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Endothelin-converting enzyme 1 (ECE-1) is a membrane-bound metalloprotease that catalyses the conversion of inactive big endothelins into active endothelins. Two different isoforms (ECE-1a and ECE-1b) have previously been identified for human ECE-1. In the present study we have cloned a novel human ECE-1 isoform, termed ECE-1c, and have thus shown for the first time the existence of three distinct ECE-1 isoforms. The three isoforms differ only in their N-terminal regions and are derived from a single gene through the use of alternative promoters. Ribonuclease protection experiments revealed that, although the relative levels of the three isoform mRNA species vary between human tissues, ECE-1c mRNA is generally the predominant isoform messenger. Immunofluorescence microscopy analysis showed distinct subcellular localizations for the three isoforms: whereas ECE-1a and ECE-1c are localized at the cell surface, ECE-1b was found to be intracellular and showed significant co-localization with a marker protein for the trans-Golgi network. We determined that the three isoforms have similar kinetic rate constants (Km, kcat and Vmax) for the processing of big endothelin 1 and that the big endothelin isoforms 1, 2 and 3 are cleaved with similar relative velocities of 1.0:0.1:0.1 by the three isoenzymes.

Modulation of the expression of the D2-dopamine receptor gene is involved in several pathological and developmental circumstances. The gene and the corresponding promoter regions of the rat D2 receptor were isolated and partly characterized to study its regulation. The rat D2-receptor gene spans at least 50 kb, and possesses eight exons; its organization was compared to those of the other dopamine-receptor genes in a phylogenetic perspective. The gene contains two transcription-start sites: the major one is located about 320 bp upstream from the 3' end of the first exon, and a minor site is 70 bp further upstream. Transient-expression assays with fusion constructs comprising fragments of the D2-promoter region and the luciferase reporter gene confirmed the existence of two independent, TATA-lacking promoters. Both promoters separately induced transcription of the luciferase gene in C6 glioma, primary fibroblasts, GH3 and MMQ pituitary cell lines, among which only the MMQ cells normally express the D2 receptor. Transcription is enhanced by the reunion of the two promoters, and modified by the addition of upstream sequences. Thus the 1-kb promoter region analysed does not contain all the elements necessary to confer tissue-specific expression of the gene, but does carry some positive and negative regulatory elements, which remain to be characterized.

Expression of the gene encoding the serotonin 5-HT1A receptor was investigated in the brain of rats from embryonic day (ED) 10 to postnatal day 18 using PCR. The 5-HT1A receptor transcripts were first detected as early as ED 12. Their concentration increased to a maximum at ED 15 and decreased progressively to very low levels just before birth (ED 20). In 18-day-old rats, the brain stem levels of 5-HT1A receptor mRNA were higher than at ED 20 but still markedly lower than at ED 15. The high rate of expression of the 5-HT1A receptor gene for a limited period during the fetal life further supports that this receptor might be involved in the trophic action of serotonin during brain maturation.

By means of immunocytochemistry, a central neuronal network containing a prolactin-like substance has been described in the rat. In order to demonstrate the synthesis of this peptide in these cells, we examined the presence of prolactin messenger RNA (PRL mRNA) in several brain samples including the pituitary gland. Amplification of the PRL mRNA was performed by the polymerase chain reaction technique, followed by southern blotting and hybridization with a specific oligonucleotide. Results showed the presence of the expected cDNA (468 bp) in the hypothalamus. Another cDNA with a lower molecular weight was also observed.