Scholarly article on topic 'Rapid functional upregulation of vasocontractile endothelin ETB receptors in rat coronary arteries'

Rapid functional upregulation of vasocontractile endothelin ETB receptors in rat coronary arteries Academic research paper on "Basic medicine"

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{"Endothelin b receptor" / Endothelin-1 / Coronary / "Smooth muscle" / Vasoconstriction / "Extracellular signal-regulated kinase" / "Sarafotoxin 6c"}

Abstract of research paper on Basic medicine, author of scientific article — Gry Freja Skovsted, Anne Fog Pedersen, Rikke Larsen, Majid Sheykhzade, Lars Edvinsson

Abstract Aims Endothelin ETB receptors mediate under normal physiological conditions vasorelaxation in coronary arteries. However, vasocontractile ETB receptors appear in coronary arteries of ischemic heart disease patients. Interestingly, organ culture of isolated coronary arteries also induces upregulation of vasocontractile ETB receptors. This study examines the early time course and mechanism behind upregulation of contractile ETB receptors in isolated rat coronary arteries during short-term organ culture. Main methods Coronary artery segments were mounted in wire-myographs and incubated in physiological saline solution. Contractions were measured after exposure to the specific ETB receptor agonist Sarafotoxin 6c (S6c) and the endogenous agonists endothelin-1 and endothelin-3. Protein localization and levels of ETB and phosphorylated-extracellular-signal-regulated-kinase-1/2 (ERK1/2) were examined by immunohistochemistry. Key findings Fresh arteries showed negligible vasoconstriction to S6c. However, incubation for only 4 and 7h increased S6c contractions two- and seven-fold, respectively. Furthermore, 7h incubation enhanced vasocontractile responses to endothelin-3 and increased ETB receptor density in vascular smooth muscle cells. ERK1/2 was activated rapidly after start of incubation. Moreover, incubation with either the transcriptional inhibitor actinomycin D or the mitogen-activated-protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 attenuated contractile ETB receptor upregulation. U0126 attenuated ETB receptor protein levels after 24h of incubation. Significance Coronary arteries rapidly upregulate vasocontractile ETB receptors during organ culture via transcriptional mechanisms and MEK-ERK1/2 signalling. This model may mimic the mechanisms seen in ischemic conditions. Furthermore, these findings have important experimental implications in tissue bath experiments lasting for more than 4h.

Academic research paper on topic "Rapid functional upregulation of vasocontractile endothelin ETB receptors in rat coronary arteries"

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Rapid functional upregulation of vasocontractile endothelin ETB receptors in rat coronary arteries

Gry Freja Skovsted a,b* Anne Fog Pedersen a, Rikke Larsen a, open access

Majid Sheykhzade b, Lars Edvinsson a

a Department of Clinical Experimental Research, Glostrup Research Institute, University of Copenhagen, Glostrup Hospital, Ndr. Ringvej 69, DK-2600 Glostrup, Copenhagen, Denmark b Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2,2100 Copenhagen, Denmark

ARTICLE INFO ABSTRACT

Aims: Endothelin ETB receptors mediate under normal physiological conditions vasorelaxation in coronary arteries. However, vasocontractile ETB receptors appear in coronary arteries of ischemic heart disease patients. Interestingly, organ culture of isolated coronary arteries also induces upregulation of vasocontractile ETB receptors. This study examines the early time course and mechanism behind upregulation of contractile ETB receptors in isolated rat coronary arteries during short-term organ culture.

Main methods: Coronary artery segments were mounted in wire-myographs and incubated in physiological saline solution. Contractions were measured after exposure to the specific ETB receptor agonist Sarafotoxin 6c (S6c) and the endogenous agonists endothelin-1 and endothelin-3. Protein localization and levels of ETB and phosphorylated-extracellular-signal-regulated-kinase-1/2 (ERK1/2) were examined by immunohistochemistry. Key findings: Fresh arteries showed negligible vasoconstriction to S6c. However, incubation for only 4 and 7 h increased S6c contractions two- and seven-fold, respectively. Furthermore, 7 h incubation enhanced vasocontractile responses to endothelin-3 and increased ETB receptor density in vascular smooth muscle cells. ERK1/2 was activated rapidly after start of incubation. Moreover, incubation with either the transcriptional inhibitor actinomycin D or the mitogen-activated-protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 attenuated contractile ETB receptor upregulation. U0126 attenuated ETB receptor protein levels after 24 h of incubation.

