Báo cáo khoa học: Biochemical characterization of human umbilical vein endothelial cell membrane bound acetylcholinesterase ppt

Biochemical characterization of human umbilical vein
endothelial cell membrane bound acetylcholinesterase
´
˜
Filomena A. Carvalho1, Luıs M. Graca2, Joao Martins-Silva1 and Carlota Saldanha1
¸
´
1 Instituto de Biopatologia Quımica, Faculdade de Medicina de Lisboa ⁄ Unidade de Biopatologia Vascular, Instituto de Medicina Molecular,
Lisbon, Portugal
´
2 Departamento de Ginecologia ⁄ Obstetrıcia, Hospital de Santa Maria de Lisboa, Lisbon, Portugal

Keywords
acetylcholinesterase; biochemical
characterization; cellular membrane; human
endothelial cells
Correspondence
F. Almeida Carvalho, Instituto de
´
Biopatologia Quımica, Faculdade de
Medicina de Lisboa ⁄ Unidade de
Biopatologia Vascular, Instituto de Medicina
´
Molecular, Edifıcio Egas Moniz, Avenue Prof
Egas Moniz, 1649–028 Lisbon, Portugal
Tel: + 351 21 7985136
Fax: +351 21 7999477
E-mail: fi
(Received 15 July 2005, revised 25 August
2005, accepted 2 September 2005)
doi:10.1111/j.1742-4658.2005.04953.x

Acetylcholinesterase is an enzyme whose best-known function is to hydrolyze the neurotransmitter acetylcholine. Acetylcholinesterase is expressed in
several noncholinergic tissues. Accordingly, we report for the first time the
identification of acetylcholinesterase in human umbilical cord vein endothelial cells. Here we further performed an electrophoretic and biochemical
characterization of this enzyme, using protein extracts obtained by solubilization of human endothelial cell membranes with Triton X-100. These
extracts were analyzed under polyacrylamide gel electrophoresis in the presence of Triton X-100 and under nondenaturing conditions, followed by
specific staining for cholinesterase or acetylcholinesterase activity. The gels
revealed one enzymatically active acetylcholinesterase band in the extracts
that disappeared when staining was performed in the presence of eserine
(an acetylcholinesterase inhibitor). Performing western blotting with the
C-terminal anti-acetylcholinesterase IgG, we identified a single protein
band of approximately 70 kDa, the molecular mass characteristic of the
human monomeric form of acetylcholinesterase. The western blotting with
the N-terminal anti-acetylcholinesterase IgG antibody revealed a double
band around 66–70 kDa. Using the Ellman’s method to measure the cholinesterase activity in human umbilical vein endothelial cells, regarding its
substrate specificity, we confirmed the existence of an acetylcholinesterase
enzyme. Our studies revealed a predominance of acetylcholinesterase over
other cholinesterases in human endothelial cells. In conclusion, we have
demonstrated the existence of a membrane-bound acetylcholinesterase in
human endothelial cells. In future studies, we will investigate the role of
this protein in the endothelial vascular system.

Acetylcholine (ACh) is an important neurotransmitter
that plays a key role in the neuronal cholinergic system. Among the various components of the neuronal
cholinergic system, acetylcholinesterase (AChE, acetylcholine acetylhydrolase, EC 3.1.1.7) plays an essential
role in the cholinergic neurotransmission system. The

primary function of AChE is to hydrolyse and thus
terminate the action of the acetylcholine [1]. Therefore,
most studies of AChE have been focused on its function. However during the past decades it has been as

well perceived that AChE and several of the components of the neuronal cholinergic system are not only

Abbreviations
ACh, acetylcholine; AChE, acetylcholinesterase; AcLDL, acetylated low density lipoprotein; ASCh, acetylthiocholine; BODIPY FL AcLDL,
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-S-indacene-3-propionic acid conjugate; BuChE, butyrylcholinesterase; BW 284c51, 1,5-bis(4allyldimetylaminopropyl) pentan-3-one dibromide; BuSCh, butyrylthiocholine; ChAT, choline acetyltransferase; DFP, di-isopropylfluorophosphate; DTNB, 5,5¢-dithiobis(2-nitrobenzoic acid); HUVECs, human umbilical vein endothelial cells; IL-1b, interleukin-1b;
VEGF, vascular endothelial growth factor; vWf, von Willebrand factor.

