Naunyn-Schmied Arch Pharmacol (2009) 379:201–215 DOI 10.1007/s00210-008-0340-5

ABSTRACT

Abstracts of the Farmacologiedagen 2007

1 LIGHT-INDUCED PHOTORELAXATION OF CORONARY ARTERIES: DO S-NITROSOTHIOLS FUNCTION AS EDHF? W.W. Batenburg, M.J. Eikmann, S.N.A. Ramzan, R. de Vries, A.H.J. Danser Dept. of Pharmacology, Erasmus MC, The Netherlands Light-induced relaxation is believed to depend on S-nitrosothiols. Snitrosothiols may serve as endothelium-derived hyperpolarizing factors (EDHF) mediating the relaxation of porcine coronary arteries (PCAs) to bradykinin (BK). Here, we compared light- and BKinduced PCA relaxation. Light relaxed preconstricted PCAs by 73± 4%. Relaxations diminished during repetitive exposure to light, especially when the periods of darkness between the exposures were <10 min. Thus, re-loading of the storage pools occurs in the absence of light. The S-nitrosothiol-depleting agent ethacrynic acid reduced light-induced relaxation, confirming its S-nitrosothiol dependency. BK relaxed PCAs by 62±4%. Endothelium removal, NO synthase inhibition, and Ca2+-dependent K+(KCa) channel blockers diminished the response to BK, but not to light. Removal of NO and inhibition of Na+–K+ATPase abolished the response to BK and light. Inhibition of guanylyl cyclase abolished the response to light and blocked the response to BK by >50%. In conclusion, photorelaxation largely depends on the release of stored S-nitrosothiols from non-endothelial cells, possibly vascular smooth muscle cells, whereas endothelial KCa channel activation appears to underlie the BK-induced release of Snitrosothiols. Both types of relaxation involve the NO-guanylyl cyclase-cGMP pathway and Na+–K+ ATPase, thereby confirming that stored S-nitrosothiols may indeed function as EDHF.

2 A 80 AMINO ACID DELETION IN THE THIRD INTRACELLULAR LOOP OF A NATURALLY OCCURRING HUMAN HISTAMINE H3 ISOFORM CONFERS PHARMACOLOGICAL DIFFERENCES AND CONSTITUTIVE ACTIVITY G. Bongers1, K.M. Krueger2, T.R. Miller2, J.L. Baranowski2, B.R. Estvander2, D.G. Witte2, M.I. Strakhova2, R.A. Bakker1, M.D. Cowart2, A.A. Hancock2, T.A. Esbenshade2, R. Leurs1 1 Leiden/Amsterdam Center for Drug Research, Dept. of Medicinal Chemistry, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands 2 Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois, USA We pharmacologically characterized two naturally occurring human histamine H3 receptor (hH3R) isoforms, the hH3R(445) and hH3R

(365). These abundantly expressed splice variants differ by a deletion of 80 amino acids in the intracellular loop 3. We show that the hH3R (365) is differentially expressed compared to the hH3R(445) and has a higher affinity and potency for H3R agonists and, conversely, a lower potency and affinity for H3R inverse agonists. Furthermore, we show a higher constitutive signaling of the hH3R(365) compared to the hH3R(445) in both [35S]GTPγS binding and cAMP assays, likely explaining the observed differences in hH3R pharmacology of the two isoforms. As H3R ligands are beneficial in animal models of obesity, epilepsy and cognitive diseases such as Alzheimer’s disease and attention deficit hyperactivity disorder and currently entered in clinical trails, these differences in H3R pharmacology of these two isoforms are of great importance for a detailed understanding of the action of H3R ligands.

3 THE ROLE OF COLLAGEN BREAKDOWN PRODUCTS INOSTEOARTHRITIS S. Braber, A. Hartog, J. van Bergen-Henegouwen, A.D. Kraneveld, G. Folkerts, J. Garssen Dept. of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, The Netherlands The mechanism by which collagen breakdown products play a role in the pathogenesis of Osteoarthritis is unknown. However, the collagen degradation product proline–glycine–proline (PGP), which is homologous to key sequences of CXCL8, stimulates the CXCR1 and CXCR2 receptors and prolongs the influx of neutrophils. The present study was conducted to examine the effect of collagen breakdown products on bovine chondrocytes. Chondrocytes and explants were obtained from bovine articular cartilage slices. Isolated chondrocytes were treated with CXCL8 or PGP and/or glycine–proline–proline (GPP) to assess their potential to produce nitric oxide (NO). The effect of collagen fragments on the rate of proteoglycan release was determined in explants. RT-PCR was used to obtain the expression of the CXCR1-receptor and the hypertrophic marker matrix metalloproteinase-13 (MMP-13) in control and CXCL8-stimulated chondrocytes. Our data show that combination of PGP with GPP causes a dosedependent increase in proteoglycan release from explants. Furthermore, a small increase was observed in the NO production in explants stimulated with PGP. After 5 days incubation without stimulation or in the presence of CXCL8, the CXCR1 gene expression was down regulated in chondrocytes. Finally, both CXCL8 increased MMP-13 expression in chondrocytes. Collectively our data show that collagen products are able to induce activation of the chondrocyte, but further investigations are needed to clarify the way of action.

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DOES ALDOSTERONE MEDIATE ITS CARDIAC EFFECTS VIA MINERALOCORTICOID RECEPTORS AND IS CORTICOSTERONE THE ENDOGENOUS AGONIST OF THESE EFFECTS IN VIVO? W. Chai, A.H.J. Danser Dept. of Pharmacology, Erasmus MC, Rotterdam, the Netherlands

INTERACTION OF IMMUNOSUPPRESSANTS WITH METHOTREXATE TRANSPORT MEDIATED BY HUMAN MULTIDRUG RESISTANCE TRANSPORTERS MRP2 AND MRP4 A.A.K. El-Sheikh, J.J.M.W. van den Heuvel, J.B. Koenderink, F.G.M. Russel Dept. of Pharmacology and Toxicology, NCMLS, Radboud University Nijmegen Medical Centre, The Netherlands

In the rat heart, aldosterone increases left ventricular pressure (LVP) and decreases coronary flow (CF). These effects appeared to occur in a mineralocorticoid receptor (MR)-independent manner since the MR antagonists spironolactone and eplerenone did not block them. However, the in vitro effectiveness of these drugs has been questioned, and the open-ring, water-soluble antagonist canrenoate might be more appropriate to test the role of MR in vitro. Furthermore, given the low cardiac levels of the cortico-sterone-degrading enzyme 11βhydroxysteroid dehydrogenase type 2 (11βHSD2), corticosterone rather than aldosterone might be the endogenous agonist of the cardiac MR. To evaluate these concepts, we compared the effects of aldosterone and corticosterone in the rat Langendorff heart in the absence or presence of canrenoate and the 11βHSD2 inhibitor carbenoxolone. Aldosterone increased LVP and decreased CF. Canrenoate increased LVP and CF but did not block the effects of aldosterone. Corticosterone increased LVP and, unlike aldosterone, increased CF. Carbenoxolone did not potentiate corticosterone. In conclusion, the lack of effect of canrenoate towards aldosterone in the heart reinforces the idea that the positive inotropic and vasoconstrictor effects of aldosterone occur in a MR-independent manner. Corticosterone is unlikely to be the endogenous agonist of these effects in vivo.

5 INSULIN-INDUCED LAMININ EXPRESSION PROMOTES A HYPERCONTRACTILE AIRWAY SMOOTH MUSCLE PHENOTYPE B.G.J. Dekkers, D. Schaafsma, J. Zaagsma, H. Meurs Dept. of Molecular Pharmacology, University of Groningen, The Netherlands Airway smooth muscle (ASM) plays a key role in airway hyperresponsiveness and remodeling in asthma. Insulin has been shown to induce a functional hypercontractile phenotype characterized by increased contractile responses and contractile protein expression, as well as decreased mitogenic capacity. Laminin, an extracellular matrix protein, has also been found to promote a contractile phenotype. Using cultured bovine tracheal smooth muscle (BTSM) strips, we investigated the role of laminin in the induction of a hypercontractile phenotype by insulin. The results demonstrate that insulin-induced hypercontractility after 8 days of tissue culture was fully normalized by treatment of BTSM strips with the laminin β1-chain competing peptide Tyr–Ile–Gly–Ser–Arg (YIGSR). The inhibitory effect of insulin on platelet-derived growth factor-induced DNA-synthesis in BTSM cells was also completely abrogated by YIGSR treatment. In addition, we demonstrated that expression of laminin β1- and γ1chains in BTSM strips was increased by insulin. Signalling through PI3-kinase- and Rho-kinase-dependent pathways were required for the increase in laminin chain abundance. Collectively, our results suggest a critical role for laminin in the induction of a hypercontractile, hypoproliferative ASM phenotype by insulin (NAF grant 0.3.36).

Multidrug resistance transporters MRP2 and MRP4 confer resistance to cytostatic and immunosuppressant drugs. In addition, the expression of these efflux pumps in the apical membrane of renal proximal tubule cells can play a key role in excretion and toxicity of these drugs. Here, we studied the effect of different immunosuppressants on ATPdependent [3H]-methotrexate (0.1 μM) uptake into membrane vesicles isolated from HEK293 cells over-expressing human MRP2 and MRP4. Our results show that, at therapeutic concentrations, mycophenolic acid and tacrolimus inhibited, while mitoxantrone and dexamethasone stimulated, transport in both MRP2 and MRP4 vesicles. Cyclosporine only inhibited MRP2, while 6-mercaptopurine only inhibited MRP4-mediated transport. Cytarabine and azathioprine had no effect on either transporter. These data suggest that inhibition of the renal drug efflux pumps, MRP2 and MRP4, may play a role in immunosuppressant drug interactions and nephrotoxicity.

7 DISTINCT ROLES OF CB1 AND CB2 CANNABINOID RECEPTORS IN HUMAN BRONCHIAL EPITHELIAL CELLS C.R.S. Elzinga, S.S. Roscioni, E. Gkoumassi, H.J. van der Horn, F.J. Warnders, E.L.P. van Streun, S.A. Nelemans, H. Meurs, J. Zaagsma, M. Schmidt Dept. of Molecular Pharmacology, University of Groningen, The Netherlands We reported in human bronchial epithelial 16HBE14o− cells that activation of the cannabinoid receptors (CB1-R and CB2-R) by the endocannabinoid virodhamine modulates the cellular level of the universal second messengers cAMP and Ca2+. Such alterations might be involved in the control of cell survival and death. In the present study, the roles of CB1-R and CB2-R were studied separately using small interference RNA. Silencing of CB1-R and CB2-R reduced their expression as assessed by using specific antibodies; in contrast, expression of β-actin was not altered. Reduction in the expression of both CB1-R and CB2-R reduced virodhamine-induced Ca 2+ and cAMP increases only slightly. Silencing of CB1-R increased virodhamine-induced reduction in cell viability, whereas CB2-R silencing had the opposite effect. Such effects were paralleled by alterations in the level of extracellular signal-regulated kinases (ERK1/ 2) and Ras-like GTPases, the latter known to regulate the cellular ERK1/2 response. These data suggest that CB1-R and CB2-R signaling in 16HBE14o− cells exert anti-apoptotic and pro-apoptotic properties, respectively, and that these effects seem to involve ERK1/2 and Ras-like GTPases.

