II for the AT1 receptor. Pretreatment with the AT1 receptor antagonist L-158,809 blocked the reduction in [125I]Ang II binding by Ang-(1-7) or Ang II. Pretreatment of vascular smooth muscle cells with increasing concentrations of Ang-(1-7) reduced Ang II-stimulated phospholipase C activity; however, the decrease was significant (81.2±6.4%, P<0.01, n=5) only at 1 μmol/L Ang-(1-7). These results demonstrate that pharmacological concentrations of Ang-(1-7) in the micromolar range cause a modest downregulation of the AT1 receptor on vascular cells and a reduction in Ang II-stimulated phospholipase C activity. Because the antiproliferative and vasodilatory effects of Ang-(1-7) are observed at nanomolar concentrations of the heptapeptide, these responses to Ang-(1-7) cannot be explained by competition of Ang-(1-7) at the AT1 receptor or Ang-(1-7)-mediated downregulation of the vascular AT1 receptor.