Angiotensin-(1-7) downregulates tyrosine hydroxylase through a

M. A. LOPEZ VERRILLI, M. M. GIRONACCI

Buenos Aires

Congreso; World Congress of Cardiology; 2008

Hypothalamic norepinephrine (NE) release may regulate arterial pressure by altering sympatheticnervous system activity. It has been demonstrated that angiotensin (Ang) II enhances NEoutflow contributing to the sympathetic hyperactivity in hypertension. It has been suggestedthat the renin-angiotensin system may counteract the pressor effects of Ang II by Ang-(1?7)generation, an antihypertensive component of this system. Since Ang-(1?7) decreaseshypothalamic NE release and this effect may be correlated with a diminished NE synthesis, wehypothetize that Ang-(1?7) may downregulate tyrosine hydroxylase (TH), the rate-limiting stepenzyme in cathecolamines biosynthesis. Our aim was to investigate the effect of Ang-(1?7) onTH activity and expression at the central level. TH activity was evaluated in hypothalami fromWistar-Kyoto and spontaneously hypertensive (SHR) rats by the release of tritiated water from3H-L-tyrosine. TH expression and phosphorylation at serine (Ser) 19 and Ser-40 weredetermined by western blot in primary neuronal cultures from hypothalami of SHR rats. BasalTH enzymatic activity was significantly higher in hypothalami from SHR than in WKYnomortensive controls (825 [3H]-H2O nmol/protein mg.h in SHR vs 694 [3H]-H2Onmol/protein mg.h in WKY) (P 0,05; n10). Hypothalami preincubated with 100 nM or 1MAng-(1?7) showed a significant decrease in TH specific activity in both rat strains. Theenzymatic activity of TH is positively regulated by its phosphorylation at Ser residues by avariety of protein kinases. We investigated whether Ang-(1?7) may affect TH phosphorylationin SHR hypothalami cathelocaminergic neurons and observed that 100 nM Ang-(1?7)decreased the phosphorylation of TH at Ser-19 and Ser-40, 324% and 315%, respectively.Under depolarization with high K -which leads to an increase in Ca2 influx and theconcomitant activation of kinases which in turn may phosphorylate TH- we observed anaugmented TH phosphorylation, which was blocked by Ang-(1?7). Treatment of hypothalamicneuronal cultures from SHR with 100 nM Ang-(1?7) during 30 min caused a decrease in THendogenous expression of 313% and this effect was blocked by an AT2 receptor antagonist,and not by an AT1- or Mas receptor antagonist, suggesting the involvement of AT2 receptors.The decrease in TH levels caused by Ang-(1?7) may be due to an increased degradation of theprotein. Since the ubiquitin-proteasome system is the major pathway for protein degradation,we examined the involvement of the proteasomal pathway in the Ang-(1?7)-induced decreasein TH expression. We observed that MG132, a selective proteasome inhibitor, blocked theAng-(1?7)-mediated TH downregulation, suggesting a proteasome-dependent TH degradation.We conclude that Ang-(1?7) caused a reduction in TH activity and expression at the centrallevel. Together with the fact that the peptide induces a decrease in NE release, our studysupports a negative neuromodulator role for Ang-(1?7) on central sympathetic nervous activity.