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gy 579 (2008) 233–240www.elsevier.com/locate/ejphar

European Journal of Pharmacolo

Endothelin receptor-mediated vasodilatation: Effects of organ culture

David Nilsson a, Angelica Wackenfors a, Lotta Gustafsson a, Martin Ugander c, Per Paulsson d,Richard Ingemansson d, Lars Edvinsson a, Malin Malmsjö a,b,⁎

a Department of Medicine, Lund University Hospital, Lund, Swedenb Department of Ophthalmology, Lund University Hospital, Lund, Sweden

c Department of Clinical Physiology, Lund University Hospital, Lund, Swedend Department of Cardiothoracic Surgery, Lund University Hospital, Lund, Sweden

Received 17 May 2007; received in revised form 8 August 2007; accepted 25 September 2007Available online 3 October 2007

Abstract

Culture of intact arteries is a frequently employed experimental model for investigating the mechanisms governing the regulation of vascularendothelin receptors. Endothelin type A (ETA) and type B (ETB) receptors on vascular smooth muscle cells are up-regulated in organ culture andthe enhanced vasoconstriction mimics the changes that occur in cardiovascular disease. The effect of organ culture on endothelial dilatoryendothelin ETB receptors is not known. We hypothesize that organ culture decreases the endothelin receptor-mediated dilatation and that this isone possible mechanism by which the effects of the endothelin in blood vessels are altered during culture.

Porcine coronary arteries were studied before and after 24 h of culture, using in vitro pharmacology and immunofluorescence. Sarafotoxin 6cand endothelin-1 were used to examine the endothelin ETA and ETB receptor effects, and the antagonists, Nω-nitro-L-arginine (L-NOARG) fornitric oxide (NO), indomethacin for prostaglandins and charybdotoxin in combination with apamin for endothelium-derived hyperpolarizing factor(EDHF), were used to study the endothelium-derived dilatory mediators.

Organ culture induced up-regulation of the sarafotoxin 6c (ETB receptor agonist) and endothelin-1 (ETA receptor agonist) elicitedvasoconstriction. The sarafotoxin 6c contraction was stronger after endothelium denudation, suggesting endothelium-dependent dilatation. Theendothelin-1 contraction was not affected by endothelium denudation. The increase in sarafotoxin 6c contraction after removal of the endotheliumwas more pronounced before than after organ culture, suggesting down-regulated endothelial endothelin ETB receptors. Also, theimmunofluorescence staining intensities for endothelial endothelin ETB receptors were higher before than after organ culture. Pre-incubationwith inhibitors for dilatory mediators suggested that both NO and EDHF play a vasodilatory role, while prostaglandins are not involved.

In conclusion, endothelial endothelin ETB receptors induce NO and EDHF mediated vasodilatation in porcine coronary arteries. In organculture, endothelial endothelin ETB receptors are down-regulated, mimicking the changes that occur in cardiovascular disease. Down-regulation ofendothelial endothelin ETB receptors may in part explain the increased endothelin ETB receptor-mediated vasoconstriction frequently studied inorgan culture.© 2007 Elsevier B.V. All rights reserved.

Keywords: Endothelin; Endothelium; Coronary artery; Vascular; Vasodilatation

1. Introduction

The endothelium displays a variety of biological functions,such as regulating the underlying smooth muscle cells and

⁎ Corresponding author. Experimental Vascular Research, BMC A13, LundUniversity Hospital, SE-221 84 Lund, Sweden. Tel.: +46 733 565 650; fax: +4646 222 0616.

E-mail address: malin.malmsjo@med.lu.se (M. Malmsjö).

0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2007.09.031

vascular tone by releasing endothelium-derived dilatory factorssuch as nitric oxide (NO), prostaglandins and endothelium-derived hyperpolarizing factor (EDHF), as well as vasocontractilefactors such as endothelin-1 (ET-1). ET-1 is one of themost potentvasoconstrictors known and is the most abundant peptide subtypeof the endothelin family in the human cardiovascular system(Inoue et al., 1989;Yanagisawa et al., 1988). ET-1 has been shownto play an important role in the regulation of normalcardiovascular functions, such as controlling the basal coronary

234 D. Nilsson et al. / European Journal of Pharmacology 579 (2008) 233–240

artery tone. An increased circulating level of ET-1 has beenassociated with cardiovascular pathologies including hyperten-sion, and has been suggested as a prognostic marker in ischaemicheart disease and congestive heart failure (Mundhenke et al.,1999; Neunteufl et al., 2002). Besides its vasoactive properties,ET-1 also acts as a mitogen of vascular smooth muscle cells, bystimulating extracellular matrix synthesis and attracting mono-cytes in the process of atherosclerosis (Achmad and Rao, 1992;Alberts et al., 1994; Rizvi et al., 1996; Weissberg et al., 1990).

