Neuromodulatory role for angiotensin-(1-7) on norepinephrine uptake in neurons from spontaneuosly hypertensive rats

M. A. LOPEZ VERRILLI, M. RODRÍGUEZ FERMEPÍN, B. E. FERNANDEZ, M. M. GIRONACCI

Belo Horizonte, Brasil

Congreso; XVIIIth Scientific Sessions of the Inter-American Society of Hypertension; 2009

Introduction Hypothalamic norepinephrine (NE) release contributes to arterial pressure regulation by altering sympathetic nervous system activity. It has been suggested that the renin-angiotensin system may counteract Ang II pressor effect by Ang-(1-7) generation, an antihypertensive component of this system. We have determined that, in contrast to Ang II, Ang-(1–7) reduces NE synthesis and release in isolated hypothalami from spontaneously hypertensive rats (SHR) and in this way it may decrease sympathetic activity. Objective Since Ang-(1-7) decreases NE release and this effect may be correlated with an augmented NE removal from the synaptic cleft, our aim was to investigate the effect of Ang-(1–7) on neuronal NE uptake and norepinephrine transporter (NET) expression at the central level. NET is responsible for the uptake of neuronally released NE into presynaptic noradrenergic neurons, thus inactivating NE. Material and methods Neuronal 3H-NE uptake was measured in isolated hypothalami from 3-month-old normotensive controls (WKY) and SHR rats in the absence (basal) or presence of 0.1-1 μM Ang-(1-7). NET expression was determined by western blot in primary neuronal cultures from hypothalami of newborn WKY and SHR rats. Results No significant difference between basal neuronal NE uptake from WKY and SHR hypothalami were observed. Ang-(1-7) (0.1-1 μM) did not modify NE neuronal uptake in both strains There was no significant difference in NET expression in hypothalamic neuronal cultures from SHR compared to WKY. The addition of 0.1 μM Ang-(1-7) to neuronal cultures from SHR hypothalami caused an increase in NET expression after 3 and 5 h of incubation (40+7% and 30+9%, respectively). To examine the receptor subtypes involved in the stimulatory effect of Ang-(1-7) on NET expression, specific Mas, AT1- and AT2-receptors antagonists were added. 1μM [D-Ala7] Ang-(1-7), a Mas receptor antagonist, blocked the effect of Ang-(1-7), suggesting the involvement of Mas receptor. Neither 1μM PD123319, an AT2 receptor antagonist, nor 1μM losartan, an AT1 receptor antagonist, modified the action of Ang-(1-7) on NET expression in SHR neuronal cultures. 1 μM actinomycin-D or 0.5-1 μM cycloheximide, blocked the stimulatory effect of 0.1 μM Ang-(1-7) on NET expression, suggesting that Ang-(1-7) stimulates NET gene transcription and translation. Conclusion Our results showed that Ang-(1-7) does not evoke an acute effect on neuronal NE uptake. Conversely, Ang-(1-7) induces a long-term effect by increasing NET expression through stimulation of NET gene transcription and translation and this effect is coupled to Mas receptor activation. Our study, together with the fact that the peptide induces a decrease in NE synthesis and release, supports a negative neuromodulatory role for Ang-(1-7) on central sympathetic nervous activity.