Journal of the Pancreas Open Access

Keywords

Anoxia; Endothelins; Ischemia; Islets of Langerhans Transplantation; Receptors, Endothelin

Abbreviations

ET: endothelin; FCS: fetal calf serum; HBSS: hanks balanced salt solution; HIF: hypoxia-inducible factor; MAP: mitogen activated protein; PCR: polymerase chain reaction; PMA: phorbol 12- myristate-13-acetate

INTRODUCTION

The success of clinical islet transplantation for the cure of patients with type 1 diabetes mellitus is limited, despite the recent progress made with an improved islet isolation process or immunosuppressive regimen [1, 2, 3]. A widespread application of this attractive alternative to standard insulin therapy or pancreas transplantation is limited by donor organ shortage and the requirement of 2-3 donor pancreata per recipient to achieve insulin independence. A key factor for the need of such substantial amounts of donor tissue is early graft loss due to inadequate engraftment early after transplantation. It is suspected that 50-70% of the total transplanted islet mass is lost or nonfunctional as a result of limited blood supply and subsequent hypoxia, the instant bloodmediated inflammatory reaction (IBMIR), the energy status of the islets, the balance of proand anti-apoptotic mediators and graft rejection [4, 5, 6]. Transplanted islets are trapped in the distal branches of the portal system, resulting in local ischemia until neovascularization of the grafted islets has been completed [7].

Local ischemia may trigger the release of vasoactive mediators, of which endothelin-1 (ET-1) and nitric oxide are the most potent in controlling hepatic blood flow. It has been demonstrated that an imbalance in hepatic vasoregulatory gene expression leads to microvascular dysfunction and therefore contributes to ischemia/reperfusion injury in organ transplantation [8, 9]. Among others, endothelins (ET-1, 2 or 3), a family of 21 amino acid residue peptides, are the most potent vasoconstrictors known. ET exerts its biological activity by binding to different cell surface receptors, ET_A or ET_B. Both ETreceptors belong to the G-protein-coupled receptor family which leads to the activation of diverse downstream kinases [10]. Besides vasoconstriction, ET mediation of several other biological activities has been detected, e.g. the production of cytokines, cell growth, cell adhesion and thrombosis, the release of nitric oxide and prostacyclin, the stimulation of insulin release and other functions [10, 11, 12, 13, 14, 15]. ET-1 has also been described as exerting anti-apoptotic effects in the endothelium and cardiomyocytes [16, 17]. Selective ET_A-receptor antagonists have been shown to mitigate hepatic ischemia/ reperfusion injury and to exert beneficial effects in experimental pancreas and liver

transplantation by improving microcirculation and reducing tissue injury [18, 19]. Therefore, the use of selective ET_A-receptor antagonists in islet transplantation could potentially improve hepatic microcirculation and counteract the near-ischemic conditions which are generated locally through the embolization of the portal branches by the islet graft. However, treatment with an endothelin receptor antagonist might affect islet cell biology directly, provided that endothelin receptors are also expressed in pancreatic islets. Depending on the receptor subtype expression on islet cells, endothelin may provoke either beneficial or detrimental biological effects which are independent of its vasoactive properties but crucial to the engraftment process [10]. Recent studies on the expression of endothelin and its receptors in pancreatic islets are incomplete and inconclusive [20, 21]. In order to evaluate potential therapeutic strategies in islet transplantation, it is essential to obtain a better understanding of the endothelin system in the endocrine cells of pancreatic islets. The present study focuses on the precise localization of ET-1 and the two main receptors, ET_A and ET_B, in endocrine cells of human and rat pancreatic islets and in islets transplanted into the liver of syngeneic rats. Binding studies with radiolabeled ET-1 and non-labeled competitors specific for either receptor were performed to elucidate the expression of ET_A- and ET_B-receptors. We also studied the functionality of endothelin receptors in isolated rat islets by their ability to induce phosphorylation of p42/44 mitogenactivated protein (MAP) kinase upon stimulation with ET-1. Quantitative polymerase chain reaction (PCR) was performed to study the regulation of endothelin and its receptors under hypoxic conditions at an oxygen tension (1% O2) comparable to the transplant site in the liver [22]. Our results demonstrate the presence of a functional and hypoxically regulated endothelin system in the endocrine cells of pancreatic islets which suggests a biological role other than the regulation of endothelial function.

SUBJECTS, MATERIALS AND METHODS

LiberaseTM and DNAse were obtained from Roche Molecular Biochemicals (Basel, Switzerland). Cell culture media, Hanks’ balanced salt solution (HBSS), Roswell Park Memorial Institute 1640 (RPMI 1640), Connaught Medical Research Laboratories 1066 (CMRL 1066), GlutaMax, sodium pyruvate, antibiotic-antimycotic solution were all obtained from Life Technologies, Gibco BRL (now: Invitrogen AG, Basel, Switzerland). ET_A-receptor antagonist (BQ123), ET_B-receptor antagonist (BQ788), and ET_B-receptor agonist (IRL1620) were purchased from Calbiochem (Juro Supply, Switzerland).

Cell Lines and Culture Conditions

Rat beta-cell lines INS-1 (kindly provided by C. Wollheim, Geneva, Switzerland) and RINm5f (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI 1640 supplemented with 1 mM sodium pyruvate, 10% fetal calf serum (FCS) (PAA, Vienna, Austria), 1 mM GlutaMax and antibiotic-antimycotic solution (100 U/mL penicillin G sodium, 100 U/mL streptomycin sulfate and 0.25 μg/mL amphotericin B as fungizone in 0.85% saline) and 50 μM betamercaptoethanol. The cells were incubated in a humidified incubator at 37°C, 5% CO2/95% air.

