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

Endothelin-1 stimulates its own synthesis in human endothelial cells.

We studied whether endothelin-1 (ET-1) would affect its own synthesis. Human umbilical cord vein endothelial cells in methionine-poor culture medium c...
387KB Sizes 0 Downloads 0 Views