Specific binding of nitroglycerin to coronary artery microsomes: Evidence of a vascular nitrate binding site

ABSTRACT. The vasodilating action of organic nitrates, such as nitroglycerin (NTG), is thought to be mediated through metabolic conversion to nitric oxide (NO) in vascular smooth muscle. Although the pertinent enzyme(s) that carries out this crucial step has not been identified, previous studies have shown that the primary enzymatic site is located within cellular membrane fractions. In these studies, we examined the binding of [¹⁴C]NTG to microsomal fractions from bovine coronary arteries. Specific binding was linearly related to protein concentration, and binding equilibrium was reversible, reached equilibrium within 1 hr, and remained stable for 4 hr at 25°. Competition experiments with unlabeled NTG demonstrated the presence of two binding sites of differing affinities (high-affinity site: B_max 24.1 ± 0.9 pmol/mg protein, K_d 554 ± 22 pM; low-affinity site: B_max 79.0 ± 2.9 pmol/mg protein, K_d 151 ± 3 μM). Both of the thiol alkylators 1-chloro-2,4-dinitrobenzene and N-ethylmaleimide were found to inhibit [¹⁴C]NTG binding, as well as enzymatic generation of NO from NTG, in a concentration-dependent manner. Competition of [¹⁴C]NTG was also observed with five other organic nitrate vasodilators, and the degree of competition was linearly related to the in vitro vaso-relaxing potencies of these agents. Parallel experiments also showed that in the absence of thiol cofactor, the enzymatic production of NO from NTG was antagonized competitively by less potent organic nitrates. Intact blood vessel experiments using rat aorta also showed that the presence of isosorbide dinitrate (ISDN), at concentrations that did not induce relaxation alone, caused a slight but significant shift in the relaxation potency of NTG (EC₂₅ 9 ± 2 versus 28 ± 7 nM, in the presence and absence of 0.3 μM ISDN, respectively; P < 0.05). These results demonstrate the presence of specific binding of organic nitrates to microsomal proteins in vascular smooth muscle, and the observed binding is apparently related to enzymatic conversion to NO and the vasodilating properties of these compounds.