Hypothalamic norepinephrine (NE) release regulates arterial pressure by altering sympathetic nervous system activity. Because angiotensin (Ang) (1–7) decreases hypothalamic NE release and this effect may be correlated with a diminished NE synthesis, we hypothesize that Ang‐(1–7) down‐regulates tyrosine hydroxylase (TH), the rate‐limiting enzyme in catecholamines biosynthesis. We investigated the effect of Ang‐(1–7) on centrally TH activity and expression. TH activity was evaluated by the release of tritiated water from 3H‐l‐tyrosine. TH expression and phosphorylation were determined by western blot. Hypothalami from normotensive or spontaneously hypertensive rats pre‐incubated with Ang‐(1–7) showed a significant decrease in TH specific activity. Ang‐(1–7) caused a decrease in TH phosphorylation at Ser19 and Ser40 residues. The heptapeptide induced a decrease in TH expression that was blocked by an AT2 receptor antagonist and not by an AT1 or Mas receptor antagonist, suggesting the involvement of AT2 receptors. The proteasome inhibitor MG132 blocked the Ang‐(1–7)‐mediated TH reduction. In addition, Ang‐(1–7) increased the amount of TH–ubiquitin complexes, indicating that the Ang‐(1–7)‐mediated TH degradation involves ubiquitin conjugation prior to proteasome degradation. We conclude that Ang‐(1–7) down‐regulates TH activity and expression centrally leading to a decrease in the central NE system activity.

It has been shown that angiotensin (ANG)-(1-7) activates nitric oxide synthase (NOS) in isolated ventricular myocytes from normotensive rats. Since many ANG-(1-7) actions are enhanced in situations of increased ANG II activity, as in hypertension, in this study we investigated the in vivo effect of ANG-(1-7) on NOS activity and expression of endothelial (eNOS), neuronal (nNOS), and inducible NOS (iNOS) in ventricles from spontaneously hypertensive rats (SHR). Rats were subjected to a 60-min ANG-(1-7) infusion (0.35 nmol/min); controls received saline. NOS activity was measured using the NADPH diaphorase histochemical method and by the conversion of L-[(14)C]arginine to citrulline, and NOS phosphorylation and expression were determined using Western blotting. In SHR, ANG-(1-7) infusion diminished mean arterial pressure from 180 ± 9 to 146 ± 9 mmHg (P < 0.05), and this effect was prevented by nitro-l-arginine methyl ester (l-NAME), a NOS inhibitor. In addition, NOS activity and eNOS phosphorylation were increased by ANG-(1-7) infusion. Ventricular eNOS and nNOS expression were increased 67.4 ± 6.4 and 51 ± 10%, respectively, by ANG-(1-7), whereas iNOS was not changed. In another set of experiments, we evaluated the mechanism by which ANG-(1-7) modifies NOS activity. Isolated ventricle slices preincubated with ANG-(1-7) showed an increase in NOS activity and eNOS phosphorylation, which was blocked by an AT(2) and a bradykinin B(2) receptor antagonist, but not by the Mas receptor antagonist. Our results show that in rats in a hypertensive state, ANG-(1-7) infusion upregulates cardiac NOS expression and activity through an AT(2)- and bradykinin-dependent mechanism. In this way ANG-(1-7) may elicit its cardioprotective action and contribute to some of the counterregulatory AT(2) receptor effects that oppose the AT(1) receptor-mediated effects.