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A NEW ANIMAL MODEL FOR COW’S MILK ALLERGY: USEFULL TOOL FOR EPITOPE TESTING E.C.A.M. van Esch1, B. Schouten1, G.A. Hofman1, L.E.M. Willemsen1, T. van Baalen2, L.M.J. Knippels2, J. Garssen1,2 1 Pharmacology and Pathophysiology, Dept. Pharmaceutical Sciences, Utrecht University 2Immunology, Numico Research BV, Wageningen

CAVEOLIN-1 IS REQUIRED AND SUFFICIENT FOR SMOOTH MUSCLE SPECIFIC PROTEIN EXPRESSION R. Gosens1,2, G. Dueck1, G. Stelmack1, X.Q. Liu1, H. Unruh1, William T Gerthoffer3, J. Zaagsma2, H. Meurs2, A..J. Halayko1 1 Dept. Physiology, University of Manitoba, Winnipeg, Canada, 2Dept. Molecular Pharmacology, University of Groningen, The Netherlands, 3 Dept. Pharmacology, University of Nevada-Reno, NV, USA.

Introduction: Cow’s milk hydrolysates contain less allergic epitopes. They are used for prevention to avoid sensitization or as therapy in allergic children. In a mouse model using a skin response as readout, the allergenicity of a partial hydrolysate (pHF) was tested in both the sensitization and the effector phase of the allergic response. This is indicative for prevention or treatment, respectively. Methods: C3H/ HeOuJ mice were sensitized orally with whey proteins using cholera toxin as adjuvant. The primary readout is an acute ear swelling response upon challenge with whey. To test the allergenicity of the pHF on effector phase, whey-sensitized mice were ear challenged with pHF. The sensitizing capacity of pHF was tested by oral sensitization with pHF using cholera toxin and subsequent ear challenge with whey. Results: Acute ear swelling correlates with specific IgE and IgG levels. Whey-sensitized mice challenged with pHF showed 60% reduction in acute skin reaction. Oral sensitization with pHF diminished ear swelling to whey by 60%. In contrast to sensitization with whey, pHF did not elicit any whey-specific IgE response. Conclusions: A partial whey hydrolysate diminished both sensitization and the effector phase in a mouse model for cow’s milk allergy. This model involves an oral route of sensitization, acute allergic skin responses as primary readout and induction of specific serum Ig’s.

Caveolae are abundant plasma membrane invaginations that are enriched in caveolin-1 (cav-1) protein. Cav-1 can serve as a scaffold for signalling molecules and exhibits autonomous signalling functions that inhibit cell proliferation. We previously showed that caveolae and cav-1 expression are highest in growth-arrested airway smooth muscle (ASM) cells with a mature, contractile phenotype. We now investigated the functional role of cav-1 in contractile protein expression. Transforming growth factor (TGF)-β induced profound increases in the contractile phenotype markers sm-α-actin and calponin but failed to induce concomitant cav-1 expression. Nonetheless, the presence of cav-1 was required as its siRNA knockdown abrogated the capacity for induction of contractile markers and associated signalling (4EBP-1 phosphorylation) by TGF-β. Conversely, adenoviral overexpression of cav-1 was sufficient to induce the expression of calponin and sm-αactin and induced spontaneous phosphorylation of 4EBP-1. Collectively, these results indicate cav-1 is required and sufficient for contractile phenotype maturation of ASM (supported by Marie Curie Fellowship OIF-008823 to RG).

11 9 RENIN INHIBITION IMPROVES CORONARY ENDOTHELIAL FUNCTION IN SHR J.H.M. van Esch, R. van Veghel, I.M. Garrelds, A.H.J. Danser Dept. of Pharmacology, Erasmus Medical Center, Rotterdam, The Netherlands RAS inhibitors have been proven to be successful in lowering BP and providing end-organ protection. In this study, we compared the cardiovascular efficacy of the new rennin inhibitor aliskiren with the AT1 receptor blocker irbesartan and the ACE inhibitor captopril in SHR. SHR were implanted with telemetry transmitters and osmotic pumps filled with vehicle, captopril, ibesartan or aliskiren. HR and MAP were monitored for 3 weeks. Subsequently, the heart was removed for Langendorff studies. Captopril (3 and 6 mg kg−1 day−1), irbesartan (15 and 30 mg kg−1 day−1) and aliskiren (100 mg/kg/day) lowered MAP in a dosedependent manner (P<0.001). HR was unaffected by aliskiren and the low ibesartan dose. HR was increased (P<0.001) by the high irbesartan dose and decreased (P<0.01) by both captopril doses. The coronary flow response to Ang II was identical in all groups, whereas the response to bradykinin was increased after treatment with captopril (6 mg kg−1 day−1), irbesartan (15 and 30 mg kg−1 day−1), or aliskiren (100 mg kg−1 day−1) (P<0.05). In conclusion, aliskiren lowers blood pressure and improves coronary endothelial function in SHR to at least the same degree as ACE inhibition and AT1 receptor blockade.

HAPLOTYPES (HAP) OF THE ANGIOTENSIN II TYPE 1 RECEPTOR (AT1R)—ETHNICITY-DEPENDENT ASSOCIATION TO SYSTOLIC BLOOD PRESSURE (SBP) IN THE SUNSET DATABASE I.N. Hahntow, C.A. Teitsma, I. van Valkengoed, R.P. Koopmans, M.C. Michel Depts. Pharmacol. & Pharmacother., Int. Med. and Social Med., AMC, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands To explore the ethnicity-dependent association of AT1R HAPs with SBP, we genotyped 1,453 black, white and south Asian subjects of the cross-sectional SUNSET database. Twelve tagging SNP were selected based on own AT1R sequencing data (pilot study), HapMap data and literature. HAPs were defined by visual cluster analysis. Twenty different HAPs in exon 5 and in the promoter region with a frequency >10 were identified. HAP distribution differed qualitatively and quantitatively between ethnic groups with Blacks and South Asians exhibiting the greatest and smallest intra-group genetic variability, respectively. A combined analysis of exon 5 and promoter region identified five HAPs associated with high or low SBP (>140 or <120 mmHg after correction for age, sex, BMI, antihypertensive treatment). HAPs associated with mean corrected SBP >140 mmHg consistently contained the snp620. Among extreme HAP carriers, two HAPs were largely limited to Blacks (~90%), whereas snp1166 was overrepresented in Whites; the distribution of the other HAPs was similar across ethnic groups. We conclude that HAP/SBP associations differ between ethnic groups and that suitable tagging SNPs need to be identified for each group.

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SEPTIC SHOCK: A ROLE FOR RGS PROTEINS? M.C. Hendriks-Balk, R.Z.M. Tjon-Atsoi, P.B. van Loenen, M.-J. Mathy, M.C. Michel, S.L.M. Peters, A.E. Alewijnse Dept. of Pharmacology and Pharmacotherapy, AcademicMedical Center Amsterdam, The Netherlands

BREAST CANCER RESISTANCE PROTEIN 1 (BCRP) AND P-GLYCOPROTEIN (MDR1) ARE KEY PLAYERS IN RENAL REGENERATION AFTER ISCHEMIC INJURY M. Huls1, F. Ulloa-Montoya2, A. Luttun3, A.L. Menke4, L. van Bolderen5, R. Woestenenk4, R. R. G. Bueters1, F. G. M. Russel1, C. M. Verfaillie2, R. Masereeuw1 1 Dept. of Pharmacology and Toxicology, NCMLS, RUNMC, 2Interdepartmental Stem Cell Institute, 3Centre for Molecular and Vascular Biology, KUL, Belgium. 4Central Hematology Lab., RUNMC, 5Dept. of Radiation Oncology, RUNMC, The Netherlands.

The profound hypotension in septic shock patients is difficult to treat as they display depressed vascular responses to _-adrenergic agonists. Bacterial lipopolysaccharide (LPS) is the main trigger for most of the cardiovascular alterations occurring in septic shock. Recently, LPSinduced cardiac failure was found to be associated with upregulation of the regulator of G protein signalling (RGS) proteins RGS4 and RGS16 (Cardiovasc Res 53:156, 2002). In this study, we investigate the effects of LPS exposure on vascular contractility in general and the role of RGS proteins in the LPS-induced vascular alterations. Exposure of rat aortic rings to various LPS concentrations (1, 3, 10, 30 μg ml−1) for 22 h had differential effects on the contractile responses to agonists at four distinct G-protein coupled receptors. Phenylephrine- and angiotensin II-induced contraction was reduced, whereas serotonin-induced contraction was significantly enhanced. The endothelin-1-induced contraction was unaffected. Concomitantly, LPS treatment increased the RGS16 mRNA expression level both in aortic rings and vascular smooth muscle cells (VSMCs), but not that of RGS2, RGS3, RGS4 or RGS5. Regulation of RGS16 mRNA in VSMCs was also time- and concentration-dependent. The changes in RGS16 mRNA might contribute to the differential regulation of the contractile responses in an LPS model of septic shock.

The kidney has a high capacity to regenerate after acute kidney injury. Tubular cells or stem cells residing in the kidney or bone marrowderived stem cells might be responsible for this regeneration process. It was shown previously that the ABC transporters MDR1 and BCRP may function as regulators of stem cell biology. Both transporters are localized in the kidney and seem to be important for renal regeneration. The goal of the present study was to define the role of MDR1 and BCRP on bone marrow-derived stem cells in renal regeneration. Therefore, we performed bone marrow transplantations using wild type and MDR1 and BCRP knockout mice. We demonstrated that bone marrow-derived stem cells have the plasticity to differentiate into renal tubule cells and that MDR1 and BCRP are key players in this process. Expression of these transporters hampers renal regeneration, possibly because the differentiation state of the transplanted bone marrow cells is an important determinant in the regenerative capacity.