Inmammals, ET-1mediates its action via twoG-protein-coupledreceptor subtypes: the endothelin type A (ETA) and type B (ETB)receptors (Arai et al., 1990; Sakurai et al., 1990). The endothelinETA receptor is the dominant receptor subtype on vascular smoothmuscle cells and mediates contraction, while the endothelin ETBreceptor is located on endothelial cells and mediates vasodilatation(Maguire and Davenport, 1995). The dilatory response mediated byendothelin ETB receptors has mainly been ascribed not only to NO(Hirata et al., 1993; Namiki et al., 1992), but also to prostaglandins(de Nucci et al., 1988; Filep et al., 1991). Recent studies on ratcarotid and cerebral arteries suggest that endothelin receptors onendothelial cells might also induce dilatation via EDHF (Szok et al.,2001; Tirapelli et al., 2005). So far, no reports have been publishedon the role of EDHF in the endothelin stimulated dilatation incoronary arteries.

Endothelin ETB receptor-mediated vasoconstriction has beenfound to be increased in cerebral vasospasm, stroke, ischaemic heartdisease and atherosclerosis (Dagassan et al., 1996; Henriksson et al.,2003; Roux et al., 1995; Wackenfors et al., 2004; Wenzel et al.,1996). Culture of human coronary, human internal mammary, ratomental, rat mesenteric and rat cerebral arteries for 24–48 h inducessimilar up-regulation of endothelin ETB receptor-mediated contrac-tion (Adner et al., 1996, 1995;Möller et al., 1997;Wackenfors et al.,2004), and has therefore been suggested as an experimental modelfor studying endothelin receptor plasticity in cardiovascular disease.The increase in endothelin contraction in organ culture has beenattributed to the up-regulation of contractile endothelin receptors onsmooth muscle cells. In cardiovascular disease, endotheliumdysfunction develops and the increase in endothelin contraction isprobably due to a combination of increased contractile endothelinreceptors on smooth muscle cells and decreased dilatory endothelinreceptors on endothelial cells. The effect of organ culture on dilatoryendothelin receptors on the endothelium has not yet been evaluated.Since organ culture is a frequently utilized experimental model forstudying the regulation of endothelin receptor-mediated contraction,calls have beenmade for studies on the effects of dilatory endothelinreceptor. In the present study, the effects of organ culture on thedilatory endothelin ETA and ETB receptors and the mediators, NO,prostaglandins and EDHFwere examined by in vitro pharmacologyand immunofluorescence.

2. Materials and methods

2.1. In vitro pharmacology

2.1.1. Tissue preparationThe coronary arteries were obtained from domestic landrace

pigs. The pigs were of both genders and had a mean body weight of

50 kg. Before surgery they were fasted overnight with free access towater. Anaesthesia was induced by an intramuscular injection ofketamine (Ketaminol vet™ 100mg/ml; Farmaceutici Gellini S.p.A,Aprilia, Italy), 15mg/kg body weight, in combination with xylazine(Rompun vet.™ 20mg/ml; Bayer AG, Leverkusen, Germany), and2 mg/kg. Anaesthesia was maintained by continuous intravenousinfusion of propofol (0.1–0.2 mg/kg/min, Diprivan™ 20 mg/ml;Astra Zeneca, Sweden) in combination with intermittent fentanyl(0.02 μg/kg, Leptanal™; Lilly, France) and atracurium besylate(0.2–0.5 mg/kg, Tracrium™; Glaxo, Täby, Sweden). The heartswere removed through a midline sternotomy and immediately puton ice. The left anterior descending coronary artery was dissected,immersed in cold sterile Dulbeccos' modified Eagles' medium(DMEM) and transported to the laboratory on ice. In the laboratory,the coronary arteries were dissected free from adhering tissue, andthen cut into cylindrical segments (3–4 mm long). For certainexperiments, endothelium denudation was performed by gentlyscraping the luminal side of the artery with a piece of metal wire(Hamel et al., 1987).

2.1.2. Experimental setupThe coronary arterial segments from each pig were divided into

two groups; one was subjected to organ culture for 24 h (cultured)and the other was immediately used for pharmacological experi-ments (control). As the left anterior descending coronary arteryalters in dimensions and reactivity from proximal to distal regionsand in order to minimize the effect of regional variability, adjacentsegments were used for control and vehicle experiments,respectively. The arterial segments used for culture were placed inwells containing 1 mL DMEM and incubated at 37 °C inhumidified 5% CO2 in air, as described previously (Adner et al.,1996). Both control and cultured segments weremounted on two L-shaped metal prongs, one of which was connected to a forcedisplacement transducer for continuous recording of the isometrictension (Högestätt et al., 1983). The mounted artery segments wereimmersed in temperature-controlled (37 °C) tissue baths containingbicarbonate-based buffer solution of the following composition(mmol/l): NaCl 119; NaHCO3 15; KCl 4.6; MgCl2 1.2; NaH2PO4

1.2; CaCl2 1.5 and glucose 5.5. The buffer was continuously gassedwith 5% CO2 in O2 resulting in a pH of 7.4. The segments wereallowed to stabilize at a resting tension of 4 mN for 30–60 minbefore the experiments were started. A resting tension of 2 to 8 mNprovides propitious conditions for studying vascular contraction incoronary arteries (Saetrum Opgaard and Edvinsson, 1997; Wack-enfors et al., 2003). The contractile capacity of each artery segmentwas determined by exposure to a potassium-rich (63.5 mmol/l)buffer solution, with the same composition as the bicarbonate buffersolution, except that some of the NaCl was replaced by KCl.Dilatory responses were assessed by cumulative addition ofadenosine 5′-O-thiodiphosphate (ADPβs, 0.1 nM–0.1 mM) inarteries pre-contracted with U46619 (thromboxane analogue,0.1 mM) and abolished dilatation indicated a properly removedendothelium. Endothelium removal did not affect the contractilecapacity of the vessel (13±1 mNwith an intact and 12±2 mN witha removed endothelium (non-cultured), P=n.s.).

