Neuropeptides 46 (2012) 373–382

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Neuropeptides

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Differences in neuropeptide Y-induced secretion of endothelin-1 in leftand right human endocardial endothelial cells

Dima Abdel-Samad a, Claudine Perreault a, Lena Ahmarani a, Levon Avedanian a, Ghassan Bkaily a,Sheldon Magder b, Pedro D’Orléans-Juste c, Danielle Jacques a,⇑,1

a Department of Anatomy and Cell Biology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4b McGill University Health Centre, Royal Victoria Hospital, 687 Pine Av. West, Montreal, Quebec, Canada H3A 1A1c Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4

a r t i c l e i n f o a b s t r a c t

Article history:Received 24 July 2012Accepted 19 September 2012Available online 26 October 2012

Keywords:Neuropeptide YNPY receptorsEndocardial endothelial cellsEndothelin-1ET-1 secretionGPCRHuman endotheliumExcitation–secretion couplingNPY receptor antagonistY5 receptor

0143-4179/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.npep.2012.09.003

⇑ Corresponding author. Tel.: +1 819 564 5419.E-mail address: Danielle.jacques@usherbrooke.ca (

1 Supported by the Canadian Institutes of Health ReseStroke Foundation of Quebec (HSFQ) to Dr. Danielle Ja

The aim of the study was to test the hypothesis that neuropeptide Y (NPY) may induce endothelin-1(ET-1) secretion in left (hLEECs) and right (hREECs) human endocardial endothelial cells. Furthermore,the type of NPY receptor implicated could be different in NPY-induced secretion in hLEECs and hREECs.Using immunofluorescence coupled to real 3D confocal microscopy and ELISA, our results showed thatstimulation of secretion by NPY induced the release of ET-1 from both right and left human ventricularendocardial endothelial cells (hEECs) in a time-dependent manner. Furthermore, the secretory capacity ofhREECs was higher than that of hLEECs. In addition, our results showed that the effect of NPY on ET-1secretion in hLEECs was only due to activation of Y5 receptors. However, the effect of NPY on ET-1 secre-tion in hREECs was due to mainly Y2 and partially Y5 receptors activation. In conclusion, our results sug-gest that differences in excitation–secretion coupling exist between hREECS and hLEECs which maycontribute to the functional differences between right and left ventricular muscle. Furthermore, highNPY level contributes to ET-1 release by hEECs and Y2 and Y5 receptors antagonists may be used for reg-ulation of ET-1 secretion in the heart.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The endothelium is a highly dynamic cell layer which lines theentire circulatory system (heart and blood vessels) and plays a veryimportant role in a variety of physiological functions, includingregulation of vasomotor tone, vascular permeability and growthas well as maintenance of blood fluidity (Aird, 2004; Brutsaert,2003; Pedrazzini et al., 2003). The endothelium that has beenviewed for a long time as an inert monolayer of cells is now recog-nized as a massive, regionally-specific, multifunctional organwhose dysfunction can be a critical factor in various pathologicalconditions (Cines et al., 1998; Kuruvilla and Kartha, 2003). It hasbeen reported that both vascular endothelial as well as cardiacendothelial cells express and release a variety of cardioactive fac-tors such as endothelin-1 (ET-1), nitric oxide (NO), angiotensin II(Ang II) and prostanoids (Brutsaert, 2003; Cines et al., 1998;Kuruvilla and Kartha, 2003; Pedrazzini et al., 2003). In addition,complex interactions have been reported to exist between ET-1

ll rights reserved.

D. Jacques).arch (CIHR) and the Heart and

cques.

and the other vasoconstrictors and vasodilators at the level ofthe heart and blood vessels (Kuruvilla and Kartha, 2003; Pedrazziniet al., 2003). For example, ET-1 induces the release of NO fromendothelial cells and atrial natrieuretic peptide (ANP) from atrialtissue. Alternatively, it was demonstrated that NO inhibits the pro-duction of ET-1, whereas Ang II stimulates its release (Kuruvillaand Kartha, 2003).

Another powerful vasoconstrictor, the 36-amino acid peptideneuropeptide Y (NPY) (Pedrazzini et al., 2003), has also been re-ported to be present in vascular endothelial cells of the humanumbilical vein (HUVEC) (Ghersi et al., 2001) and rabbit centralear artery (Loesch, 2002). Moreover, our group has demonstratedthat ET-1 and its receptors, ETA and ETB (Jacques et al., 2005), aswell as NPY and its receptors, Y1 and Y2 (Jacques et al., 2003), arepresent at the level of endocardial endothelial cells (EECs) isolatedfrom both the right (REECs) and left (LEECs) heart ventricles of hu-man donors. We have equally shown that NPY, just like ET-1, isable to induce a sustained increase in the levels of cytosolic andnuclear calcium in these cells and that only right ventricular EECspossess the capacity of secreting NPY (Jacques et al., 2000, 2005).

The aim of this study was to investigate differences that mayexist in excitation–secretion coupling between right and left

374 D. Abdel-Samad et al. / Neuropeptides 46 (2012) 373–382

ventricular EECs and to look into the role of NPY receptors Y1, Y2

and Y5 in the secretory capacity of right versus left EECs. In orderto accomplish these objectives, the techniques of indirect immuno-fluorescence and 3D confocal microscopy as well as radioimmuno-assay and ELISA were used to verify if NPY is capable of modulatingthe immunoreactivity of ET-1 in both REECs and LEECs, and thesecretion of ET-1 in both cell types and the actual role of NPYreceptors Y1, Y2, and Y5 in this process.

