Here we further performed an electrophoretic and biochemical characterization of this enzyme, using protein extracts obtained by solubili-zation of human endothelial cell membranes with
Trang 1endothelial cell membrane bound acetylcholinesterase
Filomena A Carvalho1, Luı´s M Grac¸a2, Joa˜o Martins-Silva1and Carlota Saldanha1
1 Instituto de Biopatologia Quı´mica, Faculdade de Medicina de Lisboa ⁄ Unidade de Biopatologia Vascular, Instituto de Medicina Molecular, Lisbon, Portugal
2 Departamento de Ginecologia ⁄ Obstetrı´cia, Hospital de Santa Maria de Lisboa, Lisbon, Portugal
Acetylcholine (ACh) is an important neurotransmitter
that plays a key role in the neuronal cholinergic
sys-tem Among the various components of the neuronal
cholinergic system, acetylcholinesterase (AChE,
acetyl-choline 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 func-tion However during the past decades it has been as well perceived that AChE and several of the compo-nents of the neuronal cholinergic system are not only
Keywords
acetylcholinesterase; biochemical
characterization; cellular membrane; human
endothelial cells
Correspondence
F Almeida Carvalho, Instituto de
Biopatologia Quı´mica, Faculdade de
Medicina de Lisboa ⁄ Unidade de
Biopatologia Vascular, Instituto de Medicina
Molecular, Edifı´cio Egas Moniz, Avenue Prof
Egas Moniz, 1649–028 Lisbon, Portugal
Tel: + 351 21 7985136
Fax: +351 21 7999477
E-mail: filomenacarvalho@fm.ul.pt
(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 hydro-lyze 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 endo-thelial cells Here we further performed an electrophoretic and biochemical characterization of this enzyme, using protein extracts obtained by solubili-zation of human endothelial cell membranes with Triton X-100 These extracts were analyzed under polyacrylamide gel electrophoresis in the pres-ence 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 choli-nesterase 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
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(4-allyldimetylaminopropyl) pentan-3-one dibromide; BuSCh, butyrylthiocholine; ChAT, choline acetyltransferase; DFP,
di-isopropyl-fluorophosphate; 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.
Trang 2expressed by neuronal cells, but as well by other
cellular types in various organisms Altogether, these
studies led to the introduction of the concepts of
‘non-neuronal ACh’ and ‘non-‘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,
differenti-ation, cell–cell contact, immune functions, trophic
functions, and secretion [2,3] Therefore, ACh may
be regarded as an essential cellular signaling
mole-cule that contributes to the maintenance of cellular
homeostasis [2]
AChE can be differentiated from other
cholinest-erases such as the butyryl-cholinesterase (BuChE,
acyl-choline 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)
pentan-3-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
import-ant sites, such as the peripheral anionic site, a
secon-dary 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
bio-chemical and histobio-chemical characterization of human
non-neuronal AChE in several types of cells, such as
epithelial cells (airways, alimentary tract, urogenital
tract, epidermis), mesothelial cells (pleura,
pericar-dium), immune cells (human leucocytes), muscle cells,
endothelial cells and erythrocytes [2]
Importantly, different cellular types may express
dif-ferent AChE forms This may occur because AChE
mRNA can be subjected to alternative splicing in a
tis-sue 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
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 enzymat-ically active form of acetylcholinesterase in the mem-branes 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-medi-ated uptake of LDL A modified LDL, acetylreceptor-medi-ated 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 lgÆmL)1 of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-S-indacene-3-propionic acid conjugate (BODIPY FL AcLDL) One can
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.
Trang 3ize a predominantly punctuate cytoplasmic with
peri-nuclear distribution typical of AcLDL incorporation
into endothelial cells [7] In Fig 1B we also
perfor-med 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
Wille-brand factor (64.99%) The N-terminal of the
E-selec-tin was a very low expression when the HUVECS
were unstimulated Incubation of 5 h with IL1-b
300 pgÆmL)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 pgÆmL)1(C-terminal, 99.72%) The
stimula-tion with IL1-b slightly increased the von Willebrand
factor (vWf) expression (64.99% to 68.04%)
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 ace-tylcholinesterase activity and protein concentration Furthermore, we can also conclude that the most crit-ical 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 solubi-lized 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 pgÆmL)1or 500 pgÆmL)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).