The dilatory component of the ET-1 (an endothelin ETA andETB receptor agonist, 10−11–10−6 mM) and sarafotoxin 6c

Fig. 1. Concentration–response curves to increasing concentrations ofsarafotoxin 6c (A) and endothelin-1 (B) in non-cultured (control) and culturedarterial segments. Values are presented as mean values±S.E.M. Statisticalanalyses were performed using Students' t-test where pb0.05 was consideredsignificant.

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(sarafotoxin 6c, an endothelin ETB receptor agonist, 10−11–10−6 mM) induced vasoconstriction was studied by adding thesubstances at increasing concentrations, in the presence orabsence of NO, prostaglandin and EDHF inhibitors. NO wasstudied after inhibiting prostaglandins with 10 μM indomethacinand EDHF with 50 nM charybdotoxin and 1 μM apamin. EDHFwas studied in the presence of the NO synthetase inhibitor Nω-nitro-L-arginine (L-NOARG, 0.1 mM) and indomethacin, whileprostaglandins were studied in the presence of L-NOARG,charybdotoxin and apamin. The endothelin ETB receptor agonist,sarafotoxin 6c, was first added at increasing concentrations. Thearteries werewashed, the NO, prostaglandin and EDHF inhibitorswere then added again and thereafter the effects of ET-1 atincreasing concentrations were examined. At this stage theendothelin ETB receptors were desensitized (Henriksson et al.,2004; Lodge et al., 1995), allowing ET-1 to act selectively onendothelin ETA receptors. In order to further check thedesensitization of endothelin ETB receptors, the effect ofthe selective endothelin ETB receptor antagonist, BQ788(0.1 μmol/L), on the ET-1 contraction after sarafotoxin 6cdesensitization, was evaluated. The ET-1 contraction was similarboth in the presence and absence of BQ788, suggesting activationof endothelin ETA receptors solely.

2.1.3. Drugs and solutionsSerum-free DMEM (D-glucose, 1000 mg/l) contained sodium

pyruvate (100 mg/l) and was supplemented with penicillin(100 U/ml) and streptomyocin (100 μg/ml), purchased fromGibco BRL, Praisley, UK. All drugs for the in vitro pharmaco-logical experiments were purchased from Sigma Co. (USA),except for BQ788 that was purchased from Neosystem,Strasbourg, France. ET-1 and sarafotoxin 6c were dissolved in0.9% saline with 10% albumin. The other drugs were dissolved in0.9% saline.

2.2. Immunofluorescence

2.2.1. Tissue preparationThe arteries were frozen in liquid nitrogen and stored at

−80 °C until sectioning. Prior to analyzing, the arteries weresectioned into 7 μm slices using a cryostat (Microm HM500M,Germany) and placed on microscope poly-L-lysin coated glassslides (Menzel, Braunschweig, Germany), three sections on eachslide. The slides were fixed for 10 min in ice cold acetone and re-hydrated in PBS for 10 min. The slides were allowed to dry inroom temperature for 30min to 1 h andwere then stored at−20 °Cuntil further use.

2.2.2. ImmunofluorescenceEndothelin ETA and ETB receptor protein expression was

visualized using immunofluorescence. The tissues were permea-bilized and blocked in 0.2% Triton X-100/1% bovine serumalbumin (BSA) in PBS for 1 h followed by incubation overnightwith mouse anti-porcine CD31 (Serotec, Raleigh, NC, USA) 1:3and rabbit anti-human endothelin ETB receptor (IBL-10253,Immuno-Biological Laboratories Co., Gunma, Japan) 1:50 orgoat anti-human endothelin ETA receptor (R-19, Santa Cruz

Biotechnology Inc., Santa Cruz, CA, USA) 1:50 in PBS with 1%BSA. The slides were washed twice in 0.2% Triton X-100/1%bovine serum albumin (BSA) in PBS followed by incubationovernight with donkey anti-mouse Cy™5 (Jackson ImmunoR-esearch Laboratories Inc., West Grove, PA, USA) 1:200 andgoat–anti-rabbit Alexa-633 (Molecular Probes, or donkey anti-goat Cy™3 (Jackson ImmunoResearch Laboratories Inc., WestGrove, PA, USA) 1:200 or in PBS with 1%BSA. The slides werewashed twice in PBS and analyzed by confocal microscopy. Forillustration of vessel morphology, a transmission image,corresponding to each immunofluorescence image, was capturedby a high resolution CCD camera. All arteries for immunoflu-orescence were examined at the same time to minimize thevariability (Boi and Fascio, 1998; Dai and Galligan, 2006).Confocal scans were recorded with identical settings in eachexperiment and optical sections were set to 1.0 mm. Primary andsecondary antibody controls were run in parallel in eachexperiment. The staining intensities for endothelin ETA andETB receptor protein was quantified using confocal microscopyand the Image J software (http://rsb.info.nih.gov/ij/).