2. Materials and methods

2.1. Isolation of endocardial endothelial cells

hEECs were isolated from the right and left ventricles of20-week-old human fetal hearts as described previously by ourgroup (Bkaily et al., 1997; Jacques et al., 2000). The method usedfulfills the recommendations of the institutional ethical committeeregarding the use of human material. High standard of ethics wasapplied in carrying out the investigations.

In brief, hearts were isolated under asceptical conditions fromhuman donors. Following trypsin treatment, the endocardialendothelium of both ventricles was gently isolated and washedin medium M199 (Gibco BRL; Burlington, Ont.) supplemented with10% FBS (fetal bovine serum) (Gibco BRL, Burlington, Ont.). After a10-min centrifugation period at 1000 rpm, the supernatant wasdiscarded and the cellular pellet re-suspended in medium M199supplemented with 10% FBS. Finally, cells were spread onto petridishes and placed in an incubator maintained at 37 �C and 5%CO2. For producing primary cell cultures, the suspended hREECsand hLEECs were spread in culture flasks (GibcoBRL, Burlington,Ont.) and incubated under the conditions mentioned above. Uponconfluence, cells were trypsinized and reseeded in medium M199supplemented with 10% FBS (Bkaily et al., 1997).

A test for the quality and purity of hREECs and hLEECs is rou-tinely done in our lab using an FITC-conjugated fluorescent probe,namely Lectin Ulex Europeans Agglutinin, (6 lg/ml; Sigma, St-Louis, MO). This probe binds to a-linked fucose residues knownto be present on the surface of endothelial cells (Bkaily et al.,1997; Jacques et al., 2000). A second test, also routinely done forthe same purpose, consists of using antibodies directed againstvon Willebrand factor (vWF) (1:200; Sigma–Aldrich, St-Louis,MO), which is known to be produced constitutively by endothelialcells, but not by the underlying cardiomyocytes or smooth musclecells (Datta and Ewenstein, 2001; van Mourik et al., 2002). Usingsecondary rabbit anti-IgG antibodies conjugated to Alexa Fluor488 (1 lg/ml; Molecular Probes, Eugene, OR) followed by the tech-nique of indirect immunofluorescence described in Section 2.4.vWF was found to be present throughout the cytosol of EECs in aparticulate manner. Furthermore, pharmacological studies at thelevel of AngII, ET-1 and NPY showed similar results in EECs of20-week-old fetal human hearts and those from adult rats andmice (Jacques et al., 2003 and unpublished results). In addition,pharmacological and electrophysiological studies showed similarresults also in cardiomyocytes isolated from 20-week-old humanfetal hearts and those isolated from adult animals (Bkaily, 1990).Thus, as cardiomyocytes, EECs isolated from 20-week-old fetal hu-man hearts is a good model for adult EECs.

2.2. Radioimmunoassay

hEECs were cultured in 100 mm diameter petri dishes in thepresence of M199 culture medium supplemented with 10% (v/v)FBS. At confluence, cells were incubated for time intervals of 5,10, 20, 30 and 60 min with a high concentration of KCl (30 mM)at 37 �C. At the end of each incubation period, culture medium

was collected to determine the concentration of NPY secreted byhEECs. Cells were then washed with PBS (1 M, pH 7.4), gentlyscraped in the presence of 600 ll of serum-free M199 medium, col-lected and broken by sonification. The concentration of NPY in eachsample was determined using radioimmunoassay (Alpco Diagnos-tics, New Hampshire). NPY, in samples and standards, competeswith [125I]-labeled NPY for binding to antibodies. The concentra-tion of [125I]-NPY is inversely proportional to that of unlabelledNPY in both standards and samples. Antibody-bound [125I]-NPYwas then separated from the unbound fraction by using doubleantibody coupled to solid phase. Finally, the radioactivity of theantibody-bound [125I]-NPY was measured.

2.3. Pre-treatment of EECs with NPY

hEECs were cultured on 25 mm glass coverslips or in 100 mmpetri dishes at 37 �C for 24 h, after which they were exposed to dif-ferent concentrations of extracellular NPY (10�15 and 10�7 M) inthe absence and presence of different NPY antagonists, for60 min at 37 �C. In experiments using NPY receptor antagonists,the cells were first exposed to the antagonists 15 min prior to addi-tion of NPY for 60 min. In order to perform indirect immunofluo-rescence, cells were washed with phosphate-buffered saline (PBS1 M, pH 7.4) and the cells were fixed with 4% paraformaldehyde.The technique of ELISA was used to determine the concentrationof ET-1 secreted by hEECs in the culture medium collected from100 mm petri dishes.