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F. A. Carvalho et al.

expressed by neuronal cells, but as well by other
cellular types in various organisms. Altogether, these
studies led to the introduction of the concepts of ‘nonneuronal ACh’ and ‘non-neuronal cholinergic system’
to describe the activity of this system outside of the
neuronal tissue [2]. Acetylcholine, via stimulation of
nicotinic and muscarinic receptors and, possibly also
via direct protein interaction, may modulate several
cellular signaling pathways.
Non-neuronal ACh appears to regulate different
cellular functions such as proliferation, differentiation, cell–cell contact, immune functions, trophic
functions, and secretion [2,3]. Therefore, ACh may
be regarded as an essential cellular signaling molecule that contributes to the maintenance of cellular
homeostasis [2].
AChE can be differentiated from other cholinesterases such as the butyryl-cholinesterase (BuChE, acylcholine acylhydrolase, EC 3.1.1.8) on the basis of
substrate specificity, affinity for selective inhibitors and
excess substrate inhibition [1]. Importantly, AChE is

selectively inhibited by the well-studied inhibitors BW
284c51 [1,5-bis(4-allyldimethylamminopropyl) pentan3-one dibromide] and eserine [4].
Structural studies of AChE revealed that this
enzyme consists of a globular core penetrated by a
narrow groove (the ‘gorge’) at the bottom of which lies
the active site. This core includes as well other important sites, such as the peripheral anionic site, a secondary binding site [5].
The expression and activity of AChE is as well not
restricted to the neuronal cholinergic system. In fact,
several groups of researchers have addressed the biochemical and histochemical characterization of human
non-neuronal AChE in several types of cells, such as
epithelial cells (airways, alimentary tract, urogenital
tract, epidermis), mesothelial cells (pleura, pericardium), immune cells (human leucocytes), muscle cells,
endothelial cells and erythrocytes [2].
Importantly, different cellular types may express different AChE forms. This may occur because AChE
mRNA can be subjected to alternative splicing in a tissue specific manner and protein molecular aggregates
may be formed in different types of cells. Through
alternative splicing, the AChE precursor mRNA may
post-transcriptionally generate three major AChE
mRNA species. These different mRNAs encode three
different protein isoforms with different C-terminal
extensions that display different biochemical properties
and subcellular localization. These protein isoforms
are the following: (a) the synaptic AChE (AChE-S)
which is the main isoform in brain and muscle tissues
and which may appear in soluble and in insoluble
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Characterization of HUVECs membrane bound AChE

forms, as a monomer and as several polymeric forms;

(b) the erythrocytic form (AChE-E) that normally
occurs in a dimeric form and whose C-terminal is
linked to glycosylphosphatidyl inositol, that further
anchors the protein in membranes of erythrocytes; and
(c) the readthrough form (AChE-R) that seems to be
expressed as a soluble monomer, and whose expression
is induced during development or in response to stress
conditions [5].
In this study, we report the existence of an enzymatically active form of acetylcholinesterase in the membranes of the human umbilical vein endothelial cells
(HUVECs) and we have characterized its enzymatic
properties. We analyzed the enzymatic activity of this
membrane-bound endothelial AChE in extracts of
solubilized membranes of HUVECs by electrophoresis
under nondenaturating conditions, followed by specific
staining for AChE activity. We also evaluated the
AChE activity of HUVECs under different conditions,
namely substrate nature and pH.

Results
Fluorescent acetylated low density lipoprotein
(AcLDL) uptake
To identify the HUVECs of primary culture we
made a fluorescent acetylated low density lipoprotein
(AcLDL) uptake analysis. Cells acquire the cholesterol
for membrane synthesis primarily via receptor-mediated uptake of LDL. A modified LDL, acetylated
LDL is specifically incorporated by endothelial cells
[6,7]. Figure 1 illustrates the uptake of a fluorescently
labeled AcLDL by cultured HUVECs at passage 2,
after 4 h of exposure with 10 lgỈmL)1 of 4,4-difluoro5,7-dimethyl-4-bora-3a,4a-diaza-S-indacene-3-propionic
acid conjugate (BODIPY FL AcLDL). One can visual-

A

B

Fig. 1. (A) Incorporation of BODIPY FL AcLDL by primary HUVECs.
(B) Incorporation of BODIPY FL AcLDL in the cytoplasm of primary
HUVECs and DNA staining with TO-PRO. The cultured HUVECs
were at passage 2. Scale bar: 20 lm.

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Characterization of HUVECs membrane bound AChE

ize a predominantly punctuate cytoplasmic with perinuclear distribution typical of AcLDL incorporation
into endothelial cells [7]. In Fig. 1B we also performed a DNA staining with TO-PRO iodide to reveal
the location of the nuclei in these cells. These results
confirm that the cells isolated by our procedure are
endothelial cells from the vein of human umbilical
cord.
Flow cytometry of endothelial cells
The results of flow cytometry (Fig. 2) revealed that the
HUVECs showed constitutive expression of E-selectin
(C), 56.95% of positively stained cell and von Willebrand factor (64.99%). The N-terminal of the E-selectin was a very low expression when the HUVECS
were unstimulated. Incubation of 5 h with IL1-b
300 pgỈmL)1 led to a significant increase of E-selectin
expression (C-terminal, 94.56% and N-terminal,
99.95%), reaching its maximum with incubation with
IL1-b 500 pgỈmL)1 (C-terminal, 99.72%). The stimulation with IL1-b slightly increased the von Willebrand

factor (vWf) expression (64.99% to 68.04%).