15 13 HIGH CONTENT CELL-BASED ASSAYS FOR DRUG DELIVERY ON PROTEIN KINASES, NUCLEAR RECEPTORS AND G PROTEIN-COUPLED RECEPTORS R.E.M.A. van Herpen, L.H.C.J.. van Lith, E.J.P. van Doornmalen, M. Blomenröhr, C.W. Kuil, M.C.A. Ruijs, E.E.M.G. Loomans, G.J.R. Zaman Molecular Pharmacology Unit, Organon BioSciences N.V., Oss, The Netherlands. High content screening and analysis (HCS, HCA) is the process of microscopic imaging acquisition combined with algorithmic image analysis. HCA allows the evaluation of the cellular efficacy of compounds by visualizing drug target biology or relevant cellular phenotypes associated with the biology of the target. Using HCA, protein dynamics (e.g. receptor internalization, foci formation, or nuclear translocation of proteins) and protein expression levels (measuring fluorescence intensity) can be monitored for protein kinases, nuclear, and G protein-coupled receptors. Cellular imaging applications contribute to target validation and pathway analysis, primary and secondary compound screening, and cellular assay development. Therefore, HCA provides a tool that enables the profiling of compounds in biological complex systems. HCA examples will be shown for all three target classes in comparison to other assay methodologies currently used in the drug discovery process.

EVALUATION OF A NOVEL PHENOTYPING COCKTAIL FOR PHENOTYPING THREE HUMAN CYTOCHROMES P450 ISO-ENZYMES CYP2D6, CYP2C19 AND CYP3A4 IN HEALTHY VOLUNTEERS M.P. van Iersel, T. de Boer, J. B. Hak, J. W. Wieling, H. R. S. Schwietert, I. den Daas, J. Wemer Xendo Drug Development BV, Hanzeplein 1, 9713 GZ Groningen, The Netherlands Variability in drug metabolism attributes to the variability in drug response and incidence and severity of adverse events. Cytochrome P450 iso-enzymes play an important role in drug metabolism and genetic polymorphism has been identified for some of these enzymes. Poor metabolisers have low drug metabolizing enzyme activity compared to the majority of individuals defined as extensive metabolisers. This variability may result in a different pharmacokinetic, pharmacodynamic, and adverse event profile. For phenotyping procedures, drugs are either given alone or in combinations of CYPspecific substrates. Phenotype cocktails to evaluate cytochrome P450 activity have been used widely by measuring the parent compound and its metabolite(s) in plasma or in urine. Genotyped and phenotyped healthy volunteers are important in clinical pharmacological studies to help elucidate the aethiology of side effects or deviations in pharmacokinetics and/or pharmacodynamics. Phenotyping cocktails for phenotyping human cytochromes, which can be given routinely with a simple administration and sampling scheme, are essential. In this study, a phenotyping cocktail of dextromethorphan, omeprazole, and alprazolam for phenotyping healthy volunteers for three human cytochrome P450 enzymes (viz. CYP2D6, CYP2C19 and CYP3A4/5) was studied to see if the combination could be given without changing

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the activity of one of the enzymes by the concurrent drugs given together. In addition, evaluation was done to see if the phenotype indices of metabolic ratios could be obtained in urine 0–8 h and/or in a single (or as few) plasma samples (as possible) for all three substrates.

16 A NOVEL TELOMERASE-IMMORTALIZED CARDIAC FIBROBLAST CELL LINE S.K. Janhunen, H. Laeremans, S.S. Rensen, J.F.M. Smits, W.M. Blankesteijn Dept. Pharmacology and Toxicology, CARIM, University Maastricht, Maastricht, The Netherlands Specialized fibroblasts, myofibroblasts, play a pivotal role in scar formation and prevention of cardiac dilatation after myocardial infarction. The in vitro use of primary fibroblasts is limited by poor proliferation, low transfection efficiency and spontaneous differentiation into myofibroblasts. We developed and characterized a telomeraseimmortalized cardiac fibroblast cell line, 3.3D. These cells remained proliferating for >40 passages and transfection efficiency was high (>50%) vs. <5 passages and <1% transfection efficiency in parental fibroblasts. Differentiation was determined after transforming growth factor β1 (TGFβ1) and interferon-γ (IFNγ) by assaying (myo) fibroblast markers α-smooth muscle actin (αSMA), collagen type I (ColIα1), fibronectin-1 (FN1), and the EDA splice variant of fibronectin (EDA-FN) by qPCR. TGFβ1 10 ng/ml increased αSMA (by 3.6-fold, P<0.05), but not ColIα1, FN1, or EDA-FN in 3.3D cells. The induction of αSMA was confirmed by Western blotting and immunocytochemistry. In contrast to TGFβ1, IFNγ 10 ng/ml failed to increase αSMA, while it increased ColIα1 by 1.7-fold (P<0.05), FN1 by 2.0-fold (P<0.01), and EDA-FN by 1.7-fold (P<0.05). TGFβ1 and IFNγ increased proliferation (by 25% and 20%) but not migration of the 3.3D cells. The 3.3D cell line is the first telomerase-immortalized cardiac fibroblast cell line with characteristics resembling parental cardiac fibroblasts, and it can serve as a novel tool for research on infarct healing.

17 CHARACTERIZATION OF THE VASOACTIVE PROPERTIES OF S1P RECEPTORS IN RAT AORTA M. Jongsma, M.-J. Mathy, P.B. van Loenen, N. Hajji, M.C. Michel, S.L.M. Peters, A.E. Alewijnse Dept. Pharmacology & Pharmacotherapy, Academic Medical Center, Amsterdam, the Netherlands In the cardiovascular system, the bioactive sphingolipid sphingosine1-phosphate (S1P) exerts its vasoactive effect via three specific Gprotein coupled receptors, named S1P1, S1P2 and S1P3, which are differentially expressed in both vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). Combining several recently reported synthetic S1P receptor ligands, we have explored the contribution of these three receptors separately to vascular reactivity in the rat aorta. We show that, in intact isolated rat aortic rings, all agonists at the S1P1 and/or S1P3 receptor induced endothelium-dependent vasorelaxation in pre-contracted preparations. In rat aortic ECs, all S1P1 ligands either increased or decreased the forskolin-induced cAMP accumulation, making it an unlikely mediator of vasodilation. In

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VSMCs, predominantly Gq-coupled S1P receptor signaling was observed via the S1P2 receptor, resulting in increases in intracellular calcium. In conclusion, we have shown that the newly available S1P receptor agonists can act on both ECs and VSMCs in the rat aorta. Endothelial S1P1 receptor stimulation leads to relaxation. Stimulation of mainly the S1P2 receptor in VSMCs results in contraction. Because the compounds are devoid of S1P2 activity, their net effect observed on vascular tone is vasorelaxation.

18 SUPPRESSION OF HEPCIDIN EXPRESSION IN HEME OXYGENASE (HO)-1 DEFICIENCY DESPITE IRON LOAD AND CHRONIC INFLAMMATION A. Kartikasari1, F. Wagener2, A. Yachie3, E. Wiegerinck1, E. Kemna1, D. Swinkels1 Dept. of Clinical Chemistry1, Pharmacology & Toxicology2, Radboud University Nijmegen Medical Centre, Nijmegen; 3Dept. of Laboratory Sciences, Kanazawa University, Kanazawa, Japan Since HO-1 deficiency demonstrates inflammation and anemia with severe hemosiderosis, HO-1 has been proposed central in iron metabolism. Hepcidin is important in iron homeostasis and targets the iron exporter ferroportin. To investigate the mechanism underlying the dysregulation of iron homeostasis in HO-1 deficiency, the hepcidin level in the serum of a HO-1-deficient patient was measured, using SELDITOF/MS. The capability of HO-1 to modulate hepcidin and ferroportin expression was assessed in vitro, using human hepatoma and macrophage-like cell lines, respectively. In the serum, the hepcidin level was very low, while in vitro, HO-1 activity did not affect hepcidin expression. In vitro, HO-1 activity increased ferroportin expression, indicating that the previously-described HO-1-induced iron efflux is likely mediated by ferroportin. The serum of the HO-1deficient patient contained a high level of soluble transferrin receptor, indicating the presence of a strong erythropoietic activity. HO-1 deficiency promotes stress-induced hemolysis, suppresses hepcidin production, and promotes iron loading. The erythropoietic drive has overruled hepcidin-stimulatory signals from inflammation and iron overload and is likely relevant for a variety of hemolytic anemia disorders.

19 TH1 SKEWING OF THE EFFECTOR IMMUNE RESPONSE THROUGH INTESTINAL EPITHELIAL CELLS EXPOSED TO TLR/NOD2 AGONISTS S. de Kivit1, N. Korthagen1, B. Schouten1, J. Garssen1,2, L.E.M. Willemsen1 1 Pharmacology and Pathophysiology, UIPS, Utrecht University 2 Immunology, Numico-Research BV, Wageningen Background: Under normal conditions, oral tolerance is induced against harmless antigens. Allergic individuals, however, show a Th2type immune skewing. It is believed that probiotics may modulate the intestinal immune response through interaction with Toll-like receptors (TLRs) and NOD2. Since intestinal epithelial cells (IEC) form the interface between the lumen and the mucosal immune system, in this study the contribution of IEC to immune modulation through TLR/ NOD was addressed. Methods: The effects of TLR9, TLR4, TLR2 and

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NOD2 signaling through IEC were tested in vitro in a transwell system. TLR/NOD2 agonists were added apically to IEC. Healthy donor peripheral blood mononuclear cells (PBMC) were added basolateral and stimulated with anti-CD3/CD28. Alternatively, the agonists were added to PBMC in the absence of IEC. Cytokine secretion was measured after 24 and 48 h. Results: Only in the presence of IEC TLR/NOD2 were agonists able to further enhance IL-12 secretion by PBMC. Signaling through TLR4 and TLR9 resulted in suppression of IL-13 secretion. However, TLR4 signaling in the presence of IEC also increased pro-inflammatory TNF-α secretion, whereas TLR9 increased regulatory IL-10 secretion in the presence and absence of IEC. Conclusion: TLR/NOD2 agonists cause Th1 skewing and/or Th2 suppression, and IEC may add to these effects. TLR9 signaling was most promising for prevention or treatment of allergic disease.

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would affect activation of the renin–angiotensin system (RAS). Methods: Mice were treated with either isoproterenol (ISO), T09, or both. Organs were assayed for mRNA expression of various elements of the RAS. Results: T09 alone tended to decrease renal renin expression, while the ISO-induced increase of renin and ACE mRNA in the kidney was abolished by co-treatment with T09. ACE expression was also reduced in hearts of mice treated with T09 compared to control mice. Treatment of T09 resulted in decreased mRNA levels of AT1R in the kidney of treated mice compared to control mice. Conclusions: LXR-alpha activation results in a decrease of mRNA expression of renin, AT1R and ACE in heart and kidney. These findings suggest a role for LXR-alpha in RAS regulation, and salutary effects of LXR agonists may be mediated via the RAS (supported by NHF grant 2004T4).

