17th Scientific Symposium of the Austrian Pharmacological Society (APHAR)

Cannabinoid type-1 (CB1) receptor antagonists wereamong the most promising drug targets in the last decade. Theyhave been explored and found to be effective as therapeutic agentsfor obesity and related cardiometabolic problems, including e.g.dyslipidaemias, diabetes, and metabolic syndrome. However, theuse of rimonabant, the first marketed CB1 receptor antagonist, hasbeen suspended due to its anxiogenic and depressogenic sideeffects, which were present in about 20–30% of the patients, i.e. a2.5–3-fold increase compared to placebo. Since some other anti-obesity drugs like dexfenfluramine or sibutramine were alsosuspended, the unmet need for drugs that reduce body weightbecame enormous. One approach that emerged was thedevelopment of peripheral CB1 receptor antagonists that poorlycross the blood brain barrier, the second, the development ofneutral antagonists instead of inverse agonists, and the third, theselection of the patient population with reduced risk for psychiatricside effects. An analysis regarding peripheral and centralmechanisms involved in the effects of CB1 receptor antagonistsstrongly suggest that central mechanisms are more or less involvedin most cardiometabolic therapeutic effects and thus, amongpatients with unsatisfactory therapeutic response to compounds withperipheral action, centrally acting antagonists may be needed.Based on our existing knowledge concerning the role of genetic,phenotypic and environmental factors the selection of persons whoare at no or low risk for psychiatric adverse effects may be possible.Molecular mechanisms and receptors involved in the effects ofstress and anxiety-related neurocircuitries sensitive to CB1 receptorantagonists, like the serotonergic and noradrenergic systems whichregulate the synthesis of the endocannabinoid 2-arachydonoyl-glycerol mediated by 5-HT2C and α1 receptors can be identified [1].Furthermore, variants of the serotonin transporter and the CB1receptor genes have been shown to modulate stress-inducedanxiety in human studies [1]. In conclusion, development ofperipherally acting, or the use of personalised medicine for centrallyacting CB1 receptor antagonists are promising approaches withdiverse advantages.Acknowledgements: Studies were supported by EU, LSHM-CT-2004-503474, TAMOP-4.2.1.B-09/1/KMR-2010-0001 andETT318/041/2009.Reference 1. Lazary J, Juhasz G, Hunyady L, Bagdy G: Personalized medicine can pave the way for the safe use of CB1 receptor antagonists.Trends Pharmacol Sci 2011, 32:270–280. A56The double-faced role of P2X7 receptors in toxin-inducedanimal models of Parkinson’s diseaseBeáta Sperlágh, Zsuzsanna Hracskó, Mária Baranyi,CecíliaCsölle, Flóra Gölöncsér, Ágnes Kittel andEmilia MadarászDepartment of Pharmacology, Institute of Experimental Medicine,Hungarian Academy of Sciences, 1083 Budapest, Hungary;Department of Gene Technology and Developmental Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, 1083 Budapest, Hungary E-mail: [email protected] Background: Previous studies indicate a role of P2X7 receptors inprocesses that lead to neuronal death. The main objective of ourstudy was to examine whether genetic deletion or pharmacologicalblockade of P2X7 receptors influenced dopaminergic cell death invarious models of Parkinson’s disease (PD).Methods: PC12 cells and primary mesencephalic neurons wereused in culture, and the striatum and the substantia nigra wereprepared from wild-type and P2X7 receptor knockout mice.Rotenone and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)treatments were applied in vitro and in vivo to reproduceneurochemical hallmarks of PD. Receptor expression, cell survivalindicators, and endogenous biogenic amine, amino acid, adeninenucleotide and endocannabinoid contents were analyzed.Results: mRNA encoding P2X7 and P2X4 receptors was up-regulated after treatment of PC12 