F. A. Carvalho et al.

Isolation and solubilization of plasma
membranes from cultured HUVECs
Membranes from HUVECs were isolated and further
solubilized. At different stages of this procedure,
namely before and after cell lysis and after membrane
solubilization, we measured the acetylcholinesterase
activity and the protein concentration, so as to verify
the percentage of loss between the beginning and the
end of the process. In Table 1, we show that during
this process, we only had 10–15% of total loss of acetylcholinesterase activity and protein concentration.
Furthermore, we can also conclude that the most critical stage of this procedure was the solubilization of
the membranes of HUVECs.
Western blot analysis of acetylcholinesterase
To confirm that the HUVEC membrane expresses
AChE we carried out western blotting analysis for this
enzyme. First, we observed that the extract of solubilized membranes of HUVECs was resolved as a large
number of bands by SDS ⁄ PAGE with dithiothreitol

Fig. 2. Histograms showing the effect of stimulation with IL-1b (300 pgỈmL)1 or 500 pgỈmL)1) for 5 h on the expression of E-selectin (N),
E-selectin (C) and vWf on HUVECs in vitro (A–G). An unstained negative control histogram is shown (histogram H). An increase of E-selectin
(N and C) is noted compared to constitutive expression (unstimulated) of this molecule on endothelial cells. Induction of E-selectin (N and C)
expression over endothelial cells is observed after stimulation with IL-1b (A–E). The expression of wVf over unstimulated or stimulated
HUVECs was observed to be the same (F,G).

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Characterization of HUVECs membrane bound AChE

Table 1. Acetylcholinesterase activity and protein concentration on
different stages of the isolation and solubilization of the membranes of HUVEC process. The arrows indicate (a) the percentage
of loss between the beginning of the isolation process and the end
of the cell lyses; (b) the beginning of the isolation process and after
the membrane solubilization.
Before
cell lysis
[Protein] (lgỈlL)1)
ACHE (UI x 105 cells)

After
cell lysis

After membrane
solubilization

8.8
120

8.6
115a

7.8

102b

Percent loss from ‘Before’ to ‘After’ cell lysis is 2–4%. b Percent
loss from ‘Before cell lysis’ to ‘After membrane solubilization’ is
11–15%.

a

and 2-mercaptoethanol and subsequent Coomassie
blue staining (Fig. 3A). The protein extract of
membranes of HUVECs without dithiothreitol and
2-mercaptoethanol have the same profile of the bands
observed with protein reduction.
For western blotting analysis for the AChE protein,
we employed a polyclonal antibody raised towards the
protein domain corresponding to amino acids 481–614
mapping at the C-terminal of the synaptic form of
AChE (AChE-S). Besides the C-terminal extension
typical of AChE-S, this protein region also includes
the peptide between 481 and 543 amino acids that
is common to all forms of AChE. Therefore, it is

A

B

C

Fig. 3. (A) SDS ⁄ PAGE gel with Coomassie blue staining of protein extracts of solubilized membranes of HUVECS (30 lg of protein per lane),
Human recombinant AChE standard (4.5 lg of protein per lL of sample) and human erythrocyte AChE standard (0.06 lg of protein per lL of

sample), with or without protein dithiothreitol and 2-mercaptoethanol reduction. Protein molecular mass markers are in the lane with an
asterisk below. (B) Western blotting (WB) analysis with the AChE (C) antibody (H-134) raised toward the C-terminal (481–614 amino acids)
of human synaptic AChE and the AChE (N) antibody (N-19) raised toward the N-terminal of human synaptic AChE of solubilized membranes
of HUVECS (30 lg of protein per lane), human recombinant AChE standard (0.06 lg of protein per lL of sample) and human erythrocyte
AChE standard (4.5 lg of protein per lL of sample), with or without protein dithiotreitol and 2-mercaptoethanol reduction. (C) Western blotting (WB) analysis with antibodies against known membrane proteins such as, KDR and FLT-1 antibodies [rabbit anti-(human KDR) Ig and
rabbit anti-(human FLT-1) Ig; Santa Cruz Biotechnology) of protein extracts of solubilized membranes of HUVECS (30 lg of protein per lane),
with dithiothreitol protein reduction.

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Characterization of HUVECs membrane bound AChE

predictable that this antibody should recognize all the
AChE isoforms [5]. We also employed a polyclonal
antibody for the N-terminal of the protein (the last 19
amino acids of the terminal region). Accordingly, one
single band was detected at approximately 70 kDa in
the extracts of solubilized membranes of HUVECs
either with or without protein reduction when we
employed the AChE antibody for the C-terminal
(Fig. 3B). This is consistent with the expected molecular mass for the monomeric AChE protein, as further
confirmed with the human AChE standards used. A
double band at approximately 70 kDa was observed
with the AChE specific antibody for the N-terminal of
the protein.
The membrane extracts of HUVECs are enriched
with membrane proteins, as shown by western blotting

analysis with polyclonal antibodies of FLT-1 (C-17)
and KDR, the two receptors of the vascular endothelial growth factors (VEGFs; also termed VEGF-R1
and VEGF-R2, respectively). The results confirm that
there is enrichment of membrane proteins on the
extracts of HUVECs produced (Fig. 3C).
Polyacrylamide gel electrophoresis and staining
for cholinesterase activity
To study further the activity of AChE in the HUVEC
solubilized membrane extracts, we used polyacrylamide
gel electrophoresis (PAGE) with Triton X-100 under
nondenaturating conditions, followed by specific staining for cholinesterase or acetylcholinesterase activity.
When required, the staining was performed in the presence of eserine 10 lm.
As expected for the cholinesterase staining, the
HUVEC extracts showed multiple bands that were
not totally inhibited in presence of eserine (lane 3,