22 20 RENIN AND PRORENIN RELEASE BY HUMAN MASTCELLS M. Krop, I.M. Garrelds, A.H.J. Danser Dept. of Vascular Pharmacology, Erasmus MC, Rotterdam, the Netherlands Recently, a human mast cell line (HMC-1) was shown to release angiotensin (Ang) I-generating activity (AGA). We verified whether this activity represented renin, and we evaluated its regulation. Cells were incubated with the mast cell-depleting compound 48/80, the adenylyl cyclase stimulant forskolin, 8-db-cAMP, Ang II and/or the protein kinase C (PKC) inhibitor chelerythin. AGA was measured by enzyme-kinetic assay (EKA), before and after prorenin activation with trypsin. Renin was also measured directly by immunoradiometric (IR) assay. Medium of cultured cells contained AGA, which increased after treatment with trypsin. Trypsin did not affect cellular AGA. The renin inhibitor aliskiren blocked AGA with an IC50 identical to that for human renin. IR and EKA results were identical. Compound 48/80, forskolin, and cAMPAM stimulated renin (but not prorenin) release, whereas Ang II and chelerythin blocked this release. In conclusion, HMC-1 release both renin and prorenin and store renin intracellularly. Stimulation of the adenylyl cyclase-cAMP pathway enhances renin, but not prorenin release. Stimulation of the IP3-Ca2+pathway blocks renin release. Thus, renin/prorenin release from HCM-1 cells fully resembles that from juxtaglomerular cells.

21 LXR ACTIVATION REDUCES EXPRESSION OF RAS-COMPONENTS I. Kuipers, L. van Genne, D.J. van Veldhuisen, W.H. van Gilst, R.A. de Boer University Medical Center Groningen, Dept.s of Cardiology and Clinical Pharmacology, Groningen, The Netherlands Background: Liver X receptor (LXR)-alpha is a pivotal player in cholesterol metabolism. Classical activation of LXR-alpha is induced by oxidized cholesterol or synthetic agonists such as T09. Recently, LXR-alpha was shown to regulate renin expression in a cAMPresponsive manner. It remains unclear if classical LXR-alpha stimulation has ancillary effects besides lipid regulation. The aim of this study therefore was to investigate if LXR-alpha activation by T09

OVEREXPRESSION OF COMPONENTS OF THE WNT/FRIZZLED PATHWAY CAN MODULATE MYOFIBROBLAST CHARACTERISTICS H. Laeremans, S.K. Janhunen, J.F.M. Smits, W.M. Blankesteijn Dept. of Pharmacology, CARIM, Maastricht University, The Netherlands Inadequate wound healing after myocardial infarction (MI) is one of the major causes of heart failure. After MI, specialized fibroblastic cells called myofibroblasts appear in the infarct area, which may stabilize the infarct by actively contracting the infarct area and by deposition of the extracellular matrix proteins. Our laboratory has shown that components of the Wnt/Frizzled (Wnt/Fz) cascade are expressed during the wound healing after MI, particularly in the myofibroblasts. The aim of this study was to define the role of Wnt/Fz signaling in cardiac (myo)fibroblast proliferation, differentiation, and migration. To this end, we developed a telomerase-immortalized rat cardiac fibroblast cell line. These cells were transiently transfected with Fz1 or Fz2 and treated with conditioned medium containing their endogenous ligands Wnt-3a or 5a. Proliferation rates were not significantly affected by overexpression of Wnt/Fz components. In contrast, the expressions of α-smooth muscle actin (α-SMA) and fibronectin, two differentiation markers for myofibroblasts, were significantly modulated by activation of Wnt/frizzled signaling. The expression of collagenIα1 was also significantly influenced by the Wnt/Fz pathway. Migration of the cells was determined using an in vitro wound assay, in which the closure of a scratch in a confluent cell layer was followed in time. At 12 h after scratching, the gap width was reduced to 22±3% in mock-transfected cells, whereas in Fz1 and Fz2 transfected cells, gap width was 48±2% and 52±3% of the original gap, respectively (p<0.05). The results point to a functional role for Wnt/Fz signaling in the control of differentiation and migration of cardiac (myo)fibroblasts.

23 PROTEAN AGONISM AT THE DOPAMINED2 RECEPTOR J.R. Lane1, B. Powney2, A. Wise2, S. Rees2, G. Milligan1 Molecular Pharmacology Group, Institute of Biomedical and Life Sciences, University of Glasgow1, Screening & Compound Profiling, GlaxoSmithKline Research & Development, Harlow, United Kingdom2 A range of ligands displayed agonism at the long isoform of the human dopamine D2 receptor, whether using receptor-G protein fusions or

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membranes of cells in which pertussis toxin-resistant mutants of individual Gα(i)-family G proteins could be expressed in an inducible fashion. Varying degrees of efficacy were observed for individual ligands as monitored by their capacity to load [35S]GTPγS onto each of Gα(i)1, 2, 3, and (o)1. By contrast, (S)-(−)-3-(3-hydroxyphenyl)-Npropylpiperidine (S-(−)-3PPP) was a partial agonist when Gα(o)1 was the target G protein but an antagonist at Gα(i)1, 2, and 3. In ligand binding assays, dopamine identified both high- and low-affinity states at each of the D2 receptor-G protein fusion proteins. S-(−)-3PPP bound to an apparent single state of the constructs in which the D2 receptor was fused to Gα(i)1, 2, or 3. However, it bound to distinct high- and low-affinity states of the D2 receptor-Gα(o)1 fusion. Likewise, when expression of Gα(i)1, 2, 3, and (o)1 was induced, S-(−)-3PPP identified a high-affinity site at the D2 receptor only in the presence of Gα(o)1. These results demonstrate S-(−)-3PPP to be a protean agonist at the D2 receptor and may explain in vivo actions of this ligand.

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perfused guinea pig tracheal preparations obtained 24 h after the last of 12 weekly saline or allergen challenges. Multiple allergen challenge resulted in an 1.6-fold AHR. This AHR was normalized by the NOS inhibitor L-NAME, as well as the superoxide scavenger SOD, indicating the involvement of peroxynitrite—the reaction product of NO and superoxide. Arginase activity in the tracheae was 1.8-fold increased in chronic asthma and incubation with the arginase inhibitor nor-NOHA completely abolished AHR. The effect of nor-NOHA was prevented by L-NAME, indicating that norNOHA normalizes AHR by stimulating the production of bronchodilating NO. Moreover, AHR was also normalized by L-arginine, indicating substrate limitation to NOS. In conclusion, increased arginase activity contributes to AHR in chronic allergic asthma, presumably by promoting the production of both NO and superoxide by NOS due to the L-arginine limitation (supported by the Netherlands Asthma Foundation, grant 00.24).

26 24 VIRAL CHEMOKINE RECEPTOR US28 ACTIVATES ONCOGENIC SIGNALING NETWORKS E. Langemeijer, D. Maussang, R. Leurs, M.J. Smit Leiden/Amsterdam Center for Drug Research (LACDR), Division of Medicinal Chemistry, Faculty of Sciences, Vrije Universiteit Amsterdam, The Netherlands Human cytomegalovirus (HCMV), a widely spread herpesvirus, is suggested to play a role in tumor progression US28, a HCMVencoded chemokine receptor that constitutively activates Gaq/11 signaling pathways. The overall aim is to elucidate how US28 redirects cellular signaling networks to alter cellular function. We have recently shown that US28 induces tumorigenesis both in vitro and in vivo [Maussang et al., PNAS, 103(35):13068–73, 2006]. 3T3 cells stably expressing US28 were subjected to Affymetrix mouse 430 gene chips. Several genes linked to oncogenic signaling pathways were found to be up- or downregulated. Validation by means of Q-PCR (mRNA level) and Westerns (protein level) is ongoing. Currently, the Wnt (or b-catenin) signaling pathway is looked at in more detail, as genes within this pathway were found to be differentially expressed. To this end, we use the TOP-flash (Tcf–Lef luciferase) reporter assay. HEK293T cells transfected with US28 show increased, G-protein mediated, Tcf–Lef transcriptional activity. These data suggest the involvement of the b-catenin pathway in US28mediated oncogenic signaling.

ULTRASOUND AND MICROBUBBLE TARGETED DELIVERY OF THERAPEUTIC COMPOUNDS; MECHANISMS AND CELLULAR DISTRIBUTION B.D.M. Meijering, L.J.M. Juffermans, K. Kooiman, R. Musters, A. van Wamel, O. Kamp, R.H. Henning, C. Visser, W.H. van Gilst, N. de Jong, L.E. Deelman 1 Dept. of Clinical Pharmacology, University Medical Center Groningen, 2 Dept of Physiology and Cardiology, VU University Medical Center, 3 Dept. of Biomedical Engineering, Erasmus MC; 4Interuniversity Cardiology Institute of the Netherlands This study investigated the mechanisms of ultrasound and microbubble targeted delivery (UMTD) and the cellular distribution of dextran and DNA. Fluorescent dextrans and plasmid DNA were delivered to cultured bovine aorta endothelial cells with Sonovue® using 1 MHz ultrasound. Distribution of dextran and DNA, expression of GFP, Ca2+ levels (fluo-4), and lysosomes (lysotracker) were detected by confocal microscopy. Dextrans (4.4 kDa) were homogenously distributed in both cytosol and nucleus after UMTD. This was accompanied by an influx of extracellular Ca2+ independently from L-type Ca2+ channels. In contrast, dextrans (155 kDa) and plasmids were mainly found in cytosolic vesicles. Notably, almost all cells contained plasmid encoding GFP; only 5% actually expressed GFP. Vesicles of dextran did not co-localize with lysosomes directly after ultrasound or after 1.5 h. These results suggest that pore formation and endocytosis mediate UMTD. Low expression efficiency of plasmids is caused by inefficient nuclear redistribution.