2.4. Indirect immunofluorescence

hEECs, cultured on 25 mm glass coverslips, were washed twicePBS (1 M, pH 7.4) at room temperature and then fixed for 10 minwith 4% paraformaldehyde. After washing with 1 M PBS (2�), cellswere incubated with PBS containing sodium borohydride (2 mg/ml) for 10 min. Cells were then washed with 1 M PBS (2�), perme-abilized and blocked with 7% normal goat serum (NGS), 5% non-fatdried milk (NFDM) and 0.1% Triton X-100 in 1 M PBS for 30 min.Two washes with 1 M PBS were then performed and the cells wereincubated overnight at 4 �C with primary monoclonal mouse anti-ET-1 (1:100) in PBS containing 1.4% NGS, 1% NFDM and 0.1% TritonX-100. After washing with 1 M PBS (2�), cells were incubated for1 h at room temperature with secondary rabbit anti-mouse AlexaFluor 488 (1 lg/ml) in the same buffer that was used for the pri-mary antibody. Two final washes with 1 M PBS were performedand the cells were visualized using a confocal microscope.

2.5. Confocal microscopy

Dye-loaded cells were examined with a Molecular DynamicsMultiprobe 2001 confocal argon laser scanning (CSLM) systemequipped with a Nikon Diaphot epifluorescence inverted micro-scope and a 60� Nikon oil Plan achromat objective. For ET-1 fluo-rescence intensity measurement, the 488 nm argon laser line(9.0 mV) was directed to the sample via a 510 nm primary dichroicfilter and attenuated with a 1–3% neutral density filter to reducephotobleaching. Laser line intensity, photometric gain, PMT set-tings and filter attenuation were kept rigorously constant through-out the experimental procedures.

2.6. Elisa

For studies of the effect of NPY on the secretion of ET-1, hREECsand hLEECs cultured in 100 mm petri dishes, in the presence ofmedium M199 and 10% FBS, were incubated at 37 �C and 5% CO2

until confluence was reached. Upon confluence, hEECs were ex-posed to NPY (10�7 M), in the absence and presence of different

D. Abdel-Samad et al. / Neuropeptides 46 (2012) 373–382 375

NPY receptors antagonists, for time intervals of 5, 10, 20, 30 and60 min at 37 �C. At the end of each incubation period, 600 ll ofthe culture medium were collected to determine the concentrationof ET-1 secreted by hEECs using ELISA.

The ELISA kit used throughout the study is the endothelin-1Biotrak™ ELISA System provided by Amersham Biosciences (GEHealthcare Biosciences AB, Uppsala, Sweden). All the solutionsand reagents necessary for carrying out an experiment were pro-vided with the kit with the exception of the stop solution, namelysulfuric acid (H2SO4). This assay is based on a ‘sandwich’ format.Standards and samples were incubated in the wells pre-coatedwith antibody against ET-1. Any ET-1 present in the standards orsamples will be bound to the antibody in the wells, other compo-nents of the samples being removed by washing and aspiration.The bound ET-1 was detected using a Horseradish Peroxidase(HRP)-labeled Fab’ fragment of ET-1 antibody conjugate. The con-centration of antibody conjugate bound to each well was deter-mined by the addition of tetramethylbenzidine (TMB) substrate.The reaction was finally stopped by the addition of H2SO4 andthe resultant color read at 450 nm in a microplate spectrophotom-eter. The concentration of ET-1 in the samples was determinedby extrapolation from the standard curve. This kit allows forthe determination of ET-1 in the range of 1–32 fmol/well(24.8–797.4 pg/ml).

2.7. Statistics

The levels of NPY and ET-1 fluorescence intensity are repre-sented as mean values of relative fluorescence intensity. All resultsare expressed as mean ± standard error mean (SEM) for a numberof ‘n’ cells examined of minimum 3–21 different cultures from atleast three different donors unless otherwise indicated. Thenumber of cells per single experiment depends on the density ofcells in the field of vision of the microscope objective at the mo-ment of starting the experiment. Statistical significance was deter-mined using the ANOVA repeated measures test for matchedvalues followed by a Tukey–Kramer test or the student–New-man–Keuls multiple comparison test. Probability values (p) lessthan 0.05 (confidence interval of 95%) were considered assignificant.

3. Results

3.1. Effect of 10�15 and 10�7 M NPY on the relative density (by lm3) ofintracellular ET-1 in hLEECs and hREECs

Using real 3D confocal microscopy coupled to immunofluores-cence measurement, compiling all our control samples measure-ments of the level of intracellular ET-1 in this study, the level ofintracellular ET-1 in hLEECs was found to be significantly lower(p < 0.001) than that of hREECs. Treatment of hLEECs with lowand high concentrations of NPY (respectively 10�15 and 10�7 M)for the different time intervals induced significant decrease(p < 0.05) in the relative density of ET-1 only at 60 min treatmentwith the two concentrations of NPY (Figs. 1A, B and 2A, B).

On the other hand, in another series of experiments (Figs. 1D, Eand 2C, D), treatment of hREECs with 10�15 M of NPY induced a sig-nificant, decrease in the relative density of ET-1 starting at 5 minafter treatment with NPY without any further decrease till60 min (Figs. 1D and 2C, D).

Interestingly, a very high level of this peptide (10�7 M) induceda significant time-dependent decrease in the intensity level of ET-1at 20 min treatment of hREEC (Fig. 1E). This significant decreasecontinued and became more pronounced at 30 and 60 min of treat-ment with 10�7 M NPY (Fig. 1E).

