pharmacological activities at the cellular and iso- 
lated-tissue levels, little is known about its physio- 
logical significance as a regulatory system in living 
animals. Although evidence suggests that the en- 
dothelins may play important roles in certain patho- 
logies involving abnormal vascular reactivity, it is 
unknown whether the peptides actually participate 
in homeostatic regulation (of, for example, blood 
pressure) under healthy conditions. Because en- 
dothelins and their receptors are expressed in a num- 
ber of nonvascular cell types, including endocrine 
cells and neurons, it is possible that blood vessels 
may represent only one of the many stages in which 
the peptides can play a crucial regulatory role. Al- 
though a few endothelin receptor antagonists have 
become available recently, they have not proved 
useful in studying the local interaction of endoge- 
nously produced endothelins and receptors. To ap- 
proach these questions, Dr. Yanagisawa's laboratory 
has started a project to knock out endothelin and 
endothelin receptor genes by homologous recombi- 
nation in the mouse. The laboratory has cloned the 
mouse genes for the two endothelin receptor sub- 
types, called ET^ and ETg, constructed the targeting 
vectors, and is now producing homologous- 
recombinant embryonic stem cells. The work will 
be conducted as a collaboration with Dr. Robert E. 
Hammer (HHMI, University of Texas Southwestern 
Medical Center at Dallas) and Dr. Joachim Herz 
(also at Dallas), whose team has already produced 
several mouse strains harboring other targeted 
genes. 
Molecular Identification 
of Endothelin-converting Enzyme 
Like many other peptide hormones and neuropep- 
tides, endothelins are processed from the corre- 
sponding precursor (prepro-) proteins. However, 
biologically active 21 -amino acid endothelins are 
produced via a formerly unknown type of proteo- 
lytic processing: approximately 40-residue, biologi- 
cally inactive intermediates, called big endothelins, 
are first excised from the prepro-endothelins. The 
carboxyl-terminal halves of the big endothelins are 
then cleaved off between Trp21 and Val/Ile22 to 
produce the amino-terminal active peptides. This 
unusual endoproteolytic activation is catalyzed by 
endothelin-converting enzyme (s), a membrane- 
bound metalloprotease(s) that is yet to be identi- 
fied. The activity of this enzyme requires neutral pH 
and is sensitive to EDTA and the metalloprotease in- 
hibitor phosphoramidon but is apparently distinct 
from any other proteases known, including the en- 
kephalinase or neutral endopeptidase 24.11. Be- 
cause big endothelin- 1 is virtually inactive if the 
conversion is inhibited, the enzyme could also be an 
important target for the possible pharmacological 
intervention to the system. Answers to many ques- 
tions about the biosynthetic pathway and regulation 
of the endothelins await the identification of this 
protease. 
Dr. Yanagisawa's laboratory has aimed at the mo- 
lecular characterization of the converting enzyme 
by employing various approaches. To purify the en- 
zyme at the protein level, the laboratory has estab- 
lished a rapid assay for the enzyme activity and con- 
ducted a systematic search for a suitable starting 
material. The laboratory has also established cell 
lines that produce a large amount of big endothelin- 
1 without secreting a detectable level of the mature 
peptide. These cell lines should be suitable as host 
cells for the transfection of expression cDNA librar- 
ies from endothelial cells and other tissues rich in 
the converting enzyme. 
Dr. Yanagisawa is also Associate Professor of 
Molecular Genetics at the University of Texas 
Southwestern Medical Center at Dallas. 
Articles 
Arinami, T., Ishikawa, M., Inoue, A., Yanagisawa, 
M., Masaki, T., Yoshida, M.C., and Hamaguchi, H. 
1991. Chromosomal assignments of the human 
endothelin family genes: the endothelin- 1 gene 
(EDNl) to 6p23-p24, the endothelin-2 gene 
(EDN2) to lp34, and the endothelin-3 gene 
(EDN3) to 20ql3.2-ql3.3. Am J Hum Genet 
48:990-996. 
Giaid, A., Gibson, S.J., Herrero, M.T., Gentleman, S., 
Legon, S., Yanagisawa, M., Masaki, T., Ibrahim, 
N.B., Roberts, G.W., Rossi, M.L., and Polak, J.M. 
1991. Topographical localisation of endothelin 
mRNA and peptide immunoreactivity in neurones 
of the human brain. Histochemistry 95:303- 
314. 
Imai, T., Hirata, Y., Eguchi, S., Kanno, K., Ohta, K., 
Emori, T., Sakamoto, A., Yanagisawa, M., Ma- 
saki, T., and Marumo, F. 1992. Concomitant ex- 
pression of receptor subtype and isopeptide of 
endothelin by human adrenal gland. Biochem 
Biophys Res Commun 182:1115-1121. 
Ishikawa, T., Li, L.M., Shinmi, O., Kimura, S., Yana- 
gisawa, M., Goto, K., and Masaki, T. 1991 ■ Char- 
acteristics of binding of endothelin- 1 and endo- 
thelin-3 to rat hearts. Developmental changes in 
mechanical responses and receptor subtypes. 
C/rc/Jes 69:918-926. 
Miyauchi, T., Yanagisawa, M., lida, K., Ajisaka, R., 
Suzuki, N., Fujino, M., Goto, K., Masaki, T., and 
Sugishita, Y. 1992. Age- and sex-related variation 
126 
