Evidence for Intracellular Endothelin-Converting Enzyme-2
Expression in Cultured Human Vascular Endothelial Cells
Fraser D. Russell, Anthony P. Davenport
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Abstract—We have previously reported the intracellular localization of the endothelin-converting enzyme-1 (ECE-1) in
human umbilical vein endothelial cells. In the present study, we provide the first immunocytochemical and biochemical
evidence for the presence of ECE-2 in human cells. ECE activity was determined by conversion of exogenously added
big endothelin-1 (big ET-1) to ET-1 in subcellular fractions obtained by sucrose density gradient centrifugation of
human umbilical vein endothelial cell homogenates. ECE-1 and ECE-2 can be differentiated by pH dependence for
optimal activity and by sensitivity to phosphoramidon, which shows selectivity for ECE-2 over ECE-1 and PD159790,
a novel ECE-1 selective inhibitor. Optimal ECE activity was measured at pH 6.0, a value intermediate between that
reported for ECE-1 (pH 6.8) and ECE-2 (pH 5.5), indicating expression of both enzymes. At pH 6.9, conversion of big
ET-1 was inhibited markedly by 30 mmol/L PD159790 and by 100 mmol/L phosphoramidon but not by 0.1 mmol/L
phosphoramidon. In contrast, ECE activity was unaffected by 30 mmol/L PD159790 but was inhibited markedly by 0.1
and 100 mmol/L phosphoramidon at pH 5.4 (IC50 1.5 nmol/L), consistent with ECE-2 activity. Confocal microscopy
revealed a punctate pattern of ECE-2–like immunoreactive staining in the cell cytosol, suggesting localization to
secretory vesicles with a possible role in processing big ET-1 while in transit to the cell surface via the constitutive
secretory pathway. (Circ Res. 1999;84:891-896.)
Key Words: endothelin n endothelin-converting enzyme n endothelium
E
ndothelin (ET) is a potent vasoconstrictor that is cleaved
from a low-activity precursor, big ET by endothelinconverting enzymes (ECEs). Although ET has been shown to
contribute to maintenance of vascular tone in humans,1 a
pathogenic role has been implicated by its overproduction in
patients with diseases including atherosclerosis, primary pulmonary hypertension, coronary vasospasm, and congestive
heart failure.2–5 Inhibitors of the pathway leading to production of ET may therefore be of therapeutic benefit, and it is
suggested that examination of ECEs in human cells will be
useful in drug development.
Sequence identity of two ECEs (ECE-1 and ECE-2) has
been determined using molecular techniques.6 –10 The enzymes have 59% overall homology and both are membranebound, phosphoramidon-sensitive metalloproteases. Two isoforms of ECE-1 (ECE-1a and ECE-1b) are encoded by a
single gene and differ only in their cytoplasmic N-terminal
domains. Studies on a soluble construct of ECE-111 and
molecular modeling experiments12 reveal that the putative
extracellular domain contains the catalytic site for ECE
activity.
Gene knockout studies have underlined the importance of
ET in normal fetal development. Targeted null mutation in
the mouse ECE-1 gene produced embryos that exhibited
marked craniofacial and cardiac defects as well as an absence
of epidermal melanocytes and enteric neurons of the distal
gut.13 High levels of mature ET peptide were measured in
homozygous ECE-1 knockout embryos, suggesting expression of a non–ECE-1 protease, such as ECE-2.10 Whereas
both ECE-1 and ECE-2 are sensitive to phosphoramidon but
insensitive to thiorphan treatment,10 the two enzymes can be
differentiated by their pH optimum for converting enzyme
activity (ECE-2 has pH optimum of 5.5 compared with 6.8
for ECE-1) and by their sensitivity to phosphoramidon
(ECE-2 is 250-fold more sensitive to the metalloprotease
inhibitor than ECE-1). The enzymes may also be differentiated by sensitivity to newly developed ECE inhibitors that
have selectivity for ECE-1 over ECE-2.14
In previous studies, we have reported the localization of
ECE-1a and ECE-1b in human umbilical vein endothelial
cells (HUVECs).15 However, to date, there has been little
evidence for expression of other ECEs in nonbovine mammalian tissues. In the present study, we investigated ECEs
expressed in human endothelial cells using biochemical and
immunocytochemical techniques and reveal evidence for the
intracellular localization of ECE-2.
