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

LOPEZ VERRILLI, M. A.; GIRONACCI M.

Ouro Preto, Minas Gerais, Brasil

Simposio; VIIth International Symposium on Vasoactive Peptides and II Simpsósio Mineiro de Hipertensao.; 2008

Angiotensin-(1-7) inhibits tyrosine hydroxylase activity and accelerates its degradation through a proteasome-dependent pathway M. A. Lopez Verrilli, M. M. Gironacci. Instituto de Química Física Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires.   Hypothalamic norepinephrine (NE) release may regulate arterial pressure by altering sympatheticnervous 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) decreaseshypothalamic NE release andthis effect may be correlatedwith a diminished NE synthesis, we hypothetizethat Ang-(1-7) may downregulate tyrosine hydroxylase (TH), the rate-limiting step enzyme in cathecolamines biosynthesis. Our aim was to investigate 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 3H-L-tyrosine. TH expression and phosphorylation at serine (Ser) 19 and Ser-40 were determined by western blot in primary neuronal cultures fromhypothalami of SHR rats. Basal TH enzymaticactivity was significantly higher in hypothalami from SHR than in WKY nomortensive controls (82±5 vs 69±4 [3H]-H2O nmol/protein mg.h, respectively) (P< 0,05; n=10). Hypothalamipreincubated with 100 nM or 1µM Ang-(1-7) showed a significant decrease in TH specific activity in both rat strains. Phosphorylation at Ser residuespositively 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 depolarizationwith high K+ -which leads to an increase in Ca2+ influx andthe concomitant activation of kinases which in turn mayphosphorylate 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 AT2 receptorantagonist, 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. Sincethe ubiquitin-proteasome system is the major pathway for protein degradation, we examined the involvement of the proteasomal pathway in the 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 factthat the peptide induces a decrease in NE release, our study supports a negative neuromodulator role for Ang-(1-7) on NE synthesisand central sympathetic nervous activity.