Isolation o Rat Islets

Male Sprague-Dawley or Lewis rats (225-275 g from Harlan Netherlands B.V., Horst, The Netherlands) were anaesthetized with Isoflurane (Baxter A.G., Volketswil, Switzerland) and subsequently sacrificed by neck dislocation. Islet isolation and purification were performed according to a modified procedure described earlier [23]. In brief, after cannulation of the common bile duct, a 10 mL enzyme solution (LiberaseTM 0.17-0.33 mg/mL and DNAse 0.1 mg/mL in HBSS) was instilled for retrograde perfusion of the pancreas. After organ procurement, enzymatic digestion took place for 15-30 minutes at 37°C in a shaking water bath. Following a wash step in HBSS with 10% FCS and filtration through cheese cloth, islets were obtained by centrifugation in a discontinuous density gradient of Histopaque- 1077 (Sigma Diagnostics Inc., St. Louis, MO, USA) and HBSS with subsequent handpicking. Islet purity was generally greater than 95%. Isolated rat islets were cultured in RPMI 1640 as described in Cell Lines and Culture Conditions in a humidified incubator at 37°C, 5% CO2/95% air, unless specified otherwise. For transplantation purposes, isolated Lewis islets were cultured overnight, then washed three times with 10 mL of HBSS and 2% Lewis rat serum and resuspended in washing buffer and placed on ice until transplantation. Experiments under hypoxic conditions (0.8-1.2% O2) were performed in a hypoxic chamber with an intermediate floodgate system for the hermetic transport of dishes (Coy Laboratory Products, Grass Lake, MI, USA).

Rat Islet Transplantation

Inbred male Lewis rats (150-175 g) were treated with a single injection of streptozotocin (75 mg/kg i.p.). Only rats with non-fasting blood glucose of more than 15 mM for 3 consecutive days were considered diabetic. The rats were anesthetized by sevoflurane inhalation and the abdominal cavity was accessed by midline incision. The portal branches to the left, middle and right liver lobes were temporarily closed with microvascular clamps, after which 400-450 islets in a volume of 150 μL HBSS were injected into the portal vein by a 26-gauge needle, directing the islets to the caudate liver lobes. The clamps were released and the injection needle was removed. Bleeding was stopped with a cotton swap by gentle compression at the site of injection. After closing the abdomen with sutures, the recipient was allowed to recover. The caudate liver lobes were harvested under sevoflurane anesthesia 24 h after transplantation.

Isolation o Human Islets

Human islets were isolated from cadaveric pancreata of heart-beating donors at the Division of Endocrinology and Diabetes, University Hospital of Zurich, according to the method described previously [24]. Informed consent for organ donation was asked for and obtained by the donors’ relatives. The 13 donors (7 females; 6 males) had no previous history of diabetes or metabolic disorders. Their median age was 59 years (range: 40-71 years) and the median body mass index was 25.3 kg/m2 (range: 19.6- 31.5 kg/m2). Cold ischemia time until perfusion of pancreatic duct with LiberaseTM solution averaged 5.5±2.1 h (range: 2.0-9.2 h). Isolated human islets were cultured in CMRL 1066 as described in Cell Lines and Culture Conditions for 16-24 h before exposure to hypoxic culture conditions as described above.

Immunofluorescent Stainings

Tissue specimens (human pancreatic biopsies, rat pancreata and livers) were fixed in formalin solution for 24 h, embedded in paraffin and cut into 5 μm sections. After deparaffinization, the slides were placed in 10 mM citrate buffer (pH 6.0) and heated in a microwave oven for 10 minutes (600 W) for antigen retrieval. The blocking of unspecific binding was performed in 10% normal donkey serum in PBS for 30 min. Sections were incubated overnight at 4°C with following primary antibodies: Guinea pig anti-insulin (A0564), dilution 1:100; rabbit anti-glucagon (A0565), dilution 1:100; rabbit anti-somatostatin (A0566), dilution 1:300 (all from DAKO Cytomation, Glostrup, Denmark). Mouse anti-ET-1 (803-001-R100), dilution 1:100; sheep anti-ET_A-receptor (210- 507-C250), dilution 1:50; sheep anti-ET_Breceptor (210-506-C250), dilution 1:50 (all from Alexis Corporation, Lausen, Switzerland); rabbit anti-ET_A-receptor (AER- 001), dilution 1:100; rabbit anti-ET_B-receptor (AER-002, both Alomone Labs, Jerusalem, Israel), dilution 1:100. For detection, the following secondary antibodies were used: fluorescein-5-isothiocyanate- (FITC)- conjugated rabbit anti-guinea pig IgG (F0233, DAKO Cytomation, Glostrup, Denmark), dilution 1:20; Cy2TM-conjugated donkey antirabbit IgG (711-225-152), dilution 1:100; Cy3TM-conjugated donkey anti-mouse IgG (715-165-150); rhodamine-conjugated donkey anti-sheep IgG (713-295-147), dilution 1:100; Cy3TM-conjugated donkey anti-rabbit IgG (711-165-152), dilution 1:100 (all from Jackson Immuno Research Laboratories, West Grove, PA, USA). Binding of ET-1 antibody in the immunoneutralization assay was made visible using the biotin-streptavidin-based Histostain® broad-spectrum kit (Zymed, San Francisco, CA, USA) according to the manufacturer’s protocol. Slides were mounted in HydromountTM (National Diagnostics, Atlanta, GA, USA) and studied with a Zeiss Axioplan® 2 imaging microscope coupled to Axiocam® digital camera and AxioVision® software (version 2.0.5; Zeiss, Feldbach, Switzerland).

Antibody Immunoneutralization

To assess the specificity of ET-1, ET_A-, and ET_B-receptor immunostaining, antibodies were preabsorbed with the corresponding peptide for 2 h at 37°C with a 1,000-fold molar excess of either recombinant ET-1 (155-001-PC01, Alexis Corporation, Lausen, Switzerland) or the specific peptides for ET_Aand ET_B-receptor provided by the supplier (Alomone Labs, JerUSAlem, Israel).

