Molecular mechanism(s) of action of norepinephrine on the expression of the angiotensinogen gene in opossum kidney cells

Abstract

Background: Norepinephrine (NE) is the major endogenous neurotransmitter of the renal sympathetic nerves interacting with both the alpha- and beta-adrenoceptors in the renal proximal tubules. We have previously reported that isoproterenol and iodoclonidine stimulate the expression of the angiotensinogen (ANG) gene in opossum kidney (OK) proximal tubular cells via the beta1-adrenoceptor and alpha2-adrenoceptor, respectively. We hypothesized that NE may interact with the beta- and/or alpha2-adrenoceptors to stimulate the expression of the ANG gene in OK cells.

Methods: The fusion genes containing the various lengths of the 5'-flanking regulatory sequence of the rat ANG gene fused with a human growth hormone (hGH) gene as a reporter were stably transfected into the OK cells. The stimulatory effect of NE on the expression of the fusion genes was evaluated by the amount of immunoreactive hGH (IR-hGH) secreted into the culture medium.

Results: The addition of NE stimulated the expression of the fusion gene, pOGH (ANG N-1498/+18) in a dose-dependent manner. The stimulatory effect of NE was inhibited in the presence of propranolol, atenolol, Rp-cAMP, yohimbine, staurosporine, H-7 and U73122 but not in the presence of ICI 118,551 and prazosin. The addition of a combination of isoproterenol and iodoclonidine synergistically stimulated the expression of pOGH (ANG N-1498/+18) as compared to the addition of isoproterenol and iodoclonidine alone. Furthermore, the addition of NE, forskolin, 8-Br-cAMP or phorbol 12-myristate (PMA) stimulated the expression of pOGH (ANG N-806/-779/-53/+18), a fusion gene containing the putative cAMP responsive element (CRE, ANG N-806/-779) upstream of the ANG promoter (ANG N-53/+ 18) in OK 95 cells, but had no effect on the expression of fusion genes containing the mutant of the CRE.

Conclusion: These studies demonstrate that the stimulatory effect of NE on the expression of the ANG gene in OK cells may be mediated via both the beta1- and alpha2-adrenoceptors and via the CRE (ANG N-806/-779) in the 5'flanking region of rat ANG gene.