Vol. 188, No. 2, 1992 October 30, 1992
BIOCHEMICAL
INTRACELLULAR
SIGNAL
AND BIOPHYBICAL
TRANSDUCTION
RESEARCH COMMUNICATIONS Pages 565-570
FOR INTERLEUKIN-1%
INDUCED ENDOTHELIN PRODUCTION IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS Takuyuki Katabami *, Makoto Shimizu, Kazutoshi Okano, Yohko Yano, Ken Nemoto, Mika Ogura, Tatsuto Tsukamoto, Satoshi Suzuki, Kiyoshi Ohira, Yukio Yamada, Noriaki Sekita, Akihiro Yoshida and Kazuhiko Someya
Third
Department
Received
of Internal
September
2,
Medicine, Kawasaki
St. Marianna 216, Japan
University
School of Medicine,
1992
SUMMARY: The authors investigated the intracellular signal transduction for interleukin (IL)-1 &induced endothelin (ET) production by endothelial cells frog cultured human umbilical vein (HUVEC). Cultured HUVEC released immunoreactive (iR)-ET into the media in a time-dependent manner and a significant increase of iR-ET production was observed by the addition of IL- 113. The stimulating effect of IL- I l3 on iR-ET production was respectively inhibited by protein kinase C (C kinase) inhibitor (H-7), Ca-calmodulin inhibitor (W-7), cyclic AMP-dependent protein kinase (A kinase) inhibitor (H-8) and tyrosine kinase inhibitor (genistain) in a dose-dependent fashion. The data suggested that intracellular signal transduction for IL-I R-induced iR-ET production were via such pathways as C kinase, A kinase, Ca-calmodulin and tyrosine kinase in combination or independently, though possible mediation by other pathways cannot be ruled out. 0 1992 AC? Press, Inc.
Endothelin
(ET) contains 2l-amino
porcine aortic endothelial and ET-% culture
which may have different
media,
pharmacological
activities,
effects such as vasoconstriction,
atherosclerosis
and so forth.
(4), vasculitis
(6). On the other hand,
(8). IL-I
of signal transduciton elucidated.
proliferation in association
(IL)-1 B is known
In EC
of vascular smooth
of coagulation
for IL- I -induced
We therefore
investigated
with coagulation
cascade
to act on EC, lymphocytes,
may also contriubute
responses and the enhancement
been completely
have been identified.
ET may play some role in hypertension,
(5) and vasospasm
interleukin
(7) and VSMC
inflammatory mechanisms
biological
ET- I, ET-2
only ET- I has been detected (2). ET- I has a wide spectrum of
muscle cell (VSMC)
leukocytes
acid and was isolated for the first time from
cells (EC) ( I). Three distinct human ET families,
to cell proliferation, activity.
ET production
The intracellular however
have not
the effect of human recombinant
* To whom correspondence should be addressed at the Third Department Medicine, St. Marianna University School of Medicine, 2-16-1 Sugao, Kawasaki 216, Japan.
of Internal Miyamae-ku,
0006-291X/92
565
Copyright 0 I992 All rights of reproduction
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by Academic Press, Inc. in any form reserved.
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
IL-113on the ET production from cultured human umbilical vein endothelial cells (HUVEC) in an attempt to elucidate the intracellular signal transduction for IL-l& induced ET production, using various inhibitors including protein kinase C inhibitor (H7), Ca-cahnodulin inhibitor (W-7), inhibitor of prostaglandin synthesis (indomethacin), tyrosine kinase inhibitor (genistain) and cyclic AMP-dependent protein kinase inhibitor (H-8).
MATERIALS AND METHODS Cell culture: Endocell-kit U. a kit cotaining EGM-UV ( modified MCDB 131 medium supplemented with 2% fetal calf serum, 50 mg/ml gentamycin, 0.25 mg/ml amphotericin B, I pg/ml hydrocortisone, IO pg/ml endothelial growth factor, 2.5 @ml heparin and 2.5 mg protein endothelial growth supplement ) (9) (10) and HUVEC was purchased from Kurabo Co. (Osaka, Japan). Cells which between the 3rd and 4th passage, were grown in EGM-UV under 5% COd 95% air at 37OC. Medium was exchanged every other day. When cells reached a confluence, they were detached from the plates using 0.025% trypsin and 0.01% EDTA. Exueriments: 5x 103cells /well were planted on 24 well culture dishes (Becton Dickinson Co., New Jersey, USA). When cells reached a confluence, the culture medium was removed and lml of fresh serum-free medium EBM (Kurabo, Osaka, Japan) containing modified MCDB I3 1was added, and the cells were incubated in the presence or absence of 5 rig/ml human recombinant IL- I D (Genzyme Co., Boston, USA), 10-9 - IO-6 M l-(5isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, Seikagaku Kogyo, Tokyo, Japan), 5x I O-9- 5x10-6 M N-[6-(aminohexyl)]-5-chloro- I -naphthalenesulfonamide hydrochloride (W-7, Seikagaku Kogyo ), IO-9-lo-6M N-[2-(methyl-amino) ethyl]-S-isoquinolinesulfonamide dihydrochloride (H-8, Seikagaku Kogyo ), 1 - 100 pg/ml genistain (Funakosi Pharmaceutical Co., Tokyo, Japan) and 3x1!-9- 3x10-6M indomethacin (Funakosi Pharmaceutical Co.) under 5% CO2/ 95% air at 37OC for 24 h. After incubation of the cells, the culture medium was centrifuged at 1,500 rpm for 10 min and the supernatant was stored at -80 OC until the measurement of immunoreactive (iR)ET concentration. Radioimmunoassav: ET-I, 2 [12sI]-assay system, specific for ET-l and ET-2, were purchased from Amersham Japan Co. (Tokyo, Japan). Statistical analvsis: Statistical significances were analyzed by a multiple comparison ttest.
RESULTS Effect of IL- 10 on iR-ET production by HUVEC. As shown in Fig. 1, iR-ET production by HUVEC increased in a time-dependent fashion. The addition of 5 n&ml IL-10 significantly enhanced iR-ET production at l2h, 24h and 4811of incubation compared to the level on incubation medium alone at the designated incubation period (p< 0.01, p< 0.05, p< 0.001, respectively). Effect of H-7 on iR-ET production by HUVEC. As shown in Fig. 2, H-7 suppressed IL1B-stimulated iR-ET production in a dose-dependent manner and significantly suppressed the production at I O-7and 10-s M (p< 0.05, p< 0.01, respectively). Effect of W-7 on iR-ET uroduction bv HUVEC. As shown in Fig. 3, W-7 significantly enhanced iR-ET production by HUVEC at concentrations of 5x I O-9 M, 5x 10-sM and
Vol.
188,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
45. = E 3 E = = 30. m .g s 6 15. 5 i
cir
0' 1
’
’
14) ‘*
,
0
(4,
~ncut&~~
time lh)
2
48
Fig. I. Time course of iR-ET with 5 rig/ml IL- I8 ( o-e ) or represents the mean f S.E.M. Difference from control at the *** p