Significance: Coronary arteries rapidly upregulate vasocontractile ETB receptors during organ culture via transcrip-tional mechanisms and MEK-ERK1/2 signalling. This model may mimic the mechanisms seen in ischemic conditions. Furthermore, these findings have important experimental implications in tissue bath experiments lasting for more than 4 h.

© 2012 Elsevier Inc. All rights reserved.

Article history:

Received 2 November 2011

Accepted 10 February 2012

Keywords:

Endothelin b receptor

Endothelin-1

Coronary

Smooth muscle

Vasoconstriction

Extracellular signal-regulated kinase Sarafotoxin 6c

Introduction

The endothelin family of vasoactive peptides consists of endothelin-1 (ET-1), -2 (ET-2), and -3 (ET-3). The endothelins mediate their effects through two distinct G-protein coupled receptor subtypes, ETA and ETB (Arai et al. 1990a; Sakurai et al. 1990; Yanagisawa et al. 1988). Under normal physiological conditions ETA is the dominant endothelin receptor subtype expressed in vascular smooth muscle cells (VSMC), where it mediates strong vasoconstriction (Adner et al. 1998b; Wangetal. 1998). ETB receptors are under normal conditions mainly expressed on endothelial cells where they mediate vasodilatation via production and release of nitric oxide (Hirata et al. 1993) and prostacyclin (Filep et al. 1991). However, in several pathophysiological conditions ETB receptors are upregu-lated in VSMC where they mediate vasoconstriction. Upregulation of

* Corresponding author at: Department of Clinical Experimental Research, Glostrup Research Institute, University of Copenhagen, Glostrup Hospital, Ndr. Ringvej 69, DK-2600 Glostrup, Copenhagen, Denmark. Tel.: +45 38633293; fax: +45 38633983. E-mail address: grfrsk01@regionh.dk (G.F. Skovsted).

0024-3205/$ - see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2012.02.009

ETB receptors in VSMC of coronary arteries has been demonstrated in experimental congestive heart failure (Cannan et al. 1996) and in patients with ischemic heart disease (Wackenfors et al. 2004; Dagassan et al. 1996a). In addition, forearm blood flow studies have revealed increased ETB receptor-dependent vasoconstriction in patients with atherosclerosis as compared to controls (Pernow et al. 2000).

Upregulation of contractile ETB receptors in VSMC has also been demonstrated to take place in a variety of arteries during in vitro organ culture in serum-free culture medium. In left coronary arteries (LAD), it has been demonstrated that the stimulation of fresh human, rat and porcine arteries with the ETB-specific agonist S6c gives no or only a weak vasocontractile response, whereas a strong contractile ETB receptor-mediated response appears after 1-2 day of organ culture in serum-free Dulbecco's Modified Eagles Medium (DMEM) (Johnsson et al. 2008; Nilsson et al. 2008). Information about ETB receptor plasticity in other parts of the coronary arterial tree is lacking.

The molecular mechanisms underlying VSMC ETB receptor upregulation have been studied after 24-48 h organ culture in both coronary and cerebral arteries. The increased expression of contractile ETB receptor at

these time points has been shown to depend on transcriptional mechanisms and PKC and the extracellular signal-regulated kinases 1 and 2 (ERK1/2) have been identified as upstream mediators of the increased ETB receptor gene expression (Henriksson et al. 2004; Nilsson et al. 2008; Sandhu et al. 2010). However, the detailed time course of the ETB receptor upregulation in rat coronary arteries at time points earlier than 24 h after start of organ culture has hitherto not been studied. Moreover, it is not clear from the earlier reports whether the critical factor triggering the ETB receptor upregulation during organ culture of coronary arteries is serum-deprivation or low oxygen tension, both of which are prominent characteristics of the DMEM in vitro organ culture model hitherto used.

Consequently, the aim of the present study was to investigate whether an ETB receptor-mediated vasoconstrictor response developed during short-term incubation of isolated LAD as well as septal coronary artery (SCA) segments in oxygenated buffer in a myograph. Furthermore, we aimed at investigating the detailed time course and molecular mechanisms behind this putative early ETB receptor plasticity.