3.2. NPY-induced secretion of ET-1

In another series of experiments, in order to ensure that theNPY-induced decrease in the intracellular level of ET-1 is indeeddue to secretion of this peptide by the hEEC, the technique of ELISAwas used to examine if the above-mentioned decrease is accompa-nied by an increase in the extracellular level of ET-1.

For this purpose, cultured hEECs isolated from both the rightand left ventricles of human hearts were exposed to NPY(10�7 M) for intervals of 5, 10, 20, 30 and 60 min and samples ofthe extracellular medium were collected after each time periodto determine the extracellular concentration of ET-1 by ELISA.Since secretion is a calcium-dependent phenomenon (Rettig andNeher, 2002), and since our previous results showed that 10�7 Mof NPY induced a maximal effect on intracellular Ca2+ (Jacqueset al., 2003) thus a concentration of 10�7 M of NPY was chosen herefor confirmation of ET-1 secretion.

Furthermore, since a significant decrease of intracellular ET-1took place in hLEECs only at 60 min treatment with 10�7 M NPY,thus the ELISA experiments were done with 10�7 M NPY for60 min.

At the level of hLEECs, as Fig. 1C shows, exposure of these cellsto 10�7 M of NPY for time intervals of 5, 10, 20, 30 and 60 min re-sulted in an increase in the extracellular concentration of ET-1. Thisrise became significant at 10 and 60 min of treatment with NPY(60 min NPY: 15.021 pg/ml ± 3.032) as compared to the control le-vel (3.612 pg/ml ± 1.148).

Similarly, 10�7 M of NPY induced a significant rise in the extra-cellular level of ET-1 at 30 and 60 min of exposure of hREEC to NPY(Fig. 1F)(control level [ET-1]o: 4.50 pg/ml ± 0.896; 60 min [ET-1]o:17.612 pg/ml ± 3.226).

These results clearly suggest that the NPY-induced decrease inthe [ET-1]i fluorescence intensity in both hLEECs and hREECs is in-deed due to secretion of the latter into the extracellular medium.

3.3. Y1 antagonist (BIBO3304) does not prevent NPY-induced secretionof ET-1 from hLEECs and hREECs

To investigate the implication of the Y1 receptor in the NPY-in-duced decrease of the ET-1 fluorescence intensity in hEECs, cul-tured hREECs and hLEECs were first incubated for 15 minwith the Y1 receptor antagonist, BIBO3304, at a concentration(10�6 M) which is known to completely block this type of receptor(Dumont et al., 2000a,c). After this, and in presence of the antago-nist, NPY (10�7 M) was added for 5, 10, 20, 30 and 60 min. A controlsample, where hEECs were incubated with the Y1 receptor antago-nist alone for 15 min was always performed.

Measurements of the relative density of ET-1 (by lm3) in hREECsand hLEECs exposed to NPY in the presence of the Y1 receptorantagonist were taken and these values were compared to those ob-tained before using NPY alone at 10�7 M. In the presence of the Y1

receptor antagonist, BIBO3304, the decrease in the level of ET-1fluorescence intensity started to become significant at 10 min ofhREEC and hLEEC treatment with NPY as compared to its respectivecontrol. This decrease continued up till 60 min of treatment withNPY in both types of cells. Fig. 3 panels A and C summarize the re-sults and Fig. 4 shows typical examples of 60 min treatment withNPY in presence and absence of the Y1 antagonist.

To investigate the implication of the Y1 receptor in the NPY-in-duced increase of the extracellular level of ET-1 at the level ofhEECs, cultured hREECs and hLEECs were incubated with the Y1

receptor antagonist, BIBO3304 (10�6 M), for 15 min before theapplication of NPY (10�7 M) for 5, 10, 20, 30 and 60 min in the con-tinued presence of the antagonist. Samples of the extracellularmedia of hLEECs and hREECs were then collected at each timeinterval to determine the extracellular concentration of ET-1. As

Fig. 1. Effect of 10�15 and 10�7 M NPY on relative density (by lm3) of whole-cell ET-1 in hLEECs (A and B) and hREECs (D and E) as well as the effect of 10�7 M NPY on ET-1secretion (C and F) as a function of time. Histograms showing normalized measurements of ET-1 fluorescence intensities in hLEECs (A and B) and hREECs (D and E) in theabsence (ctrl.) and following 5, 10, 20, 30 and 60 min of exposure to NPY at concentrations of 10�15 M (A and C) and 10�7 M (B and D). It can be observed from panels (A, B, Dand E) that there is a dose- and time-dependent decrease in the fluorescence intensity of ET-1 in hLEECs and hREECs after their treatment with the two differentconcentrations of NPY. (C and F) show the effect of 10�7 M NPY on extracellular release of ET-1 using ELISA. Note that in hLEECs significant simultaneous intracellulardecrease and extracellular increase were only observed at 60 min treatment, unlike hREECs. Results are presented as means ± SEM. ‘n’ represents the number of different cellsin at least three different cultures with the exception of panels (C and F) where n represents the number of different experiments of different cultures. (⁄) statisticalcomparison of the ET-1 fluorescence intensity at different time intervals of exposure to NPY with respect to the control. ⁄⁄⁄p < 0.001, ⁄⁄p < 0.01 and ⁄p < 0.05 are considered assignificant.