Trang 4and 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
Table 1 Acetylcholinesterase activity and protein concentration on
different stages of the isolation and solubilization of the
mem-branes 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
After cell lysis
After membrane solubilization
ACHE (UI x 105cells) 120 115a 102b
a
Percent loss from ‘Before’ to ‘After’ cell lysis is 2–4%.bPercent
loss from ‘Before cell lysis’ to ‘After membrane solubilization’ is
11–15%.
A
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 blot-ting (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.
Trang 5predictable 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
molecu-lar 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
endo-thelial 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
stain-ing for cholinesterase or acetylcholinesterase activity
When required, the staining was performed in the
pres-ence 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,
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 choli-nesterase activity, enzymatic assays were performed with different concentrations of two choline substrates, namely acetylthiocholine (ASCh) and butyrylthio-choline (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 Sur-prisingly, 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]
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 l M 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).
Trang 6Acetylcholinesterase is an essential enzyme in the
pro-cess 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
per-formed an enzymatic and electrophoretic
character-ization 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
the angiotensin converting enzyme or increased meta-bolism of acetylated-LDL [7] In our study, we moni-tored the uptake of a fluorescent AcLDL by our cultures of HUVECs (Fig 1) and the flow cytometry analysis with HUVECS E-selectin stimulation (adhe-sion 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 Mem-brane 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 pro-teins in endothelial cells The data presented above clearly indicates that, with this procedure (see Experi-mental procedures), the membrane-bound AChE from HUVECs can be extracted to a high extent (85–90%)
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 l M and 15 m M ) at different pH buffers
(pH 7.2, 7.6 and 8.1) Inset:
Acetylcholinest-erase activity of HUVECs (whole cells) as a
function of ASCh concentrations, between
0.1 l M and 2 m M , at the same pH buffers
(n ¼ 5).
Trang 7with 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
mem-branes of HUVECs was of 13.0 UIÆmg protein)1
This value is greater than those obtained in human
cerebrospinal fluid ( 4 UIÆmg)1 [14]); in human
ocu-lar fluid ( 0.04 UIÆmg)1 [15,16]), but lower than the
results obtained for the AChE purified from human
erythrocytes (582 UIÆmg)1of AChE [17])
From the literature, we could expect this membrane
isolation and solubilization procedure to be inadequate
for measurements of AChE activity or for
determin-ation of protein concentrdetermin-ation In fact, Triton X-100
has strong UV absorbance at 280 nm due to the
pres-ence 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
X-100 in membrane solubilization does not affect
signifi-cantly the AChE activity of membranes of HUVECs
and the determination of the total protein
concentra-tion (Table 1)
Furthermore, to identify that the extract was
enriched with protein membranes, we performed
west-ern blot analysis with the specific antibodies for
endothelial membrane proteins, such as the VEGF
receptors, FLT-1 and KDR From Fig 3C, we
conclu-ded 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
cholin-esterases, 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
electro-phoresis in the presence of dithiothreitol and 2-merca-ptoethanol, and western blotting with a specific anti-AChE Ig for the C-terminal region, a single pro-tein band was observed of approximately 70 kDa This molecular mass is the expected size for the human monomeric AChE in other cell types (human erythro-cytes [21], human blood lymphoerythro-cytes [22], mouse erythrocytes [23] and cotton aphid [24]) With a
speci-fic anti-AChE Ig for the N-terminal region, a double band around 66–70 kDa corresponding of two mono-meric 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 pro-teins 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 demon-strated 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 hN-AChE-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 pep-tide at the N-terminus A novel cyclooxygenase variant includes an unusually spliced nucleotidic sequence, which encodes for an N-terminal extension of the pro-tein 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 approxi-mately 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 37C We do not know if pH 8.1 is the optimal pH for the enzymatic activity of AChE of membranes of HUVECs However
Trang 8according 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
activ-ity 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
sat-uration of AChE enzymatic activity This result is in
clear opposition with the expected inhibition by an
excess of substrate, a typical enzymatic feature
nor-mally displayed by AChE [8,9] Further studies need to
be conducted with other techniques, such as
lumines-cence, 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
non-neuronal 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
mole-cule-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
pla-centa As the placenta is not innervated by cholinergic
neurons, the ChAT is originated from non-neuronal
sources The synaptic vesicles of acetylcholine
trans-porter (VAChT) has been localized in placental cell
types [30] Therefore a cholinergic transmission in
umbilical cord could be also associated with the
func-tion 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
func-tions of an endothelial acetylcholine and of a
puta-tive endothelial cholinergic system These functions
may be related to several cellular processes such as
induction of adhesion molecules, proliferation,
angio-genesis and hemostatic control In future studies, we
will further address these issues in the context of
HUVECs
Experimental procedures
Endothelial cell isolation and culture HUVECs were isolated from human umbilical cords provi-ded by the Departments of Obstetrics of Santa Maria Hospital in Lisbon Isolation of HUVECs was performed according to the modified Jaffe’s method described previ-ously [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 mgÆmL)1of type II collagenase (Gibco Brl) in the same medium for 15 min at 37C The endo-thelial cells were collected by centrifugation and grown
in SFM-Basal Growth Medium supplemented with basic fibroblast growth factor (20 ngÆmL)1, Gibco Brl), endothelial growth factor (10 ngÆmL)1, Gibco Brl) and penicillin⁄ streptomycin solution (10 lgÆmL)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 main-tained in a humidified atmosphere of 5% (v⁄ v) CO2 in air
at 37C
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 lgÆmL)1 of BODIPY FL AcLDL (Molecular Probes, Eugene, OR, USA) in culture medium for 4 h in a humidified atmo-sphere of 5% CO2in air at 37C After the incubation, the cells were washed once with NaCl⁄ Piand fixed with 3.7% (v⁄ v) paraformaldehyde in NaCl ⁄ Pi for 10 min at room temperature [6,7] The uptake of BODIPY 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 cm2flasks to con-fluence and stimulated with IL-1b (300 and 500 pgÆmL)1) for 5 h After being washed with NaCl⁄ Pi, the cells were fixed in 4% (v⁄ v) paraformaldeyde for 10 min and permea-bilized 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 pgÆmL)1) and cells stimulated with IL-1b 300 pgÆmL)1
or 500 pgÆmL)1for experiments with different primary anti-bodies The cells were incubated with NaCl⁄ Pi 1· with 0.1% (w⁄ v) BSA at 4 C, the primary antibodies [goat poly-clonal IgG E-selectin (N), goat polypoly-clonal IgG E-selectin
Trang 9(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
per-centage 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 4C with
periodic resuspension of the cellular suspension After cell
disruption, the obtained lysate was centrifuged at 47 000 g
for 30 min at 4C 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
resuspend-ed in Tris⁄ HCl 20 mm, EGTA 0.1 mm pH 7.4 buffer,
incu-bated for 30 min at 4C and ultra-centrifuged at 100 000 g
for 30 min at 4C HUVECs membranes were then
solubi-lized with 1% (v⁄ v) Triton X-100 in Tris ⁄ HCl 0.1 m pH 8.0
buffer for 60 min at 4C Finally the detergent-solubilized
extract was ultra-centrifuged at 100 000 g for 60 min at
4C and further concentrated with a concentrator
(Eppen-dorf, 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)
gly-cerol, 4.5% (w⁄ v) sodium dodecylsulphate (SDS), 150 mm
dithiothreitol, 2-mercaptoethanol (100 lLÆmL)1sample
buf-fer) and 0.01% (w⁄ v) bromophenol blue by heating the
