Existence of a Mutation of Angiotensin AT1 Receptor Gene Promoter Region Involved in Inhibition of AT1 Receptor Gene Transcription in Spontaneously Hypertensive Rats.

The angiotensin type 1a (AT1a) receptor gene is overexpressed in the brain and peripheral tissues of spontaneously hypertensive rats (SHR). We examined whether there are mutations responsible for overexpression of the AT1a receptor gene in the SHR AT1a receptor promoter region. Genomic DNA was extracted from the livers of SHR and Wistar Kyoto rats (WKY) of Izumo strain (SHR/Izm and WKY/Izm, respectively). Fragments of the AT1a receptor gene promoter region were amplified by polymerase chain reaction (PCR). Amplified fragments were purified by agarose gel electrophoresis, and the purified fragments were cloned using pTBlue T-Vector. Sequence analysis identified one single base mutation unique to the SHR AT1a receptor gene promoter region when compared to that of WKY. The sequence of the mutation site in SHR was the same as that of Sprague Dawley rats. Using an electrophoretic mobility shift assay, we compared gel patterns formed by DNA-protein complexes using ds-oligonucleotides representing region-1624 to-1595 of the SHR and WKY AT1a receptor promoters. There were 3 major similar DNA-protein complexes against WKY and SHR oligonucleotides. In addition, the oligonucleotide bearing the SHR sequence produced an extra band. Promoter/luciferase reporter assay demonstrated that the promoter activity of SHR AT1a receptor promoters (-2050 to +57) was lower than that of WKY. These results suggest that there is one single mutation unique to the SHR AT1a receptor gene promoter region, but that the mutation is not responsible for overexpression of the AT1 a receptor gene in SHR.