376 D. Abdel-Samad et al. / Neuropeptides 46 (2012) 373–382

panel 3B shows, administration of NPY in the presence of the Y1

receptor antagonist was not able to prevent the NPY-induced in-crease in the extracellular level of ET-1 observed at the level of

hLEECs after 60 min of treatment (control value is 3.612 pg/ml ± 0.444; 60 min value NPY alone is 15.022 pg/ml ± 3.032;60 min value BIBO3304 + NPY is 11.838 pg/ml ± 2.317).

Fig. 2. 3-D fishnet plot representations of hLEECs (A–B) and hREECs (C–D) showing the intracellular immunofluorescence labeling of ET-1 in control condition (A and C) andafter 60 min of treatment with extracellular NPY (10–15 M) (B and D). Panels are 3-D fishnet plot representations of the cells using a magnification of 1.4�. Note that there isa decrease in the immunoreactivity of ET-1 after 60 min of treatment with NPY (10–15 M) as compared to control conditions. Rotation of the fishnet plot cell was done inorder to better show the maximum intensity and distribution of ET-1. The pseudocolor bar represents ET-1 fluorescence intensity ranging from 0 (absence of any fluorescencesignal) to 255 (maximum fluorescence intensity).

D. Abdel-Samad et al. / Neuropeptides 46 (2012) 373–382 377

At the level of hREECs, administration of NPY in the presence ofthe Y1 receptor antagonist, BIBO3304, resulted in a significant risein the extracellular concentration of ET-1 starting at 20 min afterthe application of NPY (control value is 4.50 pg/ml ± 0.629;20 min value is 9.877 pg/ml ± 1.855; 30 min value is 11.923 pg/ml ± 2.144 and the 60 min value is 13.818 pg/ml ± 2.194). As canbe observed from panel 3D, the Y1 receptor antagonist in presenceof NPY was not able to prevent the increase in the extracellular le-vel of ET-1 induced by NPY (10�7 M) alone at the level of hREECs(control value is 4.50 pg/ml ± 0.629; 60 min value NPY alone is17.612 pg/ml ± 3.226; 60 min value BIBO3304 + NPY is 13.818 pg/ml ± 2.194).

3.4. Blockade of Y2 receptor prevents NPY from inducing secretion ofET-1 from hREECs but not from hLEECs

In addition to demonstrating the presence of NPY and its Y1

receptor in hEECs, our group has showed recently that the Y2

receptor was also found at the level of ventricular hEECs (Abdel-Sa-mad et al., 2007). The highly selective and non-peptidic Y2 receptorantagonist, BIIE0246 (Doods et al., 1999; Dumont et al., 2000b,c),was used in this series of experiments to examine the contributionof the Y2 receptor to the NPY-induced secretion of ET-1 in hREECsand hLEECs.

In this series of experiments, cultured hLEECs and hREECs wereincubated with the Y2 receptor antagonist, BIIE0246 (10�6 M), for15 min followed by the administration of NPY (10�7 M) for 5, 10,20, 30 and 60 min. A control sample, where hREECs and hLEECswere incubated with the Y2 receptor antagonist alone for 15 minwas always performed. It should be mentioned that a concentra-tion of 10�6 M of BIIE0246 is known to completely block the Y2

receptor (Doods et al., 1999; Dumont et al., 2000b,c).

Fig. 5 panels A and C show measurements of the relative density(by lm3) of intracellular ET-1 in hLEECs and hREECs in absence andpresence of BIIE0246 (10�6 M) + NPY (10�7 M), where these valueswere compared to control. Fig. 4 shows typical examples.

The decrease in the level of intracellular ET-1 fluorescenceintensity started to become significant after 10 min of hLEEC treat-ment with NPY in the presence of BIIE0246 and continued until60 min of this treatment when compared to its respective controlFig. 3A).

In another series of experiments, using hREECs, there was nosignificant change in the relative density of intracellular ET-1 after5, 10, 20, 30 or 60 min of exposing these cells to NPY in the pres-ence of BIIE0246 when compared to its respective control. It is tobe noted, however, that NPY alone induced a significant decreasein the intracellular level of ET-1 starting at 20 min of its applicationto hREECs (not shown).

In another series of experiments, ELISA was used to determinethe extracellular concentration of ET-1 after treatment of hREECsand hLEECs with NPY (10�7 M) in the presence of BIIE0246(10�6 M) for 5, 10, 20, 30 and 60 min.

For the effect of Y2 receptor blockade in hLEECs, it can be ob-served that there was a significant rise in the extracellular concen-tration of ET-1 starting at 5 min of hLEEC exposure to NPY (10�7 M)in the presence of BIIE0246 (10�6 M) and continued to 60 min ofthis exposure (control level is 3.612 pg/ml ± 0.634 and the 5 minlevel is 6.529 pg/ml ± 0.540). It can be clearly seen from Fig. 5B thatY2 receptor blockade did not prevent the 60-min NPY-induced risein the extracellular concentration of ET-1 at the level of hLEECs(control level is 3.612 pg/ml ± 0.634; 60 min level NPY alone15.021 pg/ml ± 3.032; 60 min level BIIE0246 + NPY is 13.517 pg/ml ± 0.820).