cells with MPTP. P2X7antagonists protected against MPTPand rotenone-induced toxicityin the LDH assay, but failed to protect after rotenone treatment inthe MTT assay in PC12 cells and in primary midbrain culture. In vivoMPTP and in vitro rotenone pretreatments increased the mRNAexpression of P2X7 receptors in the striatum and substantia nigra ofwild-type mice. Basal mRNA expression of P2X4 receptors was APHAR 2011 Abstract Preview higher in P2X7 knockout mice and was further up-regulated byMPTP treatment. Genetic deletion or pharmacological inhibition ofP2X7 receptors did not change survival rate or depletion of striatalendogenous dopamine (DA) content after in vivo MPTP or in vitrorotenone treatment. However, depletion of norepinephrine wassignificant after MPTP treatment only in P2X7 knockout mice. Thebasal ATP content was higher in the substantia nigra of wild-typemice, but the ADP level was lower. Rotenone treatment elicited asimilar reduction in ATP content in the substantia nigra of bothgenotypes, whereas reduction of ATP was more pronounced afterrotenone treatment in striatal slices of P2X7-deficient mice. Althoughthe endogenous amino acid content remained unchanged, the levelof the endocannabinoid, 2-arachidonoyl glycerol (2-AG), waselevated by rotenone in the striatum of wild-type mice, an effect thatwas absent in mice deficient in P2X7 receptors.Conclusions: We conclude that P2X7 receptor deficiency orinhibition does not support the survival of dopaminergic neurons inin vivo or in vitro models of PD. Late Abstracts (APHAR) A57The G protein-coupled receptor-associated protein 1 (GASP-1)regulates rimonabant-induced downregulation of GPR55Julia Kargl, Nariman A Balenga, Jennifer L Whistler andMaria WaldhoerInstitute of Experimental and Clinical Pharmacology, Medical University of Graz, 8010 Graz, AustriaCurrent address: Molecular and Signal Transduction Section,Laboratory of Allergic Diseases, NIAID/NIH, Bethesda, MD 20892-1889, USAErnest Gallo Clinic and Research Center, University of California,San Francisco, CA 94608, USACurrent address: Hagedorn Research Institute, Novo Nordisk A/S,2820 Gentofte, DenmarkE-mail: [email protected] Background: The G protein-coupled receptor 55 (GPR55) hasrecently been suggested to be responsible for those cannabinoidresponses that could not be attributed to either the cannabinoid 1(CB1) or cannabinoid 2 (CB2) receptor. Several potent GPR55agonists were identified such as lysophosphatidylinositol (LPI) andseveral synthetic cannabinoids: One of these is rimonabant(SR141716A), an antagonist at the CB1 receptor, which showedclinical promise, but approval was revoked due to adverse events.Generally, the activity of G protein coupled receptors (GPCRs) iscoordinated by receptor signaling, receptor desensitization andreceptor resensitization. One regulatory mechanism to guaranteeappropriate GPCR expression levels in physiological conditions isthat of downregulating GPCRs via the G protein-coupled receptor-associated sorting protein 1 (GASP-1), thus leading to anattenuation of cellular signaling events. GASP-1 was originallyfound to target δ opioid receptors to lysosomes and, hence, to thedegradative pathway. It was shown that GASP-1 is a keydeterminant in the development of analgesic tolerance tocannabinoids via its role in facilitating downregulation of the CB1receptor.Methods and results: By a variety of approaches we demonstratedthat rimonabant promotes downregulation of GPR55 via GASP-1 invitro and in vivo. We show that GPR55 interacts with GASP-1 invitro and that disrupting the GPR55/GASP-1 interaction preventspost-endocytic receptor degradation, and thereby allows receptorrecycling. Together, these data implicate GASP-1 as an importantregulator of rimonabant-mediated downregulation of GPR55.Conclusions: This work provides tangible evidence that GPR55 isdegraded after prolonged agonist stimulation and that