Materials and Methods
Site-directed antisera were raised against a synthetic peptide corresponding to a deduced amino acid sequence of bovine ECE-2
Received September 21, 1998; accepted February 22, 1999.
From the Clinical Pharmacology Unit, University of Cambridge, Centre for Clinical Investigation, Addenbrooke’s Hospital, Cambridge, UK.
Correspondence to Anthony P. Davenport, Clinical Pharmacology Unit, University of Cambridge, Level 6, Centre for Clinical Investigation, Box 110,
Addenbrooke’s Hospital, Cambridge CB2 2QQ, UK. E-mail [email protected]
© 1999 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
891
892
ECE-2 in Human Endothelial Cells
(bECE-2697–716). This sequence was distinct from ECE-1 isoforms,
and no other closely related sequences were detected in SWISSPROT and TrEMBL databases. Antisera were affinity-purified using
the immobilized immunizing peptide. Antisera were tested as previously described.16 Briefly, the immune sera cross-reacted in ELISA
with 20 nmol/L of the immobilized immunizing peptide with a titer
of 13105. Preabsorbing the antisera with 1 mmol/L of bECE-2697–716,
before immunocytochemistry abolished staining within HUVECs. A
monoclonal mouse antibody raised against human von Willebrand
factor (clone F8/86) was obtained from Dako. Affinity-purified
fluoresceinated anti-rabbit IgG (H1L) and Texas Red anti-mouse
IgG (H1L) were from Vector Laboratories. Citifluor glycerol
solution was from Agar Scientific Ltd. Synthetic culture medium 199
was obtained from Life Technologies. Nonenzymatic celldissociation solution, phosphoramidon, and thiorphan were from
Sigma-Aldrich Co Ltd. PD159790 was obtained from Parke-Davis
Pharmaceutical Research Division. All other reagents were of
analytical or electron microscopy grade.
Cell Culture
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Human umbilical cords were collected from the Rosie Maternity
Hospital, Cambridgeshire, UK. Endothelial cells were obtained from
the umbilical vein by collagenase digestion as previously described17
and grown and maintained at 37°C in a CO2 incubator (95% air/5%
CO2) in medium 199 containing Glutamax (2 mmol/L), penicillin (50
IU/mL), streptomycin (50 mg/mL), Fungizone (2.5 mg/mL), endothelial cell growth supplement (100 mg/mL), and 30% FCS. Each
experiment used HUVECs that were obtained from 10 umbilical
cords and grown to confluence (4 days at 37°C) in flasks that had a
combined surface area of 250 cm2. Cells were removed using
nonenzymatic cell-dissociation solution and pelleted in a Denley
centrifuge (400g, 5 minutes, 23°C). Cells were resuspended in a cold
solution of 90% FCS, 10% DMSO, and stored at 270°C for no
longer than 1 week.
Subcellular Fractionation by Sucrose Density
Gradient Centrifugation
Stored cells were thawed, diluted with medium 199, pelleted in a
Denley centrifuge (400g, 5 minutes, 23°C), and then resuspended in
homogenate buffer (0.25 mol/L sucrose, 10 mmol/L HEPES,
1 mmol/L N-ethylmaleimide, 10 mmol/L digitonin; pH 6.9). The
cells were homogenized by 4 strokes of a glass plunger homogenizer
and then spun in an Ole Dich microcentrifuge (23500g, 5 minutes,
4°C) to remove cell debris.
The homogenate was layered onto a sucrose gradient (r51.04 to
1.30 g/mL) and spun in an SW 41 Ti Beckman preparative swinging
bucket rotor (120 000g, 3 hours, 4°C). Fractions (0.75 mL) were
removed sequentially from the top of the tube, and density was
measured directly by weighing.