RT-PCR

Total RNA isolation from cells (INS-1, RINm5f) or purified rat or human islets was performed using TriZol reagent (Invitrogen AG, Basel, Switzerland) according to the manufacturer’s guidelines. After RNA isolation, reverse transcription was carried out with the Super Script first strand synthesis system (11904-018, Invitrogen AG, Basel, Switzerland) and oligo dT priming. Subsequent PCR amplification was performed with 30-40 cycles of 30 sec at 94°C, 30 sec at 54-60°C and 1 min at 72°C (Taq DNA Polymerase 18038-042, Invitrogen AG, Basel, Switzerland). PCR products were separated on 1% agarose gel, and pictures were taken with a gel documentation system (AlphaImager, Alpha Innotech, San Leandro, CA, USA). Amplification of human beta-actin served as internal standard and expression levels were shown to be unaffected by the experimental conditions. All primers were designed to be intron-spanning. Primer sequences, PCR fragment length, and amplification conditions of rat ET-1, ET_A-, ET_B-receptor and proinsulin were as follows. ET-1: 5’-atcatctgggtcaacactc-3’ (sense), 5’- gaatctcctggctctctg-3’ (antisense), 727 bp (annealing: 50°C; 30 cycles). ET_A-receptor: 5’-ttcgtcatggtacccttcga-3’ (sense), 5’- gatactcgttccattcatgg-3’ (antisense), 546 bp (annealing: 57°C; 40 cycles). ET_B-receptor: 5’-ttcacctcagcaggattctg-3’ (sense), 5’- aggtgtggaaagttagaacg-3’ (antisense), 474 bp (annealing: 57°C; 40 cycles). Proinsulin: 5’- acaatcatagaccatcagcaagc-3’ (sense), 5’- cagttggtagagggagcaga-3’ (antisense), 354 bp (annealing: 55°C; 30 cycles). Primers sequences, PCR fragment length, and amplification conditions of human ET-1, ET_A-receptor, ET_B-receptor and beta-actin were as follows. ET-1: 5’-ttctctctgctgtttgtggctt-3’ (sense), 5’-ccagcacttcttgtctttttgg-3’ (antisense), 338 bp (annealing: 50°C; 27 cycles). ET_Areceptor: 5’-acttcagctttcaaatacattaaca-3’ (sense), 5’-ctgcttaagatgttcagtgagggc-3’ (antisense), 674 bp (annealing: 50°C; 29 cycles). ET_Breceptor: 5’-ttccaacgccagtctggcgcggtc-3’ (sense), 5’-gtcaatactcagagcacatagact-3’ (antisense), 423 bp (annealing: 50°C; 30 cycles). Betaactin: 5’-gatgacccagatcatgtttg-3’ (sense), 5’- gagcaatgatcttgatcttc-3’ (antisense), 641 bp (annealing: 50°C; 20 cycles).

Quantitative RT-PCR

Human islet RNA isolation procedure and first strand synthesis was performed as described above. Primers were designed with respect to exon/exon boundaries using Primer Express® version 2.0 (Applied Biosystems, Foster City, CA, USA). Sequence-specific fluorescent TaqManTM probes were designed for each target. Primer sequences, probe sequence and PCR fragment length, of human ET-1, ET_A-receptor, ET_B-receptor and betaactin were as follows. ET-1: 5’- gctcgtccctgatggataaag-3’ (sense), 5’- agggcttccaagtccatacg-3’ (antisense), 5’- tgtgtctacttctgccacctggacatca-3’ (probe), 101 bp. ET_A-receptor: 5’-gggatcaccgtcctcaacct-3’ (sense), 5’-cacgactccaggaggcaact-3’ (antisense), 5’-ctctgtacctgtcaacactaaga-3’ (probe), 70 bp. ET_B-receptor: 5’-ggttccaaaatggacagcag-3’ (sense), 5’-caccaatcttttgctgtcttg-3’ (antisense), 5’-acagccagaaccacagagaccaccc-3’ (probe), 189 bp. Beta-actin: 5’-tcgcctttgccgatcc-3’ (sense), 5’-cgaagccggccttgc-3’ (antisense), 5’- tcgacaacggctccggcatg-3’ (probe), 111 bp. Sequence detection of target genes was performed in an ABI Prism 7000 system (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s guidelines. The amplification parameters were 50°C for 2 min, 95°C for 10 min and 40 cycles of 95°C for 15 sec and 60°C for 1 min. Fluorescence emission from individual PCR tubes and cycle of threshold (Ct) values were collected corresponding to the PCR cycle number at which the fluorescence was detectable above an arbitrary threshold, based on baseline data within cycles 3 to 15. The arbitrary threshold was chosen to ensure that the Ct values were obtained within the exponential phase of PCR. To normalize the differences in the amount of total RNA added to the reaction, amplification of the housekeeping gene betaactin was performed as an endogenous control. Samples were run in triplicates and differences were calculated as 2-((ΔCtHypoxia) - (ΔCtNormoxia)) whereas ΔCt represents the differences of Ct-values of the transcript of interest and the endogenous control beta-actin at the respective experimental conditions. No error bars can be computed for the 24 h normoxic reference point (calibrator) due to the delta-delta ct real-time analysis method of always setting the calibrator to a value of 1.

Receptor Binding Assays

Isolated rat islets were dispersed into single cell suspension by mild trypsinization (0.005% trypsin dissolved in PBS with 0.05 mM EDTA) and subsequent mechanical dissociation by pipetting through a Pasteur pipette. Islet cells were taken up in HBSS and 0.3% bovine serum albumin (BSA) and incubated in the presence of 200 pM ¹²⁵I-ET-1 (IM223, Amersham, Buckinghamshire, United Kingdom) for 2 h at 37°C. the excess of unlabeled ET-1 (1 μM; 155-001-PC01, Alexis Corporation, Lausen, Switzerland) was used to measure unspecific binding, and total binding was assessed in the absence of unlabeled ET-1. Bound ligand was separated from unbound by centrifugation through mineral oil, after which the cell pellet was measured for radioactivity. Specific binding to ET_A- or ET_B-receptor was determined by competition binding with ET_B- receptor agonist (IRL1620, 1 μM), ET_B- receptor antagonist (BQ788, 1 μM) or ET_A-receptor antagonist (BQ123, 1 μM), respectively.

