Downregulation of the AT1A receptor by pharmacologic concentrations of Angiotensin-(1-7).
Angiotensin (Ang)-(1-7), the amino terminal heptapeptide fragment of Ang II, is an endogenous Ang peptide with vasodilatory and antiproliferative actions. Because Ang II causes vasoconstriction and promotes growth through activation of Ang type 1 (AT1) receptors, we investigated whether the actions of Ang-(1-7) are due to its regulation of these receptors. Studies were performed in CHO cells stably transfected with the AT1A receptor. Ang-(1-7) competed poorly with [125I]-Ang II for the AT1A binding site and was ineffective at shifting the IC50 for Ang II competition with [125I]-Ang II for binding to the AT1A receptor. However, if CHO-AT1A cells were pretreated with Ang-(1-7) and then treated with acidic glycine to remove surface-bound ligand, the heptapeptide caused a concentration-dependent reduction in Ang II binding, with a maximal inhibition to 67.8 +/- 4.6% of total (p < 0.05) at 1 microM Ang-(1-7) compared with a reduction to 24% of total by 10 nM Ang II. Ang-(1-7) pretreatment caused a small but significant decrease in the affinity of [125I]-Ang II for the AT1A receptor and a significant reduction in the total number of binding sites. The Ang-(1-7)-induced reduction in binding was rapid (occurring as early as 5 min after exposure to the peptide), was maintained for 30 min during continued exposure of the cells to Ang-(1-7), and rapidly recovered after removal of the heptapeptide. The AT1 receptor antagonist L-158,809 reduced the Ang-(1-7)-induced downregulation of the AT1A receptor, suggesting that interactions with AT1A receptors mediate the regulatory events. Pretreatment with 1 microM or 10 microM Ang-(1-7) significantly reduced inositol phosphate production in response to 10 nM Ang II. The decrease in binding and responsiveness of the AT1A receptor after exposure to micromolar concentrations of Ang-(1-7) suggests that the heptapeptide downregulates the AT1A receptor to reduce responses to Ang II. Because downregulation of the receptor only occurred at micromolar concentrations of the heptapeptide, our findings suggest that Ang-(1-7) is not a potent antagonist at the AT1A receptor. However, when the balance between Ang II and Ang-(1-7) is shifted in favor of Ang-(1-7), such as during inhibition of Ang-converting enzyme, some contribution of this mechanism may come into play.
Clark
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Hypertension and Vascular Disease Center, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1032, USA. maclark@wfubmc.edu
Tallant
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D000803:Angiotensin I / Q000494:pharmacology*; D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D000959:Antihypertensive Agents / Q000494:pharmacology*; D016466:CHO Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000502:physiology*; D007295:Inositol Phosphates / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology*; D011869:Radioligand Assay; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000235:genetics / Q000378:metabolism*
D000959:Antihypertensive Agents; D007295:Inositol Phosphates; D010446:Peptide Fragments; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D000803:Angiotensin I; C118790:angiotensin I (1-7)