A

F. A. Carvalho et al.

Fig. 4B). Concerning AChE staining, our gels
revealed a single enzymatically active band in the
HUVEC solubilized membrane extracts (lane 3,
Fig. 4A). This band was not detected in the gel
when staining was carried out in the presence of
eserine (lane 3, Fig. 4A), suggesting it to be specific
for AChE activity. This single band was resolved at
the same level as one of the bands observed for each
profile of the human AChE standards used (lanes 1
and 2, Fig. 4A).

Enzyme kinetics and inhibition studies
Our preliminary enzymatic experiments revealed that
HUVECs contained cholinesterase activity (data not
shown). To understand further the nature of this cholinesterase activity, enzymatic assays were performed
with different concentrations of two choline substrates,
namely acetylthiocholine (ASCh) and butyrylthiocholine (BuSCh). As shown in Fig. 5A, the results
obtained show that the cholinesterase activity present
in endothelial cells has a higher affinity for the ASCh
substrate than for the BuSCh substrate. As AChE is
specific for ACh, this substrate preference indicates a
predominance of AChE in the HUVECs. This study
was performed at pH 8.1, which was the pH value at
which the highest AChE activity values were achieved
(compared to activities obtained at pH values 7.2 and
7.6) (Fig. 5B).
At low ASCh concentrations, the AChE enzyme of
HUVECs followed Michaelis–Menten kinetics. Surprisingly, at substrate concentrations over 4 mm (at
pH 8.1, Fig. 5B), a saturation of the enzymatic activity
was observed. This is in clear contrast with the expected
inhibition observed for AChE from other sources (at
these high concentrations of substrate) [8,9].

B

Fig. 4. Polyacrylamide gel electrophoresis
(7.5%) with 0.5% Triton X-100 with
Karnovsky and Roots [32] AChE staining
(A) and with cholinesterase nonspecific
staining (B) with or without eserine 10 lM.
Lane 1, human recombinant AChE standard

(0.06 lg of protein per lL of sample); lane
2: human erythrocyte AChE standard
(4.5 lg of protein per lL of sample); lane 3,
extract of solubilized membranes of
HUVECs (8.5 lg of protein per lane).

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Characterization of HUVECs membrane bound AChE

A

B

Fig. 5. (A) Cholinesterase activity of
HUVECs (whole cells) as a function of
different ASCh and BuSCh concentrations at
pH 8.1 (n ¼ 5). (B) Acetylcholinesterase
activity of HUVECs (whole cells) as a
function of ASCh concentrations (between
0.1 lM and 15 mM) at different pH buffers
(pH 7.2, 7.6 and 8.1). Inset: Acetylcholinesterase activity of HUVECs (whole cells) as a
function of ASCh concentrations, between
0.1 lM and 2 mM, at the same pH buffers
(n ¼ 5).

Discussion
Acetylcholinesterase is an essential enzyme in the process of neurotransmission in the neuronal cholinergic
system. In addition to its expression in neurons, AChE
is widely expressed in several other types of cells. So
far, AChE expression in endothelial cells has been
detected in gerbils [10], human fetal brain microvessels
[11], newt cerebral capillaries [12] and human skin
blood vessels [13]. This study is, to our knowledge, the
first report of the molecular expression of AChE in
human endothelial cells, more precisely in human
umbilical vein endothelial cells. We have also performed an enzymatic and electrophoretic characterization of the acetylcholinesterase enzyme present in
the membranes of these human cells.
Several markers can be routinely used to confirm
that a given cell culture is of endothelial origin, such
as the presence of the factor VIII-related antigen, of
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the angiotensin converting enzyme or increased metabolism of acetylated-LDL [7]. In our study, we monitored the uptake of a fluorescent AcLDL by our
cultures of HUVECs (Fig. 1) and the flow cytometry
analysis with HUVECS E-selectin stimulation (adhesion molecule) with interleukin1-b and von Willebrand
factor (Fig. 2). From the results we could confirm that
the cells extracted from the vein of human umbilical
cords were of endothelial origin.
We prepared extracts of solubilized membranes of
HUVECs and used them in several electrophorectic
experiments to further complement our studies. Membrane isolation procedure using a nonionic detergent
in endothelial cells proved to be a very useful tool in
the course of the identification of the membrane proteins in endothelial cells. The data presented above
clearly indicates that, with this procedure (see Experimental procedures), the membrane-bound AChE from

