Nuclear angiotensin II type 2 (AT2) receptors are functionally linked to nitric oxide production

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Abstract
Expression of nuclear angiotensin II type 1 (AT1) receptors in rat kidney provides further support for the concept of an intracellular renin-angiotensin system. Thus we examined the cellular distribution of renal ANG II receptors in sheep to determine the existence and functional roles of intracellular ANG receptors in higher order species. Receptor binding was performed using the nonselective ANG II antagonist125I-[Sar1,Thr8]-ANG II (125I-sarthran) with the AT1antagonist losartan (LOS) or the AT2antagonist PD123319 (PD) in isolated nuclei (NUC) and plasma membrane (PM) fractions obtained by differential centrifugation or density gradient separation. In both fetal and adult sheep kidney, PD competed for the majority of cortical NUC (≥70%) and PM (≥80%) sites while LOS competition predominated in medullary NUC (≥75%) and PM (≥70%). Immunodetection with an AT2antibody revealed a single ∼42-kDa band in both NUC and PM extracts, suggesting a mature molecular form of the NUC receptor. Autoradiography for receptor subtypes localized AT2in the tubulointerstitium, AT1in the medulla and vasa recta, and both AT1and AT2in glomeruli. Loading of NUC with the fluorescent nitric oxide (NO) detector DAF showed increased NO production with ANG II (1 nM), which was abolished by PD and N-nitro-l-arginine methyl ester, but not LOS. Our studies demonstrate ANG II receptor subtypes are differentially expressed in ovine kidney, while nuclear AT2receptors are functionally linked to NO production. These findings provide further evidence of a functional intracellular renin-angiotensin system within the kidney, which may represent a therapeutic target for the regulation of blood pressure.