Relationship between nitroglycerin-induced vascular relaxation and nitric oxide production. Probes with inhibitors and tolerance development.

We have shown previously that nitric oxide (NO) is generated from nitroglycerin (NTG) through enzyme-mediated reactions in the bovine coronary artery smooth muscle cell, but it is not known whether this metabolic conversion plays a significant role in the pharmacologic action of NTG, viz. relaxation. In this study, we developed a technique that allowed direct measurement of NO from intact bovine coronary arterial rings that were incubated previously with NTG, and examined whether changes in NTG-induced relaxation were accompanied by parallel changes in NO generation. Co-incubation of the vascular preparations with a potent inhibitor of glutathione-S-transferases (GSTs), bromosulfophthalein (up to 200 microM), did not affect NTG-induced relaxation, nor did it alter NO generation from NTG in the preparation. In contrast, 1-chloro-2,4-dinitrobenzene (CDNB), a GST substrate, inhibited NO generation as well as the relaxation response of NTG in the intact vascular tissue preparation. CDNB, however, did not decrease the relaxant responses of nifedipine and isoproterenol. Thus, the inhibitory effect of CDNB on NTG-induced relaxation and NO production appeared specific. When bovine coronary rings were made tolerant to NTG by pretreatment with 0.44 mM NTG for 1 hr, the EC50 was shifted to the right 162-fold, and NO generation was also reduced in intact rings and tissue homogenates. However, when the homogenates were further subfractionated to microsomes and cytosols, or when homogenates were allowed to stand for a similar time period necessary for subfractionation, the difference in NO production from control versus tolerant tissue preparations disappeared. It is possible, therefore, that the NTG-induced tolerance process might have been partially reversed during this time period. Results of this study identified CDNB as an apparently specific inhibitor of NTG action, but showed that GST-mediated reactions were probably not involved in the metabolic activation of NTG. Our results also indicated that tissue NO generation from NTG was positively related to the relaxation responses generated by this nitrovasodilator.