HUVECs can be extracted to a high extent (85–90%)
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Characterization of HUVECs membrane bound AChE

with Triton X-100. This data is consistent with that
reported by Plageman et al. [8]. The specific enzymatic
activity obtained for the extract of solubilized membranes of HUVECs was of 13.0 UIỈmg protein)1.
This value is greater than those obtained in human
cerebrospinal fluid ( 4 UIỈmg)1 [14]); in human ocular fluid ( 0.04 UIỈmg)1 [15,16]), but lower than the
results obtained for the AChE purified from human
erythrocytes (582 UIỈmg)1 of AChE [17]).
From the literature, we could expect this membrane
isolation and solubilization procedure to be inadequate
for measurements of AChE activity or for determination of protein concentration. In fact, Triton X-100
has strong UV absorbance at 280 nm due to the presence of the phenyl ring on its structure, thus making
spectrophotometric protein determination difficult [18].
On the other hand, Jaganathan et al. [19] showed that
the Triton X-100 could interfere with the enzymatic
activity of BuChE and with its interaction with specific
inhibitors.
However, our data shows that the use of Triton X100 in membrane solubilization does not affect significantly the AChE activity of membranes of HUVECs
and the determination of the total protein concentration (Table 1).
Furthermore, to identify that the extract was
enriched with protein membranes, we performed western blot analysis with the specific antibodies for
endothelial membrane proteins, such as the VEGF
receptors, FLT-1 and KDR. From Fig. 3C, we concluded that achieving the solubilized extract of HUVECs
membranes was efficient, because the membrane
extract had the specific signals for each membrane

protein.
The extract of solubilized membranes of HUVECs
showed several bands in Triton X-100 nondenaturating
PAGE followed by cholinesterase staining. This could
be explained by the fact that this extract of solubilized
membranes of HUVECs was not further purified for
AChE and it should contain other membrane proteins.
There could be several types of nonspecific cholinesterases, such as BuChE, pseudocholinesterase and
plasma cholinesterase [20], whose activity should also
be revealed by cholinesterase staining and thus should
produce extra bands in the gel. When the gels were
stained specifically for AChE staining, one single band
was detected. Importantly, this band disappeared if
staining was performed in the presence of eserine.
When we performed the SDS ⁄ PAGE and Coomassie
blue staining (Fig. 3A) of the protein bands, it was
clear that the extract of solubilized HUVECs was
composed of a multitude of proteins with different
electrophoretic migrations. Using SDS ⁄ PAGE electro5590

F. A. Carvalho et al.

phoresis in the presence of dithiothreitol and 2-mercaptoethanol, and western blotting with a specific
anti-AChE Ig for the C-terminal region, a single protein band was observed of approximately 70 kDa. This
molecular mass is the expected size for the human
monomeric AChE in other cell types (human erythrocytes [21], human blood lymphocytes [22], mouse
erythrocytes [23] and cotton aphid [24]). With a specific anti-AChE Ig for the N-terminal region, a double
band around 66–70 kDa corresponding of two monomeric distinct forms of AChE was observed. Recently,
Meshorer et al. [25] reported the existence of the novel
N-AChE protein(s) containing N-terminal extensions.

The classic human AChE protein includes a 31 amino
acid residue signal peptide at its N-terminal that is
cleaved off during protein maturation. Meshorer et al.
predict that the AChE translation product would
become a transmembrane domain in a N-terminally
extended (and 16% larger) AChE variant (hN-AChE).
The N-terminus of hN-AChE on the brain AChE proteins may enable monomeric AChE-S or AChE-R to
transverse the membrane, conferring as yet undefined
physiological functions to its cytoplasmatic domains
[25,26]. Different hN-AChE extents were also demonstrated in monocytes, granulocytes and lymphocytes.
Electrostatic, as well as covalent, interactions of
hN-AChE monomers having diverse C-termini (e.g.
AChE-E and AChE-S) can potentially create hNAChE-associated multimers with complex structures.
These unusual AChE forms have been reported in
Alzheimer’s disease and in dementia [27,28]. Also,
Meshorer et al. [25] reported that the cyclooxygenase
have the same molecular behavior as AChE protein.
The classic cyclooxygenase form includes a signal peptide at the N-terminus. A novel cyclooxygenase variant
includes an unusually spliced nucleotidic sequence,
which encodes for an N-terminal extension of the protein. The resulting protein has distinct properties from
the classic form [25].
By addressing the enzymatic cholinesterase activity
in HUVECs, we can conclude that these endothelial
cells display an enzymatic activity that is approximately three times more specific for acetylthiocholine
(ASCh), an analogue of the natural substrate ACh,
than for butyrylthiocholine BuSCh. These results
suggest that the cholinesterase activity observed in
HUVECs is mostly due to AChE activity. Among the
various conditions tested, the highest AChE activity
measured in HUVECs was attained in 0.1 m phosphate

buffer pH 8.1, 10 mm 5,5¢-dithiobis(2-nitrobenzoic
acid) (DTNB) and 1 mm ASCh, at 37 °C. We do not
know if pH 8.1 is the optimal pH for the enzymatic
activity of AChE of membranes of HUVECs. However
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F. A. Carvalho et al.

