Vol. 188, No. 1, 1992 October
RESEARCH COMMUNICATIONS Pages 286-291
STIMULATES ITS HUMAN ENDOTHELIAL
Saijonmaa , Institute
OWN SYNTHESIS CELLS
for Medical Research, Tukholmankatu SF-00250 Helsinki, Finland
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
endothelial 3 have the
subsequent specific The
Abbreviations high power
used in the text: ET-l liquid chromatography.
Copyright All rights
it may systems;
is not turnover
0 1992 by Academic Press, of reproduction in any form
prostacyclin in vasculature
of phospholipase of
(12). Stimulated (13,14).
and - 3. HPLC
angiotensin 3 stimulates In
in human umbilical be mediated by ETB
of ET-l we
cord vein receptors.
by human that
(16). its effect
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