BINDING-SITES FOR I-125 ET-1, ET-2, ET-3 AND VASOACTIVE INTESTINAL CONTRACTOR ARE PRESENT IN ADULT-RAT BRAIN AND NEURON-ENRICHED PRIMARY CULTURES OF EMBRYONIC BRAIN-CELLS

Binding sites for iodinated endothelin (ET)-2, ET-3 and vasoactive intestial contractor (VIC) were visualised in the adult rat brain using quantitative autoradiography and have a similar anatomical distribution to that of ET-1 and sarafotoxin S6b. Highest densities of binding sites for all 5 labelled peptides were present in the granular layer of the cerebellum. Cross-competition experiments show that at a concentration of 1-mu-M, unlabelled ET-1, ET-2, ET-3, VIC and sarafotoxin S6b were able to compete for the binding sites detected by each of the iodinated peptides. Binding sites for the ET isoforms were also present after 7-14 days in vitro in neurone-enriched primary cultures derived from embryonic rat cerebellum (16-18 days gestation) in which more than 90% of cells stained with an anti-neurofilament antibody. Using micro-autoradiography to detect the binding sites, an average of 14% of cells in these cultures with a diameter of 9.2 +/- 0.6-mu-m were associated with high silver grain densities (> 400 grains/100-mu-m2). With some of these cells, silver grains were localised over cell bodies and branching processes characteristic of a neuronal phenotype. A second group of cells with high grain densities were more difficult to classify using morphological criteria and may be non-neuronal. The density of silver grains over the remaining cells was low (< 20 grains/100-mu-m2) and was similar to that measured in nuclear emulsion overlying cultures used to assess non-specific binding. These results indicate that binding sites for all ET peptides are present in both adult rat brain and embryonic cerebellar cultures. In these cultures, binding does not appear to be confined to a single class of cells but may be present on both neuronal and non-neuronal cells. The results suggest that one or more of the ET isoforms could have actions in the rat CNS in addition to the proposed vasoactive role in the peripheral circulation.