In case of hREECs, there was no significant change in the extra-cellular level of ET-1 following 5, 10, 20, 30 or 60 min of cell expo-

Fig. 3. Effect of Y1 receptor blockade on NPY-induced decrease in intracellular (A and C) and increase in extracellular (B and D) ET-1 in hLEECs (A and B) and hREECs (C and D).Histograms showing measurements of ET-1 fluorescence intensity levels (A and C) and extracellular concentration (B and D) of ET-1 in hLEECs (A and B) and hREECs (C and D)in the absence (ctrl.; grey) and following 60 min of exposure to NPY (10�7 M; black) or NPY (10�7 M) in presence of the Y1 receptor antagonist, BIBO3304 (10�6M; red). InhLEECs (A) as well as hREECs (C), Y1 R antagonist did not prevent the effect of NPY. Results are presented as means ± SEM. ‘n’ is the number of different experiments ofdifferent culture cells. (⁄) is p < 0.05, (⁄⁄) is p < 0.01 and (⁄⁄⁄) is p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the webversion of this article.)

378 D. Abdel-Samad et al. / Neuropeptides 46 (2012) 373–382

sure to NPY in the continued presence of BIIE0246 when comparedto the control level. Histograms in Fig. 5D clearly show how the Y2

receptor antagonist, BIIE0246, was able to prevent the 60-minNPY-induced rise in the extracellular level of ET-1 in hREECs (con-trol level is 4.50 pg/ml ± 0.717; 60 min level NPY alone 17.612 pg/ml ± 3.226; 60 min value BIIE0246 + NPY is 7.023 pg/ml ± 0.766).

3.5. Blockade of Y5 receptor prevents NPY from inducing secretion ofET-1 from both hREECs and hLEECs

The two main receptors thought to play a major role in periph-eral cardiovascular control are the Y1 and Y2 receptors (Uddmanet al., 2002). This, however, does not exclude the possibility ofinvolvement of other NPY receptors, such as the Y5 receptor. Unfor-tunately, the lack of availability of reliable antibodies directedagainst the Y5 receptor protein prevented us from investigatingits presence at the level of the endocardial endothelial cells. Fortu-nately, though, due to the recent availability of a potent andhighly-selective Y5 receptor antagonist, L-152, 804 (Kanataniet al., 2000), we were able to examine the participation of the Y5

receptor in the ET-1 secretory effect induced by NPY in right andleft ventricular hEECs.

To investigate the implication of the Y5 receptor in the decreaseof the intracellular intensity of ET-1 labeling induced by NPY, cul-

tured hLEECs and hREECs were pretreated for 15 min with the Y5

receptor antagonist, L-152,804 (10�6 M), followed by the extracel-lular application of NPY (10�7 M) for 5, 10, 20, 30 and 60 min. Acontrol sample where hEECs were incubated with the Y5 receptorantagonist alone for 15 min was also performed.

Measurements of the relative intracellular density of ET-1 (bylm3) in both hLEECs and hREECs were taken using the 3D recon-structions of cells obtained from the confocal microscopy experi-ments. These measurements were taken under control conditionsand following the exposure of hLEECs and hREECs to L-152,804(10�6 M) + NPY (10�7 M) for 5, 10, 20, 30 and 60 min. Values ob-tained were then compared with those obtained earlier whenstudying the effect of NPY alone (10�7 M) on the level of ET-1 fluo-rescence intensity.

At the level of hLEECs, blockade of the Y5 receptor resulted in nosignificant change in the relative density of ET-1 after 5, 10, 20, 30or 60 min of application of L-152,804 (10�6 M) + NPY (10�7 M) ascompared to its respective control. Meanwhile, NPY alone resultedin a significant decrease of the ET-1 relative density observed after60 min of treatment as compared to its respective control (Fig. 6A).

In the case of hREECs, administration of L-152,804 (10�6-

M) + NPY (10�7 M) for 5, 10, 20, 30 and 60 min prevented theNPY-induced effect for all time-intervals used. Fig. 6C summarizesthe results at 60 min treatment.

Fig. 4. 3-D fishnet plot representations of hLEECs (A–C) and hREECs (D–F) showing the absence of effect of Y1R antagonist (BIBO3304 10�6 M) (B and E) on NPY (10�7 M)induced decrease of intracellular immunofluorescence labeling of ET-1 in hLEECs and hREECs. Panels A and D show the density and distribution of intracellular ET-1 in controlhLEECs (A) and hREECs (D). Panel C shows the absence of effect of the Y2R antagonist (BIIE0246 10�6 M) in the presence of NPY in hLEECs whereas panel F shows the completeprevention in hREECs. The antagonist was added 15 min prior to NPY addition for 60 min. Rotation of the fishnet plot cell was done in order to better show the maximumintensity and distribution of ET-1. The pseudocolor bar represents ET-1 fluorescence intensity ranging from 0 (absence of any fluorescence signal) to 255 (maximumfluorescence intensity). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

D. Abdel-Samad et al. / Neuropeptides 46 (2012) 373–382 379

Effect of Y5 receptor blockade on ET-1 secretion was studiedusing technique of ELISA, where the same treatment is done butwith the additional step of collecting samples for the determina-tion of the extracellular concentration of ET-1. Experiments donewith the Y5 receptor antagonist, L152,804, were conducted in thesame way. Results of the ELISA experiments for the blockade ofthe Y5 receptor are presented in Fig. 6B and D corresponding tohLEECs and hREECs, respectively.