ECE Activity and the Effect of pH in Subcellular
Endothelial Fractions
Fractions were preincubated in labeling solution (10 mmol/L
HEPES, 1 mmol/L N-ethylmaleimide, 0.1% Triton X-100; pH 6.9) in
the presence of enzyme inhibitors for 1 hour at 37°C before addition
of big ET-1 (final concentration 20 nmol/L). After an additional 2
hours of incubation at 37°C, 0.1-mL aliquots were obtained, and ET
was measured by radioimmunoassay. The effect of pH (range 3.0 to
8.3) on ECE activity was determined in fractions 5 through 10
(density 1.07 to 1.19 g/mL; n54). The effect of the enzyme
inhibitors 0.1 and 100 mmol/L phosphoramidon, 30 mmol/L
PD159790, and 10 mmol/L thiorphan was investigated under conditions designed to favor ECE-1 activity (pH 6.9) or under conditions
designed to favor ECE-2 activity (pH 5.4). The IC50 value for
inhibition of ECE activity by phosphoramidon was determined by
combining fractions 5 and 6 and preincubating aliquots with the
inhibitor (0.01 to 30 nmol/L; n53). Maximal inhibition was determined using 100 mmol/L phosphoramidon, and results were analyzed using Fig P (Biosoft). The pH conditions (pH 5.0 to 7.0) had
no effect on ET-1 stability. Fractions were incubated with ET peptide
(6.7 nmol/L) for 2 hours at 37°C, and the levels of ET detected in the
culture medium were 5.9, 5.5, and 6.3 nmol/L (mean n52) at pH 5,
6, and 7, respectively.
Measurement of ET, von Willebrand Factor, and
5*-Nucleotidase Activity
ET was measured in fractions by radioimmunoassay. A 200-mL
sample of ET standard (range 0.5 to 500 fmol per tube) was
incubated overnight at 4°C with 100 mL of antibody raised against
ET at 1:10 000-fold dilution. [125I]-ET-1 ('13 000 cpm/100 mL) was
added to the supernatants, and these were incubated overnight at
4°C. Amerlex-M reagent was added (1 hour, 20°C), and the tubes
were placed in a magnetic rack. Supernatants were discarded, and
bound radioactivity in the precipitates was counted. von Willebrand
factor was measured by using an enzyme-linked immunoassay
(Imubind) (n53) with absorbance readings at 450 nm. 59Nucleotidase activity was measured spectrophotometrically at 340
nm by using a 59-nucleotidase kit (Sigma Diagnostics) (n53).
Localization of ECE-2–Like Immunoreactivity by
Using Confocal Microscopy
HUVECs were grown on glass coverslips in synthetic culture
medium 199 (n53). The cells were rinsed in 0.1 mol/L PBS, fixed,
permeabilized by incubation with methanol/acetone (1:1; 10 minutes, 220°C), rinsed in PBS, and blocked in 10% FCS for 30
minutes at 23°C. The cells were labeled with antisera raised against
ECE-2 (10 mg/mL), von Willebrand factor (1:25), or both (18 hours,
4°C). Cells were washed in PBS (635 minutes, 4°C). Immunoreactivity was detected by using fluoresceinated anti-rabbit IgG (20
mg/mL), Texas Red anti-mouse IgG (20 mg/mL), or both together.
Coverslips were washed in PBS (635 minutes, 4°C), dipped in
distilled water, and mounted onto glass microscope slides using
glycerol. Cells were viewed using a Leica TCS 4D confocal laser
scanning microscope (Leica) equipped with an argon krypton laser
source, Leica 633 oil immersion objective, and dual-channel photodetectors. Optical sections were collected at excitation wavelengths of 488 and 568 nm to image the distribution of FITC- and
Texas Red–labeled antisera, respectively. Colocalization of doublelabeled antisera was determined by electronic overlay of signals
obtained from channels 1 and 2.
Results
ECE activity was determined by conversion of exogenously
added big ET-1 to ET-1 in subcellular fractions obtained by
sucrose density gradient centrifugation of HUVEC homogenates. In the absence of big ET-1, the concentration of ET was
,150 pmol/L, reflecting low-level basal release of peptide
over the course of the experiment (Figure 1). When big ET-1
was added to the incubation solution, the concentration of ET
was highest in dense sucrose fractions (1.10 to 1.14 g/mL).
Conversion of big ET-1 was almost abolished by 100 mmol/L
phosphoramidon, a dual inhibitor of ECE and neutral endopeptidase 24.11 (NEP), but was unaffected by 10 mmol/L
thiorphan, an NEP selective inhibitor.
