The role of endothelium in β-adrenoceptor-mediated relaxation of phenylephrine-constricted rat aorta

A.E.J. Rea, A.M. Shaw & A. MacDonald, Department of Biological and Biomedical Sciences, School of Life Sciences, Glasgow Caledonian University, Glasgow, G4 0BA.

There is still controversy over the role of endothelium and nitric oxide (NO) in β-adrenoceptor-mediated vasorelaxation. The present study investigated the relationship between pre-constriction, relaxation to isoprenaline and cyclic nucleotide levels in endothelium intact (EI) and endothelium denuded (ED) rat aorta.

Male Wistar rats (200-250 g) were stunned and killed by cervical dislocation. T horacic aortae was isolated, trimmed of connective tissue, and cut into 3 mm rings. Endothelium was denuded by gentle abrasion. Rings were placed at 2.0 g resting tension for isometric recording in Kreb’s physiological salt solution gassed with 95/5% O2/CO2 at 37°C. Tissues were incubated with IBMX (10 µM) for 30 min before pre-constriction with phenylephrine (PE) and subsequent exposure to 0.1 µM isoprenaline (ISO) for 2 min. Rings were immediately submerged in liquid nitrogen and stored at -80°C prior to cyclic nucleotide extraction and estimation using an Enzymeimmunoassay (GE Healthcare). Values are mean±s.e.mean. Statistical analysis was carried out using a One-way ANOVA followed by Bonferroni’s multiple comparison test.

Addition of ISO to 2 µM PE-constricted EI rings, resulted in vasorelaxation (% relaxation, n=5: 75±4) and an increase in cAMP concentration from basal levels (pmol/mg protein, n=5: PE only, 2.6±0.4; ISO, 7.0±0.6, P<0.01) with no change in cGMP levels (fmol/mg protein, n=5: PE only, 870±50; ISO, 730±200, P>0.05).

In ED preparations, there was no change in basal cAMP levels, but cGMP levels were lower than in EI preparations (PE only, n=5: cAMP pmol/mg protein 3.2±0.6, P>0.05; cGMP fmol/mg protein 41±7, P<0.001). Pre-constriction tension with PE (2 µM) was greater than in EI rings (g, n=5: EI, 0.82±0.11; ED, 1.6±0.1, P<0.001). Addition of ISO resulted in a reduced relaxation (% relaxation, n=5: 26±8 P<0.01) although cAMP increased from basal levels to a similar extent as observed in EI (pmol/mg protein, n=5: 6.7±1.2, P<0.05) with no change in cGMP levels (fmol/mg protein, n=5: 41±3, P>0.05). Adjustment of the PE concentration (0.1 µM) to give a similar pre-constriction as in EI preparations (g, n=4: 0.89±0.05) resulted in a greater ISO relaxation (% relaxation, n=4: 79±17), similar to that in EI preparations although the cyclic nucleotide responses were not altered (cAMP, pmol/mg protein, n=5: PE only, 3.2±0.4; Iso, 6.1±0.5, P<0.01: cGMP, fmol/mg protein, n=5: PE only, 48±11; Iso, 49±8, P>0.05).

In EI preparations, addition of a high concentration of PE (100 µM), to give a supramaximal pre-constriction (g tension, n=5: 1.0±0.1), resulted in a much reduced ISO relaxation (% relaxation, n=5: 16±11) but did not affect the cyclic nucleotide responses (cAMP, pmol/mg protein, n=5: PE only, 3.4±0.3; Iso, 7.3±1.4, P<0.01: cGMP, fmol/mg protein, n=5: PE only, 1040±140; Iso, 1030±110, P>0.05).

In conclusion, the results suggest that β-adrenoceptor-mediated vasorelaxation in rat aorta is mediated by increases in smooth muscle cAMP. Endothelium promotes relaxation by increasing basal cGMP and lowering the level of pre-constriction but does not appear to be directly stimulated by β-adrenoceptor activation.