Materials and methods

Animals and isolated vessel preparation

Male Sprague-Dawley rats (Taconic, Denmark) (9-12 weeks, 260-410 g) were sedated with 70% CO2 in 30% O2 and killed by decapitation. The heart was immediately excised and placed into ice cold oxygenated physiological saline solution (PSS) composed of (in mM): 119 NaCl, 4.7 KCl, 2.5 CaCl2, 25 NaHCO3, 1.17 MgSO4, 1.18 KH2PO4, 5.5 glucose, and 0.03 EDTA, pH 7.4. The LAD and the SCA (segment length 1-2 mm) were dissected in ice cold, oxygenated PSS and mounted on a Mulvany-Halpern wire myograph (model 610 M, Danish Myo Technology A/S, Aarhus, Denmark). The myographs were connected to a PowerLab Unit (ADInstruments, UK) and responses were sampled in LabChart™ (6.0, ADInstruments). The mounted artery segments were heated to 37 °C. After 15 min equilibration, the vessels were stretched to their optimal lumen diameter Li =0.9xL-i0o, where L100 is an estimate of the diameter of the vessel under a passive transmural pressure of 100 mm Hg, in order to obtain optimal conditions for active tension development. Subsequently, the vessels were allowed to stabilize for 20-30 min. In addition, the vessels were washed every 15 min with PSS. During incubation procedure in the myograph, the vessels were kept at this standard tension. To ensure a stable pH (7.40± 0.05), all myograph baths were continuously gassed with 12% CO2 in 88% O2, pH in the myograph baths was constantly monitored with a pH meter and gas flow to each bath was carefully adjusted accordingly.

Experimental protocols

Effect of myograph incubation time on ET receptor-mediated responses

ETB receptor-mediated responses were investigated using the peptide Sarafotoxin 6c (S6c), which exhibits high selectivity for the ETB receptor over the ETA receptors. Four consecutive cumulative concentration-response curves (1 pM to 30 nM) were obtained for each arterial segment at 1V, 4, 7 or 24 h after mounting the arteries in the myograph baths. Before each concentration-response curve, the vascular smooth muscle cell contractile function was confirmed by challenging the segments two or three times with 125 mM K+ (KPSS, similar to PSS except that NaCl was exchanged for KCl on an equi-molar basis). Dual ETA and ETB-mediated responses were investigated using the endogenous ligands, ET-1 (1 pM to 30 nM) and ET-3 (1 pM to 0.3 |jM). Because ET-1 produces a long-lasting vasocontractile response, it was only possible to perform one concentration-response curve on each arterial segment. Cumulative ET-1 and ET-3 concentration-response curves were accomplished at either 1V or 7 h of incubation in the myo-graphbathinapaired parallel experimental set-up. Theeffectofthe selective ETa receptor antagonist BQ123 (10 |jM) on the ET-1 response was

evaluated by adding the antagonist 30 min before ET-1 concentration-response curves after either 1V or 7 h of incubation.

Effect of prolonged exposure to U0126 and actinomycin D

Vessel segments were incubated in PSS buffer for 7 h with either the MEK1/2 inhibitor U0126 (10 ^M), the transcription inhibitor actinomycin D (4 |aM) or equal volumes of vehicle DMSO and subsequently S6c concentration-contraction curves were recorded.

Acute effect of U0126

Vessel segments were incubated in PSS buffer for 7 h. The mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor U0126 (10 |aM) or equal volumes of vehicle DMSO was added 30 min before onset of recording of S6c concentration-contraction curves.