J. Neurochem. (2012) 120, 46–55. Abstract As angiotensin (Ang) (1–7) decreases norepinephrine (NE) content in the synaptic cleft, we investigated the effect of Ang‐(1–7) on NE neuronal uptake in spontaneously hypertensive rats. [3H]‐NE neuronal uptake was measured in isolated hypothalami. NE transporter (NET) expression was evaluated in hypothalamic neuronal cultures by western‐blot. Ang‐(1–7) lacked an acute effect on neuronal NE uptake. Conversely, Ang‐(1–7) caused an increase in NET expression after 3 h incubation (40 ± 7%), which was blocked by the Mas receptor antagonist, a PI3‐kinase inhibitor or a MEK1/2 inhibitor suggesting the involvement of Mas receptor and the PI3‐kinase/Akt and MEK1/2‐ERK1/2 pathways in the Ang‐(1–7)‐stimulated NET expression. Ang‐(1–7) through Mas receptors stimulated Akt and ERK1/2 activities in spontaneously hypertensive rat neurons. Cycloheximide attenuated Ang‐(1–7) stimulation of NET expression suggesting that Ang‐(1–7) stimulates NET synthesis. In fact, Ang‐(1–7) increased NET mRNA levels. Thus, we evaluated the long‐term effect of Ang‐(1–7) on neuronal NE uptake after 3 h incubation. Under this condition, Ang‐(1–7) increased neuronal NE uptake by 60 ± 14% which was blocked by cycloheximide and the Mas receptor antagonist. Neuronal NE uptake and NET expression were decreased after 3 h incubation with an anti‐Ang‐(1–7) antibody. Ang‐(1–7) induces a chronic stimulatory effect on NET expression. In this way, Ang‐(1–7) may regulate a pre‐synaptic mechanism in maintaining appropriate synaptic NE levels during hypertensive conditions.

Aberrations in the ubiquitin-proteasome system (UPS) are implicated in the pathogenesis of various diseases. Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis, is involved in hypertension development. In this study we investigated whether UPS regulated TH turnover in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats (SHR) and whether this system was impaired in hypertension. PC12 cells were exposed to proteasome or lysosome inhibitors and TH protein level evaluated by Western blot. Lactacystin, a proteasome inhibitor, induced an increase of 86±15% in TH levels after 30 min of incubation, then it started to decrease up to 6 h to reach control levels and finally it rose up to 35.2±8.5% after 24 h. Bafilomycin, a lysosome inhibitor, did not alter TH protein levels during short times, but it increased TH by 92±22% above basal after 6 h treatment. Before degradation proteasome substrates are labeled by conjugation with ubiquitin. Efficacy of proteasome inhibition on TH turnover was evidenced by accumulation of ubiquitinylated TH after 30 min. Further, the inhibition of proteasome increased the quantity of TH phosphorylated at Ser40, which is essential for TH activity, by 2.7±0.3 fold above basal. TH protein level was upregulated in neurons from hypothalami and brainstem of SHR when the proteasome was inhibited during 30 min, supporting that neuronal TH is also short-term regulated by the proteasome. Since the increased TH levels reported in hypertension may result from proteasome dysfunction, we evaluate proteasme activity. Proteasome activity was significantly reduced by 67±4% in hypothalamic and brainstem neurons from SHR while its protein levels did not change. Present findings show that TH is regulated by the UPS. The impairment in proteasome activity observed in SHR neurons may be one of the causes of the increased TH protein levels reported in hypertension.

