The apolipoprotein E deficient (apoE^(-/-)) mouse has been proposed as a model for human atherosclerosis owing to spontaneous formation of atherosclerotic plaques. The constrictor peptide endothelin-1 (ET-1) is up-regulated in the aorta of (apoE^(-/-)) mice with advanced atherosclerosis (Kobayashi et al., 2000). In a previous communication to the Society, we demonstrated an increased constrictor response to ET-1 in the aortae of young apoE^(-/-) mice devoid of lesions (E_MAX: apoE^(-/-); 33 ±12%, C57/BL6J; 5.5 ±1.5% KCl response; Wiley et al., 2002). Therefore the aim of this study was to determine if the increase in response to ET-1 could be attributed to an up-regulation of ET receptor expression or affinity for the ET-1 peptide.

Aortae were obtained from 24 week apoE^(-/-) C57/BL6J (n = 4) and C57/BL6J (control; n = 4) mice (either sex, 25-35g; Charles River, UK), killed with CO₂. Saturation assays were conducted on 30μm cryostat sections of aorta, incubated for 2h in Hepes buffer (Hepes; 50mM, MgCl₂; 5mM, bovine serum albumin; 0.3%, pH 7.4). [¹²⁵I]-ET-1 was incubated in the concentration range 4pM-2nM and non-specific binding was determined using un-labelled ET-1 (1 μM). Sections were washed for 15 min in Tris-HCl (Tris; 50mM, pH 7.4) at 4 °C, air-dried and apposed, with ¹²⁵I standards, to radiation-sensitive film. Autoradiographical images were analysed using computer-assisted densitometry (Quantimet 970, Leica, Bucks, UK) and the KELL suite of programmes (FigP, Biosoft, Cambs, UK) and expressed as mean ± s.e.mean. Affinity constants (K_D) were compared using the Mann-Whitney U-test. Maximum binding densities (B_MAX) were expressed as fmol/mg protein, measured using a colorimetric lowry assay (BioRad, Herts, UK), were compared using Student’s two-tailed t-test (P<0.05).

As expected, apoE^(-/-) mice had elevated cholesterol (apoE^(-/-); 3440 ±130 mg l ^-1 and control; 360 ±40 mg l ^-1, n = 4 and 4, P<0.05, Student’s t-test) and triglyceride (apoE^(-/-); 880 ±50 mg l ^-1, control; 460 ±90 mg l ^-1 n=4 and 4, P<0.05, Student’s t-test) levels compared to controls. In mouse aorta [ ¹²⁵I]-ET-1 bound with high affinity (K_D: apoE^(-/-); 0.011 ±0.003nM, control; 0.018 ±0.002nM, n = 4 and 4), although the maximum binding density was low in both groups (B_MAX: apoE^(-/-); 10.1 ±3.9fmol mg^-1 protein, control; 9.3 ±2.4fmol mg^-1 protein, n = 4 and 4). A one-site fit was preferred over a two-site fit and Hill slopes were close to unity. There were no significant differences in either affinity or maximum binding density between the two groups (P>0.05).

This is the first report to measure ET receptor density and affinity in mouse aorta. The low receptor density in comparison to other species may explain the relatively small constrictor responses observed in our previous study (Wiley et al., 2002). The absence of either any increase in receptor density or change in affinity in aortae from apoE^(-/-) mice supports the hypothesis that the heightened constrictor response is mediated by second messenger systems.

Kobayashi, T., Miyauchi, T., Iwasa, S. et al., (2000) Pathol Int 50, 929-936
Wiley, K.E. et al., (2002). Br J Pharmacol 136, 28P