Phosphorylation of p42/p44 MAP Kinase

Rat islets were isolated and incubated overnight for recovery. The islets were then washed in PBS and resuspended in RPMI 1640 supplemented with 0.5% FCS, 1 mM GlutaMax and antibiotic-antimycotic solution (100 U/mL penicillin, 100 U/mL streptomycin sulfate and 0.25 μg/mL amphotericin B as fungizone in 0.85% saline). Aliquots of 150-200 islets were incubated in the presence of ET_A- (BQ123; 2.2 μM) and/or ET_B- receptor antagonist (BQ788; 0,12 μM) 2 h prior to stimulation with ET-1 (0.01-1 μM). Ten minutes after the addition of ET-1, the islets were immediately lysed in ice cold 50 mM HEPES, pH 7.4, 1% Triton X-100, 4 mM EDTA, 1 mM NaF, 0.1 mM Na3VO4, 1 mM Na4P2O7; and 1% protease inhibitor cocktail (P8340, Sigma Diagnostics Inc., St. Louis, MO, USA) and protein concentration was determined.

Western Blot

Equal amounts of protein (20-50 μg) were separated by 10% or 15% SDS-PAGE and transferred onto a nitrocellulose membrane (BioRad, Hercules, CA, USA). The membrane was incubated overnight at 4°C with either anti-phospho-p42/p44 MAP kinase antibody (9101) or anti-p42/p44 MAP kinase antibody (9102; both from Cell Signaling Technology Inc., Danvers, MA, USA) diluted 1:1,000 in 2% fat free milk diluent (KPL, Gaithersburg, MD, USA) in TBS/0.1% Tween-20. This was followed by incubation with horseradish peroxidaseconjugated secondary antibody (goat antirabbit IgG 1:10,000; 170-6515 BioRad, Hercules, CA, USA) for 1 h at room temperature. Signals were visualized by chemiluminescent detection with an enhanced chemiluminescent reagent (ECL, Amersham, Buckinghamshire, United Kingdom) according to the manufacturer’s protocol and exposed to imaging film (Hyperfilm ECL, Amersham, Buckinghamshire, United Kingdom). Band intensities were analyzed by densitometry (AlphaImagerTM, Alpha Innotech Corporation, San Leandro, CA, USA). Phospho-p42/p44 MAP kinase signals were normalized for total p42/p44 MAP kinase.

ETHICS

Rat islets

All procedures and experimental protocols were performed in accordance with Swiss animal protection laws.

Human islets

Informed consent for organ donation was asked for and obtained from the donors’ relatives. All studies performed were in accordance with Swiss laws and medical guidelines.

STATISTICS

The results are given as mean and standard deviation (±SD) or median, interquartile, and range. Statistical comparison between the control and the treatment groups was carried out using one-way ANOVA. Dunnett’s twotailed multiple comparison test was applied when more than one group was compared with the control/reference group. The software used was GraphPad Prism 4.00 (GraphPad Software Inc., San Diego, CA, USA). Two-tailed P values of less than 0.05 were considered statistically significant.

RESULTS

Immunohistochemical Studies of the Endothelin System on Rat and Human Islets

To determine the presence of ET-1 and its receptors ET_A and ET_B on human and rat pancreatic islets, co-staining studies with islet-specific hormones were performed on whole pancreatic sections. In human pancreatic beta-cells, all three components of the endothelin system (ET-1, ET_A- and ET_Breceptor) were detected whereas alpha-cells were only positive for ET-1 (Figure 1). Somatostatin-expressing delta-cells were negative for all three components. Interestingly, co-staining patterns differed in the two species. As opposed to human pancreatic islets, the alpha-cells of rat islets were also positive for ET_B-receptor and weakly positive for ET_A-receptor while deltacells showed immunoreactivity for ET-1 (not shown). Figure 2 summarizes the differences in species-specific co-expression patterns of endothelin and its receptors with islet-specific hormones. In order to confirm the specificities of the antibodies, competition immunoassays were performed (Figure 3). ET-1 and ET_Breceptor immunoreactivity was no longer detectable after preabsorption of the antibody with a 1,000-fold molar excess of the corresponding peptide. Since competition with the ET_A-receptor specific peptide was unsatisfactory, an unspecific staining by this antibody could not be excluded. However, both ET_A-receptor antibodies derived from different suppliers were consistent in terms of their staining pattern in pancreatic islets.

ET-1 Binding Studies on Isolated Rat Islets

To demonstrate that ET_A- and ET_B-receptor immunoreactivity represents the expression of ligand binding endothelin receptors, ET-1 binding assays were performed with dispersed rat islet cells. Maximal specific binding of radiolabeled ET-1 was 52.2±11.6 fmol/mg protein with an IC50 value of 0.37 nM, indicating the presence of high-affinity binding sites (Figure 4a). Maximal inhibition of radioligand binding by an excess of the specific ET_A-receptor antagonist BQ123 (51±21%, n=3) was comparable to those of the specific ET_B-receptor agonist IRL1620 (45±22%, n=3; P=0.742) and ET_B-receptor antagonist BQ788 (62±11%, n=3; P=0.346) (Figure 4b). The inhibitory capacity of these ligands in the range of 40-60% suggests that pancreatic islets express both ET_A- and ET_Breceptors in the ratio of roughly 1:1. Ligand binding in the presence of both antagonists (BQ123/BQ788; 22±13%, n=3; P=0.173 vs. BQ123) could not be inhibited completely which might indicate the presence of a third type of endothelin-binding receptor.

ET-1 Stimulates Phosphorylation of p42/p44 MAP kinase in Isolated Rat Islets

ET-1 has been shown to activate p42 and p44 MAP kinases through a protein kinase C (PKC)-dependent pathway [25]. To test whether the ligand binding capacity in pancreatic islet cells also translates into activation of an ET-1 relevant signaling pathway, phosphorylation of MAP kinases upon stimulation with ET-1 was analyzed. While phorbol 12-myristate-13-acetate (PMA), another known activator of MAP kinase, stimulated phosphorylation of p42/p44 by more than 4-fold, the maximal response to treatment with ET-1 was at the most 2.5-fold (Figure 5). The relatively small response of ET-1 on p42/p44 phosphorylation may originate from relatively high basal MAP kinase activity in freshly isolated rat islets. This observation is consistent with a previous report, describing sustained activation of MAP kinase during the first 24 h after the isolation procedure [26]. However, preincubation with the selective ET_A or ET_Breceptor antagonist (BQ123 or BQ788) blocked the ability of ET-1 to induce phosphorylation of MAP kinase p42/p44 completely. When combined, BQ123 and BQ788 reduced the activation of MAP kinase even below the basal value of untreated control islets (43±4%, n=3 vs. 100%; P<0.001). This suggests that ET-1 expressed in pancreatic islets may act in an autocrine fashion and stimulate MAP kinase activation. Thus, the results strongly indicate that, in isolated rat islets, both ET_A- and ET_Breceptors are expressed and are biologically active.

