Nitric oxide inhibits the expression of AT1receptors in neurons

Abstract

We have previously observed an increased of angiotensin II (ANG II) type 1 receptor (AT₁R) with enhanced AT₁R-mediated sympathetic outflow and concomitant downregulation of neuronal nitric oxide (NO) synthase (nNOS) with reduced NO-mediated inhibition from the paraventricular nucleus (PVN) in rats with heart failure. To test the hypothesis that NO exerts an inhibitory effect on AT₁R expression in the PVN, we used primary cultured hypothalamic cells of neonatal rats and neuronal cell line NG108-15 as in vitro models. In hypothalamic primary culture, NO donor sodium nitroprusside (SNP) induced dose-dependent decreases in mRNA and protein of AT₁R (10⁻⁵M SNP, AT₁R protein was 10 ± 2% of control level) while NOS inhibitor N^G-monomethyl-l-arginine (l-NMMA) induced dose-dependent increases in mRNA and protein levels of AT₁R (10⁻⁵M l-NMMA, AT₁R protein was 148 ± 8% of control level). Similar effects of SNP and l-NMMA on AT₁R expression were also observed in NG108-15 cell line (10⁻⁶M SNP, AT₁R protein was 30 ± 4% of control level while at the dose of 10⁻⁶M l-NMMA, AT₁R protein was 171 ± 15% of the control level). Specific inhibition of nNOS, using antisense, caused an increase in AT₁R expression while overexpression of nNOS, using adenoviral gene transfer (Ad.nNOS), caused an inhibition of AT₁R expression in NG108 cells. Antisense nNOS transfection augmented the increase while Ad.nNOS infection blunted the increase in intracellular calcium concentration in response to ANG II treatment in NG108 cells. In addition, downregulation of AT₁R mRNA as well as protein level in neuronal cell line in response to S-nitroso- N-acetyl pencillamine (SNAP) treatment was blocked by protein kinase G (PKG) inhibitor, while the peroxynitrite scavenger deforxamine had no effect. These results suggest that NO acts as an inhibitory regulator of AT₁R expression and the activation of PKG is the required step in the regulation of AT₁R gene expression via cGMP-dependent signaling pathway.