Catecholamines and angiotensinogen gene expression in kidney proximal tubular cells

To investigate the molecular mechanism(s) of action of catecholamines on th e expression of the angiotensinogen (ANG) gene in kidney proximal tubular c ells, we used opossum kidney (OK) cells with a fusion gene containing the 5 '-flanking regulatory sequence of the rat ANG gene fused with a human growt h hormone (hGH) gene as a reporter, pOGH (rANG N-1498/+18), permanently int egrated into their genomes. The level of expression of the ANG-GH fusion ge ne was quantified by the amount of immunoreactive-hGH (IR-hGH) secreted int o the medium. The addition of norepinephrine (NE), isoproterenol (a beta (1 )/beta (2)-adrenergic receptor (AR) agonist) and iodoclonidine (an alpha (2 )-AR agonist) stimulated the expression of the ANG-GH fusion gene in a dose -dependent manner, whereas the addition of epinephrine and phenylephrine (a lpha (1)-AR agonist) had no effect. The stimulatory effect of NE was blocke d by the presence of propranolol (beta -AR blocker), atenolol (beta (1)-AR blocker), yohimbine (alpha (2)-AR blocker), Rp-cAMP (an inhibitor of cAMP-d ependent protein kinase AI & AII) and staurosporine (an inhibitor of protei n kinase C), but was not blocked by ICI 118, 551 (beta (2)-AR blocker) and prazosin (alpha (1)-AR blocker). The addition of a combination of isoproter enol and iodoclonidine or a combination of 8-Bromo-cAMP (8-Br-cAMP) and pho rbol 12-myristate (PMA) synergistically stimulated the expression of the AN G-GH fusion gene as compared to the addition of isoproterenol, iodoclonidin e, 8-Br-cAMP or PMA alone. Furthermore, the addition of NE, 8-Br-cAMP or PM A stimulated the expression of pOGH (rANG N-806/-779/-53/+18), a fusion gen e containing the putative cAMP responsive element (CRE, ANG N-806/-779) ups tream of the ANG promoter (ANG N-53/+18) in OK cells, but had no effect on the expression of fusion genes containing the mutant of the CRE. Gel mobili ty shift assays revealed that the ANG-CRE binds with the DNA-binding domain (bZIP 254-327) of the cAMP-responsive binding protein (CREB). The binding of the labeled ANG-CRE to CREB (bZIP254-327) was displaced by unlabeled ANG -CRE and the CRE of the somatostatin gene but not by the mutants of the ANG -CRE. Finally, NE stimulated the phosphorylation of CREB in OK cells. These studies demonstrate that the molecular mechanism(s) of NE action on the ex pression of the ANG gene in OK cells may be mediated via both the PKA and P KC signalling pathways and via the phosphorylation of CREB. The phosphoryla ted CREB then interacts with the CRE in the 5'-flanking region of the ANG g ene and subsequently stimulates the gene expression.