thismechanism is regulated by GASP-1. A58The D-type prostanoid (DP) receptor enhances the signaling ofchemoattractant receptor-homologous molecule expressed onTh2 cells (CRTH2)MiriamSedej, Ralf Schröder, Kathrin Bell, Wolfgang Platzer,Evi Kostenis, Ákos Heinemann and Maria WaldhoerInstitute of Experimental and Clinical Pharmacology, Medical University of Graz, 8010 Graz, AustriaInstitute of Pharmaceutical Biology, University of Bonn, 53115 Bonn, GermanyCurrent address: Hagedorn Research Institute, Novo Nordisk A/S, 2820 Gentofte, Denmark E-mail: [email protected] Background: Prostaglandin (PG) D2 is substantially involved inallergic responses and signals via the seven-transmembrane-spanning/G protein-coupled receptors, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) and D-typeprostanoid (DP) receptor. While the proinflammatory function ofCRTH2 is well recognized and CRTH2 is hence considered as animportant emerging pharmacotherapeutic target, the role of the DPreceptor in mediating the biological effects of PGD2 in allergicinflammation has remained unclear.Methods: The cross-talk of CRTH2 and DP receptors wasinvestigated using both a recombinant HEK293 cell model andhuman eosinophils in Ca mobilization assays, co-immunoprecipitation and radioligand binding assays.Results: We show that CRTH2 and DP receptors modulate eachother’s signaling properties and form CRTH2/DP heteromers withoutaltering their ligand-binding capacities. We find that the DP receptoramplifies the CRTH2-induced Ca release from intracellular storesand, coincidentally, forfeits its own signaling potency. Moreover,desensitization or pharmacological blockade of the DP receptorhinders CRTH2-mediated signal transduction. Pharmacologicalblockade of Gαq/11 proteins abolishes the Ca response to bothCRTH2 and DP agonists, while inhibition of Gαi proteins selectivelyattenuates the CRTH2-mediated response but not the DP signal.Conclusions: Our data demonstrate the capacity of DP receptorsto amplify the biological response to CRTH2 activation. Therefore,the CRTH2/DP heteromer may not only represent a functionalsignaling unit for PGD2 but also a potential target for development ofheteromer-directed therapies to treat allergic diseases. Late Abstracts (MFT) A59Effects of diclofenac on ventricular muscle repolarization:proarrhythmic implicationsNorbert Jost, Attila Kristóf, Zsófia Kohajda, Tamás Szél,Zoltán Husti, István Koncz, Victor Juhász, István Baczkó,Julius Gy Papp, András Varró and László Virág Department of Pharmacology and Pharmacotherapy, University of Szeged; Division of Cardiovascular Pharmacology, Hungarian Academy of Sciences, 6720 Szeged, Hungary E-mail: [email protected] Background: The aim of the present work was to characterize theelectrophysiological effects of the non-steroidal anti-inflammatorydrug diclofenac and to study the possible proarrhythmic potency ofthe drug in ventricular muscle.Methods: Ion currents were recorded using the voltage clamptechnique in canine ventricular cells, and action potentials (AP) APHAR 2011 Abstract Preview were recorded from canine ventricular preparations usingmicroelectrodes. The proarrhythmic potency of diclofenac wasinvestigated in an anaesthetized rabbit proarrhythmia model.Results: Diclofenac (30 μM) decreased the amplitude of rapid (IKr)and slow (IKs) delayed rectifier and L-type calcium currents (ICa)without influencing transient outward(Ito) and inward rectifier (IK1)potassium currents. The action potential was slightly lengthened inventricular muscle but shortened in Purkinje fibres by diclofenac(20 μM). The maximum upstroke velocity (Vmax) was decreased inboth preparations. Larger repolarization lengthening was observedwhen repolarization reserve was impaired by previous BaCl2application. Diclofenac (3 mg/kg) did not prolong the QTc interval,while the potassium channel blocker dofetilide (25 μg/kg)significantly lengthened QTc in anaesthetized