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The angiotensin II (Ang II)-binding sites in rat adrenal gland membranes were characterized using ¹²⁵I-radiolabelled Ang II. While Scatchard analysis identified a single population of Ang II receptor sites, isoelectric focusing (IEF) on polyacrylamide gels revealed four peaks of specific Ang II binding which migrated to isoelectric points (pI values) 6·8, 6·7, 6·5 and 6·3. In binding assays in the presence of an excess of the Ang II receptor AT₁ subtype antagonist DuP 753, a monophasic dose-dependent displacement of ¹²⁵I-labelled Ang II binding by the Ang II receptor AT₂ subtype antagonist CGP42112A was observed, and vice versa. In this system, reduction of disulphide bridges using 1 mmol dithiothreitol (DTT)/l markedly increased the number of binding sites in the adrenal zona glomerulosa without affecting receptor affinity.

Using IEF, it was found that both DuP 753 and CGP42112A were able to reduce specific binding of each of the four peaks to some extent. However, the predominant effect of DuP 753 was to reduce the labelling of the isoform at pI 6·7 substantially, while CGP42112A significantly inhibited the specific ¹²⁵I-labelled Ang II binding to the pI 6·3 isoform. When DuP 753 and CGP42112A were used together, specific binding of ¹²⁵I-labelled Ang II to the isoforms of pI values 6·8, 6·7 and 6·3 was completely eliminated. These data suggest that the four peaks of specific binding found may be composed of different isoforms of both AT₁ and AT₂ receptor subtypes and that the Ang II receptor isoforms which migrated to pI 6·7 and pI 6·3 are predominantly composed of AT₁ and AT₂ receptor subtypes respectively. Interestingly, in the presence of both antagonists, 8·7 ± 0·9% of the specific binding migrating at pI 6·5 remained unaffected. This finding suggests the presence of an additional subtype, which is neither AT₁ nor AT₂, in the rat adrenal zona glomerulosa.

In further studies, pretreatment with DTT was found to increase the specific ¹²⁵I-labelled Ang II binding of all four isoforms. Moreover, DTT also produced a further specific binding component between pI 6·5 and pI 6·7 which exhibited AT₂ subtype pharmacology in DTT-treated preparations. Since DTT has been reported to enhance only AT₂ subtype binding this also suggests that the different isoforms may contain components related to both AT₁ and AT₂ receptor subtypes.

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				D. Daniels, D. K. Yee, and S. J. Fluharty Hydromineral Neuroendocrinology: Angiotensin II receptor signalling Exp Physiol, May 1, 2007; 92(3): 523 - 527. [Abstract] [Full Text] [PDF]