In this study we showed, for the first time, the existence of a moderate density of specific angiotensin II (Ang II) binding sites (Kd=3.9+/-1.7 nM and Bmax=467.2 130.0 fmol/mg protein) in plasma membrane preparations from rat thyroid gland. Reverse transcriptase/polymerase chain reactions, using primers based on the cloned AT1 and AT2 receptor subtypes, and pharmacological characterization, using the Ang II receptor subtype antagonists Losartan and PD 123319, revealed that these Ang II binding sites match with the AT1 receptor subtypes. To obtain more information on the molecular structure of this Ang II receptor, immunoblotting analyses were carried out using a polyclonal rabbit anti-AT1 antiserum. Western analysis of fresh plasma membrane preparations from thyroid tissue showed three prominent bands of approximately 60, 45 and 40 kDa which appear to be related to different degrees of glycosylation of the receptor molecule. The functional significance of the Ang II receptors in thyroid gland is currently not known. Nevertheless, since Ang II receptors play a pivotal role in the co-ordinated actions of the renin-angiotensin system (RAS), our findings support a reciprocal regulation of thyroid function by the RAS.
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M Montiel and E Jimenez
M Montiel, S Barker, G P Vinson, and E Jiménez
ABSTRACT
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.