mRNA of Endothelin and Its Receptors in Beta-cell Lines and Isolated Rat Islets

To substantiate our immunohistological observations that all three components of the endothelin system are expressed in pancreatic beta-cells, we aimed to demonstrate the presence of ET-1, ET_A- and ET_B receptor mRNA in rat beta-cell lines by means of a reverse transcriptase polymerase chain reaction (RT-PCR). For all the PCR reactions, intron-spanning primer pairs were chosen to exclude the amplification of genomic DNA. DNA-fragments of ET-1, ET_A-receptor and ET_B-receptor from both INS-1 and RINm5f cells could be amplified, though at quite different intensities (Figure 6). Even though no quantitative assessment was performed, mRNA expression levels from rat islets were comparable to RINm5f cells, but clearly stronger when compared to the levels obtained from INS-1 cells. Different mRNA expression levels of the components of the endothelin system may simply reflect the property of the particular beta-cell line. On the other hand, it is conceivable that intraislet endothelium also contributes to the signals generated by RT-PCR.

Islet transplantation is inherently correlated to prolonged periods of ischemia as a consequence of the transient avascular state of the grafted tissue. It has previously been demonstrated that partial oxygen tension in the transplant site in the liver may be as low as 1% O2 as compared to 5-6% O2 in the native pancreas [22]. Therefore regulation of ET-1, ET_A- and ET_B-receptor transcript levels in purified isolated human islets after exposure to hypoxia (1% O2) was investigated by means of semi-quantitative and quantitative (real-time) RT-PCR (Figures 7 and 8). Hypoxic ET-1 expression levels were increased by 2.3-fold after 24 h (P<0.05) and 4.7-fold after 48 h (P<0.001) (average values; Figure 8). ET_B-receptor mRNA levels were increased by 3.3 fold (P<0.01) after 48 h of hypoxia as compared to normoxia. There was a tendency towards upregulation after 24 h of hypoxia which did not reach statistical significance. In contrast to ET-1 and ET_Breceptor, transcript levels of ET_A-receptor did not show significant changes upon hypoxic exposure.

These data indicate that hypoxia induces the expression of ET-1 and ET_B-receptor transcript levels whereas ET_A-receptor transcription levels are slightly reduced.

Immunohistochemical Studies of the Endothelin System in Rat Islets Before and After Transplantation

To investigate whether hypoxic regulation of the endothelin system also occurs in vivo, we performed immunohistochemical staining of ET-1 and its receptors (ET_A and ET_B) in islet grafts one day after intrahepatic (lobus caudatus) transplantation into syngeneic normoglycemic rats. At this stage, islets have been assumed to experience hypoxic conditions due to their avascular state. Similar to our in vitro data of hypoxic islets and compared to pancreatic sections, ET_B-receptor expression in transplanted islets appeared to be increased while ET_A-receptor immunoreactivity was completely lost (Figure 9). Immunostaining for ET-1 was no longer detectable which is discordant with the hypoxic upregulation of rat islet ET-1 transcript levels in vitro and could be attributed to transplant site microenvironmental factors other than hypoxia.

DISCUSSION

Endothelins have been shown to be involved in the physiology and pathophysiology of the vascular system, heart, lungs, kidney, brain, liver and various other tissues [10]. Endothelins mediate diverse biological effects in an autocrine or paracrine fashion which can either be beneficial or detrimental. With regard to intrahepatic islet transplantation, pre- and/or post-operative treatment with endothelin receptor antagonists would potentially improve graft perfusion and function, similar to the beneficial effects described in an experimental model involving transplantation of a pig pancreas or hepatic ischemia/reperfusion in rats [19, 27]. However, this treatment could also affect the islet graft in a negative fashion due to the blockade of the antiapoptotic effects of the endothelin system [16, 17]. The present study was performed to determine whether the endothelin system is present in the endocrine cells of pancreatic islets and to assess its regulation by hypoxia and in a transplant setting. Our results provide the necessary biological basis for the study design of endothelin agonist/antagonist treatment before and/or after islet transplantation and its putative impact on graft viability and function.

We were able to show the presence of ET-1, ET_A- and ET_B-receptors in endocrine cells of human and rat pancreata by immunohistochemical staining. In human pancreatic betacells, all three components of the endothelin system (ET-1, ET_A- and ET_B-receptor) can be detected whereas alpha cells were only positive for ET-1. Somatostatin-expressing delta-cells were negative for all three components which is in agreement with a previous study [28]. Interestingly, in contrast to human pancreatic islets, the alpha-cells of rat islets were also positive for ET_B-receptors, weakly positive for ET_A-receptors, and deltacells showed immunoreactivity for ET-1. Such species-specific differences in expression levels of the components of the endothelin system have been described elsewhere [29, 30]. Therefore, speciesdependent differences in the biological activities of the endothelin system need to be anticipated when drawing conclusions from animal experimentation for human biology. Preclinical experiments could involve transplanting human islets into non-obese diabetic severe combined immunodeficiency (NOD-SCID) mice which might provide a possible solution for the study of human islet biology in a transplant setting, but brings up the problems of xenotransplantation. As no perfect experimental model exists, biological findings need to be confirmed in human experimentation.