according to the optimal pH values for the AChE
activity in other human cells, we may admit that
pH 8.1, approximately, is also acceptable in HUVECs.
As an example, the optimal pH value for AChE activity of human erythrocytes membranes is 8.0 [21]. At
low substrate concentrations, the AChE enzyme from
HUVECs followed Michaelis–Menten kinetics. At
ASCh concentrations over 4 mm, we observed a saturation of AChE enzymatic activity. This result is in
clear opposition with the expected inhibition by an
excess of substrate, a typical enzymatic feature normally displayed by AChE [8,9]. Further studies need to
be conducted with other techniques, such as luminescence, to confirm this result.
Altogether our results demonstrate the expression
of the AChE enzyme in the membranes of endothelial
cells, more precisely in HUVECs. Currently, we do
not know what isoform of AChE is expressed on
HUVECs. Furthermore, the aggregate structure of
this enzyme in HUVEC membranes is also not
determined.
The endothelial AChE was shown to mediate the
breakdown of acetylcholine. These data raise several
questions concerning the function of this protein in the
endothelial cell, and the putative existence of a nonneuronal endothelial cholinergic system, as well as its

function within the endothelium. In a recent study,
acetylcholine was shown to mediate a small facilitator
effect on the expression of intracellular adhesion molecule-1 in HUVECs [13]. In this same study, the
authors further demonstrated the expression of the
choline acetyltransferase (ChAT) enzyme in these
endothelial cells. Additionally, the production of ACh
by HUVECs was demonstrated by the use of HPLC
techniques [29]. Also, it has been shown that there are
high amounts of acetylcholine and ChAT in the placenta. As the placenta is not innervated by cholinergic
neurons, the ChAT is originated from non-neuronal
sources. The synaptic vesicles of acetylcholine transporter (VAChT) has been localized in placental cell
types [30]. Therefore a cholinergic transmission in
umbilical cord could be also associated with the function of the AChE in HUVECs.
A comprehensive characterization of AChE and of
other cholinergic components in HUVECs will be an
important step for understanding the possible functions of an endothelial acetylcholine and of a putative endothelial cholinergic system. These functions
may be related to several cellular processes such as
induction of adhesion molecules, proliferation, angiogenesis and hemostatic control. In future studies, we
will further address these issues in the context of
HUVECs.
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Experimental procedures
Endothelial cell isolation and culture
HUVECs were isolated from human umbilical cords provided by the Departments of Obstetrics of Santa Maria
Hospital in Lisbon. Isolation of HUVECs was performed
according to the modified Jaffe’s method described previously [31].
Briefly, after several washes of the vein of umbilical cords

with SFM-Basal Growth Medium (Gibco Brl, Invitrogen
Corporates, Paisley, UK), we isolated the endothelial cells
by digestion with 1 mgỈmL)1 of type II collagenase (Gibco
Brl) in the same medium for 15 min at 37 °C. The endothelial cells were collected by centrifugation and grown
in SFM-Basal Growth Medium supplemented with
basic fibroblast growth factor (20 ngỈmL)1, Gibco Brl),
endothelial growth factor (10 ngỈmL)1, Gibco Brl) and
penicillin ⁄ streptomycin solution (10 lgỈmL)1, Gibco Brl).
Cells were cultured in culture flasks that were previously
treated with 80 lg of fibronectin (BD Biosciences, Bedford,
MA, USA) in culture medium. Cell cultures were maintained in a humidified atmosphere of 5% (v ⁄ v) CO2 in air
at 37 °C.

Fluorescent AcLDL uptake
HUVECs were seeded on 22-mm surface glass coverslips
and grown overnight. The cells were washed twice with
NaCl ⁄ Pi and were incubated with 10 lgỈmL)1 of
BODIP FL AcLDL (Molecular Probes, Eugene, OR,
USA) in culture medium for 4 h in a humidified atmosphere of 5% CO2 in air at 37 °C. After the incubation, the
cells were washed once with NaCl ⁄ Pi and fixed with 3.7%
(v ⁄ v) paraformaldehyde in NaCl ⁄ Pi for 10 min at room
temperature [6,7]. The uptake of BODIPYÒ FL AcLDL
was measured at excitation and emission wavelengths of
485 and 530 nm, respectively, using fluorescence inverted
confocal microscope LSM 510 from Zeiss (Jena, Germany).

