© 1999 American Heart Association, Inc.
Original Contribution |
Evidence for Intracellular Endothelin-Converting Enzyme-2 Expression in Cultured Human Vascular Endothelial Cells
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 apd10{at}medschl.cam.ac.uk
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Abstract |
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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 µmol/L PD159790 and by 100 µmol/L phosphoramidon but not by 0.1 µmol/L phosphoramidon. In contrast, ECE activity was unaffected by 30 µmol/L PD159790 but was inhibited markedly by 0.1 and 100 µmol/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.
Key Words: endothelin • endothelin-converting enzyme • endothelium
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endothelin (ET) is a potent vasoconstrictor that is cleaved from a low-activity precursor, big ET by endothelin-converting 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 3 4 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 7 8 9 10 The enzymes have 59%
overall homology and both are membrane-bound,
phosphoramidon-sensitive metalloproteases. Two isoforms
of ECE-1 (ECE-1
and ECE-1ß) 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-1 and
ECE-1ß 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.
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Materials and Methods |
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Site-directed antisera were raised against a synthetic peptide corresponding to a deduced amino acid sequence of bovine ECE-2 (bECE-2697–716). This sequence was distinct from ECE-1 isoforms, and no other closely related sequences were detected in SWISS-PROT 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 1x105. Preabsorbing the antisera with 1 µmol/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 (H+L) and Texas Red anti-mouse IgG (H+L) were from Vector Laboratories. Citifluor glycerol solution was from Agar Scientific Ltd. Synthetic culture medium 199 was obtained from Life Technologies. Nonenzymatic cell-dissociation 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
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 µg/mL), Fungizone
(2.5 µg/mL), endothelial cell growth supplement (100
µg/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 -70°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 µmol/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 (2x500g, 5 minutes, 4°C) to
remove cell debris.
The homogenate was layered onto a sucrose gradient
(
=1.04 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; n=4). The effect of the enzyme
inhibitors 0.1 and 100 µmol/L
phosphoramidon, 30 µmol/L PD159790, and 10
µmol/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; n=3). Maximal inhibition was
determined using 100 µmol/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 n=2) 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-µL
sample of ET standard (range 0.5 to 500 fmol per tube) was incubated
overnight at 4°C with 100 µL of antibody raised against ET at
1:10 000-fold dilution. [125I]-ET-1
(
13 000 cpm/100 µL) 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) (n=3) with absorbance readings at
450 nm. 5'-Nucleotidase activity was measured spectrophotometrically at
340 nm by using a 5'-nucleotidase kit (Sigma Diagnostics)
(n=3).
Localization of ECE-2–Like Immunoreactivity by Using Confocal
Microscopy
HUVECs were grown on glass coverslips in synthetic culture
medium 199 (n=3). The cells were rinsed in 0.1 mol/L PBS, fixed,
permeabilized by incubation with methanol/acetone (1:1;
10 minutes, -20°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 µg/mL), von Willebrand factor (1:25), or both (18
hours, 4°C). Cells were washed in PBS (6x5 minutes, 4°C).
Immunoreactivity was detected by using fluoresceinated
anti-rabbit IgG (20 µg/mL), Texas Red anti-mouse IgG (20 µg/mL), or
both together. Coverslips were washed in PBS (6x5 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 63x 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
double-labeled antisera was determined by electronic overlay of signals
obtained from channels 1 and 2.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 µmol/L phosphoramidon, a
dual inhibitor of ECE and neutral
endopeptidase 24.11 (NEP), but was unaffected by
10 µmol/L thiorphan, an NEP selective inhibitor.
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), was almost
abolished by 100 µmol/L phosphoramidon (
) but
was unaffected by 10 µmol/L thiorphan (
). Basal release of
ET-1, measured in fractions to which big ET-1 was not added, was <150
pmol/L (
). i.r. indicates immunoreactivity.
ECE activity was primarily associated with fractions 5 through 10
(density 1.07 to 1.20 g/mL) and was inhibited by 30 µmol/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 5'-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 low concentrations of von
Willebrand factor were detected in fractions 5 through 7 and
fraction 11.
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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 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 µmol/L PD159790 at pH 6.9 (mean for fractions
5 through 10 55.2%; range 45.9% to 61.5%; n=3), indicating
expression of ECE-1 (Figure 4). However,
ECE activity was largely unaffected by 30 µmol/L PD159790 at pH
5.4 (mean inhibition for fractions 5 through 10 4.3%; range 0% to
14.7%; n=4). 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.
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To confirm expression of ECE-2 in HUVECs, the sensitivity of ECE
activity to 0.1 µmol/L phosphoramidon was
examined at pH 5.4 and 6.9. At pH 6.9, ECE activity was only weakly
affected by 0.1 µmol/L phosphoramidon treatment
(mean inhibition for fractions 5 through 10 15.9%; range 0% to 28%;
n=3) (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%; n=3). No further inhibition in ECE
activity was observed when fractions were incubated with 100
µmol/L phosphoramidon at pH 5.4 (mean inhibition
66.6%; range 60.0% to 79.6%; n=3), indicating a maximal or near
maximal effect of the inhibitor when used at a
concentration of 0.1 µmol/L. The IC50
value for inhibition of ECE activity by phosphoramidon
at pH 5.4 was 1.5±0.4 nmol/L (Figure 5).
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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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the subcellular localization of ECE-1
and ECE-1ß 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. 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
5'-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
5'-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 5'-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 ECE-2 contributes to the converting enzyme activity in these cells. Although both ECE-1 and ECE-2 are phosphoramidon-sensitive, 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 µmol/L and 4 nmol/L, respectively.10 With the use of the mass action equation, 0.1 µmol/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 µmol/L with no effect on ECE-2 at 100 µmol/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 µmol/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-1 and ECE-1ß.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 correlation 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.
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Acknowledgments |
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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.
Received September 21, 1998; accepted February 22, 1999.
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  D. J. Metcalf, T. D. Nightingale, H. L. Zenner, W. W. Lui-Roberts, and D. F. Cutler Formation and function of Weibel-Palade bodies J. Cell Sci., January 1, 2008; 121(1): 19 - 27. [Abstract] [Full Text] [PDF]
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