From the Third Department of Internal Medicine, Kumamoto University
School of Medicine, Japan.
Correspondence to Shojiro Naomi, Third Department of Internal Medicine, Kumamoto University School of Medicine, 1–1-1 Honjo, Kumamoto 860, Japan.
Methods and Results—ECE-1 messenger RNA (mRNA) and protein
expression in cultured endothelial cells were assayed
by Northern and Western blotting, respectively. Incubation with ET-1
for 6 hours caused a significant decrease in ECE-1 mRNA expression. The
action of ET-1 on ECE-1 mRNA expression was antagonized by pretreatment
with BQ788, a specific ETB receptor antagonist, but not by
pretreatment with BQ123, a specific ETA receptor
antagonist. The expression of ECE-1 protein was also
inhibited at 6 hours after incubation with ET-1. The effects of ET-1 on
ECE-1 mRNA and protein expression were shown to be mimicked by
ionomycin, a calcium ionophore, but not by
12-O-tetradecanoylphorbol
13-acetate, a protein kinase C activator.
Conclusions—The present results demonstrate that ET-1
suppressed ECE-1 protein levels by inhibiting ECE-1 mRNA expression
through the ETB receptor, suggesting the existence of a feedback action
of ET-1 on ECE-1 in pulmonary endothelial
cells.
The lung is an organ that abundantly expresses
ECE-12 3 as well as preproET-1 mRNA. The
conversion of big ET-1 was reported to be more efficient in the lung
than in the kidney or mesentery in isolated perfusion of
rats.5 Increased pulmonary
production of ET-1 has been demonstrated in pulmonary
hypertension6 and acute myocardial infarction. In
such states, an elevation of ET-1 may not be beneficial to maintain
pulmonary and systemic circulation. For the counterregulatory
mechanism, ET-1 may act directly on ECE-1 expression. In the
present study, we investigated the effects of ET-1 on ECE-1
expression in cultured rat pulmonary
endothelial cells and evaluated whether those effects
might be mediated by PKC activation or intracellular calcium
accumulation.
Culture of Rat Endothelial Cells
Northern Blot Analysis
Western Blot Analysis
Statistical Analysis
As shown in Fig 2A
The ETB receptor has been reported to be coupled to phospholipase
C and could mediate phosphoinositide hydrolysis,
thereby inducing elevation of cytosolic Ca2+ in
endothelial cells.7 We showed
that ionomycin suppressed mRNA and protein expression for ECE-1. Thus,
ionomycin mimics the action of ET-1 on ECE-1 expression, supporting the
idea that elevation of cytosolic Ca2+ may be
responsible for the intracellular signaling caused by ET-1 on the
regulation of ECE-1 expression. The activation of PKC is one of the
important components of ET-1–induced intracellular signaling. Uchida
et al8 studied the regulation of ECE-1 mRNA in
bovine glomerular endothelial cells. They
found that TPA increased the expression of ECE-1 mRNA. However, they
did not measure the ECE-1 protein level. In the present study, TPA
did not change either ECE-1 mRNA expression or the protein level. This
discrepancy may be explained by the difference in cell type used. The
AP-1 site, which mediates the transcriptional response from TPA, has
been found in the human promotor regions of the ECE-1 isozyme ECE-1a
gene but not in the promotor of the ECE-1b gene.9
TRLEC-03 cells possess only ECE-1b.10 Although
the promotor region of rat ECE-1 has not been analyzed, our
data suggest that AP-1 sites on the promotor region of ECE-1b either do
not exist as they do in humans or are not involved in operating the
transcriptional response of rat ECE-1b. Collectively, our results
indicate that the inhibitory action of ET-1 on ECE-1 may
not be mediated by the activation of PKC in TRLEC-03 cells.
Miyauchi et al11 reported that in patients with
chronic hemodialysis, strikingly elevated big ET-1 levels with only
slightly elevated ET-1 levels in plasma are observed. Recently, it has
been reported that rats with hypertension induced by hepatic
overexpression of human preproET-1 have a high big ET-1 to mature ET-1
ratio.12 These findings suggest that under
certain conditions in which big ET-1 is elevated, ECE-1 can act as a
rate-limiting enzyme in the conversion of big ET-1 to mature ET-1 in
plasma.
The present results have demonstrated that ET-1 inhibits the level
of ECE-1 protein by suppressing the expression of ECE-1 mRNA through
the ETB receptor in cultured pulmonary
endothelial cells. Future analysis of the
feedback system in certain pathophysiological
conditions with elevated ET-1 levels should prove that it plays an
important role in the regulation of ECE-1 expression by ET-1 in
vivo.
Received September 30, 1997;
revision received November 10, 1997;
accepted November 13, 1997.
2.