Flow cytometry of endothelial cells and
quantitative analysis
HUVECs monolayers were grown in 25 cm2 flasks to confluence and stimulated with IL-1b (300 and 500 pgỈmL)1)
for 5 h. After being washed with NaCl ⁄ Pi, the cells were

fixed in 4% (v ⁄ v) paraformaldeyde for 10 min and permeabilized in 90% (v ⁄ v) methanol for 20 min. The experience
was carried out under different conditions such as, control
1 (only cells), control 2 (cells stimulated with IL-1b
300 pgỈmL)1) and cells stimulated with IL-1b 300 pgỈmL)1
or 500 pgỈmL)1 for experiments with different primary antibodies. The cells were incubated with NaCl ⁄ Pi 1 · with
0.1% (w ⁄ v) BSA at 4 °C, the primary antibodies [goat polyclonal IgG E-selectin (N), goat polyclonal IgG E-selectin

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Characterization of HUVECs membrane bound AChE

(C), goat polyclonal IgG vWf; Santa Cruz Biotechnology,
Inc., Santa Cruz, CA, USA] were added for 30 min at
room temperature. Then, the cells were washed with
NaCl ⁄ Pi 1 · with 0.1% (w ⁄ v) BSA at 4 °C and incubated
with secondary antibody (Alexa Fluor 488) for 30 min at
room temperature. The cells were washed and resuspended
in NaCl ⁄ Pi buffer and finally, analyzed by flow cytometry
with a BD FacsCalibur flow cytometer (BDIS, San Jose,
CA, USA) by using the same settings for all samples. Gated
cells were acquired (5000 events), and markers were set
according to negative control values to quantitative percentage of positively stained cells.

Isolation and solubilization of plasma
membranes domains from culture HUVECs
Endothelial cells in culture dishes (from passage 2 or 3)
were detached with the use of a cell scraper and further
washed twice with NaCl ⁄ Pi buffer by centrifugation for
10 min at 700 g. A total of 5 · 106 cells were subsequently

resuspended in lyses buffer (Tris ⁄ HCl 1 mm pH 7.4, EDTA
1 mm). Cell lysis was conducted for 60 min at 4 °C with
periodic resuspension of the cellular suspension. After cell
disruption, the obtained lysate was centrifuged at 47 000 g
for 30 min at 4 °C in order to isolate cell membranes.
When required, one more cycle of cell lysis ⁄ centrifugation
was performed as described above. Afterwards, the
obtained pellet of membranes was subsequently resuspended in Tris ⁄ HCl 20 mm, EGTA 0.1 mm pH 7.4 buffer, incubated for 30 min at 4 °C and ultra-centrifuged at 100 000 g
for 30 min at 4 °C. HUVECs membranes were then solubilized with 1% (v ⁄ v) Triton X-100 in Tris ⁄ HCl 0.1 m pH 8.0
buffer for 60 min at 4 °C. Finally the detergent-solubilized
extract was ultra-centrifuged at 100 000 g for 60 min at
4 °C and further concentrated with a concentrator (Eppendorf, Germany). Samples were analyzed for protein content
using the CBQCA protein quantification kit (Molecular
Probes).

Western blotting analysis of acetylcholinesterase
Samples of the extract of solubilized membranes of
HUVECs (30 lg of total protein for each lane) were treated
with Tris ⁄ HCl 80 mm pH 6.8 buffer with 16% (v ⁄ v) glycerol, 4.5% (w ⁄ v) sodium dodecylsulphate (SDS), 150 mm
dithiothreitol, 2-mercaptoethanol (100 lLỈmL)1 sample buffer) and 0.01% (w ⁄ v) bromophenol blue by heating the
mixture at 100 °C for 15 min.
Samples were loaded onto a 7.5% polyacrylamide gel
with 0.5% SDS (SDS ⁄ PAGE). We also loaded the AChE
human recombinant standard (see Results), human AChE
erythrocyte standard and the mixture of protein markers
(Precision Plus Protein Standards, 10–250 kDa) from BioRad (Richmond, CA, USA) for the estimation of the
molecular mass of proteins. The run of the gel was made

5592

F. A. Carvalho et al.

in 0.25 m Tris with 1.9 m glycin, 0.01 m EDTA and
0.017 m SDS at 80 V for the stacking gel and 100 V for
the running gel, for approximately a total of 70 min. The
gel was subsequently stained in 0.25% Coomassie blue in
50% (v ⁄ v) methanol and 10% (v ⁄ v) acetic acid for
10 min and further destained in 10% (v ⁄ v) methanol and
10% (v ⁄ v) acetic acid.
For western blotting, SDS ⁄ PAGE gels were transferred to
a nitrocellulose membrane [Protan BA 85 Cellulosenitrat(e),
Schleicher and Schuell, Dassel, Germany] using the TransBlot SD Semi-dry Transfer apparatus (Bio-Rad, Richmond,
CA, USA). Following the transfer, membranes were stained
with the 0.5% Ponceau S in 5% (w ⁄ v) trichloroacetic acid
solution for 2 min so as to control for protein transfer.
After washing out the Ponceau S staining with 1 · NaCl ⁄ Pi
buffer, blots were blocked by incubation with NaCl ⁄ Pi ⁄ 5%
(w ⁄ v) non-fat milk for 30 min at room temperature. Blots
were subsequently incubated with the AChE antibody (rabbit polyclonal IgG, AChE (H-134), Santa Cruz Biotechnology), and the AChE antibody (goat polyclonal IgG, AChE
(N-19), Santa Cruz Biotechnology) at a dilution of 1 : 500
in NaCl ⁄ Pi ⁄ 2% (w ⁄ v) non-fat milk with 0.02% (w ⁄ v)
sodium azide under gentle shaking at room temperature
overnight. In the next day, blots were then washed three
times with 2% non-fat milk in NaCl ⁄ Pi ⁄ Tween 20 (0.1%
Tween 20 in NaCl ⁄ Pi · 1) and incubated with the horseradish-peroxidase-linked secondary antibody (donkey antirabbit IgG, Santa Cruz Biotechnology) at a dilution of
1 : 3000 for 1 h at room temperature in NaCl ⁄ Pi ⁄ 2% (w ⁄ v)
milk. Finally we washed twice the blots with NaCl ⁄ Pi ⁄
Tween 20 and once only with NaCl ⁄ Pi buffer. Results were
visualized by enhanced chemiluminescence (Super-Signal
West Pico trial kit, Pierce, Rockford, IL, USA), followed by

