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Preservation of 125‐I‐Angiotensin II in brain AT‐1 receptor binding assays
Author(s) -
Karamyan Vardan T.,
Gadepalli Rama S,
Rimoldi John M.,
Speth Robert C.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a432-b
Subject(s) - radioligand , angiotensin ii , chemistry , receptor , angiotensin ii receptor type 1 , agonist , endocrinology , medicine , angiotensin iii , biochemistry , biology
Studies involving inhibition of brain aminopeptidases have sparked a debate as to whether angiotensin II (Ang II) or Ang III is the endogenous agonist of brain AT 1 receptors. In brain angiotensin receptor binding studies radioligand degradation continues to be a major problem. Our objective was to design a procedure whereby binding of 125 I‐Ang II to rat brain hypothalamic AT 1 receptors could be determined with minimal metabolic degradation. After 1 hr incubation at 24°C in 5 mM EDTA, 150 mM NaCl, 0.1 mM bacitracin and 50 mM NaPO 4 (pH 7.2) buffer (with 10 μM PD123319 added to inhibit AT 2 receptor binding) HPLC analysis indicated that only 3.1±0.8% of the bound radioligand was 125 I‐Ang II, 2.9±1.1% was 125 I‐Ang III and 82±3.2% was 125 I‐tyrosine. Specific (3 μM SI Ang II displaceable) binding was not significantly different from zero. With the addition of o‐phenanthroline (1 mM), puromycin (3 mM), phenylmethylsulfonyl fluoride (PMSF, 1 mM) and glutamate phosphonate (3 mM) to the incubation buffer bound radioligand was 70.2±0.8% 125 I‐Ang II, 15.8±2.5% 125 I‐Ang III and only 6.5±1.2% other fragments. Specific binding was 630±140 pmol/mg wet weight with K D = 3.3±1.6 nM. These results indicate that under conditions which protect 125 I‐Ang II from degradation, it binds to brain AT 1 receptors. This suggests that Ang II is capable of eliciting pressor and dipsogenic effects in the brain without the necessity of conversion to Ang III.