Vitamin C attenuates nitrate tolerance independently of its antioxidant effect

In LLC-PK1 kidney epithelial cells, a 5-h pretreatment with glyceryl trinitrate (GTN) resulted in substantial desensitization of the intracellular cyclic GMP response to a subsequent 10-min challenge with GTN (1 microM). GTN-tolerant cells were fully sensitive to the spontaneous nitric oxide (NO) donor spermine NONOate, which does not require enzymatic bioactivation. Cyclic GMP stimulation by GTN was up to 3.1-fold higher when vitamin C (1-10 mM) was present during the pretreatment period. In contrast, other oxygen radical scavengers such as tiron or dimethylsulfoxide and the NO scavenger PTIO left tolerance induction unaltered. Together, our results suggest that reactive oxygen species or NO do not contribute to the development of nitrate tolerance. Tolerance reduction by vitamin C may be due to a stabilizing effect on enzymes involved in the bioconversion of GTN to NO.