25 AIRWAY HYPERRESPONSIVENESS IN A GUINEA PIG MODEL OF CHRONIC ALLERGIC ASTHMA: CENTRAL ROLE OF ARGINASE H. Maarsingh, I.S.T. Bos, F.J. Westerhof, J. Zaagsma, H. Meurs Dept. of Molecular Pharmacology, University of Groningen, The Netherlands Using a guinea pig model of acute allergic asthma, we previously demonstrated a key role for increased arginase activity in the development of airway hyperresponsiveness (AHR) after the early, as well as late, asthmatic reaction. We now study the involvement of arginase in the development of AHR in chronic allergic asthma. To this aim, airway responsiveness to methacholine was measured in

27 CPG POTENTIATES THE EFFECTS OF CIGARETTE SMOKE ON RELEASES OF IL-8 IN NEUTROPHILS E. Mortaz, D. Raats, P. Vader, I. Adcock, K. Ito, F.P Nijkamp, G. Folkerts Division of Pharmacology and Pathophysiology, Dept. of Pharmaceutical Sciences, Utrecht, The Netherlands Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide. Smoking is considered the major cause of the disease. All smokers develop airway inflammation through oxidative stress, with macrophages, neutrophiles, and medi-

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ators (such as IL-8) involved. Previous studies have implicated a role of bacterial DNA(CpG) in the initiation of inflammation. In this study, we investigated the capacity of cigarette smoke extracts (CSE) and CpG-ODN to trigger and modulate IL-8 releases in human neutrophils. Human neutrophils were isolated from whole blood or Buffy coat. Neutrophils were exposed to the CSE (0.06 OD), CpG-ODN (3 μM) or in combination for 30 min or 9 h for studying signaling and protein production, respectively. Supernatants of cells were harvested and analyzed for IL-8 production by ELISA. The NF-κB and AP-1/c-fos activities were monitored by Western blot by using antibodies. CSE and CpG-ODN induced the release of IL-8 in neutrophils. When co-administered, release of IL-8 synergically increased, which was in correspondence with degradation of IκB-α in cytoplasm. CpGODN and CSE evoked concomitant increases in intracellular NO levels and, consequently, nuclear accumulation of c-fos and NF-κB. Pharmacological inhibition of NF-κB activity (by curcumine) or using of N-acetylcysteine (NAC) attenuated the release of IL-8. These results identify reactive oxygen dependent activation of NFκB and c-fos as an important mechanism to CSE and CpG-ODN. In conclusion, this study shows that the combination of CSE and CpGODN provide a substantial release of IL-8, which may account to exacerbation of the lung in COPD patients when they are exposed to bacterium infections

28 PORPHYRINS RESCUE CURCUMIN-INDUCED FIBROBLAST APOPTOSIS VIA THE INDUCTION OF HEME OXYGENASE-1 R. Mutsaers, A. Scharstuhl, B. Pennings, F. Russel, F. Wagener Dept. of Molecular Pharmacology & Toxicolog, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands A crucial aspect determining the outcome of scar formation is fibroblast apoptosis. We hypothesized that induction of heme oxygenase-1 (HO-1) expression and activity by FePP and CoPP could inhibit circumin-induced apoptosis, whereas inhibition of HO-activity by SnMP would aggravate the effects of curcumin. Human dermal fibroblasts were treated with 25 μM curcumin to induce apoptosis. Curcumin (25 μM) very potently induced apoptosis, as measured by flow cytometry, but resulted in similar low HO-1 expression levels and HO-activity as untreated cells. Our results show that both FePP and CoPP completely attenuated curcumin-induced apoptosis. In addition, both porphyrins caused high HO-1 expression and HO activity when administered alone or in combination with curcumin. Surprisingly, SnMP simultaneously administered with curcumin also resulted in complete inhibition of apoptosis. Although ex vivo HO activity in cell lysates was absent after treatment with SnMP, the SnMP–curcumin combination resulted in a super-induction of HO-expression, compared to SnMP treatment alone. This discrepancy suggests that SnMP gets compartmentalized in the cell and is unable to inhibit HO-1 and that so-called “leakage” occurs. Modulation of fibroblast apoptosis though porphyrin-induced HO may lead to novel therapeutic targets in wound healing.

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29 HEME INHIBITS TOLL-LIKE RECEPTOR-MEDIATED NF-ΚB SIGNALING IN CHO CELLS VIA A HEME-OXYGENASE INDEPENDENT PATHWAY S. Noordermeer1,2, F. Wagener1, G. Yang2, F. Russel1, P. Dennery2 1 Pharmacology and Toxicology, Radboud University Nijmegen Medical Center, 2Pediatrics, University of Pennsylvania, USA Toll-like receptors (TLRs) are important for the immune system as sensors and determine the defense against invading pathogens. Here, we postulate that metallo-porphyrins can modulate the TLR-signaling pathway. To study this, we used three CHO cell lines containing a NFκB reporter gene, leading to cell surface CD25 expression following NF-κB activation. Besides this negative control, the other two expressed either TLR-2 (CHO-TLR2) or TLR-4 (CHO-TLR4). All cell lines were treated with different porphyrins (heme, ZnMP, CoPP, or SnMP) in combination with known ligands for TLR-2 (Pam3Cys) or TLR-4 (LPS). Treatment of cells with TLR-ligands LPS or Pam3Cys leads to CD25 expression on the membrane in CHO-TLR4 or CHO-TLR2, respectively. Interestingly, the addition of heme or ZnMP blocks this TLR-ligand-induced CD25 expression on the membrane, whereas CoPP and SnMP demonstrate no effect. Similarly, heme blocks TLR-ligand-induced CD25 mRNA production. Thus, metallo-porphyrins differentially modulate the TLR-signaling pathway. Heme inhibits the transcription of effector genes, whereas ZnMP inhibits translation and CoPP and SnMP demonstrate no effect. Regulation of TLR-signaling using different porphyrins may provide a novel therapeutic target for immunomodulation.

30 BRADYKININ PROTECTS ENDOTHELIAL CELLS FROM ROS-INDUCED SENESCENCE H. Oeseburg, D. Iusuf, W.H. van Gilst, R.H. Henning, A.J.M. Roks Dept. of Clinical Pharmacology, University Medical Center Groningen, The Netherlands There is evidence that nitric oxide (NO) can have a protective effect against cell senescence. Bradykinin (BK) is a vasodilator drug that can stimulate the release of NO and prostaglandins (PG). We hypothesize that BK protects against endothelial senescence. Senescence was induced in BAEC cells by treating them for 1 h with H2O2 (concentrations between 10 and 35 mM). Pre-treatment with BK was performed 30 min before H2O2 treatment (10−12–10−9 mol/L). After 3 days, the amount of senescent cells was determined by β-gal senescence staining. H2O2 treatment for 1 h resulted in an increase of senescent cells in a dose-dependent manner, with a three-times increase of senescent positive cells at 25 μM. Pre-treatment with BK significantly reduced the effect of H2O2 in a dose-dependent manner, with a reduction of 40% of senescent cells at 10−9 mol/L bradykinin. The positive effect of bradykinin could be blocked with selective BK2 receptor blocker. Blocking the NO resulted in a total abolishment of the effect of BK. Blocking PG resulted in an additional increase of the number of senescent cells. Pre-treatment with bradykinin protects BAEC from oxidative stress induced senescence. The protective effect of bradykinin seems to be mediated by nitric oxide and by prostaglandins.

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EX VIVO-STIMULATION OF BONE MARROW-DERIVED MONONUCLEAR CELLS WITH ANGIOTENSIN (1–7) DOES NOT RESULT IN EXTRA BENEFIT AFTER CELL THERAPY IN HEART FAILURE RATS C. Qian, A.J.M. Roks, L. Yu, W.H. van Gilst, R.G. Schoemaker Dept. of Clinical Pharmacology, University Medical Center of Groningen (UMCG), Groningen, The Netherlands

PHENOTYPIC MODULATION OF AIRWAY SMOOTH MUSCLE BY LIPOPOLYSACCHARIDE AND CIGARETTE SMOKE T. Pera1, R. Gosens1, A.H. Lesterhuis1, A.J.M. van Oosterhout2, J. Zaagsma1, H. Meurs1 1 Dept. of Molecular Pharmacology; 2Lab. of Allergology & Pulmonary Diseases, University of Groningen, The Netherlands

We and others have recently demonstrated the beneficial effects of angiotensin (1–7) [Ang-(1–7)] on cardiovascular systems and its stimulative effects on blood or bone marrow progenitor cells both in vivo and in vitro. However, whether the ex vivo effects of Ang-(1–7) on bone marrow-derived mononuclear cells (BM-MNCs) would benefit cell therapy in vivo remains unknown. Using cell culture and pharmacological treatments, we examined effects of Ang-(1–7) on BM-MNCs in vitro. For in-vivo experiments, BM-MNCs were isolated from R26 human alkaline phosphatase (hPAP) transgenic rats (F344 background). After pretreated by Ang-(1–7) for 7 days, transgenic BmMNCs (5×105/200 μl) were intracoronarily transported into inbred F344 rats with acute myocardial infarction. Ang-(1–7) (10−8 mol/L) significantly enhanced the proliferation, differentiation, and function of the cultured BM-MNCs/EPCs during ex vivo expansion, which is antagonized by A-779 (10−7 mol/L), but not PD123319 (10−7 mol/L). Intracoronary transplantation of either untreated or Ang-(1–7) pretreated BM-MNCs/EPCs markedly attenuated cardiac remodeling and partly improved cardiac function. Furthermore, the recruitment of hPAP + cells was inversely correlated with LVEDP (R2 =0.617, P<0.001). However, no difference in therapeutic potential was observed between untreated and Ang-(1–7) pretreated BM-MNCs.

Chronic obstructive pulmonary disease (COPD) is an inflammatory disease characterized by a progressive decline in lung function. One of the main features of COPD is airway remodelling, characterized by increased airway smooth muscle (ASM) mass and fibrosis. The mechanisms underlying this airway remodelling are currently unknown. A major cause of COPD is cigarette smoke (CS). In animal models, inhalation of lipopolysaccharide (LPS) – a major constituent of CS – has been shown to induce various inflammatory and pathological changes closely mimicking COPD, including airway remodelling. Using cultured bovine tracheal smooth muscle (BTSM) cells and tissue, we studied the effects of LPS and CS extract (CSE) on BTSM proliferation and contractility. Both LPS (1 ng/ml–10 μg/ml, 28 h) and CSE (1–15%, 1 h) induced a profound and dose-dependent increase in DNA synthesis in cultured BTSM cells, associated with activation of ERK and p38 MAPkinases. Moreover, 8 days of culturing of BTSM strips with LPS (1 μg/ml) or CSE (15%, 1 h daily) caused a significant decrease of maximal KCl-induced smooth muscle contraction. In conclusion, both LPS and CSE may induce a phenotypic shift of airway smooth muscle to a proliferative, hypocontractile phenotype, which could be involved in airway remodelling in COPD (supported by Boehringer Ingelheim).

32 HUMAN NEUTROPHILS FROM SMOKERS VS. NON-SMOKERS PRODUCE MORE MMP’S, ELASTASE AND IL-8 AFTER STIMULATION WITH CIGARETTE SMOKE S.A. Overbeek, E. Mortaz, F.P. Nijkamp, G. Folkerts Dept. of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, The Netherlands. Neutrophils contribute to the destruction of the lungs in chronic obstructive pulmonary diseases by the release of proteases, elastases, and interleukine-8 (IL-8). The aim of this study was to investigate whether the basal production of these mediators is altered in smokers compared to healthy volunteers. Furthermore, production of proteases, elastases, and IL-8 of neutrophils from healthy subjects and smokers were determined upon stimulation with cigarette smoke extract (CSE). Blood was obtained from volunteers (n=9 per group) and neutrophils were purified by Ficoll technique. Neutrophils were incubated with or without CSE (OD=0.06) for 9 h, and accordingly, metalloproteinases (MMP)-2 and -9, elastase, and IL-8 were measured with ELISA. The basal production of metalloproteinase MMP-2 and -9 (P<0.02), elastase (P<0.002) and IL-8 (P=0.03) was higher in smokers compared to the healthy subjects. CSE activated the neutrophils in both groups; however, neutrophils from smokers produced significant higher levels of MMP-2 and -9 (P<0.02), elastase (P<0.005) and IL-8 (P<0.01). Collectively, our data show that neutrophils from smokers produce more mediators with or without stimulation with CSE in vitro. This increased mediator release may make smokers prone to develop lung emphysema in the future.