ECE activity was primarily associated with fractions 5
through 10 (density 1.07 to 1.20 g/mL) and was inhibited by
30 mmol/L PD159790, the ECE-1 selective inhibitor, at pH
6.9 (Figure 2). Little or no activity was observed in fractions
1 through 4 and fraction 11. A sharp peak of 59-nucleotidase
activity, a plasma membrane marker, was detected in fractions 6 through 8. Only low-level activity was detected in
fractions 1 through 5 and 9 through 11. von Willebrand
factor, a storage granule glycoprotein, was mainly associated
with fractions 1 through 4 and fractions 8 through 10. Only
Russell and Davenport
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Figure 1. Representative plot showing ECE activity in subcellular fractions of human umbilical vein homogenates isolated by
sucrose density gradient centrifugation. Enzymatic activity, measured by conversion of 20 nmol/L big ET to ET in each fraction
(M), was almost abolished by 100 mmol/L phosphoramidon (Œ)
but was unaffected by 10 mmol/L thiorphan (F). Basal release of
ET-1, measured in fractions to which big ET-1 was not added,
was ,150 pmol/L (E). i.r. indicates immunoreactivity.
low concentrations of von Willebrand factor were detected in
fractions 5 through 7 and fraction 11.
Because predominant ECE activity was detected in fractions 5 through 10, these were used in subsequent experiments. The pH for optimal ECE activity was found to be 6.0
Figure 2. Representative plots showing comparison of ECE
activity (A), 59-nucleotidase activity (59-ND) (B), and von Willebrand factor (vWF) (C) in subcellular endothelial fractions
obtained by sucrose density gradient centrifugation. ECE activity was primarily associated with fractions 5 through 10 and was
inhibited by 30 mmol/L PD159790, the ECE-1 selective inhibitor,
at pH 6.9. A sharp peak of 59-nucleotidase activity, a plasma
membrane marker, was detected in fractions 6 through 8. von
Willebrand factor was mainly associated with fractions 1
through 4 and fractions 8 through 10.
April 30, 1999
893
Figure 3. Effect of pH on ECE activity in endothelial cell fractions obtained by sucrose density gradient centrifugation. Optimum ECE activity was obtained at pH 6.0 in all fractions examined. Values are mean6SEM, n54.
in each of these samples (Figure 3), a value intermediate
between that reported for ECE-1 and ECE-2. Experiments
were carried out at pH 6.9, which favors ECE-1 activity, and
at pH 5.4, which favors ECE-2 activity, to characterize
enzymes that contribute to conversion of exogenously added
big ET-1 in HUVECs. ECE activity was markedly inhibited
using 30 mmol/L PD159790 at pH 6.9 (mean for fractions 5
through 10 55.2%; range 45.9% to 61.5%; n53), indicating
expression of ECE-1 (Figure 4). However, ECE activity was
largely unaffected by 30 mmol/L PD159790 at pH 5.4 (mean
Figure 4. Effect of pH on sensitivity of ECE activity to
30 mmol/L PD159790 and 0.1 mmol/L phosphoramidon. ECE
activity was inhibited by 30 mmol/L PD159790 at pH 6.9 but
was insensitive to 30 mmol/L PD159790 at pH 5.4 (A). ECE
activity was only weakly affected by 0.1 mmol/L phosphoramidon treatment at pH 6.9 but was inhibited markedly at pH 5.4
(B). No further inhibition in ECE activity was observed when
fractions were incubated with 100 mmol/L phosphoramidon at
pH 5.4 (not shown). Values are mean6SEM with significance
defined by P,0.05 using an unpaired t test. The number of
experiments for each treatment is shown in a box at the base of
each bar.
894
ECE-2 in Human Endothelial Cells
Figure 5. Inhibition of ECE-2 activity by phosphoramidon at pH
5.4. ECE activity was determined by measuring conversion of 20
nmol/L big ET-1 to ET-1. Values are expressed as mean6SEM,
n53.
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
inhibition for fractions 5 through 10 4.3%; range 0% to
14.7%; n54). The detectable activity measured in the presence of the ECE-1 inhibitor at pH 5.4 is consistent with
expression of a converting enzyme that is distinct from
ECE-1.
To confirm expression of ECE-2 in HUVECs, the sensitivity of ECE activity to 0.1 mmol/L phosphoramidon was
examined at pH 5.4 and 6.9. At pH 6.9, ECE activity was only
weakly affected by 0.1 mmol/L phosphoramidon treatment
(mean inhibition for fractions 5 through 10 15.9%; range 0%
to 28%; n53) (Figure 4). However, when experiments were
repeated at pH 5.4, sensitivity was increased (mean inhibition
for fractions 5 through 10 70.5%; range 60.4% to 81.4%;
n53). No further inhibition in ECE activity was observed
when fractions were incubated with 100 mmol/L phosphoramidon at pH 5.4 (mean inhibition 66.6%; range 60.0% to
79.6%; n53), indicating a maximal or near maximal effect of
the inhibitor when used at a concentration of 0.1 mmol/L. The
IC50 value for inhibition of ECE activity by phosphoramidon
at pH 5.4 was 1.560.4 nmol/L (Figure 5).
