We have previously shown in a rat model of hypertension, where adenovirus transfer of the prepro-ET-1 gene increased plasma ET-1 elevating systemic blood pressure, there was a significant compensatory downregulation in ET_A receptor density correlating with reduced function in peripheral vessels but no change in ET_B receptors (Télémaque-Potts et al2002). Our aim was to determine the relationship between ET_B and ET_A receptors (Davenport 2002) by comparing the density of each subtype in ET_B knockout mice, where plasma ET levels are also elevated.

Following euthanasia, sections (15µm) were cut from the brains of 6 male (+/+) control, aged 29±3 days and 7 male ET_B receptor knockout mice (-/-), aged 25±2 days (Hay et al 2001). Competition binding assays were carried out (Davenport et al 2002) using a fixed concentration of [¹²⁵I]-ET-1 (0.1 nM,) and increasing concentrations of BQ-3020 (20 pM-100 µM) for one hour. One µM ET-1 defined the non-specific binding (NSB). Binding parameters were calculated using non-linear iterative curve fitting programmes (KELL, Biosoft, Cambridge, U.K.) and compared using Student's t-test. To further confirm the absence of ET_B receptor expression, longitudinal adjacent sections of the remaining whole torso, cut from the midline were incubated with either 0.25 nM [¹²⁵I]-BQ3020 or with 0.25 nM [¹²⁵I]-PD151242, to visualise the ET_B and ET_A sub-types respectively. The autoradiographical distribution of receptor sub-types was also compared by incubating [¹²⁵I]-ET-1 (0.1 nM) with 0.1 µM BQ123 to block ET_A binding and visualise ET_B receptors. ET_A receptors were visualised by incubating adjacent sections with 0.1 µM BQ3020. In autoradiographical images of whole body sections of control mice, [¹²⁵I]-BQ3020 binding localised to ET_B receptor-rich organs in peripheral tissues including kidney and brain as expected but could not be detected in these tissues in ET_B deficient animals, confirming gene deletion.

In control brains, unlabelled BQ3020 competed for [¹²⁵I]-ET-1 biphasically (Table 1) with a more abundant high affinity site corresponding to the ET_B receptors and a low affinity, micromolar ET_A site, giving a selectivity for BQ3020 of over 200 fold. In the knockout mice however, BQ3020 did not compete for the high affinity ET_B site in agreement with the autoradiographical data. There was no change in the affinity for the ET_A receptor compared with control but importantly ET_A receptor density was significantly reduced by 45%.

Table 1

These results reveal deletion of the ET_B sub-type is associated with a reduction in both central and peripheral ET_A number and may reflect an unsuspected role for ET_B receptors in regulating development or expression of the ET_A sub-type. The density of the ET_A receptors was downregulated by nearly half in the brain. Since ET-1 does not cross the blood-brain barrier, these results suggest an unexpected modulation of ET_A receptors by ET_B in the CNS.

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Hay, D.W., et al. ( 2001). Br J Pharmacol; 132:1905-15
Télémaque-Potts S., et al. (2002). Clin Sci;103 Suppl 48:357S-62S