exposure to Super RX Fugi Medical X-ray film (Fugifilm,
Tokyo, Japan) and subsequent development.

Polyacrylamide gel electrophoresis and staining
for cholinesterase activity
Polyacrylamide gel electrophoresis under nondenaturating
conditions was done on 7.5% slab gels with 0.5% Triton
X-100 in glycine ⁄ Tris buffer 50 mm at pH 8.5 with a
Mighty Small II SE 245 apparatus (Hoefer Scientific Instruments, San Francisco, CA, USA). The pre-run of the gel
was made at 75 V for 1 h at room temperature. All samples
analyzed were loaded onto the gel in a volume of 2.5 lL
per well in 50% (w ⁄ v) sucrose and 0.01% (w ⁄ v) bromophenol blue. Solubilized HUVECs membrane extracts were
loaded at a protein content of 8.5 lgỈlane)1. The human
recombinant acetylcholinesterase and the human erythrocytes acetylcholinesterase standards (both from Sigma
Chemical Co., St. Louis, MO, USA) were loaded onto the
gel at a protein content of 0.06 and 4.5 lg per lane. The
run of the gel was made at 100 V for 3 h in glycine ⁄ Tris
buffer 50 mm pH 8.1 with 0.5% (v ⁄ v) Triton X-100.

FEBS Journal 272 (2005) 5584–5594 ª 2005 FEBS


F. A. Carvalho et al.

Staining for cholinesterase activity was done by shaking
the gel at 30 °C, for 30 min in 20 mm phosphate buffer
pH 7.0 with 2% (v ⁄ v) a-naphthyl-acetate 30 mm in acetone.
Afterwards, fast Red TR salt (0.5 mgỈmL)1; Sigma) was
added to the gel and cholinesterase activity was revealed by
the appearance of red bands on the gel [10]. For the specific

staining of the acetylcholinesterase activity we used the
Karnovsky and Roots staining procedure [32]. Thus, the gel
was incubated with 67 mm phosphate buffer at pH 6.1,
acetylthiocholine 2 mm, sodium citrate 5 mm, copper(II)
sulfate 3 mm and potassium hexacyanoferrate (III) 0.5 mm,
by shaking the gel at room temperature for 2 h or until the
bands appeared on the gel. In AChE inhibition studies, we
incubated the gel with eserine at 10 lm in phosphate buffer
0.1 m at pH 8.1 for 30 min at room temperature during the
staining of the gel.

Enzyme assays
Acetylcholinesterase activity was assayed by the use of the
Ellman’s method [33]. Briefly, we assayed the AChE activity of 4 · 105 cells (whole cells) in the presence of an acetylthiocholine substrate and 10 lm DTNB in 0.1 m phosphate
buffer pH 8.1. One unit (UI) of AChE activity represents
the amount of enzyme, which hydrolyses 1 lm of acetylthiocholine (ASCh) per minute, at 37 °C. The absorbance
was monitored at 412 nm using a Genesys 10 UV spectrophotometer (ThermoSpectronic). We used a pH 8.1 phosphate buffer, as it was the one at which we had the highest
AChE activities among the pH values tested (7.2, 7.6 and
8.1).
To study the substrate affinity of the AChE present
in the HUVEC, we used ASCh and butyrylthiocholine
(BuSCh) at concentrations between 0.1 and 15 mm. The
Ellman’s method was also used to measure the AChE activity present in the different extracts obtained during isolation and solubilization of membranes of HUVECs.

Acknowledgements
The authors would like to acknowledge the Department of Obstetrics on Santa Maria’s Hospital of Lisbon for providing the human umbilical cords that were
essential for this work, with the previous consent of
the pregnant ladies. Also we would like to thank
Dr Ana Luı´ sa Caetano for assistance with the flow
ˆ

cytometry experiments and Professor Angelo Calado
for helping with immunoblotting experiences.

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