To determine the localization of ECE-2 in HUVECs,
permeabilized cells were labeled with antisera raised against
bovine ECE-2 and human von Willebrand factor. Confocal
microscopy showed a differential distribution for the two
antibodies. A punctate pattern of immunofluorescence staining was observed for ECE-2 within the cell cytosol, whereas
von Willebrand factor was detected in discrete, rod-shaped
structures resembling endothelial cell storage granules called
Weibel-Palade bodies (Figure 6).
Discussion
Although the subcellular localization of ECE-1a and ECE-1b
has previously been reported in HUVECs,15 there is less
evidence for expression of other ECEs in nonbovine mammalian tissues. Important biochemical information has been
obtained from studies using cells transfected with cDNA
encoding ECE-2. However, it is not known whether ECE-2 is
expressed in native endothelial cells or, indeed, whether the
enzyme behaves similarly in native endothelial cells. In the
present study, we have identified expression of ECE-2 in
human endothelial cells and report on its biochemical characteristics and subcellular localization.
Figure 6. Confocal microscopy of permeabilized HUVECs
double-labeled with antisera raised against bovine ECE-2 (A)
and human von Willebrand factor (B). A punctate pattern of
ECE-2 staining was detected (arrows), whereas von Willebrand
factor was observed in rod-shaped structures (arrowheads).
Electronic overlay of the 2 images showed that ECE-2 (green)
does not colocalize with the storage granule glycoprotein von
Willebrand factor (red) in these cells (C). Scale bar540 mm.
ECEs are characterized by their sensitivity to phosphoramidon, a dual ECE and NEP inhibitor, and can be distinguished
from NEP by the NEP selective inhibitor thiorphan. ECE
activity was measured in fractions of HUVEC homogenates
obtained by sucrose density gradient centrifugation by measuring conversion of exogenously applied big ET-1 to the
mature peptide using radioimmunoassay. In all subcellular
fractions examined, conversion of big ET-1 was catalyzed by
a phosphoramidon-sensitive, thiorphan-insensitive protease
consistent with that of an ECE.
The localization of ECE activity was compared with that of
59-nucleotidase activity, a plasma membrane marker and von
Willebrand factor, an endothelial cell storage granule glycoprotein. The two distinct peaks of von Willebrand factor in
the subcellular fractions are consistent with the presence of
the glycoprotein in the cytosol (buoyant fractions with density ,1.06 g/mL) and storage granules (dense fractions
'1.12 g/mL).18 It is possible that some of the von Willebrand
factor detected in the cytosolic fractions was derived from
vesicles or storage granules that degraded during the fractionation procedure. Negligible levels of 59-nucleotidase activity
were detected in fraction 10, which contained both ECE
activity and von Willebrand factor, thus providing evidence
for localization of ECE to a nonplasmalemmal compartment,
possibly endothelial cell–specific storage granules called
Weibel-Palade bodies.15 The colocalization of ECE and
59-nucleotidase in fractions 6 through 8 suggests conversion
of big ET-1 by ECE expressed on the plasma membrane but
may also include activity derived from ECE within intracellular compartments located in the plasma membrane– enriched fraction.
The effect of pH on ECE activity has been reported in
membranes obtained from Chinese hamster ovary (CHO)/
ECE-110,19 or CHO/ECE-2 cells,10 with optimal activity
determined at pH 6.8 and pH 5.5 for ECE-1 and ECE-2,
respectively. In the present study, optimal ECE activity was
observed at pH 6.0, a value intermediate between that
reported for ECE-1 and ECE-2. This finding may reflect
subtle species differences between bovine and human ECEs,
or, alternatively, it may indicate expression of both ECE-1
and ECE-2 in HUVECs.
