BIOCHEMICAL
Vol. 188, No. 1, 1992 October
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 286-291
15, 1992
ENDOTHELIN-1
Outi
STIMULATES ITS HUMAN ENDOTHELIAL
Minerva
Received
Tuulikki
Saijonmaa , Institute
August
31,
OWN SYNTHESIS CELLS
Nyman,
and
Frej
IN
Fyhrquist
for Medical Research, Tukholmankatu SF-00250 Helsinki, Finland
2,
1992
SUM MARY We studied whether endothelin-1 (ET-l) would affect its own synthesis. Human umbilical cord vein endothelial cells in methionine-poor were treated with synthetic culture medium containing [35 S] methionine ET-l was performed with ET-l or ET-3. Immunoprecipitation of 35 S -labeled rabbit ET-l antiserum. ET-l caused an 40 f 4 % (mean f SEM) increase of immunoprecipitable 35 S -labeled ET-l as confirmed by its elution point in reversed phase high power liquid chromatography (HPLC). ET-3 caused a 23 + 2 % increase in ET-1 concentration. Amplification of cDNA by PCR showed cord vein endothelial cells. We both ET-1 and ETR receptor mRNAs in human endothelial cells. This conclude that ET-l increases its own synthesis in suggests a positive autocrine feed-back action of ET-l on its own synthesis, an effect which is probably mediated by non-specific ETR receptors. Q 1992
Endothelin-1
(ET-l)
endothelial 3 have the
cells been
only
is
(1).
Three
found
distinct
secreted
role
in
vasoconstrictive
powerful
by cloning
isoform
important
a
substances
such
mechanism
of action
mediated
opening
of
subsequent specific The
by
two
mechanism
channels
production
activation (7-11).
Abbreviations high power
used in the text: ET-l liquid chromatography.
Copyright All rights
coupled
of ET-l
may
to
and ET-3
286
is
may
play
an
to
release from
of
it may systems;
phospholipase species
= Endothelin-1
C and
described
is not turnover
its other
endothelial but
transduction
Also
in endothelium
Inc. reserved.
of
and ETET-l
is unclear,
has been
phosphoinositide
$4.00
0 1992 by Academic Press, of reproduction in any form
by ET-l
ET-2
by
However, addition
the (3-6)
signal
and
production
0006-291X/92
be
prostacyclin in vasculature
hydrolysis A2
In
stimulates
intracellular
of phospholipase of
and
(2).
Endothelin-1
tone.
ET-l
of ET-l
distinct
calcium
cells.
produced
ET-l,
genes
vascular vessels
as EDRF
phosphoinositol activation
endothelial
on blood
peptide
of endothelin;
endothelin
modulating
effect
cells. be
isoforms
human
by
vasoactive The
vasoconstrictor
clear. pathway
and tissue
(12). Stimulated (13,14).
and - 3. HPLC
=
Vol.
188,
The
No.
1, 1992
production
angiotensin 3 stimulates In
the
BIOCHEMICAL
of
II,
ET-l
and vasopressin
the release
present
is
study
in human umbilical be mediated by ETB
of ET-l we
show
cord vein receptors.
AND
BIOPHYSICAL
RESEARCH
stimulated
by
substances
(14,15).
Recently
it has
by human that
endothelin-1
endothelial
MATERIALS
endothelial
cell
such been
cells
stimulates culture.This
COMMUNICATIONS
as
shown,
thrombin, that
ET-
(16). its effect
own
synthesis
is likely
to
AND METHODS
Cell culture. Endothelial cells were prepared from human umbilical cord veins according to Jaffe et al (17). Veins were cannulated, washed with phosphate buffered saline and treated with 0.5 % collagenase (Sigma, St Louis, MO, USA) in phosphate buffered saline for 15 min in 37 oC water bath. Cells were grown in 0.2 % gelatin (Sigma) coated cell culture flasks (Costar, Cambridge, USA) in Medium 199 (Gibco Laboratories, Inc., Belmont, California, USA) supplemented with 20 % fetal calf serum (Gibco), 20 pg/ml endothelial cell growth supplement (Sigma), 12 U/ml heparin (Sigma), 100 U/ml G-penicillin , 100 pg/ml streptomycin (Gibco) and 2 mM glutamine (Gibco) at 37 OC in humidified 5 % carbon dioxide in air. Medium was changed every other day. Cells were tested for viability with Trypan Blue.
ET-1 synthesis. The synthesis of ET-l was studied by incorporation of [35S] methionine (Amersham, Buckinghamshire, UK) into ET-l. Endothelial cells were subcultured on 6-well dishes (Nunc, Roskilde, Denmark). At confluence medium was removed, the cells were rinsed with Minimum Essential Medium ( MEM, Gibco) and 2 ml of 80 % methionine free MEM supplemented with 30 pCi [35 S] methionine/ml were added and the incubation was continued for 24 h. Control, ET-l (1.25 rig/ml, Peptide Institute, Barnet, UK), or ET-3 (1.25 rig/ml, Peptide Institute), treated cultures were run in parallel. Thrombin (2 U/ml, Sigma), and nitroglycerine (100 ug/ml, Orion, Espoo, Finland) treated cultures were used as a positive and negative controls, respectively. After incubation period the peptides in the media were extracted with Bondelut Cl8 OH columns (Analytichem International, Harbor City, USA) as described earlier (18) and the eluates were lyophilized. For immunoprecipitation of [3 5S] ET-l the lyophilized samples were dissolved in 200 1.11 of 50 mM phosphate buffer pH 7 containing 1 mM Na2 EDTA, 0.2 mM cystine, 0,l g/l merthiolate, 1 g/l Triton X-100 and 1 g/l BSA and incubated at 4 oC overnight with 5 pl of ET-l antiserum (18) or with 5 pl of normal rabbit serum (nonspecific binding). The ET antiserum used showed 100 % cross-reaction with ET-2 and ET-3, and