Fig. 6B summarizes the effect of Y5 blockade on ET-1 secretionby NPY at 60 min treatment. It can be clearly observed that treat-ment of hLEECs with L-152,804 (10�6 M) + NPY (10�7 M) for60 min was able to completely inhibit the NPY-induced rise in

the extracellular level of ET-1 (control level is 3.612 pg/ml ± 0.416; level at 60 min NPY alone is 15.022 pg/ml ± 3.032; le-vel at 60 min L-152,804 + NPY is 5.447 pg/ml ± 0.866).

In the case of hREECs, it can be observed from Fig. 6D that de-spite the significant difference between the 60-min effect of NPY(10�7 M) alone as compared to that of L-152,804 (10�6 M) + NPY(10�7 M), the rise induced by the latter in the extracellular concen-tration of ET-1 was still highly significant when compared to thecontrol (control level is 4.5 pg/ml ± 0.567; level at 60 min L-152,804 + NPY is 10.309 pg/ml ± 0.963). This suggests the partialimplication of the Y5 receptor in the NPY-induced secretion ofET-1 at the level of hREECs.

Fig. 5. Effect of Y2 receptor blockade on NPY-induced decrease in intracellular (A and C) and increase in extracellular (B and D) ET-1 in hLEECs (A and B) and hREECs (C and D).Histograms showing measurements of ET-1 fluorescence intensity levels (A and C) and extracellular concentration (B and D) of ET-1 in hLEECs (A and B) and hREECs (C and D)in the absence (ctrl.; grey) and following 60 min of exposure to NPY (10�7 M; black) or NPY (10�7 M) in presence of the Y2 receptor antagonist, BIIE0246 (10�6 M; red). InhLEECs (A and B) the Y2 receptor antagonist did not prevent the effect of NPY, while in hREECs (C and D) the Y2 receptor antagonist completely prevented the effect of NPY.Results are presented as means ± SEM. ‘n’ is the number of different experiments of different culture cells. (⁄) is p < 0.05, (⁄⁄) is p < 0.01 and (⁄⁄⁄) is p < 0.001. (For interpretationof the references to colour in this figure legend, the reader is referred to the web version of this article.)

380 D. Abdel-Samad et al. / Neuropeptides 46 (2012) 373–382

4. Discussion

Our results clearly showed that NPY induced secretion of ET-1in both hLEECs and hREECs. The measured low level of decreaseof intracellular ET-1 when compared to the amount of releasedpeptide could be due in part to the fact that the measured intracel-lular ET-1 is actually the result of released minus the de novo syn-thesized peptide. In addition, our results indicate that the level ofcirculating NPY at both the exit (hREECs) and entry (hLEECs) ofthe circulation would contribute to ET-1 secretion. Thus, a cross-talk between these two peptides takes place in hEECs in general.Furthermore, our results showed that the hEECs are among the ma-jor contributors to ET-1 release.

Elevated NPY concentrations were reported in cases of hyper-tension and high plasma NPY immunoreactivity was detected inpatients with acute myocardial ischemia (Ullman et al., 1994a)and congestive heart failure (Ullman et al., 1994b). Moreover, ET-1 is also known to be involved in cardiac pathophysiological states,since the production of ET-1 was reported to be increased in casesof acute myocardial ischemia and myocardial infarction (Shahet al., 1996). Increased plasma ET-1 was also observed in cardiacfailure and seems to be of bad prognosis in congestive heart failure

(Kuruvilla and Kartha, 2003). Both NPY and ET-1 are also hypertro-phic factors for the cardiac muscle. Furthermore, this stimulatoryeffect of NPY on ET-1 secretion by venous and arterial EECs mayexplain the role that this type of cells plays as a tuning device forthe level of circulating ET-1. Thus NPY is one of the controllers ofthe tuning system of ET-1 in the circulating blood.

Our results showed that at a concentration near the physiolog-ical circulating NPY, this peptide induced secretion of ET-1. Hence,upon contact of NPY present in the venous blood with the rightendocardial endothelium, hREECs may release ET-1 into the circu-lation. Then hLEECs would adjust the level of circulating ET-1through uptake (Gray and Webb, 1996). Our results confirmedwhat was reported previously (Jacques et al., 2003) that hREECshad a higher level of ET-1 when compared to hLEECs. This couldprobably explain the higher secretory capacity of hREECs thanhLEECs. The latter type of cell seems to have more of a tuning rolefor the circulating arterial ET-1.