ECE-2 mRNA has been detected in cultured HUVECs,10
and so in this study, we investigated the possibility that
Russell and Davenport
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ECE-2 contributes to the converting enzyme activity in these
cells. Although both ECE-1 and ECE-2 are phosphoramidonsensitive, thiorphan-insensitive proteases, the two enzymes
can be differentiated by the aforementioned pH dependence
for optimal activity10,19 and by differential sensitivity to
phosphoramidon, which has IC50 values for inhibition of
ECE-1 and ECE-2 of 1 mmol/L and 4 nmol/L, respectively.10
With the use of the mass action equation, 0.1 mmol/L
phosphoramidon would be expected to inhibit only 10% of
ECE-1 but .95% of ECE-2. A novel series of quinazoline
inhibitors of ECE can also be used to differentiate the two
enzymes.14 PD069185 inhibits ECE-1 with an IC50 value of
1.1 mmol/L with no effect on ECE-2 at 100 mmol/L.
PD159790, which was used in the present study, has slightly
lower potency at ECE-1 than PD069185 but has no cellular
toxicity at concentrations up to 100 mmol/L.14 The higher
sensitivity of big ET-1 conversion to ET-1 by PD159790 in
fractions 5 through 10 at pH 6.9 compared with pH 5.4 is
indicative of expression of ECE-2 in human endothelial cells.
This hypothesis is supported by the higher sensitivity of big
ET-1 conversion by phosphoramidon, which was observed at
pH 5.4 compared with pH 6.9. The IC50 value determined for
phosphoramidon at pH 5.4 (IC50 1.5 nmol/L) is similar to that
previously reported for ECE-2–transfected CHO cells (IC50 4
nmol/L).10
Several findings have recently indicated the possible existence
of other converting enzymes in mammalian tissues. Neuronal
cells, which lack detectable levels of ECE-1 mRNA,19 produce
ET-3,20,21 suggesting the existence of a big ET-3–specific
converting enzyme. Interestingly, a novel ECE (ECE-3) was
purified by SDS-PAGE from bovine iris microsomes that has
specificity for big ET-3.22 Although we cannot exclude the
possibility that ECE-3 is expressed in human endothelial cells,
this is probably unlikely. Whereas ECE-3 has specificity for big
ET-3, ET-1 is the predominant mature peptide isoform in human
endothelial cells with no detectable production of ET-3 reported.23,24 In addition, optimal ECE-3 activity was observed at pH
6.6,22 which is higher than the pH determined for optimal ECE
activity in HUVECs, suggesting the presence of an ECE that
does not have the characteristics of either ECE-1 or ECE-3.
Findings from experiments carried out using CHO/ECE-2
cells transfected with a prepro–ET-1 construct suggest that
ECE-2 may convert endogenous big ET-1 to the biologically
active peptide in acidic intracellular vesicles of the secretory
pathway and not on the cell surface.10 The punctate pattern of
ECE-2–like immunoreactive staining in the HUVEC cytosol
would be consistent with expression of ECE-2 in secretory
vesicles and suggests the involvement of ECE-2 in the basal
release of ET via the constitutive secretory pathway. The
immunofluorescence staining did not colocalize with von
Willebrand factor in storage granules previously shown to
express ECE-1a and ECE-1b.15 This suggests a distinct role
for ECE-2 in conversion of big ET-1 to ET-1 in human
endothelial cells within the constitutive secretory pathway.
It remains to be determined whether ECE-2, which has acidic
pH optimum, has a pathogenic role in diseases in which cellular
pH is reduced, eg, ischemic heart disease. Lactate accumulation
and a concomitant intracellular acidosis (pH 5.8) has been
detected in hearts subjected to global ischemia,25 and a correla-
April 30, 1999
895
tion between myocardial ischemia and increased plasma levels
of ET is now well established.3,26 In conclusion, the findings of
the present study indicate expression of ECE-2 in human
endothelial cells. It is suggested that an inhibitor that is nonselective for ECE-1 and ECE-2 may be beneficial in reducing ET
production in diseases in which a pathogenic role of the peptide
has been implicated. The findings also suggest that an effective
converting enzyme inhibitor will be one that can access intracellular vesicles and is therefore permeable to the plasma
membrane.
Acknowledgments
This work was supported by grants from the British Heart Foundation, Isaac Newton Trust, and the Royal Society. We thank the staff
at the Rosie Maternity Hospital (Cambridge, UK) for permission to
collect umbilical cords and Dr Jeremy Skepper for assistance with
confocal microscopy.
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Evidence for Intracellular Endothelin-Converting Enzyme-2 Expression in Cultured
Human Vascular Endothelial Cells
Fraser D. Russell and Anthony P. Davenport
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Circ Res. 1999;84:891-896
doi: 10.1161/01.RES.84.8.891
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