Decreased arteriolar tetrahydrobiopterin is linked to superoxide generation from nitric oxide synthase in mice fed high salt

Abstract

Objective: Impaired endothelium-dependent arteriolar dilation in mice fed high salt (HS) is due to local oxidation of nitric oxide (NO) by superoxide anion (O(2) (-)). We explored the possibility that "uncoupled" endothelial nitric oxide synthase (eNOS) is the source of this O(2) (-).

Methods: Levels of L-arginine (L-Arg), tetrahydrobiopterin (BH(4)), and O(2) (-) (hydroethidine oxidation) were measured in spinotrapezius muscle arterioles of mice fed normal salt (0.45%, NS) or (4%, HS) diets for 4 weeks, with or without dietary L-Arg supplementation. The contribution of NO to endothelium-dependent dilation was determined from the effect of N(omega)-nitro-L-arginine methyl ester (L-NAME) on responses to acetylcholine (ACh).

Results: Arterioles in HS mice had lower [BH(4)] and higher O(2) (-) levels than those in NS mice. ACh further increased arteriolar O(2) (-) in HS mice only. L-Arg supplementation prevented the reduction in [BH(4)] in arterioles of HS mice, and O(2) (-) was not elevated in these vessels. Compared to NS mice, arteriolar ACh responses were diminished and insensitive to L-NAME in HS mice, but not in HS mice supplemented with L-Arg.

Conclusions: These findings suggest that eNOS uncoupling due to low [BH(4)] is responsible for O(2) (-) generation and reduced NO-dependent dilation in arterioles of mice fed a HS diet.

Figure 1

Arteriolar ethidium bromide fluorescence after exposure of the spinotrapezius muscle to hydroethidine in mice fed normal salt or high salt diet, with or without L-arginine (L-Arg) supplementation. Measurements were made in otherwise untreated muscles (Control) or in muscles also exposed to ACh (1×10^-5M). Top: representative fluorescence images for each experimental group. Arrow in each image indicates position of vessel wall. Bottom: mean fluorescence values for each group. Values in parentheses represent number of vessels studied in each group. *P < 0.05 vs. Control value in untreated NS mice. † P < 0.05 vs. Control in same diet group.

Figure 2

Effect of L-arginine supplementation (L-Arg) on arteriolar wall L-arginine levels in mice fed normal salt or high salt diet. * P < 0.05 vs. untreated group. In Figures 2-5, n= number of samples, with each sample comprised of muscle vessels pooled from 5-6 mice.

Figure 3

Arteriolar wall BH₄ content in mice fed normal salt or high salt diet, with or without L-Arg supplementation. * P < 0.05 vs. untreated and L-Arg supplemented normal salt mice.

Figure 4

Ratio of BH₂ content to BH₄ content in arterioles from mice fed normal salt or high salt diet, with or without L-Arg supplementation. * P < 0.05 vs. untreated and L-Arg supplemented normal salt mice.

Figure 5

GTP cyclohydrolase-1 expression in arterioles from mice fed normal salt or high salt diet, with or without L-Arg supplementation.

Figure 6

Arteriolar dilation in response to iontophoretic application of ACh in mice fed normal salt (NS) or high salt (HS) diet, with or without L-Arg supplementation. * P < 0.05 vs. both NS groups and the HS group supplemented with L-arginine. n= number of vessels (1 vessel studied per mouse).

Figure 7

Magnitude of responses to ACh, under normal superfusate (Control) and in the presence of N^ω-nitro-L-arginine methyl ester (L-NAME, 10^-4M), in mice fed NS or HS diet, with or without L-Arg supplementation. Number of vessels for each group same as in Figure 6. * P < 0.05 vs. Control.

Figure 8

Arteriolar dilation in response to iontophoretic application of sodium nitroprusside (SNP) in mice fed NS or HS diet, with or without L-Arg supplementation. n= number of vessels (1 vessel studied per mouse).