Endothelin receptor expression on pancreatic islet cells was confirmed by binding studies with radiolabeled ligand and competition by unlabeled ET-1 or receptor-specific antagonists. The binding capacity for ET-1 is comparable to other tissues such as the liver or heart, both of which have also been shown to express ET-receptors on parenchymal cells [31, 32]. Both the relatively high ET-1 binding capacity in islet tissue and the presence of ET_A- and ET_B-receptor mRNA in beta-cell lines (RINm5f, INS-1) strongly supports the immunohistochemical finding of non-endothelial expression of the two receptor subtypes in pancreatic islets. ET_Aand ET_B-receptors are expressed at a ratio of nearly 1:1 based on competition binding experiments with the specific antagonists BQ123 or BQ788, respectively. The presence of both receptor subtypes in isolated islets was confirmed by the ability of either of the antagonists to prevent ET-1-stimulated phosphorylation of p42/p44 MAP kinases, both of which are downstream mediators of endothelin signaling [33]. The finding of endothelin receptor expression on endocrine cells of pancreatic islets supports previous observations by which endothelin stimulates insulin secretion in isolated islets [34, 35]. It was suggested that stimulation of insulin secretion was mediated indirectly through the activation of glucagons-producing alpha-cells [21]. However, the evidence shown for ETreceptor expression on beta-cells and the ability of endothelin to elicit a raise in intracellular Ca2+ suggests a direct insulinotropic effect of endothelin on pancreatic beta-cells [36, 37].

Endothelins do not only regulate vascular homeostasis [38], but also mediate cell proliferation [39, 40] and are involved in the promotion or prevention of apoptosis [17, 41]. Several biological properties of endothelin may therefore also have a direct impact on islet cell survival and function after intrahepatic transplantation. For example, in adipocytes or rat lung epithelial cells, ET-1 reduces tumor necrosis factor-alpha (TNFalpha)- induced expression of inducible nitric oxide synthase (iNOS) [42, 43]. TNF-alpha in combination with other proinflammatory cytokines is released either through the activation of Kupffer cells or by islet resident macrophages known to promote beta-cell dysfunction and apoptotic beta-cell death via synthesis of nitric oxide [44, 45]. ET-1 mediates cytoprotection through the inhibition of iNOS, which is especially relevant in tissue inflammation, considering the fact that proinflammatory cytokines stimulate the expression of this hormone [46, 47].

Another beneficial function of ET-1 could be the ability to stimulate angiogenesis through enhanced expression of the vascular endothelial growth factor (VEGF) [48, 49]. ET-1 stabilizes hypoxia-inducible factor-1- alpha (HIF-1-alpha) which then promotes the transcription of the VEGF gene. Both factors, VEGF and HIF-1-alpha, are expressed in islet cells and could therefore not only stimulate islet revascularization but also provide cytoprotection from hypoxia-related graft injury [50, 51]. The majority of the abovedescribed biological activities of ET-1 will be potentially advantageous to islet survival and engraftment, especially in the posttransplantation period where near-ischemia may be prevalent in the islet graft [52, 53]. In vitro, hypoxia stimulates the expression of ET-1 and ET_B-receptor mRNA whereas ET_Areceptor steady state transcript levels are slightly reduced. This could be confirmed only partially in our syngeneic rat islet transplantation model where ET_B-receptor staining intensity seemed to be increased while ET_A-receptor and ET-1 immunoreactivity was strongly reduced. This discrepancy between in vitro and in vivo regarding the level of ET-1 expression may be explained by the more complex biology present in a transplantation setting. A very similar regulation pattern of the endothelin system was also found in a rat liver model of 1 h ischemia followed by 6 h of reperfusion [54]. While, in the liver, the described specific regulation pattern of the endothelin system may warrant a balanced action of vasodilators and vasoconstrictors under ischemic conditions, its involvement in ischemic islets remains speculative. It is conceivable that, in transplanted islets, altered expression of the endothelin system reflects a biological response by which cytoprotection is promoted through upregulation of the ET_Breceptor and microcirculation is improved by downregulation of ET-1.

In summary, our study clearly demonstrates the presence of ET-1 and its ET_A and ET_B receptors in non-endothelial, hormoneproducing cells of pancreatic islets. Its oxygen-dependent expression strongly suggests a rescue mechanism by which islet cells adapt to hypoxic conditions. These findings are of importance with respect to a potential therapeutic application of ET-1 antagonists in islet transplantation as has been used successfully in other organ transplant models. Further investigation is needed to shed light on the question as to whether the potential benefits of antagonizing endothelin in clinical islet transplantation outweigh the potent angiogenic and cytoprotective effect of endothelin demonstrated in other tissues.

Acknowledgements

Funding of this project by the Olga Mayenfisch, Hartmann-Müller, Hermann-Klaus, the Swiss National Foundation (grant 32-67132.01) and partial funding by the Theodor and Ida Herzog-Egli, the Julius Muller foundation and a private sponsor of the UBS is gratefully acknowledged. We thank The Electron Microscopy Institute of the University of Zurich, in particular Dr. M Höchli for his support and work with the confocal microscope. We also thank Trinh Lu for technical support as well as the members of the division of Endocrinology and Diabetology for helpful discussions.