Shimada K, Takahashi M, Tanzawa K. Cloning and
functional expression of endothelin-converting enzyme from rat
endothelial cells. J Biol Chem. 1994;269:18275–18278.
3.
Xu D, Emoto N, Giad A, Slaughter C, Kaw S, DeWit D,
Yanagisawa M. ECE-1: a membrane-bound metalloprotease that catalyzes
the proteolytic activation of big endothelin-1. Cell. 1994;78:473–485.[Medline]
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4.
Emoto N, Yanagisawa M. Endothelin-converting enzyme-2
is a membrane-bound, phosphoramidon-sensitive
metalloproteinase with acidic pH optimum. J Biol Chem. 1995;270:15262–15268.
5.
Hisaki K, Matsumura Y, Maekawa H, Fujita K, Takaoka M,
Morimoto S. Conversion of big ET-1 in the rat lung: role of
phosphoramidon-sensitive endothelin-1-converting
enzyme. Am J Physiol. 1994;266:H422–H428.
6.
Yoshibayashi M, Nishioka K, Nakao K, Saito Y,
Matsumura M, Ueda T, Temma S, Shirakami G, Imura H, Mikawa H. Plasma
endothelin concentrations in patients with pulmonary
hypertension associated with congenital heart defects.
Circulation. 1991;84:2280–2285.
7.
Luscher TF, Oemar BS, Boulanger CM, Hahn AWA.
Molecular and cellular biology of endothelin and its receptors.
J Hypertens. 1993;11:7–11.[Medline]
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8.
Uchida K, Uchida S, Nitta K, Yumura W, Nihei H.
Regulated expression of endothelin converting enzymes in
glomerular endothelial cells (GEN).
J Am Soc Nephrol. 1997;8:580–585.[Abstract]
9.
Valdenaire O, Rohrbacher E, Mattei M-G. Organization
of the gene encoding the human endothelin-converting enzyme (ECE-1).
J Biol Chem. 1995;270:29794–29798.
10.
Shimada K, Takahashi M, Ikeda M, Tanzawa K.
Identification and characterization of two isoforms of an
endothelin-converting enzyme-1. FEBS Lett. 1995;371:140–144.[Medline]
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11.
Miyauchi T, Suzuki N, Kurihara T, Yamaguchi I,
Sugishita Y, Matsumoto H, Goto T, Masaki T. Endothelin-1 and
endothelin-3 play different roles in acute and chronic alterations of
blood pressure in patients with chronic hemodialysis. Biochem
Biophys Res Commun. 1991;178:276–281.[Medline]
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12.
Niranjan V, Telemaque S, deWit D, Gerard RD, Yanagisawa
M. Systemic hypertension induced by hepatic overexpression of human
preproendothelin-1 in rats. J Clin Invest. 1996;98:2364–2372.[Medline]
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© 1998 American Heart Association, Inc.
Brief Rapid Communications
Endothelin-1 Inhibits Endothelin-Converting Enzyme-1 Expression in Cultured Rat Pulmonary Endothelial Cells
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—The lung expresses
large amounts of endothelin-converting enzyme-1 (ECE-1), which
catalyzes a step in the biosynthesis of potent vasoactive endothelin-1
(ET-1) from the inactive intermediate big ET-1. Because there has been
no report concerning a possible relationship between ET-1 and ECE-1, we
investigated the effects of ET-1 on ECE-1 expression in cultured rat
pulmonary endothelial cells.
Key Words: endothelin • endothelium • receptors
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Endothelin-1 is a
potent vasoconstrictor peptide produced in vascular
endothelial cells and is involved in
pathophysiological conditions such as acute renal
failure,1 pulmonary hypertension, and
systemic hypertension. ETA receptors are present on the vascular
smooth muscle and mediate direct vasoconstriction, whereas ETB
receptors are present on endothelial cells and
produce vasodilation via the endothelin-induced release of nitric oxide
and prostacyclin. ET-1 is initially synthesized as preproET-1, which is
processed to an inactive intermediate big ET-1. Mature ET-1 is produced
from big ET-1. The final step is catalyzed by the endothelin-converting
enzyme. An ECE-1 has recently been cloned2 3 and
is the major form of ECE in most tissues. A more recently cloned
ECE-24 is a homologous protein that differs from
ECE-1 in sensitivity to phosphoramidon, in optimal pH,
and in tissue distribution and quantity.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Materials
ET-1 was purchased from Peptide Institute. TPA and
ionomycin were from Sigma Chemical Co. BQ123 and BQ788 were kindly
provided by Banyu Pharmaceutical Co Ltd (Tokyo). cDNA for ECE-1
representing the region between nucleotides 437
and 4391 of rat ECE-1 cDNA and AEC27–121,2 a
monoclonal antibody against rat ECE-1, were kindly donated by Dr K.
Tanzawa (Sankyo Pharmaceutical Co Ltd, Tokyo).