34 ACTIVITY OF SPLEEN TYROSINE KINASE (SYK) IS REQUIRED FOR DEVELOPMENT OF COLITIS IN MICE D. Raats1, A. Rijnierse1, E. Herlaar2, R. Singh2, D.G. Payan2, J. Schmitz2, N. Bunnett3, A.D. Kraneveld1 1 Dept. of Pharmacology & Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands, 2 Rigel Pharmaceuticals, South San Francisco, California, USA and 3 Dept.s of Surgery & Physiology, University of California, San Francisco, CA, USA In the intestine, mast cell products mediate allergic diseases and contribute to inflammatory bowel disease and irritable bowel syndrome. The non-receptor tyrosine kinase syk is essential for immunoreceptor signaling, including mast cell Fc receptors and B and T cell receptors. We hypothesized that inhibition of syk suppresses mast cell-dependent intestinal inflammation and functional disorders. Mice were sensitized with protein (ovalbumin, ip) or chemical (DNFB, skin sensitization) antigens, and challenged with antigen orally (ovalbumin) or intrarectally (DNFB) and treated with the specific syk-inhibitor R788. In ovalbumin-sensitized and -challenged mice, treatment with R788 resulted in reduced mast cell activation and reduced myeloperoxidase levels. In DNFB-sensitized mice syk inhibition by R788 resulted in the prevention of mast cell degranulation and consequently reduced diarrhea formation, colonic patch hypertrophy and leukocyte infiltration 72 h after antigen-specific challenge. The results obtained indicate that syk inhibition prevents mast cell degranulation, suppresses inflammation, and reduces functional disturbances. Thus, antagonists of syk tyrosine kinase

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represent a potential novel therapy for inflammatory and functional disorders of the intestine.

35 INDICATION FOR CLINICAL RELEVANCE OF IMMUNOGLOBULIN-FREE LIGHT CHAINS IN INFLAMMATORY BOWEL DISEASE AND IRRITABLE BOWEL SYNDROME A. Rijnierse, F.A. Redegeld, B.R. Blokhuis, M.W. Van der Heijden, A.A. Te Velde, I. Pronk, D.W. Hommes, J. Santos, M. Guilarte, F.P. Nijkamp, A.S. Koster, A.D. Kraneveld Division of Pharmacology & Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands Inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) are interrelated intestinal disorders both associated with increased numbers of mast cells and mast cell activation. Previously, we have shown that immunoglobulin-free light chains (IgLC) are able to mediate experimental colonic hypersensitivity responses via mast cell activation. Here, we demonstrate increased serum concentrations of IgLC of patients suffering from IBD, which are reduced in patients treated with immunosuppressive drugs. Furthermore, we demonstrate that IgLC can be found in the intestinal mucosa and inflammatory lesions in biopsies of IBD patients associated with the presence of plasma cells and mast cells. Increased serum concentrations of IgLC and increased presence of IgLC in intestinal biopsies are also observed in patients suffering from IBS. We propose increased levels of IgLC as potential biomarkers of and as therapeutic targets for IBD or IBS.

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37 VASCULAR REACTIVITY AFTER SHORT-TERM HEMORRHAGIC SHOCK I.V. Samarska1, H. Buikema1, A.H. Epema2, L.P.H.J. Aarts2, R.H. Henning1 Dept. Clinical Pharmacology1 and Dept. Anesthesiology2, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. The aim of the present study was to investigate the changes in vascular reactivity following a 90-min hemorrhagic shock in two types of general anesthesia. Methods: Descending thoracic aortic rings, about 2 mm in length, were mounted on two 200-μm stainless wires in the jaws of a wire myograph. Results: The maximal contractions to phenylefrine (PE) were increased significantly (P<0.001) in shock-mice anesthetized with isoflurane. At the same time, there were no significant differences in concentration-dependent responses to PE of mice aortic rings from groups, where nitrous oxide was used as anesthetic agent. Acetylcholine caused relaxation of mouse aorta, which was reversed to contraction at the concentration of 1 μM–0.1 mM. The reactions to acetylcholine were abolished by the COX inhibitor indomethacin (1 μM). Conclusions: Short-term hemorrhagic shock affects vasomotor responses. Shock under isoflurane increases vascular contraction to phenylephrine. Acetylcholine initiates both contractile and relaxant responses in mouse aorta. It seems that COX-derived metabolites can mediate the contractions evoked by acetylcholine.

38 36 EPAC AND THE PULMONARY SYSTEM S.S. Roscioni1, C.R.S. Elzinga1, I.S.T. Bos1, F.J. Warnders1, H.J. Van der Horn1, A.J. Halayko2, H. Meurs1, M. Schmidt1 1 Dept. Molecular Pharmacology, University of Groningen, The Netherlands and 2Dept. Physiology, University of Manitoba, Winnipeg, Canada From the year of its discovery until now, exchange protein directly activated by cAMP (Epac) has been deeply investigated and widely recognized as a key molecule in distinct pathways. By catalyzing the GDP/GTP exchange on Ras-like small GTPases, Epac is able to transduce cAMP-related signals and to modulate cellular responses, including integrin-mediated cell adhesion and endothelial barrier functioning through effectors such as phospholipase C-ɛ and extracellular signal-regulated kinases (ERKs). Here, we report that cultured human airway smooth muscle (hTERT) and human bronchial epithelial (HBE) cells express Epac1 and Epac2 and key effectors such as Rap1, Rap2 and R-Ras. Activation of Epac proteins with 8-pCPT-2’-OMecAMP in hTERT and HBE cells induces GTP-loading of Rap and RRas, the latter known to regulate cell adhesion and cell migration. Epac also slightly activate matrix-metalloproteinase-9, a potential regulator of tissue remodelling, and was found to modulate ERK and c-Jun kinases, known to have cell proliferative and cell survival effects. Collectively, these data point at Epac as a potential multifunctional target for future therapeutical applications in lung pathophysiology.

GENE THERAPY WITH 2,3-INDOLEAMINE DIOXYGENASE REDUCES ACUTE REJECTION FOLLOWING ALLOGENEIC KIDNEY TRANSPLANTATION M. Sandovici1, L.E. Deelman1, H. van Goor2, D. de Zeeuw1, R.H. Henning1 1 Dept. of Clinical Pharmacology, UMC Groningen, 2Dept. of Pathology and Laboratory Medicine, UMC Groningen 2,3-Indoleamine dioxygenase (IDO), the rate limiting enzyme in the tryptophan catabolism, has recently emerged as an important immunosuppressive molecule, involved in the regulation of both physiologic (maternal tolerance) and pathologic (neoplasia, autoimmune diseases) processes. Here, we investigate the effects of adenovirus-mediated IDO gene therapy on acute rejection of the renal graft. The experiments were performed in a rat model (Fisher to Lewis) of acute renal transplant rejection. Adenovirus-(Ad)IDO (n=7/group), Adluciferase (n=5/group), or saline (n=7/group) was injected into the renal artery of the donor kidney before transplantation. Rats were killed after 6 days. Successful IDO gene delivery was confirmed with PCR and Western blot. Endogenous and trangene IDO expression was analysed with immunohistochemistry. Plasma creatinine for the saline injected group, Ad-luciferase group, and Ad-IDO were 222±68, 254±81, and 94± 26 μmol/l, respectively. The present study demonstrates for the first time that IDO (over)expression in the renal graft improves renal function and morphology in a clinically relevant model of acute rejection.

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N-ACETYLCYSTEINE PREVENTS CIGARETTE SMOKE (CS)-INDUCED OVER EXPRESSION OF TLR4 ON MONOCYTE-DERIVED MACROPHAGES (MDMS) H. Sarir, E. Mortaz, K. Karimi, F. P. Nijkamp, G. Folkerts1 1 Dept. of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, The Netherlands

THE PIG’S MDR1, THE FIRST IN A LINE OF PORCINE ABC-DRUG TRANSPORTERS? J.A. Schrickx, J. Fink-Gremmels Dept. Veterinary Pharmacology, Pharmacy and Toxicology, Faculty Veterinary Medicine, Utrecht University, The Netherlands

Background: CS, a complex of over 4,700 chemical compounds, includes high concentrations of reactive oxygen species (ROS) that stimulates various immune cells. Macrophages are key cells in tissue destruction associated with CS-induced lung emphysema. Human macrophages express Toll-like receptors, and the activation leads to the production of pro-inflammatory mediators. Objective: Whether CS could modulate TLR4 surface and mRNA expression in MDMs and whether reactive oxygen species were involved was investigated. Methods: MDMs were obtained from human monocytes and were exposed to CS extract for 4 and 24 h. Expression of TLR4 was detected by FACS, and mRNA expression was measured by Q-PCR. To study the functional response of this receptor, the amount of IL8 was determined by ELISA after stimulation by LPS and CS extracts. Results: TLR4 surface expression was decreased and mRNA expression increased 4 h after CS exposure. Twenty four hours after CS exposure, TLR4 surface expression was increased. Interestingly, N-acetylcysteine inhibited modulation of TLR4 expression and IL8 production by CS. Conclusion: These findings suggest that TLR4 expression is regulated by ROS in cigarette smoke. TLR4 may be involved in the pathogenesis of COPD.

40 A PREBIOTIC OLIGOSACCHARIDE MIXTURE INHIBITS PATHOPHYSIOLOGICAL CHANGES IN MICE ORALLY SENSITIZED AGAINST CASEIN OR WHEY PROTEINS B. Schouten1, B.C.A.M. van Esch1, G.A. Hofman1, L.E.M. Willemsen1, J. Garssen1,2 1 Pharmacology and Pathophysiology, UIPS, Utrecht University, 2 Immunology, Numico-Research BV, Wageningen Oligosaccharides in human milk and are believed to have a positive effect on gut health. Oligosaccharides promote the growth of lactobacilli and bifidobacteria, which have direct immunomodulatory capacities. Therefore, we have tested a 2% oligosaccharide mixture of fructo-, galacto- and acidic-oligosaccharides (GOS, FOS and AOS) that mimicks the oligosaccharide composition in human milk, in mice orally sensitized against casein and whey proteins. Female C3H/ HeOuJ mice were sensitized weekly (six times) and fed with the control or prebiotic diet, starting 2 weeks prior to the first sensitization. Acute allergic skin reactions, serology and contractility of the intestine were analyzed. In the whey- and casein-sensitized animals, the allergic skin reaction was significantly reduced in the prebiotic group. Casein- and/or whey-specific IgG1 and IgE levels were unaltered, while in whey-sensitized animals, IgG2a levels were enhanced by the diet. The hypocontractility of the colon of the caseinsensitized mice was restored to normal levels by the prebiotic diet. In conclusion, oligosaccharides reduce systemic and local allergic symptoms when provided during the sensitization phase.