Conflict of interest

The authors have no potential conflicts of interest

References

1. Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000; 343:230-8. [PMID 10911004]
2. Ryan EA, Lakey JR, Rajotte RV, Korbutt GS, Kin T, Imes S, et al. Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol. Diabetes 2001; 50:710-9. [PMID 11289033]
3. Ryan EA, Lakey JR, Paty BW, Imes S, Korbutt GS, Kneteman NM, et al. Successful islet transplantation: continued insulin reserve provides long-term glycemic control. Diabetes 2002; 51:2148- 57. [PMID 12086945]
4. Korsgren O, Nilsson B, Berne C, Felldin M, Foss A, Kallen R, et al. Current status of clinical islet transplantation. Transplantation 2005; 79:1289-93. [PMID 15912090]
5. Lehmann R, Zuellig RA, Kugelmeier P, Baenninger PB, Moritz W, Perren A, et al. Superiority of small islets in human islet transplantation. Diabetes 2007; 56:594-603. [PMID 17327426]
6. Bharat A, Saini D, Benshoff N, Goodman J, Desai NM, Chapman WC, Mohanakumar T. Role of intraislet endothelial cells in islet allo-immunity. Transplantation 2007; 84:1316-23. [PMID 18049117]
7. Ricordi C, Strom TB. Clinical islet transplantation: advances and immunological challenges. Nat Rev Immunol 2004; 4:259-68. [PMID 15057784]
8. Kim YH, Lee SM. Role of Kupffer cells in the vasoregulatory gene expression during hepatic ischemia/reperfusion. Arch Pharm Res 2004; 27:111-7. [PMID 14969349]
9. Scommotau S, Uhlmann D, Löffler BM, Breu V, Spiegel HU. Involvement of endothelin/nitric oxide balance in hepatic ischemia/reperfusion injury. Langenbecks Arch Surg 1999; 384:65-70. [PMID 10367633]
10. Kedzierski RM, Yanagisawa M. Endothelin system: the double-edged sword in health and disease. Annu Rev Pharmacol Toxicol 2001; 41:851-76. [PMID 11264479]
11. Cunningham ME, Huribal M, Bala RJ, McMillen MA. Endothelin-1 and endothelin-4 stimulate monocyte production of cytokines. Crit Care Med 1997; 25:958-64. [PMID 9201047]
12. Shichiri M, Hirata Y, Nakajima T, Ando K, Imai T, Yanagisawa M, et al. Endothelin-1 is an autocrine/paracrine growth factor for human cancer cell lines. J Clin Invest 1991; 87:1867-71. [PMID 2022753]
13. Fernandez-Patron C, Zouki C, Whittal R, Chan JS, Davidge ST, Filep JG. Matrix metalloproteinases regulate neutrophil-endothelial cell adhesion through generation of endothelin-1[1-32]. FASEB J 2001; 15:2230-40. [PMID 11641250]
14. Hirata Y, Emori T, Eguchi S, Kanno K, Imai T, Ohta K, Marumo F. Endothelin receptor subtype B mediates synthesis of nitric oxide by cultured bovine endothelial cells. J Clin Invest 1993; 91:1367-73. [PMID 7682570]
15. D'Orléans-Juste P, Télémaque S, Claing A, Ihara M, Yano M. Human big-endothelin-1 and endothelin-1 release prostacyclin via the activation of ET1 receptors in the rat perfused lung. Br J Pharmacol 1992; 105:773-5. [PMID 1324048]
16. Ogata Y, Takahashi M, Ueno S, Takeuchi K, Okada T, Mano H, et al. Antiapoptotic effect of endothelin-1 in rat cardiomyocytes in vitro. Hypertension 2003; 41:1156-63. [PMID 12668584]
17. Shichiri M, Kato H, Marumo F, Hirata Y. Endothelin-1 as an autocrine/paracrine apoptosis survival factor for endothelial cells. Hypertension 1997; 30:1198-203. [PMID 9369276]
18. Palmes D, Budny TB, Stratmann U, Herbst H, Spiegel HU. Endothelin-A receptor antagonist reduces microcirculatory disturbances and transplant dysfunction after partial liver transplantation. Liver Transpl 2003; 9:929-39. [PMID 12942454]
19. Witzigmann H, Ludwig S, Armann B, Gäbel G, Teupser D, Kratzsch J, et al. Endothelin(A) receptor blockade reduces ischemia/reperfusion injury in pig pancreas transplantation. Ann Surg 2003; 238:264-74. [PMID 12894021]
20. Yamamoto T, Uemura H. Distribution of endothelin-B receptor-like immunoreactivity in rat brain, kidney, and pancreas. J Cardiovasc Pharmacol 1998; 31 Suppl 1:S207-11. [PMID 9595439]
21. Brock B, Gregersen S, Kristensen K, Thomsen JL, Buschard K, Kofod H, Hermansen K. The insulinotropic effect of endothelin-1 is mediated by glucagon release from the islet alpha cells. Diabetologia 1999; 42:1302-7. [PMID 10550413]
22. Carlsson PO, Palm F, Andersson A, Liss P. Markedly decreased oxygen tension in transplanted rat pancreatic islets irrespective of the implantation site. Diabetes 2001; 50:489-95. [PMID 11246867]
23. Gotoh M, Maki T, Kiyoizumi T, Satomi S, Monaco AP. An improved method for isolation of mouse pancreatic islets. Transplantation 1985; 40:437- 8. [PMID 2996187]
24. Lehmann R, Weber M, Berthold P, Züllig R, Pfammatter T, Moritz W, et al. Successful simultaneous islet-kidney transplantation using a steroid-free immunosuppression: two-year follow-up. Am J Transplant 2004; 4:1117-23. [PMID 15196070]
25. Bogoyevitch MA, Parker PJ, Sugden PH. Characterization of protein kinase C isotype expression in adult rat heart. Protein kinase C-epsilon is a major isotype present, and it is activated by phorbol esters, epinephrine, and endothelin. Circ Res 1993; 72:757-67. [PMID 8443867]
26. Paraskevas S, Aikin R, Maysinger D, Lakey JR, Cavanagh TJ, Hering B, et al. Activation and expression of ERK, JNK, and p38 MAP-kinases in isolated islets of Langerhans: implications for cultured islet survival. FEBS Lett 1999; 455:203-8. [PMID 10437773]
27. Peralta C, Bulbena O, Bargalló R, Prats N, Gelpí E, Roselló-Catafau J. Strategies to modulate the deleterious effects of endothelin in hepatic ischemiareperfusion. Transplantation 2000; 70:1761-70. [PMID 11152109]
28. Kakugawa Y, Giaid A, Yanagisawa M, Baynash AG, Melnyk P, Rosenberg L, Duguid WP. Expression of endothelin-1 in pancreatic tissue of patients with chronic pancreatitis. J Pathol 1996; 178:78-83. [PMID 8778321]
