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Molecular and Cellular Biochemistry

, Volume 146, Issue 1, pp 79–89 | Cite as

Stable expression of a functional rat angiotensin II (AT1A) receptor in CHO-K1 cells: Rapid desensitization by angiotensin II

  • Thomas J. Thekkumkara
  • Jing Du
  • David E. Dostal
  • Thomas J. Motel
  • Walter G. Thomas
  • Kenneth M. Baker
Article

Abstract

The octapeptide angiotensin II mediates the physiological actions of the renin-angiotensin system through activation of several angiotensin II receptor subtypes; in particular the AT1. In many tissues, the presence of multiple angiotensin II receptor subtypes, together with a low number of receptors, makes it difficult to study biological responses to physiological concentrations (10−11–10−9 M) of angiotensin II. Also, cultured cells show diminished angiotensin II receptor binding with respect to time in culture and passage number. To address these problems, we expressed the recombinant AT1A receptor in CHO-K1 cells. The stably transfected receptor was characterized using radioligand binding studies and functional coupling to cytosolic free calcium. Radioligand binding of [125I] angiotensin II to the angiotensin II receptor was specific, saturable, reversible and modulated by guanine nucleotides. Like the endogenous AT1A receptor, reported in a variety of tissues, the specific, noncompetitive, nonpeptide AII receptor antagonist, EXP3174, blocked binding of [125I] angiotensin II to the transfected receptor. Scatchard analysis demonstrated that the transfected receptor had a dissociation constant of 1.9 nM with a density of 3.4 pmol/mg protein.

An important feature of many of the responses to angiotensin II is the rapid desensitization that occurs following agonist occupancy and the development of tachyphylaxis. In AT1A receptor transfected CHO-K1 cells, angiotensin II (10−9 M) stimulated a rapid increase in cytosolic free calcium that was completely desensitized within 50 sec following receptor occupancy. Agonist induced desensitization was unaffected when receptor internalization was blocked by pretreatment with concanavalin A or incubation at 4°C, and no changes in AT1A receptor affinity or number were observed. Receptor desensitization was also unaffected by inhibition or activation of protein kinase C. Thus, we have established a permanent, high-level transfectant of the AT1A receptor in CHO-K1 cells and have shown that these receptors rapidly desensitize following exposure to physiological concentrations of agonist. The mechanism of rapid desensitization is not related to receptor sequestration, internalization or controlled by PKC phosphorylation. This provides an excellent model for studying AII actions mediated through a specific receptor subtype, at subnanomolar concentrations.

Key words

angiotensin II receptors angiotensin II desensitization internalization stable expression intracellular calcium (Ca2+

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Copyright information

© Kluwer Academic Publishers 1995

Authors and Affiliations

  • Thomas J. Thekkumkara
    • 1
  • Jing Du
    • 1
  • David E. Dostal
    • 1
  • Thomas J. Motel
    • 1
  • Walter G. Thomas
    • 1
  • Kenneth M. Baker
    • 1
  1. 1.Geisinger ClinicWeis Center for ResearchDanvilleUSA

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