2.3. Calculations and statistics

Calculations and statistical analysis were performed usingGraphPadPrism 4.0 software (GraphPad Software, Inc., SanDiego, CA, USA). The negative logarithm of the drugconcentration that elicited 50% contraction (pEC50) wasdetermined by linear regression analysis using the values

Fig. 2. Concentration–response curves to increasing concentrations ofsarafotoxin 6c in arteries with intact (control) and removed endothelium(endothelium denuded). Experiments were performed in (A) non-cultured and(B) cultured arterial segments. Values are presented as mean values±S.E.M.Statistical analyses were performed using Students' t-test where pb0.05 wasconsidered significant.

Fig. 3. Confocal images showing representative examples of immunofluores-cence staining for endothelin ETB (top panels, red) and endothelin ETA receptors(bottom panels, green), before (0 h) and after culture (24 h). The images arecaptured to show both the endothelial cell (EC) and smooth muscle cell (SMC)layer, where the vessel lumen is on the top of the images. To the right of eachimmunofluorescence image is a transmission image for illustration ofmorphology. Note how the endothelin ETB receptor fluorescence is less intensein the endothelial cell layer, after culture, while the staining intensity forendothelial endothelin ETA receptors remains unchanged.

236 D. Nilsson et al. / European Journal of Pharmacology 579 (2008) 233–240

immediately above and below half-maximum response. Themaximum contraction was calculated in percent of contractionstimulated by 63.5 mmol/L potassium, and maximum dilatationwas calculated as a percentage of the corresponding pre-contraction with U46619 (0.1 mM). The maximum contractionor dilatation has been referred to as Emax. The immunofluores-cence staining intensities will be referred to as arbitraryfluorescence units (FU). The experiments were performed inarteries from 6–10 pigs for each substance, and statisticalsignificance was defined as pb0.05, using Student's t-test.Values are presented as means±S.E.M.

2.4. Ethics

The study was approved by the Ethics Committee of LundUniversity in Sweden.

3. Results

3.1. Contractile endothelin ETA and ETB receptors on smoothmuscle cells

The endothelin ETB receptor-mediated contractile responsewas studied using the selective receptor agonist sarafotoxin 6c.The ET-1-induced vasoconstriction was studied after desensi-tizing the endothelin ETB receptors with sarafotoxin 6c prior toadding ET-1, leaving only endothelin ETA receptors to respond

(Henriksson et al., 2004; Lodge et al., 1995). The ET-1 andsarafotoxin 6c stimulated vasoconstrictions were stronger afterculture (Fig. 1). Immunofluorescence experiments demonstrat-ed increased endothelin ETA and ETB receptors staining on thevascular smooth muscle cells after organ culture (Fig. 3). Thepotassium-contraction was not affected by organ culture (13±1 mN before and 14±2 mN after organ culture, P=n.s.),indicating unchanged smooth muscle cell function.

3.2. Dilatory endothelin receptors on endothelial cells

First we tried to analyze the ET-1 and sarafotoxin 6cdilatation after U46619 pre-contraction. No dilatory responsewas observed when sarafotoxin 6c was added at increasingconcentrations, presumably because of its strong vasocontrac-tile properties. In a second series of experiments, the ET-1 andsarafotoxin 6c dilatation was studied by means of alteredvasoconstriction. In non-cultured arteries, the contractilesarafotoxin 6c response was stronger after removal of theendothelium (Fig. 2A), thus indicating endothelin ETB receptormediated, endothelium-dependent dilatation. After organ cul-ture, the contractile sarafotoxin 6c response was similar inarteries with and without an intact endothelium (Fig. 2B). Takentogether, these results suggest down-regulated endothelialdilatory endothelin ETB receptors during culture.

The ET-1 contraction was not altered by removal of theendothelium, before or after culture (Emax=132±11% withendothelium and 117±10% without endothelium in non-

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cultured arteries and Emax=147±39% with endothelium and135±18% without endothelium in cultured arteries), suggestingthe absence of an endothelium dependent, endothelin ETAreceptor-mediated dilatation.

Immunofluorescence staining intensity for endothelin ETB

receptors on endothelial cells was lower after culture (FU=82±21% before and 36±8% after culture, Fig. 3), supporting the invitro pharmacology results that the endothelial endothelin ETB

receptors are down-regulated during culture. Faint staining wasalso observed for endothelin ETA receptors on endothelial cells.This was not altered by organ culture (FU=42±17% before and36±8% after culture, Fig. 3).