González GE, Seropian IM, Krieger ML, Palleiro J, Lopez Verrilli MA, Gironacci MM, Cavallero S, Wilensky L, Tomasi VH, Gelpi RJ, Morales C. Effect of early versus late AT1 receptor blockade with losartan on postmyocardial infarction ventricular remodeling in rabbits. Am J Physiol Heart Circ Physiol 297: H375-H386, 2009. First published May 8, 2009 doi:10.1152/ajpheart.00498.2007.-To characterize the temporal activation of the renin-angiotensin system after myocardial infarction (MI) in rabbits, we examined cardiac ANG II type 1 receptor (AT1R) expression and ANG II levels from 3 h to 35 days. The effects of losartan (12.5 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 ) on functional and histomorphometric parameters when treatment was initiated early (3 h) and late (day 15) post-MI and maintained for different periods of time [short term (4 days), midterm (20 days), and long term (35 days)] were also studied. AT1R expression increased in the MI zone at 15 and 35 days (P Ͻ 0.05). ANG II levels increased (P Ͻ 0.05) in the non-MI zone at 24 h and in the MI zone as well as in plasma at 4 days and then progressively decreased until 35 days. The survival rate was significantly lower in untreated MI and early long-term-treated animals. Diastolic pressure-volume curves in MI at 35 and 56 days shifted to the right (P Ͻ 0.05). This shift was even more pronounced in long-term-treated groups (P Ͻ 0.05). Contractility decreased (P Ͻ 0.05 vs. sham) in the untreated and long-term-treated groups and was attenuated in the midterm-treated group. The early administration of losartan reduced RAM 11-positive macrophages from 4.15 Ϯ 0.05 to 3.05 Ϯ 0.02 cells/high-power field (HPF; P Ͻ 0.05) and CD45 RO-positive lymphocytes from 2.23 Ϯ 0.05 to 1.48 Ϯ 0.01 cells/HPF (P Ͻ 0.05) in the MI zone at 4 days. Long-term treatment reduced the scar collagen (MI: 70.50 Ϯ 2.35% and MI ϩ losartan: 57.50 Ϯ 2.48, P Ͻ 0.05), determined the persistency of RAM 11-positive macrophages (3.02 Ϯ 0.13 cells/HPF) and CD45 RO-positive lymphocytes (2.77 Ϯ 0.58 cells/HPF, P Ͻ 0.05 vs. MI), and reduced the scar thinning ratio at 35 days (P Ͻ 0.05). Consequently, the temporal expressions of cardiac AT1R and ANG II post-MI in rabbits are different from those described in other species. Long-term treatment unfavorably modified post-MI remodeling, whereas midterm treatment attenuated this harmful effect. The delay in wound healing (early reduction and late persistency of inflammatory infiltrate) and adverse remodeling observed in long-term-treated animals might explain the unfavorable effect observed in rabbits.angiotensin II type 1 receptor blockers MYOCARDIAL INFARCTION (MI) leads to global structural alterations that involve infarcted as well as noninfarcted areas in a process collectively known as ventricular remodeling (26). The initial phase of remodeling, which starts in the acute phase of the MI, is associated with geometric changes and dilation of the MI zone, as a consequence of injured wall thinning (14, 26). Shortly afterward, this dilation may progress to the whole ventricle, and...

Since angiotensin (Ang) (1-7) injected into the brain blocked Ang II pressor actions in rats made hypertensive by aortic coarctation (CH), we examined systemic and tissue angiotensin peptide levels, specifically concentrating on the hypothalamic Ang-(1-7) levels. Plasma, heart and kidney isolated from CH rats showed increased levels of Ang I, Ang II and Ang-(1-7) compared with the normotensive group, with Ang II being the predominant peptide in heart and kidney. In the hypothalamus, equimolar amounts of Ang II and Ang-(1-7) were found in the sham group, whereas only Ang-(1-7) levels increased in CH rats. We conclude that aortic coarctation activates systemic and tissue renin-angiotensin system. The increased central levels of Ang-(1-7) in the CH rats suggest a potential mitigating role of this peptide in central control of the hypertensive process.

The aqueous and organic extracts of Acacia visco Lor. Ap Griseb (Fabaceae) were tested for antiinflammatory activity in experimental models in rat. Besides, the free-radical scavenging capacity of extracts from A. visco was determined. The extracts revealed antiinflammatory effect against carrageenan-induced oedema, phospholipase A 2 -induced oedema, cotton pellet-induced granuloma and they did not show acute toxic effect. Among the class of compounds characterized from A. visco leaves, the triterpenoid 20(29)-lupen-3b-ol (lupeol), 12-ursen-3b-ol (a-amyrin) and 12-oleanen-3b-ol (b-amyrin) may be mainly responsible for the pharmacological activities.

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