The pig is an experimental animal in a wide range of research areas: among others, pharmacokinetics; physiology; cardiovascular research; and, last but not least, veterinary medicine. Recently, we have functionally characterized the pig’s MDR1 (ABCB1) by an ex vivo lymphocyte assay. Expression was measured by quantitative RT-PCR and immunohistochemistry. Lymphocytes were isolated from peripheral blood by a Ficoll density gradient, incubated with Rhodamine123 to allow cellular uptake, and subsequently incubated with a range of drugs. Typical MDR1 inhibitors such as PSC833, ketoconazole, and verapamil, as well as drugs known to be substrates for human MDR1, decreased Rh123 efflux at concentrations that are in the same order of magnitude as previously reported for human MDR1. ABCB1 mRNA expression was detected in all tested organs and was highest in the adrenal gland. Moreover, a high expression was measured in the intestines but a low expression was found in the kidneys. Data analysis revealed that pigs have likely only one MDR1 gene, while rodents have two. MDR1protein was detected in the intestinal epithelium, lung bronchi(oles), vasculature, and bile duct epithelium, but was absent in the biliary canalicular membrane and kidney tubule epithelial cells. Our data suggest comparable expression patterns and characteristics of MDR1 in porcine and human tissues. These findings lead to an improved understanding of data obtained from kinetic studies in the pig, particularly drug distribution across biological barriers, and indicate that the pig is one of the most suitable animal models for human drug research.

42 THE EFFECT OF CHRONIC LEPTIN EXPOSURE ON THE REACTIVITY OF ISOLATED RAT CAROTID ARTERIES S. van Stijn, S.H. Schirmer*, M.-J. Mathy, M.C. Michel, A.E. Alewijnse, S.L.M. Peters Depts. of Pharmacology & Pharmacotherapy and *Cardiology, Academic Medical Center, Amsterdam, The Netherlands Besides its main function to decrease food intake by a central mechanism, leptin also has profound peripheral actions, including influences on vascular tone. We investigated the effects of 24 h of leptin (0.3, 1 and 3 μg/ml) treatment on vasodilator and constrictor responses in isolated rat carotid arteries. The relative dilatory responses to methacholine were not substantially altered in preparations exposed to leptin. However, in the leptin-exposed segments, the contractile response to 1 μM phenylephrine used for pre-constriction was significantly lower and proved to be unstable. Indeed, when constructing concentration response curves for phenylephrine, leptin concentration-dependently lowered Emax values. The decreased responses to phenylephrine in leptin-treated preparations were not prevented by endothelium removal. However, concomitant inhibition of NO synthases by means of LNAME completely restored the contractile responses to phenylephrine. From this study, we conclude that chronic leptin treatment most likely results in an increased expression of iNOS in the vascular smooth muscle cells. The dilatory effects of the increased NO production by iNOS partially counteracts the phenylephrine-induced vasoconstriction.

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the treatment of social dysfunctions; furthermore, they extend the involvement of opioid–cannabinoid interactions in the modulation of drug-directed behavior and food-directed behavior to another natural reinforcer, i.e. social interaction.

BINDING CHARACTERISTICS OF FREE IMMUNOGLOBULIN LIGHT CHAINS (FLC) TO ANTIGEN AND IN VITRO ANALYSIS OF THE FLC ANTAGONIST F991 M. Thio, M. Fischer1, B. Blokhuis, F.P. Nijkamp, F.A. Redegeld Division of Pharmacology & Pathophysiology, 1Div. of Med. Chemistry, UIPS, Utrecht University, The Netherlands. Recently, we showed that immunoglobulin free light chains (FLC) are capable of inducing hypersensitivity responses in skin and airways. We demonstrated that mast cells bind FLC and hypothesize the presence of a FLC-specific receptor. Crosslinking of surface-bound FLC induces mast cell activation and degranulation. Mast cells play key roles in various pathologies, such as atopic asthma and various chronic inflammatory diseases. In many of these disorders, increased levels of FLC are detected. Inhibition of FLC-induced mast cell activation therefore provides a novel therapeutic strategy to treat chronic inflammatory diseases. In intact antibodies, both heavy and light chains cooperate in binding to an antigen. Only few studies focused on the capability of FLC to interact with antigen. In this study, we measured the binding characteristics of FLC to antigen using surface plasmon resonance (SPR) technology. Real-time binding of mouse FLC to antigen-coated sensor chips was measured and compared to IgE. Our studies reveal substantial and specific binding of FLC to cognate antigen. Our SPR data further showed that the FLC antagonist F991 did not interfere with antigen binding of FLC. F991 most likely inhibits the binding of FLC to their putative receptors, as was demonstrated by FACS analysis of the binding of FLC to cultured mouse mast cells in this study. Our results demonstrate that FLC indeed are able to bind to the appropriate antigen and strengthen our previous results that antigen crosslinking of FLC can induce hypersensitivity responses in vivo.

45 THE SPHINGOSINE-1-PHOSPHATE TYPE 3 RECEPTOR AND LIGAND-DIRECTED SIGNALING J. van Unen, M. Jongsma, M.C. Michel, S.L.M. Peters, A.E. Alewijnse Dept. of Pharmacology and Pharmacotherapy, Academic Medical Center, Amsterdam, The Netherlands Ligand-direct signaling, also termed functional selectivity, refers to the phenomenon that different ligands can induce differential activation of signal transduction pathways of a particular receptor due to the unique, ligand-specific receptor conformations these ligands induce. In this study, we investigated ligand-directed signaling at the sphingosine-1phosphate type 3 (S1P3) receptor. Stimulation of the S1P3 receptor resulted in the inhibition of cAMP accumulation and the release of intracellular calcium. Efficacy and potency at both pathways were determined for several commercially available S1P3 ligands. The order of potency for the tested ligands was comparable for both signaling responses, but marked differences were observed in their efficacy. For example, VPC23153 was a highefficacy agonist in the cAMP assay (α=0.7±0.1, n=9) but was a weak partial agonist in the calcium assay (α=0.3±0.1, n=3). In conclusion, we have shown that several S1P3 ligands differentially activate Gi and Gq-coupled signal transduction pathways. The phenomenon of ligand-directed signaling, which has already been described for various other G-protein coupled receptors, is thus also important for the S1P3 receptor.

46 44 CROSS-TALK BETWEEN ENDOCANNABINOID AND OPIOID SYSTEMS IN THE MODULATION OF SOCIAL BEHAVIOR IN ADOLESCENT RATS V. Trezza1, L.J.M.J. Vanderschuren1 1 Dept. Pharmacology and Anatomy, Rudolf Magnus Institute of Neuroscience, Utrecht During adolescence, rats display a characteristic form of social interaction termed social play behavior, which is highly rewarding and essential for social and cognitive development. The brain opioid system, which closely interacts with the endocannabinoid system in the regulation of reward processes, has been strongly implicated in the modulation of social play behavior. Cannabinoid neurotransmission may therefore influence social play in concert with the opioid system. To study the role of endocannabinoid neurotransmission in the modulation of social play behavior, we used adolescent male Wistar rats that were briefly socially isolated before testing. We found that directly stimulating cannabinoid receptors using the CB1 cannabinoid receptor agonist WIN55,212–2 reduced social play. However, URB597, which inhibits hydrolysis of the endocannabinoid anandamide, enhanced social play. The opioid receptor antagonist naloxone reversed the effects induced by URB597 on social play. In addition, the wellknown stimulatory effect of morphine on social play was attenuated by the CB1 cannabinoid receptor antagonist SR141716A. These results suggest that endocannabinoid degradation inhibitors hold promise for

SALMETEROL AND FLUTICASON BY INCREASING OF TRANSLOCATION OF GLUCOCORTICOID RECEPTORS SUPPRESS CIGARETTE SMOKE-INDUCED IL-8 PRODUCTION BY NEUTROPHIS M.M. Vaezirad, E. Mortaz, A.D. Kraneveld, F.P. Nijkamp, G. Folkerts Div. Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, The Netherlands Background: Chronic obstructive pulmonary disease (COPD) is a major public health problem. It is the fourth leading cause of chronic morbidity and mortality in the USA. Cigarette smoking is a major known risk factor. Airway inflammation in COPD involves IL-8, which are generally considered to be important mediators in neutrophil recruitment. The combination of inhaled corticosteroids and longacting beta2-adrenoceptor agonists is increasingly used as maintenance therapy in patients with COPD. Objective: In this study, we studied the modulatory effects of salmeterol and fluticasone on release of IL-8 and proteases induced by cigarette smoke extracts (CSE) in neutrophils. Methods: polymorphonuclear neutrophils (PMN) were isolated from human Buffy coat or whole blood and activated with CSE for 9 h. To test of the effects of fluticasone and salmeterol, cells were preincubated with these drugs and then stimulated with CSE. IL-8 production, MKP-1, corticosteroid receptor (GR) expression, and translocation of GR, P65 were determined by ELISA and Western blot methods, respectively. Results: We found that CSE increase production of IL8 by concentration manner and combinations of drugs have inhibitory effects rather single on IL-8 release. Besides, inhibitory effects of the drugs were paralleled with increase translocation of GR through the

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nucleus. Conclusion: This study shows that the combination therapy for inhibition of IL-8 and proteases provides a more suppressive effect rather single therapy.

We conclude that agonist-induced desensitization of β3-adrenoceptor signalling is cell type-specific. It does not involve up-regulation of a Gi protein but, apparently, a reduced adenylyl cyclase function.

