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Hindlimb resistance vessels of nitrate-tolerant rats exhibit endothelial dysfunction
Glyceryl trinitrate (GTN) is used in the treatment of cardiovascular diseases. However, chronic GTN therapy often leads to the development of tolerance to its hemodynamic and antianginal effects. Previous studies have demonstrated that in addition to GTN tolerance, endothelial dysfunction also develops in aortic segments following prolonged exposure to GTN in vivo (Molina et al. 1987; Münzel et al. 1995). Whether this phenomenon occurs in other parts of the vasculature is unknown. The aim of this study was to determine whether continuous GTN exposure induced endothelial dysfunction in parallel with GTN tolerance in resistance vessels. GTN -tolerance was induced in vivo in male Sprague-Dawley rats (250-275 g). Animals in the GTN-treated group (n=7) were exposed to a continuous source of GTN via subdermal implantation of transdermal GTN patches for 48 hours (0.4mg/hr GTN) as described previously (Ratz et al. 2002). Control animals (n=6) received drug-free patches for the same time interval. A third group of recovery animals (n=7) was exposed to GTN for 48 hours followed by a 48 hour nitrate-free period. Animals were anaesthetized with sodium pentobarbital (100 mg/kg i.p.) and the abdominal aorta proximal to the iliac bifurcation was cannulated with a double lumen cathether according to the procedure of Hale et al. (2001). The right hindlimb vascular bed was perfused at a constant flow rate of 1.5 ml/min/100g body weight with oxygenated dextran-Tyrode’s solution, and the perfusion pressure monitored. The preparation was contracted submaximally with the α1-agonist, methoxamine, and concentration-response curves were obtained for GTN (0.1nM- 20uM) to assess vascular smooth muscle function and for the endothelium-dependent vasodilator, acetylcholine (ACh, 1pM- 10 uM) to assess endothelial function. The EC50 and Emax values were calculated from the concentration response curves and analyzed using one-way analysis of variance at a significance level of p<0.05 and Student-Neuman Keuls post-hoc tests. GTN-treated animals exhibited an 8.6-fold increase in the EC50 value for the GTN-induced decrease in perfusion pressure (p<0.001), and a significant decrease in the Emax (p<0.05) compared to control animals, indicating that the animals were tolerant to the vasodilator effects of GTN. Endothelial dysfunction also occurred in nitrate-tolerant animals; the EC50 for ACh-induced vasodilation was increased 6.8-fold (p<0.01), although the Emax was not significantly altered. There were no significant differences for ACh EC50 and ACh Emax between control and recovery groups, indicating that GTN-induced impairment of endothelial cell function was reversible. However, there was a difference in GTN EC50 between recovery and control groups (p<0.05). Thus, although there was some reversal of GTN tolerance in the recovery group, a nitrate-free interval greater than 48 hours is likely required to see a complete reversal of tolerance. We conclude that endothelial dysfunction due to prolonged nitrate exposure is a generalized phenomenon, and affects endothelium-dependent responses not only in conductance vessels, but also in the resistance vasculature. Supported by The Heart and Stroke Foundation of Ontario and the Canadian Institutes of Health Research.
Molina et al. 1987. J. Cardiovasc. Pharmacol. 10:371-378. |
