Developmental expression of endothelin receptors in cerebellar neurons differentiating in culture

Abstract We describe the identification and expression of endothelin (ET) receptor subtypes in differentiating cultured cerebellar neurons. Using [ 125 I]ET-1 and the subtype-selective ligands BQ-123 and sarafotoxin 6c as selective ligands for the ET A and ET B receptors, respectively, we found that cerebellum from 8-day-old rats displayed only the ET B receptor subtype. We next cultivated cerebellar granule cell neurons to study ET receptor differentiation between 2 and 22 days in vitro. Using the above reagents, we found that while unlabeled ET-1 displayed monophasic competition curves, BQ-123 and sarafotoxin 6c displayed partial displacement curves, indicating the presence of both ET A and ET B receptors on these neurons. The proportion of ET B receptors gradually decreased from day 2 onwards and the proportion of ET A receptors gradually increased. On days 2, 3, 4, and 5 of culture, the ET B :ET A receptor ratios were 90:10, 70:30, 60:40, and 40:60, respectively. There was no further change in receptor subtype ratio beyond day 5 and up to day 22. Northern blot analysis showed that ET B receptor message expression was 6.9-fold higher than that of ET A receptor expression on day 2, but steadily decreased with time, whereas ET A receptor message expression was minimal on day 2 and maximal by day 3 and 4. By day 7, receptor message was of equal abundance, which was in good agreement with the binding studies. This novel, developmentally regulated process predicts the existence of endogenous mediators of neuronal ET receptor expression.