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CIGARETTE SMOKE-INDUCED LUNG EMPHYSEMA: C57BL/6 VERSUS BALB/C K.A.T. Verheyden, A.H. van Houwelingen, I. van Ark, F.P. Nijkamp, G. Folkerts Dept. Pharmacology & Pathophysiology, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, PO Box 80082, 3508 TB Utrecht, The Netherlands2

BLUNTING OF THE CARDIOPROTECTIVE EFFECT OF ASPIRIN BY IBUPROFEN: WHAT IS THE EVIDENCE? R. van Westrhenen1, C.C. Gispen-de Wied1, J.F.F. Lekkerkerker1 Medicines Evaluation Board (CBG), The Hague

Background: The most important cause for developing lung emphysema is cigarette smoke (CS). The alveoli are destroyed and, hence, the alveolar septa disappear. This process is irreversible. There is much discussion in the literature on whether lung emphysema is a Th1- or Th2-driven disease. Objective: A more Th1 mouse strain: the C57BL/6 was compared with a more Th2 mouse strain: the BALB/c in the development of CSinduced lung emphysema. Methods: Mice were exposed to CS or normal air (controls) for 12 weeks. Airway and blood cells, right ventricular hypertrophy, airway reactivity, lung cytokines, and lung emphysema were measured. Results: In the C57BL/6 mice, the CS induced lung emphysema that was associated with hypertrophy of the right heart ventricular. Airway hyperreactivity, broncho-alveolar cells, and the mean linear intercept (Lm) were increased. The fat percentage was decreased. In the BALB/c mice, only broncho alveolar cells and hypertrophy were increased. Conclusion: Based on the results, it is more likely that the development of lung emphysema is a more Th1 mediated disease since an increase in Lm was only observed in the C57BL/6 mice and not in the BALB/c mice.

Recently, it has been suggested that the cardioprotective effect of aspirin may be blunted by simultaneous use of ibuprofen, potentially increasing cardiovascular morbidity and mortality. The FDA has issued a warning against the concomitant use of these two medications, suggesting to dose aspirin at least 30 min before and a maximum of 8 h after ibuprofen. The validity of this measure taken was challenged by investigating scientific literature for evidence of a possible interaction between aspirin and ibuprofen. A literature search was performed using the search terms “aspirin” and “ibuprofen” in PubMed. In two of the three identified short-term ex vivo studies, an interaction effect between aspirin and ibuprofen on platelet function was demonstrated, while in a third study, such an interaction was not demonstrated. In an additional clinical study, mortality was significantly higher in subjects using aspirin and ibuprofen together, compared with aspirin use alone. However, potential confounding effects of, e.g., smoking were not controlled in this study. It is known that the antiplatelet effect of aspirin is not absolute in all patients and some patients experience thromboembolic events despite aspirin use. These patients are clinically called aspirin-resistant. A strategy to investigate the potential risk of simultaneous use of aspirin and ibuprofen for cardiovascular morbidity and mortality will be proposed, as well as how to identify aspirin resistancy.

50 48 AGONIST-INDUCED DESENSITIZATION OF HUMAN Β3-ADRENOCEPTORS EXPRESSED IN CHO OR HEK293 CELLS W. Vrydag, A.E. Alewijnse, M.C. Michel Dept. Pharmacology and Pharmacotherapy, Academic Medical Center, Amsterdam, The Netherlands β3-Adrenoceptor agonists are in clinical development for the treatment of depression and voiding disorders, which require longterm treatment. Previous studies have reported that β3-adrenoceptor signalling may be rather resistant to agonist-induced desensitization upon expression in CHO cells, whereas desensitization may occur in HEK293 cells. Therefore, we have compared desensitization in stably transfected CHO and HEK293 cells. Unless otherwise indicated, cells were treated for 24 h with 10 μM isoprenaline or vehicle. After washing three times with buffer, cAMP accumulation in response to freshly added agonist was measured using the LANCE® kit (Perkin Elmer). Isoprenaline pre-treatment had no major effects on subsequent cAMP responses to isoprenaline in CHO cells but reduced the maximum isoprenaline response in HEK293 cells by about 70% without major changes of agonist potency. This desensitization was time- and concentration-dependent. It was similarly observed in pertussis toxin-treated cells. Moreover, isoprenaline pre-treatment also decreased forskolin-induced cAMP accumulation.

OLANZAPINE-INDUCED WEIGHT GAIN: DEVELOPING AN ANIMAL MODEL USING MALE RATS E.M. van der Zwaal, M.C.M. Luijendijk, S.E. la Fleur, R.A.H. Adan Rudolf Magnus Institute of Neuroscience, Dept. of Pharmacology and Anatomy, UMCU, The Netherlands Olanzapine is one of the most commonly prescribed antipsychotic drugs, which causes significant weight gain as a side effect, increasing the risk for diabetes and cardiovascular disease. The mechanisms responsible for the weight gain caused by olanzapine are not understood. The drug has an affinity for dopamine, 5-HT, histamine, muscarinic, and alpha-adrenergic receptors, and gaining insight into which of these receptors is responsible may be crucial to developing novel antipsychotics that are equally therapeutically effective but less prone to cause weight gain. We are currently developing an animal model for olanzapineinduced weight gain using male Wistar rats to study in detail the effects on eating behaviour, food preference, and locomotor activity. Because the half-life of olanzapine in male rats is approximately 2 1/ 2 h, compared to over 20 h in humans, we decided to use osmotic minipumps to administer an olanzapine-solution continuously for 4 weeks in different paradigms. Retrospectively, we identified problems concerning the long-term stability of olanzapine in solution. Nevertheless, preliminary results indicate a reduction of locomotor activity and an increase in food intake, meal size, and duration, as well as changes in diurnal feeding. After having identified further the effects of olanzapine administration on different aspects of energy balance, we plan to use this model to identify the receptors responsible for these effects.

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Author index Aarts LPHJ (37) Adan RAH (50) Adcock I (27) Alewijnse AE (12, 17, 42, 45, 48) Ark I van (47) Baalen T van (8) Bakker RA (2) Baranowski JL (2) Batenburg WW (1) Bergen-Henegouwen J van (3) Blankesteijn WM (16, 22) Blokhuis B (43) Blokhuis BR (35) Blomenröhr M (13) Boer RA de (21) Boer T de (15) Bolderen L van (14) Bongers G (2) Bos IST (25, 36) Braber S (3) Bueters RRG (14) Buikema H (37) Bunnet N (34) Chai W (4) Cowart MD (2) Daas I den (15) Danser AHJ (1, 4, 9, 20) Deelman LE (26, 38) Dekkers BGJ (5) Dennery P (29) Doornmalen EJP (13) Dueck G (10) Ebenschade TA (2) Eikmann MJ (1) El-Sheikh AAK (6) Elzinga CRS (7, 36) Epema AH (37) Esch BCAM van (40) Esch ECAM van (8) Esch JHM van (9) Estvander BR (2) Fink-Gremmels J (41) Fischer M (43) Fleur SE la (50) Folkerts G (3, 27, 32, 39, 46, 47) Garrelds IM (9, 20) Garssen J (3, 8, 19, 40) Genne L van (21) Gerthoffer WT (10) Gijzen K (27) Gilst WH van (21, 26, 30, 31) Gispen-de Wied CC (49) Gkiko M (27) Gkoumassi E (7) Goor H van (38) Gosens R (10, 33) Grandoch M (13) Guilarte M (35) Hahntow IN (11) Haiji N (17)

Hak JB (15) Halayko AJ (10, 36) Hancock AA (2) Hartog A (3) Heijden MW van der (35) Hendriks-Balk MC (12) Henning RH (26, 30, 37, 38) Herlaar E (34) Herpen REMA van (13) Heuvel JJMW van den (6) Hofman GA (8, 40) Hom HJ van der (7) Hommes DW (35) Horn HJ van der (36) Houwelingen AH van (47) Huls M (14) Iersel MP van (15) Ito K (27) Iusuf D (30) Janhunen SK (16, 22) Jong N de (26) Jongsma M (17, 45) Juffermans LJM (26) Kamp O (26) Karimi K (39) Kartikasari A (18) Kemna E (18) Kivit S de (19) Knippels LMJ (8) Koenderink JB (6) Kooiman K (26) Koopmans RP (11) Kornowski A (18) Korthagen N (19) Koster AS (35) Kraneveld AD (3, 34, 35, 46) Krop M (20) Krueger KM (2) Kuil CW (13) Kuipers I (21) Laeremans H (16, 22) Lane JR (23) Langemeijer E (24) Lekkerkerker JFF (49) Lesterhuis AH (33) Leurs R (2, 24) Lith LHCJ van (13) Liu XQ (10) Loenen PB van (12, 17) Loomans EEMG (13) Luijendijk MCM (50) Luttun A (14) Maarsingh H (25) Masereeuw R (14) Mathy M-J (12, 17, 42) Maussang D (24) Meijering BDM (26) Menke AL (14) Meurs H (5, 7, 10, 25, 33, 36) Michel MC (11, 12, 17, 42, 45, 48)

Miller TR (2) Milligan G (23) Mortaz E (27, 32, 39, 46) Musters R (26) Mutsaers R (28) Nelemans SA (7) Nelissen RLH (12) Nijkamp FP (27, 32, 35, 39, 43, 46, 47) Noordermeer S (29) Oeseburg H (30) Oian C (31) Oosterhout AJM van (33) Overbeek SA (32) Payan DG (34) Pennings B (28) Pera T (33) Peters SLM (12, 17, 42, 45) Powney B (23) Pronk I (35) Raats D (27, 34) Ramzan SNA (1) Rees S (23) Redegeld FA (35, 43) Rensen SS (16) Rijnierse A (34, 35) Roks AJM (30, 31) Roscioni SS (7, 36) Ruijs MCA (13) Russel F (28, 29) Russel FGM (6, 14) Samarska IV (37) Sandovici M (38) Santos J (35) Sarir H (39) Schaafsma D (5) Scharstuhl A (28) Schirmer SH (42) Schmidt M (7, 36) Schmitz J (34) Schoemaker RG (31) Schouten B (8, 19, 40) Schrickx JA (41) Schwietert HRS (15) Singh R (34) Smit MJ (24) Smits JFM (16, 22) Stelmack G (10) Stijn S van (42) Strakhova MI (2) Streun ELP van (7) Swinkels D (18) Teitsma CA (11) Thio M (43) Tjon-Atsoi RZM (12) Trezza V (44) Ulloa-Montoya F (14) Unen J van (45) Unruh H (10) Vaezirad MM (46)

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Vader P (27) Valkengoed I van (11) Vanderschuren LJMJ (44) Veghel R van (9) Velde AA te (35) Veldhuisen DJ van (21) Verheyden KAT (47) Verfaillie CM (14) Visser C (26) Vries R de (1)

Vrydag W (48) Wagener F (18, 28, 29) Wamel A van (26) Wanders FJ (7, 36) Wemer J (15) Westerhof FJ (25) Westrhenen R van (49) Wiegerinck E (18) Wieling JW (15) Willemsen LEM (8, 19, 40)

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Wise A (23) Witte DG (2) Woestenenk R (14) Yachie A (18) Yang G (29) Yu L (31) Zaman GJR (13) Zaagsma J (5, 7, 10, 25, 33) Zeeuw D de (38) Zwaal EM van der (50)