Identification and characterization of angiotensinIV binding sites in rat neurone and astrocyte cell cultures.
This study demonstrates the existence of the putative receptor for the hexapeptide (3-8) fragment of angiotensin II (AngIV) on rat astrocytes and neurons grown in cell culture. Binding of 125I-AngIV was saturable and distinct from that of the AngII receptor subtypes. Equilibrium binding was attained in 15 min in astrocytes and 75 min in neurons at 22 degrees C. The bound peptide was confirmed by HPLC to be intact AngIV while the bound peptide was substantially degraded, even in the presence of peptidase inhibitors. Scatchard analysis of equilibrium binding was consistent with a two binding site model, revealing a high affinity and a low affinity binding site in both cell types. In neurons, the respective association constants (Ka) were 2.72 +/- 0.23 nM-1 and 727 +/- 354 nM-1, with associated receptor densities of 109.30 +/- 58.87 and 1723 +/- 1167 fmol/mg protein. Similar analyses in astrocytes gave Kas of 5.71 +/- 2.85 nM-1 and 277 +/- 205 nM-1, and respective densities of 191.1 +/- 90.1 and 1425 +/- 1250 fmol/mg protein. However, the quantitative reliability of these binding isotherms may be influenced by the degration of unbound peptide. Competitive binding analysis was used to determine the specificity of the receptor site, with the relative order of affinities being AngIV > AngIII > AngII(4-8), and no displacement by AngII, Iosartan and PD123319 in either neurons or astrocytes. Autoradiography with 125I-AngIV performed on neuronal cultures demonstrated that binding was confined to a subpopulation of the total cells. These data support the existence of a specific binding site for AngIV in both neurons and astrocytes, consistent with the properties of binding reported previously in the brain, and distinguish this site from the AngII receptor subtypes.
Greenland
K
K
Department of Physiology and Pharmacology, University of Queensland, Australia.
Wyse
B
B
Sernia
C
C
D000804:Angiotensin II / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D001345:Autoradiography; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D007457:Iodine Radioisotopes; D007700:Kinetics; D009474:Neurons / Q000378:metabolism*; D051381:Rats
D007457:Iodine Radioisotopes; D000804:Angiotensin II; C012877:angiotensin II, des-Asp(1)-des-Arg(2)-Ile(5)-