3.3. Endothelium-derived mediators involved in the endothelinETB dilatation

In non-cultured arteries, the sarafotoxin 6c contraction wassignificantly stronger when only prostaglandins were made

Fig. 4. Concentration–response curves to increasing concentrations of sarafotoxin 6cafter inhibiting prostaglandins with 10 μMindomethacin and EDHFwith 50 nMcharybof L-NOARG, charybdotoxin and apamin (B and E), while EDHFwas studied in the pand F). Values are presented as mean values±S.E.M. Statistical analyses where perfo

available for dilatation (pre-incubated with L-NOARG, char-ybdotoxin and apamin, Fig. 4), indicating that prostaglandinsplay a minor vasodilatory role in coronary arteries. In non-cultured arteries, in which only NO or EDHF was madeavailable for dilatation, sarafotoxin 6c induced weakervasoconstriction, indicating that these mediators have avasodilatory role (Fig. 4). In cultured arteries, the contractionwas not affected by the presence of inhibitors for NO,prostaglandins or EDHF (Fig. 4).

3.4. Endothelial cell function

ADPβs elicited considerable vasodilatation after pre-incu-bation with U46619 (Rmax=81±6% of U46619 pre-constric-tion). After removal of the endothelium, ADPβs caused nodilatation indicating that the response was endotheliumdependent. After organ culture the dilatory response toADPβs was diminished (Rmax=44±12% of U46619 pre-

in non-cultured (control) and cultured porcine coronary arteries. NO was studieddotoxin and 1μMapamin (A andD). Prostaglandinswere studied in the presenceresence of the NO synthetase inhibitor L-NOARG (0.1 mM) and indomethacin (Crmed using Students' t-test where pb0.05 was considered significant.

238 D. Nilsson et al. / European Journal of Pharmacology 579 (2008) 233–240

constriction, pb0.01), presumably due to reduced endothelialfunction.

4. Discussion

4.1. Main findings

In the present study we have shown for the first time thatsarafotoxin 6 stimulated dilatation is decreased after organculture. Down-regulation of endothelial endothelin ETB receptorsmay in part explain the increase in endothelin ETB receptor-mediated vasoconstriction frequently seen in organ culture. Caremust therefore be undertaken when interpreting the results oforgan culture experiments, and it may be necessary to evaluate theendothelium and smooth muscle cell function separately whenelucidating endothelin receptor-mediated responses.

4.2. Contractile endothelin receptors in organ culture

Changes in endothelin receptor expression have been shown totake place in several pathophysiological conditions, such asatherosclerosis (Dagassan et al., 1996), ischaemic stroke (Henriks-son et al., 2003; Patel et al., 1996) and ischaemic heart disease(Wackenfors et al., 2004). In these conditions, endothelin ETA andETB receptors on vascular smoothmuscle cells have been observedto be up-regulated. Endothelin receptors on vascular smoothmuscle cells are both mitogenic, leading to atherosclerosis, andvasocontractile, which may lead to the elevated vascular tonefrequently observed in cardiovascular disease. The endothelinreceptor regulation has previously been studied by means of organculture. Culture of artery segments induces similar up-regulation ofcontractile endothelin receptors to that seen in cardiovasculardisease (Wackenfors et al., 2004). Endothelin activity depends on abalance between the smooth muscle and endothelial effects.Endothelin receptors on smooth muscle cells mediate vasocon-striction, while endothelin receptors on endothelial cells mediatedilatation. The enhanced vasocontractile effect of sarafotoxin 6cduring organ culture might be the result of both an increase in thenumber of contractile endothelin receptors in smooth muscle and adecrease in dilatory endothelin receptors on endothelial cells,although this possibility has not yet been elucidated. Since organculture is a commonly practiced model for studying endothelinreceptor regulation, we think that it is important to analyze theeffect of organ culture on the dilatory effects of endothelin receptorson the endothelium. The long term aim is to find out how topharmacologically interact with the endothelin receptor regulationand hopefully identify new therapeutical targets.Organ culture is anin vitro model in which inhibitors of different signal-transductionpathways can be examined in detail. One step in this long term planis tomake a detailed analysis of the effect on endothelial endothelinreceptors by organ culture, which is done in the current study.

4.3. The method of studying endothelin dilatation

Dilatation is commonly studied in blood vessels that have beenpre-constricted by e.g. U46619 or norepinephrine. After reachingmaximal contraction, a dilatory agent is added and the

concentration-dependent dilatation studied. In the current exper-imental setup, endothelin did not elicit vasodilatation in pre-contracted arteries. Presumably, a strong vasoconstrictor likeendothelin will mainly elicit contraction by activation of receptorson smooth muscle cells. There are two ways of establishingwhether this is the case. The first is to use a perfusion system inwhich the pharmacological agent is added to the perfusate andthus acts only via the endothelium. This can be done in cerebralarteries where the blood–brain barrier prevents the substancefrom reaching the smooth muscle cell layer (Szok et al., 2001).The second is to examine the contractile response and the way inwhich it is affected by removal of the endothelium or by inhibitorsof dilatory mediators. Increased contraction will then reflectdilatation. This was the method chosen for the current study.

