Angiotensin-(1-7) downregulates tyrosine hydroxylase through a proteasome-dependent pathway

M.A. LOPEZ VERRILLI; GIRONACCI M.

Buenos Aires, Argentina

Congreso; World Congress of Cardiology; 2008

Angiotensin-(1-7) downregulates tyrosine hydroxylase through a proteasome-dependent pathway María Alejandra Lopez Verrilli, Mariela 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 sympathetic nervous system activity. It has been demonstrated that angiotensin (Ang) II enhances NE outflow contributing to the sympathetic hyperactivity in hypertension. 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 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 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 from hypothalami of SHR rats. Basal TH enzymatic activity was significantly higher in hypothalami from SHR than in WKY nomortensive controls (82±5 [3H]-H2O nmol/protein mg.h in SHR vs 69±4 [3H]-H2O nmol/protein mg.h in WKY) (P< 0,05; n=10). Hypothalami preincubated with 100 nM or 1µM Ang-(1-7) showed a significant decrease in TH specific activity in both rat strains. The enzymatic activity of TH is positively regulated by its phosphorylation at Ser residues by a variety of protein kinases. We investigated whether Ang-(1-7) may affect TH phosphorylation in SHR hypothalami cathelocaminergic neurons 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+ -which leads to an increase in Ca2+ 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 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 the protein. 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 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. We conclude that Ang-(1-7) caused a reduction in TH activity and expression at the central level. 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 central sympathetic nervous activity.