Effects of flavonoids on glyceryl trinitrate tolerance

Abstract

The development of tolerance is a major limitation to the clinical application of the vasodilator glyceryl trinitrate (GTN) for chronic management of angina pectoris. It is associated with an increased oxidative stress. In view of the relaxation and antioxidant properties of flavonoids, it is hypothesized that the naturally occurring compounds may have the potentials to prevent and/or reverse GTN tolerance. The present study examined this hypothesis in isolated rat aortae of male Sprague Dawley rats (8 weeks old).

In study I, the properties of GTN-induced relaxation and tolerance was studied. GTN-induced relaxation was entirely mediated through activation of soluble guanylyl cyclase (sGC). Prior exposure of isolated aortae to GTN (3×〖10〗^(-5)M) for an hour resulted in reduced relaxation to subsequent additions of GTN; the impaired relaxation was more prominent in aortae without endothelium. While sodium nitroprusside, deta-NONOate and acetylcholine also caused relaxation by activating sGC, their responses were not altered by prior exposure to GTN. Therefore, it appears that sGC activity and its downstream signaling was not affected during GTN tolerance. The ability of apocynin (an antioxidant), but not the combination of DETCA and tiron (which eliminate superoxide anions), to prevent GTN tolerance suggests a possible involvement of peroxide compounds in its development.

In study II, the effects of pre- and post-treatment of aorta with 10 flavonoids on GTN tolerance were examined. Among them, only apigenin, when applied before and present during the GTN incubation, prevented the development of tolerance. In aortic rings that were exposed previously to GTN, not only apigenin but also kaempferol, quercetin and genistein significantly improved the subsequent relaxations to GTN. Structure activity relationship analysis revealed that the C2=C3 functionality may be essential for reversing GTN tolerance, while the positioning of B-ring in C3 or the presence of a bulky glycoside group at C3 would greatly reduce this function.

Apigenin is the only one of the 10 flavonoids that can both prevent and reverse GTN tolerance. In study III, its effects on the enzymes that were proposed to be responsible for the bioactivation and hence tolerance of GTN, namely aldehyde dehydrogenase (ALDH) and glutathione S-transferase (GST), were examined. ALDH inhibitor attenuated GTN–induced relaxation in rings without and with GTN tolerance; its inhibitory effect was minimally prevented by pre-incubation with apigenin. Moreover, apigenin pre-incubation had no effect on ALDH activity, which was reduced during GTN tolerance. These results suggest that apigenin did not prevent GTN tolerance through activation of ALDH. Prior exposure to GST inhibitor also resulted in reduced relaxation to GTN; this inhibition was reversed in aortae treated with apigenin after GST exposure. As such, GST inhibition may account for the development of tolerance and its reversal may underlie the restoration of relaxation to GTN by the flavonoid in tolerant aorta.

Taken in conjunction, the results reported in this thesis suggest that apigenin is effective in preventing and reversing GTN tolerance. GST but not ALDH in vascular smooth muscle appears to be involved in the beneficial effect of apigenin. Word count: