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 ¸ ´ Instituto de Biopatologia Quımica, Faculdade de Medicina de Lisboa ⁄ Unidade de Biopatologia Vascular, Instituto de Medicina Molecular, Lisbon, Portugal ´ 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: filomenacarvalho@fm.ul.pt (Received 15 July 2005, revised 25 August 2005, accepted 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 5584 FEBS Journal 272 (2005) 5584–5594 ª 2005 FEBS 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 FEBS Journal 272 (2005) 5584–5594 ª 2005 FEBS 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 illustrates the uptake of a fluorescently labeled AcLDL by cultured HUVECs at passage 2, after 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 (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 Scale bar: 20 lm 5585 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 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 Histograms showing the effect of stimulation with IL-1b (300 pgỈmL)1 or 500 pgỈmL)1) for 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) 5586 FEBS Journal 272 (2005) 5584–5594 ª 2005 FEBS F A Carvalho et al Characterization of HUVECs membrane bound AChE Table 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 (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 FEBS Journal 272 (2005) 5584–5594 ª 2005 FEBS 5587 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 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 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 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) 5588 FEBS Journal 272 (2005) 5584–5594 ª 2005 FEBS F A Carvalho et al Characterization of HUVECs membrane bound AChE A B Fig (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 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 FEBS Journal 272 (2005) 5584–5594 ª 2005 FEBS 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%) 5589 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 ( 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 mm ASCh, at 37 °C We not know if pH 8.1 is the optimal pH for the enzymatic activity of AChE of membranes of HUVECs However FEBS Journal 272 (2005) 5584–5594 ª 2005 FEBS 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 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 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 FEBS Journal 272 (2005) 5584–5594 ª 2005 FEBS Characterization of HUVECs membrane bound AChE 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 mgỈmL)1 of type II collagenase (Gibco Brl) in the same medium for 15 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 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 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 h After being washed with NaCl ⁄ Pi, the cells were fixed in 4% (v ⁄ v) paraformaldeyde for 10 and permeabilized in 90% (v ⁄ v) methanol for 20 The experience was carried out under different conditions such as, control (only cells), control (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 · with 0.1% (w ⁄ v) BSA at °C, the primary antibodies [goat polyclonal IgG E-selectin (N), goat polyclonal IgG E-selectin 5591 Characterization of HUVECs membrane bound AChE (C), goat polyclonal IgG vWf; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA] were added for 30 at room temperature Then, the cells were washed with NaCl ⁄ Pi · with 0.1% (w ⁄ v) BSA at °C and incubated with secondary antibody (Alexa Fluor 488) for 30 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 or 3) were detached with the use of a cell scraper and further washed twice with NaCl ⁄ Pi buffer by centrifugation for 10 at 700 g A total of · 106 cells were subsequently resuspended in lyses buffer (Tris ⁄ HCl mm pH 7.4, EDTA mm) Cell lysis was conducted for 60 at °C with periodic resuspension of the cellular suspension After cell disruption, the obtained lysate was centrifuged at 47 000 g for 30 at °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 at °C and ultra-centrifuged at 100 000 g for 30 at °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 at °C Finally the detergent-solubilized extract was ultra-centrifuged at 100 000 g for 60 at °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 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 The gel was subsequently stained in 0.25% Coomassie blue in 50% (v ⁄ v) methanol and 10% (v ⁄ v) acetic acid for 10 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 so as to control for protein transfer After washing out the Ponceau S staining with · NaCl ⁄ Pi buffer, blots were blocked by incubation with NaCl ⁄ Pi ⁄ 5% (w ⁄ v) non-fat milk for 30 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 : 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 : 3000 for 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 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 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 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 mm, sodium citrate mm, copper(II) sulfate mm and potassium hexacyanoferrate (III) 0.5 mm, by shaking the gel at room temperature for 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 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 · 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 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 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Biochem Pharmacol 7, 88–95 FEBS Journal 272 (2005) 5584–5594 ª 2005 FEBS ... 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... 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. .. 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