Our results showed that at the level of hREECs, blockade of theY1 receptor did not have any effect on the NPY-induced decrease ofintracellular ET-1 level. This suggests that the Y1 receptor is per-haps not implicated in the NPY-induced secretory effect on ET-1in this type of cells. In order to confirm the results obtained by

Fig. 6. Effect of Y5 receptor blockade on NPY-induced decrease in intracellular (A and C) and increase in extracellular (B and D) ET-1 in hLEECs (A and B) and hREECs (C and D).Histograms showing measurements of ET-1 fluorescence intensity levels (A and C) and extracellular concentration (B and D) of ET-1 in hLEECs (A and B) and hREECs (C and D)in the absence (ctrl.; grey) and following 60 min of exposure to NPY (10�7 M; black) or NPY (10�7 M) in presence of the Y5 receptor antagonist, L-152,804 (10�6 M; red). InhLEECs (A and B) the Y5 receptor antagonist completely prevented the effect of NPY, while in hREECs (C and D), the Y5 receptor antagonist partially prevented the release ofET-1 into the extracellular milieu by NPY. Results are presented as means ± SEM. ‘n’ is the number of different experiments of different culture cells. (⁄) is p < 0.05, (⁄⁄) isp < 0.01 and (⁄⁄⁄) is p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

D. Abdel-Samad et al. / Neuropeptides 46 (2012) 373–382 381

indirect immunofluorescence, we performed ELISA, the results ofwhich revealed that NPY elicited a rise in the extracellular concen-tration of ET-1 even after blockade of the Y1 receptor. These resultsclearly suggest that the Y1 receptor is not implicated in the secre-tion of ET-1 induced by NPY at the level of hREECs. These resultswere surprising especially since activation of this receptor subtypewas reported by our group to induce, in addition to the Y2 receptor,an increase in the intracellular calcium level in human hREECs (Jac-ques et al., 2003). This suggests that the absence of contribution ofY1 to NPY-induced secretion of ET-1 from hREECs could be due, inpart, to the insufficient increase of intracellular Ca2+ by Y1 receptoractivation. This shoud be verified in future studies. However,blockade of the Y2 receptor in hREECs prevented the NPY-inducedsecretion of ET-1 but not from hLEECs. These results clearly showthat the Y2 receptor is indeed implicated in mediating the secretoryeffect of NPY on ET-1 at the level of human hREECs but not hLEECs.These results also suggest that Y2 receptors’ activation is impli-cated in release of ET-1 at the exit of the venous circulation. Inaddition, these results suggest that normal or abnormal Y2 recep-tor functioning and/or density may indirectly, via ET-1 secretion byEECs, modulate the functioning of the right ventricule in health anddisease particularly in right ventricular hypertrophy. This lattershould be verified in the future.

It is well known in the literature that the Y1 receptor is the mainreceptor present in blood vessels and it is found in the heart aswell. So it was conventionally thought that this receptor is the ma-jor subtype implicated in peripheral cardiovascular control. Our re-sults, therefore, are unique since they show that the Y1 receptor isnot the main and/or major pathway by which NPY exerts its phys-iological and pathological actions in EECs and related adjacent cellssuch as cardiomyocytes, but that the Y2 receptor as well as proba-bly Y5 receptor pathways are highly and equally important. Inaddition, they show for the first time that Y2 receptor activationis implicated in the NPY-induced excitation–secretion coupling inhEECs which could affect not only the contractility of the subjacentventricular cardiomyocytes, but also contribute to the regulation ofthe normal functioning of the human heart. The contribution of Y2

receptor to ET-1 release by NPY only at the hREECs level demon-strates that these two types of EECs are different. This differencebetween right and left ventricular EECs regarding the excitation–secretion coupling response to NPY could, therefore, be due, atleast in part, to the presence or absence and/or differential densityof the Y2 receptors. This should be clarified in the future.

In contrast to the results obtained in hREECs, our results inhLEECs demonstrated that neither the single block of the Y1 northat of the Y2 receptor had any influence on the NPY-induced

382 D. Abdel-Samad et al. / Neuropeptides 46 (2012) 373–382

release of ET-1. Therefore, it is only logical to suggest that otherNPY receptors, such as the Y5 receptor could be implicated in theabove-mentioned effect.

As expected, at the level of hLEECs, blockade of Y5 receptor re-versed the effect of NPY-induced ET-1 secretion in this type of cells.These results show that the main receptor mediating the excita-tion–secretion coupling of NPY in hLEECs is the Y5 receptor. Fur-thermore, our results also showed that this receptor is at leastpartially, but significantly contributing also to NPY-induced secre-tion in hREECs. This new and important role of the Y5 receptor inmediating the NPY-induced secretion of ET-1 in left ventricularEECs is consistent with the reports available in the literature aboutits implication in cardiac hypertrophy (Pellieux et al., 2000; Bell etal., 2002). This is because ET-1, similar to NPY, is known to be a po-tent hypertrophic factor of the heart, so it is possible that NPYmediates its hypertrophic action on the myocardium, in part viaits induction of ET-1 release from endocardial endothelial cells.

In conclusion, the results obtained in this study clearly demon-strate that a dialog indeed exists between the systems of NPY andET-1 at the level of human endocardial endothelial cells. The exis-tence of interactions or crosstalk between two cardioactive media-tors is not uncommon. The existence of such crosstalk in theendocardial endothelium, which is known for its very close proxim-ity to the underlying myocardium, serves to endorse the importantand rather indispensable role that these cells play in regulating car-diac growth, contractile performance and rhythmicity.

Acknowledgements

This study was supported by a Grant from the Canadian Insti-tutes of Health Research (CIHR) and Quebec Heart and StrokeFoundation (QHSF) to Dr Danielle Jacques.

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