4.4. Endothelium dysfunction in organ culture

The present results show that the endothelium functiondiminishes during organ culture and that the dilatory endothelinETB receptors on the endothelium are down-regulated. Down-regulation of endothelial endothelin ETB receptors may in partexplain the increased endothelin ETB receptor-mediatedvasoconstriction frequently seen in organ culture. Usingacetylcholine, we have previously demonstrated that organculture stimulates the development of endothelium dysfunction(Alm et al., 2002) in a similar way to that in cardiovasculardiseases (de Meyer, 2000). In the current experiments, we choseto examine the endothelium function further by using ADPβs.ADPβs is a strong vasodilator that has proven especiallyprominent in the arterial coronary circulation (Malmsjo et al.,2000). ADPβs dilatation decreased during organ culture,suggesting reduced endothelium function. The dilatory effectsof ADPβs disappeared after removal of the endothelium,indicating that this dilatation was endothelium dependent.

4.5. Dilatory endothelin receptors in cardiovascular disease

Only a limited number of studies exist where the role ofendothelial endothelin receptors in cardiovascular disease hasbeen investigated. However it was shown that NO release inresponse to stimulation of endothelin ETB receptors wasreduced in renal rat vessels with hypertension, diabetes mellitusand hypercholesterolemia (Kakoki et al., 1999). Similarly, weshow that endothelial endothelin ETB receptors are down-regulated in organ culture. Attenuation of the vasodilatoryproperty of the endothelium in organ culture was verified in thepresent experiment by using the vasodilator ADPβs and hasbeen shown before using acetylcholine in rat mesenteric arteries(Alm et al., 2002). Organ culture may serve as a suitableexperimental model in which to study the endothelial endothelinETB receptor regulation in detail in order to further elucidate themechanisms involved in endothelium dysfunction.

4.6. Dilatory mediators

Only few reports exist on the dilatory effect of endothelinreceptors on the endothelium (Climent et al., 2005; D'Orleans-

239D. Nilsson et al. / European Journal of Pharmacology 579 (2008) 233–240

Juste et al., 2002; Szok et al., 2001; Ushio-Fukai et al., 1992).These studies have mainly reported an NO mediated, but alsominor prostaglandin and EDHF mediated dilatory effects afteractivation of endothelin ETB receptors on the endothelium. Inthe present study, the dilatory mechanisms of endothelinreceptors were examined in the porcine coronary circulation.Previous studies on the coronary circulation have shown thatstimulation of endothelin ETB receptors results in NO andprostaglandin mediated dilatation that was endothelium depen-dent (Climent et al., 2005; Ushio-Fukai et al., 1992). Hitherto,no reports have been published on the role of EDHF in theendothelin stimulated dilatation in coronary arteries. Our resultssuggest that NO and EDHF are the principal endothelium-dependent mediators inducing endothelin ETB receptor-medi-ated dilatation in porcine coronary arteries.

4.7. Conclusions

Culture of intact arteries has been suggested as anexperimental model suitable for elucidating the mechanismsgoverning the regulation of vascular endothelin receptors.Endothelin ETA and ETB receptors on vascular smooth musclecells are up-regulated in organ culture and the enhancedvasoconstriction mimics the changes that occur in cardiovas-cular disease. The effects of organ culture on dilatory endothelinETA and ETB receptors on the endothelium, and the possiblecontribution of endothelium dysfunction to the increase inendothelin-induced vasoconstriction, have been sought after.We have shown that endothelial endothelin ETB receptors aredown-regulated, mimicking the changes that occur in cardio-vascular disease. Down-regulation of endothelial endothelinETB receptors may in part explain the increase in endothelinETB receptor-mediated vasoconstriction frequently seen inorgan culture.

Acknowledgements

This study was supported by the Åke Wiberg Foundation, theM. Bergvall Foundation, Anna Lisa and Sven-Eric Lundgrensfoundation for medical research, the Anders Otto SwärdsFoundation / Ulrika Eklunds Foundation, the Swedish MedicalAssociation, the Royal Physiographic Society in Lund, theSwedishMedical ResearchCouncil, the Crafoord Foundation, theSwedish Heart–Lung Foundation, the Swedish GovernmentGrant for Clinical Research and the Swedish HypertensionSociety.

References

Achmad, T.H., Rao, G.S., 1992. Chemotaxis of human blood monocytes towardendothelin-1 and the influence of calcium channel blockers. Biochem.Biophys. Res. Commun. 189, 994–1000.

Adner, M., Erlinge, D., Nilsson, L., Edvinsson, L., 1995. Upregulation of a non-ETA receptor in human arteries in vitro. J. Cardiovasc. Pharmacol. 26,S314–S316.

Adner, M., Cantera, L., Ehlert, F., Nilsson, L., Edvinsson, L., 1996. Plasticity ofcontractile endothelin-B receptors in human arteries after organ culture. Br.J. Pharmacol. 119, 1159–1166.

