Functional characterization and expression of endothelin receptors in rat carotid artery: involvement of nitric oxide, a vasodilator prostanoid and the opening of K+ channels in ETB-induced relaxation

Abstract

We aimed to functionally characterize endothelin (ET) receptors in the rat carotid artery. mRNA and protein expressions of both ETA and ETB receptors, evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Western immunoblotting, were detected in carotid segments. Immunohistochemical assays showed that ETB receptors are expressed in the endothelium and smooth muscle cells, while ETA receptors are expressed only in the smooth muscle cells. In endothelium-denuded vessels, levels of ETB receptor mRNA were reduced. Vascular reactivity experiments, using standard muscle bath procedures, showed that ET-1 induces contraction in endothelium-intact and -denuded carotid rings in a concentration-dependent manner. Endothelial removal enhanced ET-1-induced contraction. BQ123 and BQ788, selective antagonists for ETA and ETB receptors, respectively, produced concentration-dependent rightward displacements of the ET-1 concentration-response curves. IRL1620, a selective agonist for ETB receptors, induced a slight vasoconstriction that was abolished by BQ788, but not affected by BQ123. IRL1620-induced contraction was augmented after endothelium removal. ET-1 concentration dependently relaxed phenylephrine-precontracted rings with intact endothelium. The relaxation was augmented in the presence of BQ123, reduced in the presence of BQ788 and completely abolished after endothelium removal. IRL1620 induced vasorelaxation that was abolished by BQ788 and endothelium removal, but not affected by BQ123. Preincubation of intact rings with N(G)-nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), indomethacin or tetraethylammonium (TEA) reduced IRL1620-induced relaxation. The combination of L-NAME, indomethacin and TEA completely abolished IRL1620-induced relaxation while sulfaphenazole did not affect this response. 4-aminopyridine (4-AP), but not apamin, glibenclamide or charybdotoxin, reduced IRL1620-induced relaxation. The major finding of this work is that it firstly demonstrated functionally the existence of both ETA and ETB vasoconstrictor receptors located on the smooth muscle of rat carotid arteries and endothelial ETB receptors that mediated vasorelaxation via NO-cGMP pathway, vasodilator cyclooxygenase product(s) and the activation of voltage-dependent K+ channels.

Figure 1

Concentration–response curves for ET-1 obtained in isolated rat carotid rings in the presence (Endo+) and absence (Endo−) of endothelium. Values are means±s.e.m.; n=9 for Endo+ and n=6 for Endo− rings.

Figure 2

Representative RT–PCR products of 20 ng total RNA extracted from carotid arteries of Wistar rats. The bar graphs show the relative absorbance values of ET_A and ET_B receptor bands in endothelium intact (Endo+) or denuded (Endo−) rings (a) and the relative absorbance values of eNOS bands (b). ET_A and ET_B values were normalized to the corresponding GAPDH values, used as internal standard. Results are reported as means±s.e.m. and are representative of three experiments.

Figure 3

Representative Western immunoblotting products of 20 μg total protein extracted from endothelium-intact carotid arteries of Wistar rats. The bar graphs show the relative absorbance values of ET_A and ET_B receptor bands. Values were normalized by the corresponding COX-1 bands, used as internal standard. Results are reported as means±s.e.m. and are representative of five experiments.

Figure 4

Representative immunohistochemical photomicrographs of ET_A (a) and ET_B (b) receptors in rat carotid artery sections. Arrows indicate expression of ET_A receptor in smooth muscle cells and ET_B in both endothelial and smooth muscle cells.

Figure 5

Concentration–response curves for ET-1 obtained in endothelim-denuded rat carotid rings in the absence or presence of different concentrations of BQ123 (a) or BQ788 (b). Values are means±s.e.m. Six preparations were used in each group.

Figure 6

Concentration–response curves for IRL1620 obtained in endothelium-denuded (Endo−) and endothelium-intact (Endo+) rat carotid rings in the absence or presence of BQ123 (3 μM) and BQ788 (3 μM). Values are means±s.e.m. of 5–7 independent preparations.

Figure 7

Relaxation responses induced by ET-1 (a) and IRL1620 (b) on rat carotid rings precontracted with phenylephrine. The concentration–response curves for both agonists were obtained in endothelium-denuded (Endo−) and endothelium-intact rings (Endo+) in the absence or presence of BQ123 (3 μM) and BQ788 (3 μm). Values are means±s.e.m. of 5–8 independent preparations.

Figure 8

Relaxation responses induced by IRL1620 on endothelium-intact rat carotid rings precontracted with phenylephrine. The concentration–response curves were obtained in the absence (control) or in the presence of L-NAME (100 μM), ODQ (1 μM), indomethacin (10 μM) (a), TEA (10 mM), sulfaphenazole (10 μM) (b), 4-AP (1 mM), glibenclamide (3 μM), apamin (1 μM) or charybdotoxin (0.01 μM) (c). The rings were pre-incubated with the drugs for 30 min. Values are means±s.e.m. of 6–8 independent preparations.