Brain ETA receptors are downregulated in ETB-deficient mice but are unaltered in endothelial cell-specific ETB knock-out

1R.E. Kuc, 1J.J. Maguire, 2N.F. Kelland, 2A.J. Bagnall, 2Y.V. Kotelevtsev 2D.J. Webb & 1A.P. Davenport. 1Clinical Pharmacology Unit, University of Cambridge, Level 6 Centre for Clinical Investigation, Box 110 Addenbrooke’s Hospital, Cambridge, CB2 2QQ, UK. 2Centre for Cardiovascular Science, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, EH8 9XD, UK

ETB receptors predominate in the brain and are expressed by neurons, astrocytes and endothelial cells, with a small proportionET A receptors present on neurones and smooth muscle cells of intracerebral vessels. We have previously shown that in the brain of homozygous globally ETB deficient mice (ETB-/-), ETA receptor density is significantly downregulated in the brain by 45% ( Davenport and Kuc, 2003). Since ET-1 does not cross the blood-brain barrier, these results cannot be explained by a compensatory downregulation in response to increased circulating levels of ET-1 and suggest an unexpected modulation of ETA receptors by ETB in the CNS. In order to determine the contribution by endothelial cell ETB receptors, we have characterised ETA and ETB receptor distribution by both radioligand binding and immunocytochemistry in the endothelial cell specific ETB receptor knockout mouse (EC-specific KO) (Kelland et al. 2005). Floxed ETB mice (FF/--) were crossed with Tie2-Cre mice to generate an endothelial cell (EC)-specific ETB knockout mouse (FF/Tie2-Cre) (Kelland et al. 2005). Immunohistochemistry using selective antisera ( Davenport and Kuc, 2005a), confirmed ETB receptors could not detected in EC in the periphery but expression was unaltered in other cell types. Following CO2 euthanasia, sections were cut from the brains of 3 FF/Tie2-Cre or control male mice, aged 8-16 weeks, 26-35g. Competition binding assays were carried out using a fixed concentration of [125I]-ET-1 (0.1 nM) and increasing concentrations of BQ-3020 (20 pM-100 μM). Binding parameters were calculated with iterative non-linear curve-fitting programs (KELL suite, Biosoft, Cambridge, UK, Davenport and Kuc, 2005b). Data for each experiment are mean ± s.e.mean, n=3 animals. In brains from both control and FF/Tie2-Cre mice, BQ3020 competed for [125I]-ET-1 binding biphasically with a more abundant high affinity site corresponding to the ETB receptors and a low affinity, ET A site was detected. There was no significant difference in the affinity of ETB receptors: 53 ±7 nM in control and 55 ±6 nM in brains of FF/Tie2-Cre mice. No change in the density of ETB receptor could be detected in the EC-specific KO (150 ±15 fmol/mg protein) versus control (146 ±14 fmol/mg protein) reflecting low numbers of EC in brain compared to other cell types. Unlike the ETB-/- mice, deficient in ETB receptor expression in all cells, ETA receptor density was not reduced in EC-specific KO (7 ±2 fmol/mg protein) versus control (7 ±1 fmol/mg protein). These results suggest EC ETB receptors can be excluded from modulating ETA receptor expression in the brain, which in globally ETB deficient (ETB-/-) mice may be the result of interaction between ETB receptors expressed by other cell types such as neurones or glia.

Davenport AP, Kuc RE. J Cardiovasc Pharmacol 44 Suppl 1:S 276-8, 2004.
Kelland NF, Bagnall AJ, Gulliver-Sloan FH, Kuc RE, Maguire JJ, Davenport AP, Gray GA, Kotelevtsev YV, Webb DJ. Vol2Issue4abst022P
Davenport A.P. & Kuc R.E. (2005a). In:Methods Mol Biol. 306:93-120, 2005.
Davenport AP, Kuc RE. (2005b)Methods Mol Biol. 306:155-72, 2005.