Endothelium function and maximal contractile capacity

Endothelium function was determined on all artery segments by assessing the relaxation to the acetylcholine receptor agonist carba-chol (10 |aM) after obtaining steady-state pre-contraction tone with prostaglandin F2a (PGF2a) (10 |aM). Endothelial function at 100% was defined as a carbachol induced relaxation to baseline of the PGF2a induced contraction. Maximal relaxation was determined by exposing the vessels to calcium-free buffer solution (Ca2+-free PSS) buffer. After the third concentration-response curve, the arteries maximal contractile capacity of the arteries was assessed by addition of a cocktail solution (KPSS with 10 |jM PGF2a and 10 |jM 5-hydroxytryptamine (5-HT)) (Fig. 1 supplementary material). Maximal contractile capacities were calculated as the difference between maximal tension induced by cocktail solution and maximal relaxation induced by Ca2+-free PSS. All contractile responses are expressed as percentage of the maximal contraction induced by cocktail solution. Emax represents the maximum contractile response elicited by an agonist, and EC50 (as negative logarithm: pEC50) represents the agonist concentration eliciting 50% of the maximum response. Basal vascular tone was determined as the difference between tension of vessels when kept in PSS and calcium-free PSS. Only segments with a basal tone < 35% of the maximal contractility were included.

¡mmunohistochemistry

Rat coronary arteries were isolated and incubated for V, 1V, 4, 7 or 24 h in oxygenated PSS buffer containing either U0126 (10 ^M) or equal volumes of vehicle DMSO. The immunostaining protocol is described in details in supplementary materials. In short, the vessels were snap-frozen in Tissue TEK® (Gibco) and sectioned with a thickness of 10 pmona cryostat. Following fixation, permeabilization, and blocking, the sections were incubated with primary antibodies to actin (mouse anti-actin, 1:500, Abcam, UK) and to either ETB receptors (sheep anti-ETB receptor, 1:250, Alexis Biochemicals, UK) or phosphorylated ERK1/2 (rabbit anti-phosphor-p44/42 MAPK, 1:200, Cell Signaling, USA). The following secondary antibodies were used: donkey anti-mouse dylight™ 549, donkey anti-sheep dylight™ 488, and donkey anti-rabbit dylight™ 488 (all from Jackson ImmunoRe-search, UK (1:200)). Immunoreactivity was visualized and photographed with a Nikon confocal microscope (EZ-c1, Germany) at the appropriate wavelengths.

Data analyses

Data were analysed using GraphPad Prism© software (GraphPad Software Inc., USA) and are expressed as mean±SEM (n equals the number of animals). All contractile responses from the in vitro pharmacology experiments are expressed as percentage of the maximal contraction induced by cocktail solution (KCl, 125 mM, PGF2a. 0.01 mM, and serotonin 0.01 mM). Emax represents the maximum contractile response elicited by an agonist and EC50 (as negative logarithm: pEC50) represents the agonist concentration required to

produce 50% of the maximum response. Two-way ANOVA with repeated measurements and Bonferroni multiple comparisons posttest were used to compare concentration-response curves obtained in coronary arteries. .Student's t-test (for paired or unpaired groups as appropriate) was used on experiment comparing only two mean values. One-way ANOVA Bonferroni multiple comparisons post-test was used to compare significant differences between immunoreactiv-ity results. All statistical analyses were considered significant when p < 0.05.

Chemicals

All drugs and chemicals for myograph studies were acquired from Sigma-Aldrich unless otherwise indicated. ET-1 and S6c were obtained from NeoMPS (Strassbourg, France). Actinomycin D, U0126, and BQ788 were prepared as stock solutions in DMSO in concentrations of 10 mM, 20 mM, and 1.8 mM, respectively. S6c and ET-1 were dissolved in 0.1% BSA in distilled water to reach stock concentrations at 0.1 mM. PBS tablets (Invitrogen) was dissolved in distilled water reaching following concentrations: 0.14 M NaCl, 0.01 M PO4 buffer, 0.003 M KCl.

Results

The average diameters of LAD segments (340 ± 16 |am) were larger than those of SCA segments (260±13 |jm). However, maximal contractile capacity (KPSS with 10 ^M PGF2a and 10 ^M 5-HT) was not significantly different between LAD (6.4 ±0.8 mN/mm) and SCA (7.0 ± 0.5 mN/mm) segments. In most experiments, LAD had a slightly higher basal tone compared to SCA, and since the basal tone in LAD was highly dependent on the pH in the buffer, the pH was kept tightly in a range between 7.35 and 7.45.

