Dietary Salt, Oxygen Radicals, and the Microvascular Endothelium:

Reactive oxygen species and their effect on vascular regulation

    In rats fed a high-salt diet for 2-4 weeks, we have found that the endothelium-dependent dilation of skeletal muscle arterioles in response to acetylcholine (ACh) or increased hemodynamic shear stress is greatly reduced and no longer altered by inhibiting the production of nitric oxide (NO). This suggests that the contribution of NO to these responses has been eliminated. We are currently exploring the possible role of reactive oxygen species in this reduced contribution of NO to endothelium-dependent dilation. We have found that during exposure to superoxide dismutase (SOD) + catalase to scavenge superoxide anion, resting arteriolar diameters and ACh-induced dilation are unchanged in rats fed a normal diet. However, SOD + catalase completely reverses the blunted responses to ACh in rats fed high salt. We observed this same effect during exposure to the more membrane-permeable superoxide anion scavenger 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) + catalase (Cat) (see figure below left).

    To quantify the oxidative state of the microvascular wall, the tissue was exposed to tetranitroblue tetrazolium (TNBT), which forms insoluble blue/black formazan deposits when reduced by reactive oxygen species. Using image analysis, we found evidence of significant formazan deposits in both the arteriolar and venular walls of rats fed high salt (below right). As also shown, exposure of the muscle to TEMPO + catalase prevented this formazan formation in high-salt rats, which verifies the specificity of these measurements as an index of oxidant activity.

    Taken together, these findings strongly suggest that (1) ingestion of a high salt diet reduces the endothelium -dependent dilation of striated muscle arterioles by eliminating local NO activity, and (2) the loss of this NO activity is related to the generation of reactive oxygen species in the arteriolar wall. We are currently trying to gain more insight into the mechanism by which high salt intake can lead to the generation of reactive oxygen species. Recent Western analysis suggests that high salt intake does not change the expression of antioxidant enzymes in the microvascular wall (see bottom figure). We are currently trying to determine if the activity of these enzymes, or the expression and/or activity of oxidant-generating enzymes (i.e., xanthine oxidase and NAD(P)H oxidase) is altered in rats fed high salt.

Western Blots illustrating the expression of different antioxidant enzymes in the microvascular wall