Alberts, G.F., Peifley, K.A., Johns, A., Kleha, J.F., Winkles, J.A., 1994.Constitutive endothelin-1 overexpression promotes smooth muscle cellproliferation via an external autocrine loop. J. Biol. Chem. 269, 10112–10118.

Alm, R., Edvinsson, L., Malmsjo, M., 2002. Organ culture: a new model forvascular endothelium dysfunction. BMC Cardiovasc. Disord. 2, 8.

Arai, H., Hori, S., Aramori, I., Ohkubo, H., Nakanishi, S., 1990. Cloning andexpression of a cDNA encoding an endothelin receptor. Nature 348, 730–732.

Boi, S., Fascio, U., 1998. A method of quantitative measurement of fluorescenceintensity by confocal laser scanning microscopy. J. Comput.-Assist.Microsc. 10, 163–166.

Climent, B., Fernandez, N., Sanz, E., Sanchez, A., Monge, L., Garcia-Villalon,A.L., Dieguez, G., 2005. Enhanced response of pig coronary arteries toendothelin-1 after ischemia–reperfusion. Role of endothelin receptors, nitricoxide and prostanoids. Eur. J. Pharmacol. 524, 102–110.

Dagassan, P.H., Breu, V., Clozel,M., Kunzli, A., Vogt, P., Turina,M., Kiowski,W.,Clozel, J.P., 1996. Up-regulation of endothelin-B receptors in atherosclerotichuman coronary arteries. J. Cardiovasc. Pharmacol. 27, 147–153.

Dai, X., Galligan, J.J., 2006. Differential trafficking and desensitization ofhuman ET(A) and ET(B) receptors expressed in HEK 293 cells. Exp. Biol.Med. (Maywood) 231, 746–751.

de Meyer, G.H., 2000. AG: nitric oxide and vascular endothelium dysfunction.Nitric oxide; biology and pathobiology, pp. 547–568.

de Nucci, G., Thomas, R., D'Orleans-Juste, P., Antunes, E., Walder, C., Warner,T.D., Vane, J.R., 1988. Pressor effects of circulating endothelin are limited byits removal in the pulmonary circulation and by the release of prostacyclinand endothelium-derived relaxing factor. Proc. Natl. Acad. Sci. U. S. A. 85,9797–9800.

D'Orleans-Juste, P., Labonte, J., Bkaily, G., Choufani, S., Plante,M., Honore, J.C.,2002. Function of the endothelin(B) receptor in cardiovascular physiology andpathophysiology. Pharmacol. Ther. 95, 221–238.

Filep, J.G., Battistini, B., Cote, Y.P., Beaudoin, A.R., Sirois, P., 1991.Endothelin-1 induces prostacyclin release from bovine aortic endothelialcells. Biochem. Biophys. Res. Commun. 177, 171–176.

Hamel, E., Assumel-Lurdin, C., Edvinsson, L., Fage, D., MacKenzie, E.T.,1987. Neuronal versus endothelial origin of vasoactive acetylcholine in pialvessels. Brain Res. 420, 391–396.

Henriksson, M., Stenman, E., Edvinsson, L., 2003. Intracellular pathwaysinvolved in upregulation of vascular endothelin type B receptors in cerebralarteries of the rat. Stroke 34, 1479–1483.

Henriksson, M., Xu, C.B., Edvinsson, L., 2004. Importance of ERK1/2 inupregulation of endothelin typeB receptors in cerebral arteries. Br. J. Pharmacol.142, 1155–1161.

Hirata, Y., Emori, T., Eguchi, S., Kanno, K., Imai, T., Ohta, K., Marumo, F.,1993. Endothelin receptor subtype B mediates synthesis of nitric oxide bycultured bovine endothelial cells. J. Clin. Invest. 91, 1367–1373.

Högestätt, E.D., Andersson, K.E., Edvinsson, L., 1983. Mechanical propertiesof rat cerebral arteries as studied by a sensitive device for recording ofmechanical activity in isolated small blood vessels. Acta Physiol. Scand.117, 49–61.

Inoue, A., Yanagisawa, M., Kimura, S., Kasuya, Y., Miyauchi, T., Goto, K.,Masaki, T., 1989. The human endothelin family: three structurally andpharmacologically distinct isopeptides predicted by three separate genes.Proc. Natl. Acad. Sci. U. S. A. 86, 2863–2867.

Kakoki, M., Hirata, Y., Hayakawa, H., Tojo, A., Nagata, D., Suzuki, E., Kimura,K., Goto, A., Kikuchi, K., Nagano, T., Omata, M., 1999. Effects ofhypertension, diabetes mellitus, and hypercholesterolemia on endothelintype B receptor-mediated nitric oxide release from rat kidney. Circulation99, 1242–1248.

Lodge, N.J., Zhang, R., Halaka, N.N., Moreland, S., 1995. Functional role ofendothelin ETA and ETB receptors in venous and arterial smooth muscle.Eur. J. Pharmacol. 287, 279–285.

Maguire, J.J., Davenport, A.P., 1995. ETA receptor-mediated constrictorresponses to endothelin peptides in human blood vessels in vitro. Br. J.Pharmacol. 115, 191–197.

