Temporal role of endothelium derived NO vs EDHF in vasodilator responses to acetylcholine in rat mesenteric arteries

L.S Harrington1,2, M.J Carrier2, C.J. Garland3, J.A Mitchell 1 Cardiothoracic Pharmacology, Unit of Critical Care Medicine, The National Heart and Lung Institute, Imperial College, Dovehouse Street, London, SW3 6LY. 2 CVIR, William Harvey Institute, QMUL Charterhouse Square, London, EC1M 6BQ. 3Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 6AD, UK

The relative contribution to vasodilator responses from endothelium-derived NO, compared to EDHF, has been suggested to decline as artery size decreases. However, in most experimental approaches acute responses of blood vessels to endothelial cell agonists such as acetylcholine are studied. Although it is thought that under these conditions EDHF is the predominate pathway, agonists such as acetylcholine can stimulate the release of NO for prolonged periods (Mitchell et al., 1992). Therefore, in the current study we have investigated the relative contribution from NO, compared to EDHF pathways, over the course of 20-30 minutes, in 2nd and 3rd order mesenteric arteries from the rat.

Male Wistar rats (200-250g) were killed by lethal exposure to CO2. 2nd and 3rd order mesenteric arteries were mounted in isometric wire myographs, normalized and the presence of endothelium confirmed as described previously (Harrington and Mitchell, 2004). Arteries were pre-contracted with an EC80 concentration of U46619 (3 x 10-7M) before the addition of a maximal concentration of acetylcholine (3 x 10-6M). The vasodilator effects of acetylcholine were monitored for 30 minutes. In some experiments L-NAME (10-3M), which inhibits NOS, or TRAM 34 (10-6M) plus apamin (5 x 10-7M), which inhibit EDHF, were added alone or in combination, 30 minutes before U46619. In either 2nd or 3rd order mesenteric vessels acetylcholine induced a vasodilator response which lasted for at least 20 minutes (Figure 1). When vessels were pre-treated with L-NAME the duration of the vasodilator response induced by acetylcholine was dramatically reduced (Figure 1). Pre-treatment of tissues with TRAM34 plus apamin had no detectable effect on vasodilator responses (Figure 1). However, when vessels were pre-treated with L-NAME, and TRAM34 plus apamin, dilator responses induced by acetylcholine were blocked (Figure 1).

Figure 1. Effect of L-NAME (10-3M) or TRAM34 (10-6M) plus apamin (5x 10-7M) on the duration of vasodilator response induced by acetylcholine in 2nd (A) or 3rd (B) order mesenteric arteries from the rat. *** indicates significance of p<0.001 by two way ANOVA; n=6. • Control, n L-NAME, L-NAME plus TRAM and Apamin, m TRAM plus apamin.

These data suggest that when the endothelium is activated, NO release can be sustained for prolonged periods, and at a time when EDHF effects decline. This is similar to the relationship between the release of NO and prostacyclin from the endothelium (Mitchell et al., 1992).

Mitchell JA, de Nucci G, Warner TD, Vane JR. (1992) Br J Pharmacol. 105(2):485-9
Harrington LS and Mitchell JA (2004) Br J Pharmacol 143: 611-6