mixture at 100C 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
Bio-Rad (Richmond, CA, USA) for the estimation of the
molecular mass of proteins The run of the gel was made
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 Trans-Blot 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 (rab-bit polyclonal IgG, AChE (H-134), Santa Cruz Biotechno-logy), 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 horse-radish-peroxidase-linked secondary antibody (donkey anti-rabbit 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⁄ Pibuffer 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 Instru-ments, 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) bromo-phenol blue Solubilized HUVECs membrane extracts were loaded at a protein content of 8.5 lgÆlane)1 The human recombinant acetylcholinesterase and the human erythro-cytes 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
Trang 10Staining for cholinesterase activity was done by shaking
the gel at 30C, 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 mgÆmL)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
activ-ity of 4· 105cells (whole cells) in the presence of an
acetyl-thiocholine 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
acetyl-thiocholine (ASCh) per minute, at 37C The absorbance
was monitored at 412 nm using a Genesys 10 UV
spectro-photometer (ThermoSpectronic) We used a pH 8.1
phos-phate 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
activ-ity present in the different extracts obtained during
isola-tion and solubilizaisola-tion of membranes of HUVECs
Acknowledgements
The authors would like to acknowledge the
Depart-ment of Obstetrics on Santa Maria’s Hospital of
Lis-bon 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
cytometry experiments and Professor Aˆngelo Calado
for helping with immunoblotting experiences
References
1 Massoulie` J, Sussman J, Bon S & Silman I (1993)
Struc-ture and function of acetylcholinesterase and
butyryl-cholinesterase Brain Res 98, 139–146
2 Wessler I, Kirckpatrick CJ & Racke´ K (1999) The choli-nergic ‘pitfall’: acetylcholine, a universal cell molecule in biological systems, including humans Clin Exp Pharma-col Physiol 26, 198–205
3 Wessler I, Kirckpatrick CJ & Racke´ K (1998) Non-neuronal acetylcholine, a locally acting molecule widely distributed in biological systems: expression and function in humans Pharmacol Ther 77, 55– 79
4 Mikalsen A, Andersen RA & Alexander J (1986) Use of ethopropazine and BW 284c51 as selective inhibitors for cholinesterases from various species Comp Biochem Physiol 83, 447–449
5 Grisaru D, Sternfeld M, Eldor A, Glick D & Soreq
H (1999) Structural roles of acetylcholinesterase var-iants in biology and pathology Eur J Biochem 264, 672–686
6 Scoumanne A, Kalamati T, Moss J, Powell JT, Gosling
M & Carey N (2002) Generation and characterisation
of human saphenous vein endothelial cell lines Athero-sclerosis 160, 59–67
7 Voyta JC, Via DP, Butterfield CE & Zetter BR (1984) Identification and isolation of endothelial cells based on their uptake of acetylated-low density lipoprotein J Cell Biol 99, 2034–2040
8 Plageman LR, Pauletti GM & Skau KA (2002) Charac-terization of acetylcholinesterase in Caco-2 cells Exp Biol Med 227, 480–486
9 Sharma S & Rathaur S (1999) Characterization of secre-tory acetylcholinesterase from Setaria cervi microfilariae:
a potential antigen for diagnosis of human filariasis Trop Med Int Health 4, 341–348
10 Lan CT, Shich JY, Wen CY, Tan CK & Ling EA (1996) Ultrastructural localization of acetylcholinester-ase and choline acetyltransferacetylcholinester-ase in oligodendrocytes, glioblasts and vascular endothelial cells in the external cuneate nucleus of the gerbil Anat Embryol (Berlin)
194, 177–185
11 Kasa P, Pakaski M, Joo F & Lajtha A (1991) Endo-thelial cells from human brian microvessels may be cholinoceptive, but not synthesize acetylcholine
J Neurochem 56, 2143–2146
12 Ciani F & Franceschini V (1984) Ultrastructural study and cholinesterase activity of paired capillaries in the newt brain J Hirnforsch 25, 11–20
13 Kirckpatrick CJ, Bittinger F, Nozadze K & Wessler I (2003) Expression and function of the non-neuronal cholinergic system in endothelial cells Life Sci 72, 2111–2116
14 Rao RV, Gnanamuthu C & Balasubramanian AS (1989) Human cerebrospinal fluid acetylcholinesterase and butyrylcholinesterase: evidence for identity between the serum and cerebrospinal fluid butyrylcholinesterase Clinica Chimica Acta 183, 135–146