29. Cernacek P, Strmen J, Levy M. Acute renal effects of endothelin-A blockade: interspecies differences. J Cardiovasc Pharmacol 1998; 31 Suppl 1:S269-72. [PMID 9595457]
30. Fukuroda T, Kobayashi M, Ozaki S, Yano M, Miyauchi T, Onizuka M, et al. Endothelin receptor subtypes in human versus rabbit pulmonary arteries. J Appl Physiol 1994; 76:1976-82. [PMID 8063659]
31. Housset C, Rockey CD, Bissell DM. Endothelin receptors in rat liver: lipocytes as a contractile target for endothelin 1. Proc Natl Acad Sci U S A 1993; 90:9266-70. [PMID 8415690]
32. Modesti PA, Vanni S, Paniccia R, Perna A, Maccherini M, Lisi G, et al. Endothelin receptors in adult human and swine isolated ventricular cardiomyocytes. Biochem Pharmacol 1999; 58:369-74. [PMID 10423180]
33. Wang Y, Rose PM, Webb ML, Dunn MJ. Endothelins stimulate mitogen-activated protein kinase cascade through either ET_A or ET_B. Am J Physiol 1994; 267:C1130-5. [PMID 7943276]
34. Gregersen S, Thomsen JL, Hermansen K. Endothelin-1 (ET-1)-potentiated insulin secretion: involvement of protein kinase C and the ET(A) receptor subtype. Metabolism 2000; 49:264-9. [PMID 10690956]
35. Gregersen S, Thomsen JL, Brock B, Hermansen K. Endothelin-1 stimulates insulin secretion by direct action on the islets of Langerhans in mice. Diabetologia 1996; 39:1030-5. [PMID 8877285]
36. Kawanabe Y, Okamoto Y, Enoki T, Hashimoto N, Masaki T. Ca(2+) channels activated by endothelin-1 in CHO cells expressing endothelin-A or endothelin-B receptors. Am J Physiol Cell Physiol 2001; 281:C1676- 85. [PMID 11600432]
37. Iwamuro Y, Miwa S, Zhang XF, Minowa T, Enoki T, Okamoto Y, et al. Activation of three types of voltage-independent Ca2+ channel in A7r5 cells by endothelin-1 as revealed by a novel Ca2+ channel blocker LOE 908. Br J Pharmacol 1999; 126:1107-14. [PMID 10204997]
38. Wright CE, Fozard JR. Regional vasodilation is a prominent feature of the haemodynamic response to endothelin in anaesthetized, spontaneously hypertensive rats. Eur J Pharmacol 1988; 155:201-3. [PMID 2907490]
39. Mazzocchi G, Rossi GP, Rebuffat P, Malendowicz LK, Markowska A, Nussdorfer GG. Endothelins stimulate deoxyribonucleic acid synthesis and cell proliferation in rat adrenal zona glomerulosa, acting through an endothelin A receptor coupled with protein kinase C- and tyrosine kinase-dependent signaling pathways. Endocrinology 1997; 138:2333-7. [PMID 9165019]
40. Komuro I, Kurihara H, Sugiyama T, Yoshizumi M, Takaku F, Yazaki Y. Endothelin stimulates c-fos and c-myc expression and proliferation of vascular smooth muscle cells. FEBS Lett 1988; 238:249-52. [PMID 3139457]
41. Cattaruzza M, Dimigen C, Ehrenreich H, Hecker M. Stretch-induced endothelin B receptor-mediated apoptosis in vascular smooth muscle cells. FASEB J 2000; 14:991-8. [PMID 10783154]
42. Mérial-Kieny C, Lonchampt M, Cogé F, Verwaerde P, Galizzi JP, Boutin JA, et al. Endothelin-1 inhibits TNF alpha-induced iNOS expression in 3T3- F442A adipocytes. Br J Pharmacol 2003; 139:935-44. [PMID 12839867]
43. Markewitz BA, Michael JR, Kohan DE. Endothelin-1 inhibits the expression of inducible nitric oxide synthase. Am J Physiol 1997; 272:L1078-83. [PMID 9227507]
44. Nussler AK, Carroll PB, Di Silvio M, Rilo HL, Simmons RL, Starzl TE, Ricordi C. Hepatic nitric oxide generation as a putative mechanism for failure of intrahepatic islet cell grafts. Transplant Proc 1992; 24:2997. [PMID 1466032]
45. Stevens RB, Lokeh A, Ansite JD, Field MJ, Gores PF, Sutherland DE. Role of nitric oxide in the pathogenesis of early pancreatic islet dysfunction during rat and human intraportal islet transplantation. Transplant Proc 1994; 26:692. [PMID 7513466]
46. Saleh D, Furukawa K, Tsao MS, Maghazachi A, Corrin B, Yanagisawa M, et al. Elevated expression of endothelin-1 and endothelin-converting enzyme-1 in idiopathic pulmonary fibrosis: possible involvement of proinflammatory cytokines. Am J Respir Cell Mol Biol 1997; 16:187-93. [PMID 9032126]
47. Yoshizumi M, Kurihara H, Morita T, Yamashita T, Oh-hashi Y, Sugiyama T, et al. Interleukin 1 increases the production of endothelin-1 by cultured endothelial cells. Biochem Biophys Res Commun 1990; 166:324-9. [PMID 2405848]
48. Matsuura A, Yamochi W, Hirata K, Kawashima S, Yokoyama M. Stimulatory interaction between vascular endothelial growth factor and endothelin-1 on each gene expression. Hypertension 1998; 32:89-95. [PMID 9674643]
49. Pedram A, Razandi M, Hu RM, Levin ER. Vasoactive peptides modulate vascular endothelial cell growth factor production and endothelial cell proliferation and invasion. J Biol Chem 1997; 272:17097-103. [PMID 9202027]
50. Gorden DL, Mandriota SJ, Montesano R, Orci L, Pepper MS. Vascular endothelial growth factor is increased in devascularized rat islets of Langerhans in vitro. Transplantation 1997; 63:436-43. [PMID 9039936]
51. Moritz W, Meier F, Stroka DM, Giuliani M, Kugelmeier P, Nett PC, et al. Apoptosis in hypoxic human pancreatic islets correlates with HIF-1alpha expression. FASEB J 2002; 16:745-7. [PMID 11923216]
52. Carlsson PO, Liss P, Andersson A, Jansson L. Measurements of oxygen tension in native and transplanted rat pancreatic islets. Diabetes 1998; 47:1027-32. [PMID 9648824]
53. Carlsson PO, Palm F, Andersson A, Liss P. Chronically decreased oxygen tension in rat pancreatic islets transplanted under the kidney capsule. Transplantation 2000; 69:761-6. [PMID 10755523]
54. Sonin NV, Garcia-Pagan JC, Nakanishi K, Zhang JX, Clemens MG. Patterns of vasoregulatory gene expression in the liver response to ischemia/reperfusion and endotoxemia. Shock 1999; 11:175-9. [PMID 10188769]