Impaired flow-induced dilation of coronary arterioles of dogs fed a low-salt diet: roles of ANG II, PKC, and NAD(P)H oxidase

Abstract

Low-salt (LS) diet has been considered to be beneficial in the prevention and treatment of hypertension; however, it also increases plasma angiotensin (ANG) II and may cause adverse cardiovascular effects, such as endothelial dysfunction. We assessed endothelial function of coronary arterioles and vascular superoxide production, as a function of LS diet. Dogs were fed with LS (0.05% NaCl) or a normal-salt (NS, 0.65% NaCl) diet for 2 wk. There were threefold increases in plasma ANG II, associated with a 60% reduction in flow-induced dilation (FID) in coronary arterioles of LS compared with NS dogs. In vessels of NS dogs, FID was primarily mediated by nitric oxide (NO), as indicated by an eliminated FID by N(ω)-nitro-l-arginine methyl ester (l-NAME). In vessels of LS dogs, however, FID was eliminated. Administration of apocynin, a NAD(P)H oxidase inhibitor, partially restored FID and additional l-NAME eliminated FID. Generation of superoxide, measured with dihydroethidium, was significantly greater in vessels of LS than in NS dogs, which was further increased in response to ANG II or phorbol 12,13-dibutyrate, an agonist of protein kinase C (PKC). The enhanced superoxide was normalized by apocynin, losartan (a blocker of angiotensin type 1 receptor), and chelerythrine chloride (an antagonist of PKC). Western blotting indicated an upregulation of gp91(phox) and p47(phox), associated with increased expression of phosphorylated PKC in vessels of LS dogs. In separate experiments, dogs were fed simultaneously with LS and losartan (LS + Losa) for 2 wk. There was a significant increase in plasma ANG II in LS + Losa dogs, which, however, was associated with normal FID and gp91(phox) expression in coronary arterioles. In conclusion, LS led to endothelial dysfunction, as indicated by an impaired flow-induced dilation caused by decreasing NO bioavailibility, a response that involves angiotensin-induced activation of PKC that, in turn, activates vascular NAD(P)H oxidase to produce superoxide.

Fig. 1.

Flow-induced dilation of coronary arterioles of normal-salt (NS, n = 5; A) and low-salt (LS, n = 7; B) diet dogs in the control condition and after incubating vessels with apocynin (APO, 10⁻⁵ M), or apocynin plus N^ω-nitro-l-arginine methyl ester (l-NAME, 3 × 10⁻⁴ M) for 30 min. Changes in diameter were normalized to passive diameter (PD) of the vessels. *Significant difference from the curve of APO. C and D: sodium nitroprusside (SNP) and adenosine (ADO)-induced dilations in coronary arterioles of NS and LS dogs.

Fig. 2.

Generation of superoxide in coronary arterioles of dogs using high-performance liquid chromatography (HPLC)/fluorescence detector of 2-hydroxyethidium (A and B) and confocal fluorescent image of superoxide by dihydroethidium (C and D). A and B: HPLC traces and summary data in control and in the presence of apocynin in NS (n = 5) and LS (n = 7) dogs. MV, millivolt. *Significant difference from NS dogs. #Significant difference from the corresponding control. C and D: original and summary data of confocal fluorescent images for superoxide production (red color) in the endothelium and smooth muscle layers in NS (n = 5) and LS (n = 5) dogs. EC, endothelial cells; SMC, smooth muscle cells. *Significant difference from NS dogs.

Fig. 3.

Protein expression of gp91^phox and p47^phox in coronary arterioles of dogs. Densitometry data were summarized from two blots. *Significant difference from NS dogs. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 4.

Superoxide production assessed by HPLC/fluorescence detector of 2-hydroxyethidium in coronary arterioles of NS and LS dogs in control (C) and in response to ANG II (10⁻⁷ M) and ANG II plus apocynin (10⁻⁵ M), losartan (Losa, 2 × 10⁻⁶ M), or chelerythrine chloride (CLT, 2 × 10⁻⁶ M) (A); and in control and in response to phorbol 12,13-dibutyrate (PDBu, 10⁻⁵ M) and PDBu plus CLT (B) (n = 5–7 dogs/group). *Significant difference from NS dogs. #Significant difference from corresponding controls. †Significant difference from those treated with ANG II alone or PDBu alone.

Fig. 5.

Protein expression of angiotensin type 1 (AT₁) receptor (A and B) and protein kinase C (PKC) and phosphorylated PKC (p-PKC-βII/Ser⁶⁶⁰) (C and D) in coronary arterioles of dogs. Densitometry data were summarized from three blots. *Significant difference from NS dogs.

Fig. 6.

A: flow-induced dilation of coronary arterioles of LS diet dogs treated with losartan (Losa, 2 mg·kg⁻¹·day⁻¹ orally) (n = 2 dogs/8 arterioles) in the control condition and after incubating vessels with apocynin (10⁻⁵ M) or apocynin plus l-NAME (3 × 10⁻⁴ M) for 30 min. Changes in diameter were normalized to passive diameter of the vessels. *Significant difference from the curve of apocynin. B: 2-hydroxyethidium was determined in coronary arteries of LS + Losa dogs (n = 2 dogs/5–7 arteries) in the absence and in the presence of PDBu and PDBu plus losartan (Losa) or CLT. *Significant difference from the absence of PDBu. #Significant difference from PDBu. C and D: protein expression of gp91^phox in coronary arterioles of NS-, LS-, and LS- plus losartan-treated dogs. *Significant difference from NS. #Significant difference from LS.