Time course of contractile ETB receptor upregulation

To investigate the time course of incubation-induced upregulation of contractile ETB receptors, we investigated contractile responses to the ETB-specific agonist S6c after various time periods of incubation. S6c-mediated contraction was negligible after 1Vi h incubation of LAD and SCA (Emax = 9.5±3.8% and 6.3 ±4.4% of maximal contraction, respectively), but the response successively increased with time. The S6c-induced contraction increased by approximately twofold after 4 h incubation in both LAD (Emax 20.1 ±4.5%, p>0.05) and SCA (11.3 ±4.6%, p>0.05), and by approximately seven-fold after 7 h of incubation (LAD Emax = 68.6±4.2% and SCA Emax = 45.4±5.2%, p<0.001) (Fig. 1A and B). The response increased only slightly further after 24 h of incubation in LAD and SCA. There was no change in pEC50 over time despite the successive increases in Emax. Contractile responses to K+ (125 mM) and to the cocktail (KPSS, PGF2a and 5-HT) were not significantly altered at the time points studied (supplementary data). The S6c-induced concentration-response curves recorded after 7 h incubation were unaffected by whether the vessels had been stimulated with S6c in previous concentration-response curves or just incubated without stimulation, indicating that incubation-induced upregulation of the contractile ETB response is not affected by stimulation of the ETB receptors with S6c during the incubation.

To investigate the importance of the enhanced ETB-mediated contractility for the contractile responses to endogenous endothelin receptor agonists, we determined ET-1 and ET-3-induced contractile responses before or after incubation for 7 h. The contractile response to ET-1 was unchanged by incubation for 7 h in both LAD and SCA (Fig. 1B and E). In contrast, 7h incubation resulted in a leftwards shifted of ET-3 concentration-response curve in both LAD and SCA. This was more pronounced in LAD, in which the pEC50 shifted from

7.2 ±0.03 to 9.7 ±0.7 compared to SCA, where pEC50 shifted from

6.3 ± 0.3 to 7.5 ± 0.1 (Fig. 1C and F).

In order to ascertain whether the observed development of an ETB receptor-mediated contractile response was an indirect effect of disturbed endothelium-mediated vasodilatation in the arterial segments during incubation, the presence of intact functional endothelium in the arteries was tested by precontracting the arteries with PGF2a (10 |jM) and subsequently induce endothelium-mediated vasodilatation by application of carbachol (10 ^M). There was no significant difference between endothelium-mediated relaxation in LAD (53 ±8%) and SCA (62 ±7%). Furthermore, there was no correlation between endothelium function and S6c-induced vasoconstriction after 7 h incubation in neither LAD nor SCA (supplementary data), indicating that the observed enhanced ETB receptor-mediated contractility is not dependent on changes in endothelium function.

Involvement of contractile ETB receptors in ET-1 induced contraction

The specific competitive ETA receptor antagonist BQ123 (10 ^M) was added 30 min before recording of ET-1 concentration-response curves at either 1Vi h or 7 h of incubation. BQ123 induced a parallel and rightward shift of the ET-1 concentration-response curve in both coronary artery segments after 1Vi h of incubation (LAD pEC50 = 8.9 ± 0.1 and SCA pEC50 = 8.5 ± 0.1 without BQ123, and LAD pEC50 = 8.3 ± 0.3 and SCA pEC50 = 7.6 ± 1.5 with BQ123) without significant alteration in the maximal contractile responses (Fig. 2A and D). This indicates that after 1Vi h of incubation the ET-1 induced contraction is mediated by ETA receptors. In SCA BQ123 induced a parallel rightward shift of ET-1 concentration-response curve after 7 h of incubation (Fig. 2E). In contrast in LAD after 7 h of incubation an ETA receptor blocking resulted in a biphasic ET-1 concentration-response curve (Fig. 2B). These results indicate that, although the shape of ET-1 induced contraction-response curve does not change after incubation (Fig. 1B), the ET-1 induced contraction after 7 h of incubation involves both ETA and ETB receptor in LAD.