rabbits. Thecombination of diclofenac and dofetilide significantly prolongedQTc.Diclofenac alone did not induce torsades de pointes ventriculartachycardia (TdP) while TdP incidence following dofetilide was 20%.However, the combination of diclofenac and dofetilide led to asignificant increase in the incidence of TdP.Conclusions: The results indicate that diclofenac, at therapeuticconcentration and even at high dose, does not increase the risk ofarrhythmia in normal heart. However, high dose drug treatment mayenhance the proarrhythmic risk in the heart when the repolarizationreserve is reduced.Acknowledgements: This work was funded by grants from OTKA(CNK-77855, K-82079) and the National Development Agency(TÁMOP-4.2.1/B-09/1/KONV-2010-0005). A60Properties of the transient outward, ultra-rapid delayed rectifierand acetylcholine-sensitive potassium currents in isolatedatrial myocytes from dogs: sinus rhythm and tachypacedmodel of permanent atrial fibrillationZsófia Kohajda, Attila Kristóf, Péter P Kovács, Claudia Corici,László Virág, Viktor Juhász, Zoltán Husti, István Baczkó,András Varró and Norbert Jost Department of Pharmacology and Pharmacotherapy, University ofSzeged; Division of Cardiovascular Pharmacology, HungarianAcademy of Sciences, 6720 Szeged, Hungary E-mail: [email protected] Background: Atrial fibrillation (AF) is a common and severearrhythmia, which largely affects quality of life. State-of-the-arttreatment of AF still relies heavily on pharmacological modalities.Therefore, the aim of the present study was to investigate andcompare the properties of three repolarizing currents whichcontribute to AF-induced remodeling, i.e. the transient outward (Ito),ultra-rapid delayed rectifier (IKur) and acetylcholine-sensitivepotassium currents (IK,ACh) in isolated atrial myocytes obtained fromnormal (SR) and tachypaced model of permanent atrial fibrillation(ATR) dogs.Methods: The tachypaced atrial fibrillation model was performed indogs. Transmembrane ionic currents were investigated by applyingthe whole-cell patch clamp technique at 37°C, and ECG wasrecorded in conscious dogs.Results: In all atrial canine myocytes, we have identified an Itocurrent sensitive to 4-aminopyridine (4-AP; 3 mM). The currentinactivation was best fitted by two exponentials. The Ito current wasslightly downregulated in ATR cells when compared with thatrecorded in SR cells. The IKur current, measured as sustainedcurrent (Isus), was upregulated in ATR dogs. However, the selectiveIKur blocker 4-AP (50 μM) did not block either Isus or IKur „like tail”currents, which questions the reliability of these results. IK,ACh wasactivated by the cholinergic agonist carbachol (CCh; 2 μM). In SR,CCh activated a large current either at inward or outward directions.The selective IK,ACh blocker tertiapin (10 nM) blocked the CCh-induced current by 57%. In atrial myocytes from ATR dogs we couldmeasure the presence of a constitutively active IK,ACh, which couldbe blocked by 26 % with 10 nM tertiapin. However, in ATR atrialmyocytes, CCh in addition could also activate a significant ligand-dependent and tertiapine-sensitive IK,ACh current. Tertiapineffectively prevented burst-induced AF in conscious ATR dogs.Conclusions: The presence of the constitutively activated IK,ACh inatrial myocytes from ATR dogs shows that electrical remodelingdeveloped in our model; this was further supported by theinducibility of AF by rapid atrial bursts in these dogs. The IK,AChcurrent (both ligand-dependent and constitutively active currents)seems to play a significant role in the canine atrial electricalremodelling, and may be a promising drug target for suppressingAF.Acknowledgements: This work was funded by grants from OTKA(CNK-77855, K-82079) and the National Development Agency(TÁMOP-4.2.1/B-09/1/KONV-2010-0005). APHAR 2011 Abstract Preview APHAR 2011 Abstract Preview Author Index (Numbers refer to Abstract No.)