Malmsjo, M., Hou, M., Harden, T.K., Pendergast, W., Pantev, E., Edvinsson, L.,Erlinge, D., 2000. Characterization of contractile P2 receptors in humancoronary arteries by use of the stable pyrimidines uridine 5′-O-thiodiphosphateand uridine 5′-O-3-thiotriphosphate. J. Pharmacol. Exp. Ther. 293, 755–760.

240 D. Nilsson et al. / European Journal of Pharmacology 579 (2008) 233–240

Möller, S., Edvinsson, L., Adner, M., 1997. Transcriptional regulated plasticityof vascular contractile endothelin ET(B) receptors after organ culture. Eur. J.Pharmacol. 329, 69–77.

Mundhenke,M., Schwartzkopff, B., Kostering,M.,Deska,U.,Klein, R.M., Strauer,B.E., 1999. Endogenous plasma endothelin concentrations and coronarycirculation in patients with mild dilated cardiomyopathy. Heart 81, 278–284.

Namiki, A., Hirata, Y., Ishikawa, M., Moroi, M., Aikawa, J., Machii, K., 1992.Endothelin-1- and endothelin-3-induced vasorelaxation via commongeneration of endothelium-derived nitric oxide. Life Sci. 50, 677–682.

Neunteufl, T., Berger, R., Pacher, R., 2002. Endothelin receptor antagonists incardiology clinical trials. Expert Opin. Investig. Drugs 11, 431–443.

Patel, T.R., Galbraith, S., McAuley, M.A., McCulloch, J., 1996. Endothelin-mediated vascular tone following focal cerebral ischaemia in the cat.J. Cereb. Blood Flow Metab. 16, 679–687.

Rizvi,M.A.,Katwa, L., Spadone,D.P.,Myers, P.R., 1996. The effects of endothelin-1 on collagen type I and type III synthesis in cultured porcine coronary arteryvascular smooth muscle cells. J. Mol. Cell. Cardiol. 28, 243–252.

Roux, S., Loffler, B.M., Gray, G.A., Sprecher, U., Clozel, M., Clozel, J.P., 1995.The role of endothelin in experimental cerebral vasospasm. Neurosurgery37, 78–85 (discussion 85–76).

Saetrum Opgaard, O., Edvinsson, L., 1997. Mechanical properties and effects ofsympathetic co-transmitters on human coronary arteries and veins. BasicRes. Cardiol. 92, 168–180.

Sakurai, T., Yanagisawa, M., Takuwa, Y., Miyazaki, H., Kimura, S., Goto, K.,Masaki, T., 1990. Cloning of a cDNA encoding a non-isopeptide-selectivesubtype of the endothelin receptor. Nature 348, 732–735.

Szok, D., Hansen-Schwartz, J., Edvinsson, L., 2001. In depth pharmacologicalcharacterization of endothelin B receptors in the rat middle cerebral artery.Neurosci. Lett. 314, 69–72.

Tirapelli, C.R., Casolari, D.A., Yogi, A., Montezano, A.C., Tostes, R.C., Legros,E., D'Orleans-Juste, P., de Oliveira, A.M., 2005. Functional characterizationand expression of endothelin receptors in rat carotid artery: involvement ofnitric oxide, a vasodilator prostanoid and the opening of K+ channels inETB-induced relaxation. Br. J. Pharmacol. 146, 903–912.

Ushio-Fukai, M., Nishimura, J., Aoki, H., Kobayashi, S., Kanaide, H., 1992.Endothelin-1 inhibits and enhances contraction of porcine coronary arterialstrips with an intact endothelium. Biochem. Biophys. Res. Commun. 184,518–524.

Wackenfors, A., Ingemansson, R., Malmsjo, M., 2003. Endothelin receptors inendothelium-denuded human coronary artery bypass grafts and coronaryarteries. Ann. Thorac. Surg. 75, 874–881.

Wackenfors, A., Emilson, M., Ingemansson, R., Hortobagyi, T., Szok, D., Tajti,J., Vecsei, L., Edvinsson, L., Malmsjo, M., 2004. Ischemic heart diseaseinduces upregulation of endothelin receptor mRNA in human coronaryarteries. Eur. J. Pharmacol. 484, 103–109.

Weissberg, P.L., Witchell, C., Davenport, A.P., Hesketh, T.R., Metcalfe, J.C.,1990. The endothelin peptides ET-1, ET-2, ET-3 and sarafotoxin S6b are co-mitogenic with platelet-derived growth factor for vascular smooth musclecells. Atherosclerosis 85, 257–262.

Wenzel, R.R., Duthiers, N., Noll, G., Bucher, J., Kaufmann, U., Luscher, T.F.,1996. Endothelin and calcium antagonists in the skin microcirculation ofpatients with coronary artery disease. Circulation 94, 316–322.

Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui,Y., Yazaki, Y., Goto, K., Masaki, T., 1988. A novel potent vasoconstrictorpeptide produced by vascular endothelial cells. Nature 332, 411–415.