Involvement of gene transcription and MEK1/2 activity in upregulation of contractile ETB receptor

The increase in S6c induced contraction during 7 h incubation was inhibited by incubation in the presence of either the transcriptional inhibitor actinomycin D (4 ^M) or the MEK1/2 inhibitor U0126 (10 |jM) (Fig. 2C and F). Segments incubated with vehicle for 7h showed Emax of 56.7 ± 2.2% and 51.7 ± 3.6% in LAD and SCA, respectively. Segments incubated for 7 h in the presence of actinomycin D displayed significantly reduced Emax values at 27.2± 5.3% (LAD) and 15.2 ±6.5% (SCA). Segments incubated for 7 h in the presence of U0126 also showed significantly reduced Emax values at 32.9 ±6.0% (LAD) and 16.7 ±3.9% (SCA). These findings indicate that both transcriptional mechanisms and intracellular signalling via MEK1/2 is involved in the upregulation of contractile ETB receptors.

In order to determine whether U0126 (10 |jM) had a direct attenuating effect on S6c-induced contraction, the MEK1/2 inhibitor was added 30 min before S6c concentration-response curves were obtained (after 7 h incubation). U0126 did not affect the S6c induced contraction compared to control segments, which had been given an equal volume DMSO (data not shown), indicating that the above described effect of 7 h incubation in the presence of U0126 is not the result of an acute effect of U0126 on ETB-mediated contraction.

Immunohistochemistry

Specific antibodies towards the ETB receptor visualised the proteins localized in VSMC after incubation (Fig. 3A). The ETB receptor protein expression increased significantly after 7 h (208 ± 29%, p<0.05) and 24 h incubation (385±54%, p<0.001) as compared to non-incubated arteries (100±14%).

Fig. 1. Log concentration-response curves to Sarafotoxin 6c (S6c) in rat (A) LAD (n = 4-14) and (D) SCA (n = 3-14). Log concentration-response curves to endothelin-1 (ET-1) in rat (B) LAD (n = 9-11) and (E) SCA (n = 9-10). Log concentration-response curves to endothelin-3 (ET-3) in rat (C) LAD (n = 6) and (F) SCA (n = 6). The experiments were performed after 1V (circles), 4 (squares), 7 (triangles), and 24 (cross) h of incubation in PSS buffer. Values plotted as means ±SEM. (*** p<0.001, ** p<0.01,*p<0.05), time of incubation vs. 1V h, two-way ANOVA with repeated measurements and Bonferroni multiple comparisons test were used.

In order to evaluate the involvement of MEK1/2 signalling in the increased ETB receptor expression, we examined the effect of the selective MEK1/2 inhibitor U0126. Incubation with U0126 (10 цМ) significantly attenuated ETB receptor expression after 24 h incubation (198 ±12%, p<0.05) compared to segments incubated for 24 h in the presence of the DMSO (385 ± 54%) (Fig. 3A). U0126 had no significant effect on the increased ETB receptor expression after 7 h incubation.

To demonstrate activation of the MEK-ERK1/2 signalling pathway during incubation, coronary arteries were incubated for 0, V, 1V, 4, 7, and 24 h in PSS with and without U0126 (10 цМ) (Figs. 3B) and the levels of phosphorylated ERK1/2 in the segments were determined by immunohistochemistry. ERK1/2 phosphorylation increased significantly after 1V h (219±40%) and 4h incubation (404 ±28%) as compared to non-incubated arteries (100± 18%). After 7 h and 24 h the level of phosphorylated ERK1/2 decreased. Incubation of the coronary arteries with 10 цМ U0126 significantly abolished ERK1/2 phosphorylation at all incubation times, except for 24 h of incubation, where the level of phosphorylated ERK1/2 was negligible.

Discussion

The present study demonstrates for the first time that only 7 h incubation of isolated coronary artery segments in oxygenated physiological buffer in a myograph induces a significant upregulation of contractile ETB receptors. This rapid upregulation of ETB receptors is important to keep in mind, if tissue bath experiments on coronary

arteries persist for more than 4 h, due to a potentially interference of these emerging ETB receptors. This rapid functional upregulation is associated with increased ETB receptor immunoreactivity in VSMC and is abolished by the transcription inhibitor actinomycin D and the MEK1/2 inhibitor U0126.

