The octapeptide, angiotensin-ll (Ang II) is one of the most potent vasoactive substances known and regulates a variety of physiological responses, including fluid homeostasis, aldosterone production, renal function and contraction of vascular smooth muscle (VSM). Over-expression of the angiotensin receptor-1 (AT1R) gene produces hypertension especially in female transgenic mice. These studies have suggested that increased transcription of the hAT1 R gene may lead to hypertension. We have therefore analyzed the role of single nucleotide polymorphisms (SNPs) in the 5' flanking region of the hAT1R gene in hypertension. We have found that hAT1 R gene promoter has a haplotype block of at least five SNPs consisting of T/A at - 777, T/G at -680, A/C at -214, G/C at -213, and A/G at -119. Our studies have shown that variants -777T, - 680T, -214A, -213G, and -119A always occur together (creating haplotype-l containing TTAGA and haplotype-ll containing AGCCG respectively). Our studies have shown that haplotype-l of the hAT1R gene is associated with hypertension in Caucasian women and transient transfection of reporter construct containing haplotype-l of the hAT1R gene has increased promoter activity in adrenal cortical cells and VSMC as compared to haplotype-ll. Our gel shift assays have shown that: (a) transcription factor C/EBP binds more strongly to an oligonucleotide containing -119A (haplotype-l) as compared to -119G (haplotype- ll) and (b) transcription factor USF (which binds to an E-box motif CANNTG) binds strongly to an oligonucleotide containing nucleoside A and G at -214 and -213 (haplotype-l) as compared to the same oligonucleotide containing nucleoside C at -213 and -214 (haplotype-ll). We will therefore analyze the effect of haplotypes -I and II on transcriptional regulation of the hAT1R gene in an in-vitro system using adrenocortical and vascular smooth muscle cells, examine the effect of haplotypes-l and II on hAT1 R mRNA level and on blood pressure using male and female transgenic mice, and examine the effect of IL-6, glucocorticoids, and estrogens on hAT1R mRNA level in transgenic mice containing haplotype I and II of the hAT1 R gene, and to correlate potential changes in mRNA levels with blood pressure in transgenic mice. ? ? ? ? ?