The SV40-transformed rat vascular endothelial
cell line in lung, TRLEC-03,2 was established at
the Institute of Cytosignal Research, Inc (Tokyo, Japan) and was kindly
donated by Dr S. Tsurufuji. TRLEC-03 cells were cultured in RPMI 1640
medium supplemented with 10% fetal calf serum, 100 U/mL penicillin,
and 100 µg/mL streptomycin at 37°C in a humidified atmosphere of
95% air/5% CO2. The cells used in the study
were taken from the 5th to 20th passages. Confluent cells
(107) in 100-mm collagen-coated dishes were
preincubated in serum-free RPMI overnight. Those cells received 8 mL of
fresh, serum-free RPMI before application of the PKC
activator TPA or the calcium ionophore ionomycin. ET-1
(10-7 mol/L) was applied at time zero with or
without pretreatment of the ETA receptor antagonist (BQ123)
or ETB receptor antagonist (BQ788) for 30 minutes. Samples
were obtained for RNA or protein analysis at 1, 3, 6, and 20
hours. At each time point, samples were also obtained from cells not
exposed to the interventional compounds as time controls.
Total RNA was isolated from endothelial
cells with the acid guanidinium thiocyanate–phenol-chloroform method.
Total RNA (20 µg) was size fractionated by 1.2% agarose gel
electrophoresis and transferred to a nylon membrane. The rat ECE cDNA
probe was labeled with [32P]dCTP (3000 Ci/mmol,
Amersham International PLC) by the random primed labeling method. The
RNA immobilized on the membrane was hybridized with the
labeled probes in the presence of 50% formamide, 5x Denhardt's
solution, 100 µg/mL salmon sperm DNA, 0.5% SDS, and 5x SSPE buffer
(1x SSPE buffer: 150 mmol/L NaCl, 10 mmol/L
NaHPO4, 1 mmol/L EDTA, pH 7.4) for 20 hours
at 42°C. Autoradiography was performed with an
intensifying screen at -80°C. The results were quantified by
densitometric scanning. To control for variability in the loaded
quantity of RNA, membranes were probed with GAPDH cDNA and used to
normalize ECE-1 mRNA.
The confluent cells were collected with scrapers. After
centrifugation, precipitated cells were
homogenized in 10 vol of homogenization
buffer (20 mmol/L Tris/HCl, pH 7.5, 5 mmol/L
MgCl2, 0.1 mmol/L PMSF, 20 µmol/L
pepstatin A, and 20 µmol/L leupeptin) by use of a Polytron
homogenizer. The homogenates were
centrifuged at 1000g for 10 minutes. The
supernatants were centrifuged at 100 000g for 30
minutes at 4°C. The membrane proteins (1 µg) obtained were
subjected to a 4% to 20% gradient SDS–polyacrylamide gel.
The separated proteins were transferred to polyvinylidene membranes
(Millipore Co). The resultant blots were incubated with a purified
monoclonal antibody against rat ECE-1, AEC27–121, at 4°C overnight
followed by the goat anti-mouse IgG antibody coupled with horseradish
peroxidase for 1 hour. The blots were activated with an
enhanced chemiluminescence kit (Amersham International PLC) and exposed
to hyperfilm.
Results are expressed as mean±SEM. Statistical analyses
were performed by ANOVA and Fisher's least significant difference post
hoc test. Values of P<.05 were considered to be
statistically significant.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
ECE-1 mRNA levels were measured at different time points up
to 20 hours after the addition of ET-1
(10-7mol/L). Fig 1A
shows that the ECE-1 message that was
normalized as a ratio of ECE-1 mRNA to GAPDH mRNA decreased after 6
hours of exposure to ET-1 (P<.05 versus control; n=5). We
tested whether the inhibitory effect of ET-1 on ECE-1 mRNA
expression was mediated by either the ETA or ETB receptor. Fig 1B shows
that ET-1 inhibited ECE-1 mRNA to 62±10% of control levels
(P<.05 versus control; n=5) at 6 hours after incubation.
ECE-1 mRNA expression by ET-1 remained suppressed to 57±10% of
control (P<.05 versus control; n=5) by preincubation with
BQ123 (10-6 mol/L). Preincubation with BQ788
(10-6 mol/L) abolished ET-1–induced inhibition
in ECE-1 mRNA expression to 85±12% of control (P=NS versus
control; n=5). TPA (5x10-7 mol/L) did not alter
ECE-1 mRNA expression up to 20 hours after incubation (data not shown).
Ionomycin (5x10-6 mol/L) decreased ECE-1 mRNA
expression at 3, 6, and 20 hours' incubation to 79±7%, 39±5%, and
44±12% of control, respectively (Fig 1C).