Mounting evidence suggests that under different pathological circumstances including atherosclerosis (Dagassan et al. 1996a; Pernow et al. 2000), ischemic heart disease (Dimitrijevic et al. 2009; Wackenfors et al. 2004), and focal cerebral ischemia (Stenman et al. 2002), the balance of vasoconstriction and relaxation by endothelin receptor activation is skewed towards a contractile phenotype with increased levels of vasoconstrictive ETB receptors in VSMCs. In vitro organ culture of isolated artery segments (Adner et al. 1998a) induces upregulation of contractile endothelin receptors resembling the pattern seen in cardiovascular diseases (Wackenfors et al. 2004), indicating that organ culture can be used as a convenient in vitro model to study this pathophysiological phenomenon. Previous studies have shown that fresh isolated rat coronary arteries display negligible ETB receptor-mediated contractions (Goodwin et al. 1999; Harrison et al. 1992), whereas a strong ETB receptor-mediated contractile response appears after 24 h of organ culture in serum-free DMEM (Eskesen and Edvinsson 2006; Ghorbani et al. 2010; Johnsson et al. 2008). In the present study, we studied for the first time the early time course of contractile ETB receptor upregulation during incubation in aerated buffer in a myograph. We demonstrate a rapid and strong increase in S6c mediated contractile responses in coronary arteries already after 7 h of incubation, which is a considerably more

Fig. 2. Log concentration-response curves to endothelin-1 (ET-1) with and without the specific ETA receptor antagonist BQ123 (10 |iM) after 1V h of incubation in rat (A) LAD and (D) SCA and after 7 h incubation in PSS (B) LAD and (E) SCA. The specific ETA receptor antagonist BQ123 (10 |iM, n = 3) was added 30 min before initiation of concentration-response curve. Values plotted are expressed as means ± SEM. The maximal contractile responses to ET-1 were significantly attenuated after 7 h incubation in the presence of either the transcription inhibitor, actinomycin D (4 |iM) (n = 5) or the MEK1/2 inhibitor U0126 (10 |iM) (n = 7) in both (B) LAD and (D) SCA. Values plotted are expressed as means ± SEM. (*** p<0.001, ** p<0.01,*p<0.05), treatment group vs. DMSO, two-way ANOVA with repeated measurements and Bonferroni multiple comparisons test were used.

rapid response than what has earlier been demonstrated in both mes-enteric (Moller et al. 2002) and cerebral arteries (Henriksson et al. 2003). The specific involvement of ETB receptors in the rapidly upre-gulated contractile response in coronary arteries was confirmed by the demonstration of a strong parallel rightwards shift of the S6c concentration-response curves using the competitive selective ETB receptor antagonist BQ788 (Ishikawa et al. 1994).

The ETA receptor has a higher affinity for endothelin-1 than the two other agonists, with an affinity order of endothelin-1 >endothelin-2>endothelin-3, while the ETB receptor exhibits similar affinities for all three isopeptides (Arai et al. 1990b; Sakurai et al. 1990). Earlier studies of the selectivity of endogenous endothelins for ETA and ETB receptor revealed that ET-1 and ET-2 bind with equal selectivity to the ETA and ETB receptors, whereas ET-3 is the only one of the endothelin isoforms which distinguishes between the receptors. ET-3 has a higher selectivity for the ETB receptor than the ETA receptor (Davenport 2002). Incubation of coronary arteries did not change the contractile responses to ET-1, which is in accordance with earlier studies (Wackenfors et al. 2004; Johnsson et al. 2008). However, the specific ETA receptor antagonist BQ123 induced parallel rightward shift of the ET-1 concentration-response curve after 1V h of incubation, and interestingly induced a bi-phasic ET-1 response in LAD incubated for 7 h. This indicates that the ETA receptor dominates the vasocontractile response to ET-1 in rat coronary arteries, whereas, the ETB receptor may be involved in the ET-1 induced contraction after 7 h of incubation. Studies have shown that atherosclerotic human coronary arteries have upregulated ETB receptor

(Dagassan et al. 1996b) Based on our results, one could imagine that a local selective blockade of ETA receptor in atherosclerotic coronary arteries with upregulated ETB receptors will only displace a part of the endothelin-1 induced contraction. Whereas, targeting both receptors with a dual ETA/B receptor blockade could be more efficient.

Interestingly, we also demonstrate for the first time that the contractile responses to ET-3 is significantly enhanced by incubation for 7 h, which is in accordance with the selectivity of this ligand for ETB over ETA receptor.

