MEE T I N G REP 0 R Endothelin: a knockout in London Bruno Battistini, Renia Botting and Timothy D. Warner At a recent conference* on endothelin, all the major areas of endothelin physiology and pharmacology, and the roles of the endothelins in health and disease, were discussed. In ad- dition, attention was focused on the application of the technique of gene knockout on endothelins. Gene targeting Knockout of the endothelin 1 gene in mice (homozygous ET-l-null mice) causes severe craniofacial and thoracic blood vessel malformations (Y. Kurihara, University of Tokyo), suggesting an important role for endothelin 1 (ET-1) in the normal development of the pharyngeal arches, heart and large blood vessels 1. This appears to be dependent upon the action of ET-1 on endothelin ET A receptors, as the frequency and sever- ity of these malformations are in- creased by treatment with either the ET A receptor antagonist BQ123 or antibodies against the ET A receptor, and are mimicked by knockout of the etA. Homozygous ET-l-null and ET A- null mice die soon after birth but ET-l-heterozygotic mice are viable. Studies on these animals show them to have lower tissue and plasma lev- els of ET-1 than wild-type mice (T. Kuwaki, University of Tokyo), but unexpectedly elevated blood press- ures. This increase in blood pressure should not be taken to indicate that ET-1 has a hypotensive role in the adult animal as it may be explained by changes in the chemoreceptor reflex and increases in the sym- pathetic drive, as observed in the heterozygote mice (Kuwaki). Indeed, overexpression of ET-1 in the rat elevates blood pressure (S. T616maque, Howard Hughes Medical Institute, Dallas). Interestingly, mice lacking endo- thelin 3 (homozygous ET-3-null mice) unlike ET-l-null mice, are viable at birth, although they die at three to four weeks of age as a result of toxic megacolon (A. Baynash, Howard Hughes Medical Institute, Dallas). These mice have spotted coats (Fig. 1) as a result of regional lack of epidermal melanocytes, which is an identical phenotype to that found in the lethal spotted-rat model of Hirschsprung disease. Similar changes were seen in homo- zygous ETB-null mice (K. Hosoda, Kyoto University) indicating a role for endothelin 3 (ET-3) and ET B receptors in human Hirschsprung disease. Together these results show, unexpectedly, that ET-1 and ET-3, acting on ET A and ET B receptors, respectively, are essential for the development of neural crest derived structures. Endothelin converting enzymes: cloned and expressed A recent major advance in the field of endothelin research has been the purification and cloning of endothe- lin converting enzyme (ECE), which transforms big endothelin into its active form2-7. It is apparent that there is not a single endothelin con- verting enzyme but a group of related proteins, named ECE-la, ECE-lb and ECE-2, which have dif- ferent preferences for big ET-1, big ET-2 and big ET-3 as substrates (Table 1). In general terms these ECEs are type II integral membrane-bound proteases with considerable struc- tural homology to endopeptidase N.E.P. 24.11 and Kelt blood group protein, and consist of a single trans- membrane domain, conserved Zn2÷- binding motifs, N-glycosylation sites, a short N-terminal cytoplasmic tail and a large extracellular portion that contains the catalytic domain. The ECE-la enzyme is expressed and localized in the vascular endothelial cells of all organs and in airway, gas- trointestinal, and urinary epithelial cells (F. Bayan, Montreal General Hospital). Gel filtration suggests that ECE-la has a molecular weight of T 250-280 kDa (Refs 6, 8), whereas SDS-PAGE under reducing condi- tions reveals a single band at 120-130 kDa (Ref. 6) suggesting that ECE-la may exist as a heterodimer. ECE-lb is present in human renal adenocarcinoma cells, which pro- duce ET-2 (Ref. 7) (K. Yorimitsu, Chiba University School of Medi- cine), and also in human umbilical vein endothelial cells, bovine adrenal cortex and bovine aortic endothelial cells (O. Valdenaire, Hoffmann- La Roche, Basel). Development of ECE inhibitors Because the production of the mature endothelins requires the action of ECE(s), these are attractive targets at which to aim pharmacolo~- cal interventions. Phosphoramidon inhibits both the conversion of exogenously adminis- tered big ET-1 and the de novo pro- duction of ET-1. Phosphoramidon analogues have, therefore, been stu d- ied in the hope of producing selective ECE inhibitors. For example, removal of the rhamnose moiety of phospho- ramidon followed by the addition of 2-(2-naphthyl)ethyl to phosphonyl- L-Leu-L-Trp increases the potency against ECE sixfold, but decreased it twofold against N.E.P. 24.11 (A. Jertg, Ciba-Geigy, Summit). However, per- haps of more interest is the develop- ment of CGS26303 and CGS263c)3, which are dual N.E.P. 24.11 and ECE inhibitors (S. De Lombaert, Ciba- Geigy, Summit), and inhibit the in vivo hypertensive effects of ex- ogenous administration of big ET-1 and reduce the blood pressure of spontaneously hypertensive rats (De Lombaert). Endothelin receptors In mammals two endothelin recep- tors have been cloned and expressed: the ET A receptor, rank order of potency of ET-1 = ET-2 > ET-3, and the ET B receptor, rank order of potency of ET-1 = ET-2 = ET-3. Despite this, evidence from the use of selective and nonselective endothelin receptor antagonists has been re- ported to suggest the existence of subtypes of the ET Breceptor9. There B. Bonistini, PostdoctoralFellow, R. Boning. Information Scientist, and T. D. Wemor, Lecturer. William Harvey Research Institute, St Bartholomew's Hospital Medical College, Charterhouse Square, London.UK ECIM 6BQ *4th International Conference on Endothelin, 23-26 April 1995, London, UK. ©1995, Elsevier Science Ltd TiPS-July 1995 (Vol. 16) 2 1 7