Angiotensin-(1-7) and low-dose angiotensin II infusion reverse salt-induced endothelial dysfunction via different mechanisms in rat middle cerebral arteries

Abstract

The goals of this study were to 1) determine the acute effect of ANG-(1-7) on vascular tone in isolated middle cerebral arteries (MCAs) from Sprague-Dawley rats fed a normal salt (NS; 0.4% NaCl) diet, 2) evaluate the ability of chronic intravenous infusion of ANG-(1-7) (4 ng·kg(-1)·min(-1)) for 3 days to restore endothelium-dependent dilation to acetylcholine (ACh) in rats fed a high-salt (HS; 4% NaCl) diet, and 3) determine whether the amelioration of endothelial dysfunction by ANG-(1-7) infusion in rats fed a HS diet is different from the protective effect of low-dose ANG II infusion in salt-fed rats. MCAs from rats fed a NS diet dilated in response to exogenous ANG-(1-7) (10(-10)-10(-5) M). Chronic ANG-(1-7) infusion significantly reduced vascular superoxide levels and restored the nitric oxide-dependent dilation to ACh (10(-10)-10(-5) M) that was lost in MCAs of rats fed a HS diet. Acute vasodilation to ANG-(1-7) and the restoration of ACh-induced dilation by chronic ANG-(1-7) infusion in rats fed a HS diet were blocked by the Mas receptor antagonist [D-ALA(7)]-ANG-(1-7) or the ANG II type 2 receptor antagonist PD-123319 and unaffected by ANG II type 1 receptor blockade with losartan. The restoration of ACh-induced dilation in MCAs of HS-fed rats by chronic intravenous infusion of ANG II (5 ng·kg(-1)·min(-1)) was blocked by losartan and unaffected by d-ALA. These findings demonstrate that circulating ANG-(1-7), working via the Mas receptor, restores endothelium-dependent vasodilation in cerebral resistance arteries of animals fed a HS diet via mechanisms distinct from those activated by low-dose ANG II infusion.

Fig. 1.

Response of isolated middle cerebral arteries from rats fed a normal salt diet to ANG-(1-7) (10⁻¹⁰–10⁻⁵ M; n = 8) before and after acute addition of the Mas receptor antagonist d-ALA (10 μM; n = 6), the AT₁ receptor antagonist losartan (10 μM; n = 6), or the AT₂ receptor antagonist PD-123319 (1 μM; n = 6) to the tissue bath. *P < 0.05, inhibitor vs. control.

Fig. 2.

Response to acetylcholine (10⁻¹⁰–10⁻⁵ M) (A) and sodium nitroprusside (10⁻¹²–10⁻⁴ M) (B) in isolated middle cerebral arteries from rats fed a high-salt diet (n = 7 to 8) vs. rats fed a high-salt diet and receiving a 3-day infusion of ANG-(1-7) (4 ng·kg⁻¹·min⁻¹; n = 7) or a 3-day infusion of isotonic saline (n = 6 to 7). *P < 0.05, high-salt ANG-(1-7) infused vs. high-salt and high salt-saline infused.

Fig. 3.

Response to acetylcholine (10⁻¹⁰–10⁻⁵ M) (A) and sodium nitroprusside (10⁻¹²–10⁻⁴ M) (B) in isolated middle cerebral arteries from rats fed a normal salt diet (n = 6) vs. normal salt-fed rats receiving a chronic 3-day infusion of ANG-(1-7) (4 ng·kg⁻¹·min⁻¹; n = 7) or 3-day isotonic saline infusion (n = 5 to 6).

Fig. 4.

Response to acetylcholine (10⁻¹⁰–10⁻⁵ M) in isolated middle cerebral arteries from rats fed a high-salt diet and receiving a 3-day infusion of ANG-(1-7) (4 ng·kg⁻¹·min⁻¹; n = 7) ± the nitric oxide synthase inhibitor N^ω-nitro-l-arginine methyl ester (l-NAME, 100 μM; n = 7) in the perfusate and superfusate. Data from the ANG-(1-7) infused group are replotted from Fig. 2A. *P < 0.05, l-NAME treated vs. ANG-(1-7) infused.

Fig. 5.

Response to acetylcholine (10⁻¹⁰–10⁻⁵ M) in isolated middle cerebral arteries from rats fed a high-salt diet and receiving a chronic (3 days) intravenous infusion of ANG-(1-7) (n = 7) or a simultaneous infusion of ANG-(1-7) + d-ALA (n = 6) (A), an infusion of ANG-(1-7) (n = 7) or a simultaneous infusion of ANG-(1-7) + losartan (n = 8) (B), or an infusion of ANG-(1-7) (n = 7) or a simultaneous infusion of ANG-(1-7) + PD-123319 (n = 10) (C). Data from the ANG-(1-7) infused control group are replotted from Fig. 2A. *P < 0.05, ANG-(1-7) + inhibitor vs. ANG-(1-7) infused.

Fig. 6.

Response to acetylcholine (10⁻¹⁰–10⁻⁵ M) in isolated middle cerebral arteries from rats fed a high-salt diet receiving an infusion of isotonic saline (n = 6), an infusion of ANG II (5 ng·kg⁻¹·min⁻¹; n = 6), or a simultaneous infusion of ANG II + d-ALA (n = 6) or ANG II + losartan (n = 6). *P < 0.05, ANG II infused and ANG II + d-ALA coinfused vs. saline infused and ANG II + losartan coinfused.

Fig. 7.

A: representative bright-field and fluorescent images of basilar artery cross sections (10 μm) from high salt-fed rats receiving a 3-day infusion of either isotonic saline or ANG-(1-7). The vessels were stained with dihydroethidium (5 μM) to assess vascular superoxide levels. B: vascular superoxide levels quantified as brightness values in dihydroethidium-stained basilar artery cross sections from high salt-fed rats receiving a 3-day infusion of either isotonic saline (n = 6) or ANG-(1-7) (n = 5). Data are expressed as means (brightness values) ± SE. *P < 0.05, ANG-(1-7) infused vs. saline infused.

Fig. 8.

A: representative bright-field and fluorescent images of basilar artery cross sections (10 μm) from normal salt-fed rats receiving a 3-day infusion of either isotonic saline or ANG-(1-7). The vessels were stained with dihydroethidium (5 μM) to assess vascular superoxide levels. B: vascular superoxide levels quantified as brightness values in dihydroethidium-stained basilar artery cross sections from normal salt-fed rats receiving a 3-day infusion of either isotonic saline (n = 6) or ANG-(1-7) (n = 6). Data are expressed as means (brightness values) ± SE.