The enhanced contractile responses to S6c after 24 h organ culture of both cerebral and mesenteric arteries has been shown to be the result of a transcriptional upregulation of ETB receptors with increased ETB receptor mRNA levels (Moller et al. 2002; Henriksson et al. 2003) and increased ETB receptor protein after organ culture (Johnsson et al. 2008). In the present study, we likewise demonstrated by immunohistochemistry that ETB receptor protein levels were increased after 7 and 24 h of incubation of the coronary arteries. In order to investigate if the rapid upregulation of contractile ETB receptors in coronary arteries during 7 h incubation occurs via transcrip-tional mechanisms, we studied the effects of the transcriptional inhibitor actinomycin D on S6c-induced contractions after 7 h incubation. Actinomycin D abolished the functional ETB receptor upregula-tion after 7 h incubation in both LAD and SCA. Interestingly, the inhibitor was not able to completely reverse the ETB receptor-mediated response to the levels observed in fresh segments, indicating that the increased ETB receptor activity after 7 h incubation is

Fig. 3. Quantifications and montages of immunohistochemistry intensities of ETB receptors (A) and pERK (B) in the smooth muscle cell layer of LAD before culture, 0 h, after culture (0.5 h, 1.5 h, 4 h), 7 h and 24 h and in the presence of U0126 (10 |jM) or an equal volume DMSO using confocal microscopy. Note the almost complete inhibition of pERK1/2 fluorescent intensity, using U0126. The staining intensities are expressed as a ratio of intensity before culture = 100% (mean±SEM). Number of animals in the staining n = 4-6. One Way ANOVA with Newman-Keuls multiple comparison test (** p<0.01, *** p<0.001) and Student's t-test. (# p<0.05) were used.

not solely due to transcriptional ETB receptor upregulation, but may involve other mechanisms as well.

ERK1/2 has previously been shown to be an upstream mediator of ETB receptor upregulation in cerebral and mesenteric arteries (Henriksson et al. 2004; Uddman et al. 2003). In the present study, the level of phosphorylated ERK1/2 increased very early during the incubation and peaked after 4 h of incubation. Incubation with the MEK1/2 inhibitor U0126 (10 цМ) completely abolished the phosphorylation of ERK1/2, decreased the S6c-mediated vasoconstriction after 7 h incubation, and significantly decreased ETB receptor protein expression after 24 h incubation. Together; these findings strongly suggest that signalling via the MEK-ERK1/2 pathway is involved in functional ETB receptor upregulation during organ culture of coronary arteries. However, since the S6c-mediated vasoconstriction after 7 h incubation was not completely abolished in presence of U0126, this initial functional upregulation might involve additional mechanisms. Studies have shown that even fresh coronary arteries from healthy rats contain ETB receptor protein in VSMC (Wendel-Wellner et al. 2002; Wendel et al. 2005). Hence, we speculate that a part of the contractile ETB receptor upregulation may involve post-translational modifications of already expressed ETB receptors.

Conclusion

In conclusion, our results demonstrate that incubation of both LAD and SCA rat coronary arteries rapidly increases the ETB receptor-mediated contraction as well as upregulation of ETB receptor protein levels. These findings may have important experimental implications in tissue bath experiments lasting for more than 4 h, due to the possible interference of emerging ETB receptors. We demonstrate involvement of transcriptional mechanisms and ERK1/2 activity in the ETB receptor upregulation process. By using the specific MEK1/2 inhibitor U0126, the upregulation of contractile ETB receptors can be prevented by treatment with inhibitors of the central signal transduction pathways underlying the upregulation process. The here employed organ culture model represents a model

situation with no-flow and serum starvation as seen in ischemic conditions. Thus, despite the obvious simplicity of the model system, our results suggest that ERK1/2-mediated upregulation of vascular contractile ETB receptors may play a pivotal role in the pathogenesis of ischemic heart diseases, and that inhibition of the underlying signal transduction may be a therapeutic strategy to avoid this pathological response of the coronary vasculature.

Supplementary materials related to this article can be found online at doi:10.1016/j.lfs.2012.02.009.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Acknowledgments

Thanks to Professor Karin Warfvinge (www.sciencesupport.se) and PhD Gro Klitgaard Povlsen for fruitful discussions and valuable comments on the manuscript.

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