View larger version (24K):
[in a new window]
Figure 1. Northern blot analysis demonstrating
effects of ET-1 on ECE-1 mRNA expression in cultured
endothelial cells. A, Representative
Northern blot analysis (top) of ECE-1 and GAPDH mRNA. Lanes 1,
2, 4, 6, and 8 show control cells (C) before and after incubation for
1, 3, 6, and 20 hours. Lanes 3, 5, 7, and 9 show cells (ET-1) incubated
with ET-1 (10-7 mol/L) for 1, 3, 6, and 20 hours. Graph
(bottom) shows abundance of ECE-1 mRNA relative to GAPDH mRNA as
corrected for respective time controls (100%). *P<.05
compared with respective time controls. B, The effect of either the ETA
or ETB receptor antagonist on ET-1–induced suppression of
ECE-1 mRNA. Bar graph shows ECE-1 mRNA abundance relative to GAPDH
mRNA. Values are expressed as percent of time controls (100%).
*P<.05 compared with controls. 
P<.05
compared with BQ123. C, Effect of ionomycin on the expression of ECE-1
mRNA. Graph shows abundance of ECE-1 mRNA relative to GAPDH mRNA as
corrected for respective time controls (100%). *P<.05,
**P<.01 compared with respective time controls., ECE-1 protein
level decreased to 71±12% of control (P<.05 versus
control; n=6) after incubation with ET-1 for 6 hours. ECE-1 protein
level did not change significantly after 6 hours' incubation with TPA
(Fig 2B). As with the action on ECE-1 mRNA expression, ionomycin
inhibited the ECE-1 protein level to 73±11% of control
(P<.05 versus control; n=8) at 6 hours after the incubation
(Fig 2B).
View larger version (38K):
[in a new window]
Figure 2. Western blot analysis demonstrating
effects of ET-1, TPA, and ionomycin on ECE-1 protein level in cultured
rat endothelial cells. A,
Representative Western blot analysis (top) of
ECE-1 protein. Lane 1 shows control cells (C). In lane 2, cells were
stimulated with ET-1 (10-7 mol/L) for 6 hours. Bar graph
(bottom) shows ECE-1 protein abundance quantified by scanning
densitometry. Values are expressed as percent of time controls (100%).
*P<.05 compared with controls. B,
Representative Western blot analysis (top) of
ECE-1 protein. Lane 1 shows control cells (C). In lane 2, cells were
stimulated with TPA (5x10-7 mol/L) for 6 hours. In lane
3, cells were stimulated with ionomycin (I) (5x10-6
mol/L) for 6 hours. Bar graph (bottom) shows ECE-1 protein abundance.
*P<.05 compared with controls.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
We used SV40-transformed rat pulmonary
endothelial cells in which the mRNA and protein for
ECE-1 were abundantly expressed.2 To prove the
existence of a feedback action between ET-1 and ECE-1, we first
obtained data showing that ECE-1 mRNA expression was suppressed at 6
hours after treatment with ET-1. Similar suppression occurred in the
ECE-1 protein level at 6 hours after incubation with ET-1. These
results indicate that the reduction in the ECE-1 protein level is
reflected by changes in the transcription and/or mRNA stability of
ECE-1. Because endothelial cells possess the ETB
receptor dominantly, we hypothesized the inhibitory action
of ET-1 on ECE-1 expression could be mediated by the ETB receptor. The
present results revealed that the specific ETB receptor
antagonist BQ788 antagonized the inhibitory
action of ET-1 on ECE-1 mRNA expression. These results are the first to
demonstrate that ET-1 can affect ECE-1 expression through the ETB
receptor.
Selected Abbreviations and Acronyms
ECE-1
=
endothelin-converting enzyme-1
ET-1
=
endothelin-1
ETA
=
endothelin A
ETB
=
endothelin B
PKC
=
protein kinase C
TPA
=
12-O-tetradecanoylphorbol 13-acetate
TRLEC-03
=
SV40-transformed rat vascular endothelial cell line in
lung
Acknowledgments
Our work was supported by a grant-in-aid for scientific research
in Japan (grant No. B 07457242 and No. 09470238), a grant-in-aid for
exploratory research in Japan (No. 09877220), and a grant from the
Houansha Foundation. The authors acknowledge Drs K. Shimada, M.
Takahashi, and K. Tanzawa (Sankyo Co Ltd, Tokyo) for technical support
and advice. The authors are indebted to Y. Kitamoto and H. Nonoguchi
for their helpful discussion.
Footnotes
Guest editor for this article was Joseph Loscalzo, MD, Boston University Medical Center, Boston, Mass.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
1.
Tomita K, Ujiie K, Nakanishi T, Tomura S, Matsuoka
O, Ando K, Schichiri Y, Hirata Y, Marumo F. Plasma endothelin levels in
patients with acute renal failure. N Engl J
Med. 1989;321:1127. Letter.[Medline]
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