Angiotensin-(1-7) inhibits tyrosine hydroxylase activity and accelerates its degradation through a proteasome-dependent pathway

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

Ouro Preto, Brasil

Simposio; VII International Symposium of Vasoactive Peptides; 2008

Hypothalamic norepinephrine (NE) release may regulate arterial pressure by altering sympathetic nervous system activity. It has been suggested that 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) decreases hypothalamic NE release and this effect may be correlated with a diminished NE synthesis, we hypothetize that Ang-(1-7) may downregulate tyrosine hydroxylase (TH), the rate-limiting step enzyme in cathecolamines biosynthesis. Our aim was toinvestigate the effect of Ang-(1-7) on TH activity and expression at the central level. TH activity was evaluated in hypothalami from Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats by the release of tritiated water from ^3 H-L-tyrosine. TH expression and phosphorylation at serine (Ser) 19 and Ser-40 were determined by western blot in primary neuronal cultures from hypothalami of SHR rats. Basal TH enzymatic activity was significantly higher in hypothalami from SHR than in WKY nomortensive controls (82±5 vs 69±4 [^3 H]-H2O nmol/protein mg.h, respectively) (P< 0,05; n=10). Hypothalami preincubated with 100 nM or 1µM Ang-(1-7) showed a significant decrease in TH specific activity inboth rat strains. Phosphorylation at Ser residues positively regulates TH enzymatic activity. We investigated whether Ang-(1-7) may affect TH activation in SHR hypothalami neuronal cultures and observed that 100 nM Ang-(1-7) decreased the phosphorylation of TH at Ser-19 and Ser-40, 32±4% and 31±5%, respectively. Under depolarization with high K^+ -whichleads to an increase in Ca^2+ influx and the concomitant activation of kinases which in turn may phosphorylate TH- we observed an augmented TH phosphorylation, which was blocked by Ang-(1-7). Treatment of hypothalamic neuronal cultures from SHR with 100 nM Ang-(1-7) during 30 min caused a decrease in TH endogenous expression of 31±3% and thiseffect was blocked by an AT_2 receptor antagonist, and not by an AT1- or Mas receptor antagonist. The decrease in TH levels caused by Ang-(1-7) may be due to an increased degradation of the protein. Since the ubiquitin-proteasome system is the major pathway for protein degradation, we examined the involvement of the proteasomal pathway inthe Ang-(1-7)-induced decrease in TH expression. We observed that MG132, a selective proteasome inhibitor, blocked the Ang-(1-7)-mediated TH downregulation, suggesting a  proteasome-dependent TH degradation. Together with the fact that the peptide induces a decrease in NE release, our study supports a negative neuromodulator role for Ang-(1-7)on NE synthesis and central sympathetic nervous activity.