key: cord-0009669-1bk71pox
authors: nan
title: Oral Communications Tuesday 15 July Respiratory and pulmonary pharmacology (14.30‐15.30)
date: 2008-06-28
journal: Fundam Clin Pharmacol
DOI: 10.1111/j.1472-8206.2008.00593.x
sha: bf3d91b38ac4515fd692da62ee7e19b18608b42a
doc_id: 9669
cord_uid: 1bk71pox

nan

. Effects of AF on IFNs production in patients with URVI measured by ELISA IFN production Anaferon, n = 93 Placebo, n = 46 baseline 2-3 days baseline 2-3 days Serum IFNa, pg/mL 44.9 ± 3.5 66.3 ± 3.5* 40.8 ± 4.7 34.5 ± 4.6 Serum IFNc, pg/mL 59.6 ± 2.9 78.9 ± 3.6* 54.4 ± 4.7 54.0 ± 5.4 Spontaneous IFNa production, pg/mL 77.3 ± 4.5 91.6 ± 4.6* 73.3 ± 5.9 77.3 ± 7.3 Spontaneous IFNc production, pg/mL 44.9 ± 2.5 63.5 ± 2.9* 43.3 ± 4.1 44.3 ± 3.8 Mitogen-induced IFNa production, pg/mL 103.5 ± 6.0 143.2 ± 8.3* 107.9 ± 8.9 97.0 ± 11.7 Mitogen-induced IFNc production, pg/mL 91.2 ± 6.6 162.5 ± 22.0* 86.4 ± 6.5 78.9 ± 5.9

The data represent the mean values ± SE; *P < 0.001 vs. placebo.

Effect of etanercept in a strain-dependent mouse model of allergeninduced airway hyperresponsiveness and lung inflammation J Nasra, K Tomlinson, L Berry, M Airey, S Shaw, R Foulkes, N Gozzard, R Palframan UCB, Slough, UK The aim of these studies was to develop an allergen-induced murine model of airway inflammation, characterised by airway hyperresponsiveness (AHR) and an upregulation of TH 1 and TH 2 cytokines. The role of TNF in this model was investigated using etanercept; a recombinant human soluble TNF receptor fusion protein reported to have efficacy in patients with persistent asthma and known to neutralise mouse TNF. C57bl/6 or Balb/c mice (male, 20-25 g, n = 8/group) were sensitised subcutaneously on day 0 with saline or 100 lg of house dust mite [Dermatophagoides pteronyssinus (Der p)] extract in complete Freund's adjuvant. On day 14, mice were exposed to saline or Der p (100 lg or 25 lg) via intranasal instillation. 48 hours post-challenge, non-invasive whole body plethysmography was used to assess AHR stimulated by doubling concentrations of aerosolised methacholine (MCh, 3.125-50 mg/ml). Bronchoalveolar lavage fluid (BALF) was retrieved for measurement of inflammatory cells and cytokine profile. In subsequent studies using Balb/c mice (n = 10/group), the effect of etanercept (30 mg/kg, i.v.) or vehicle (PBS, i.v.) , administered on days 13 and 14, was assessed. Allergeninduced AHR was more profound in Balb/c as compared to C57bl/6 mice. Immunisation and challenge of Balb/c mice with Der p induced AHR (> 2-fold increase in Penh at all concentrations of MCh), whereas AHR in C57bl/6 mice was less robust (> 2-fold increase only at 6.25 and 12.5 mg/ml MCh). The AHR was accompanied by BALF neutrophilia (Balb/c: 83.66 ± 17.31 x 10 4 cells/mL; C57bl/ 6: 47.10 ± 21.62 x 10 4 cells/ml) and eosinophilia (Balb/c: 17.51 ± 5.07

x 10 4 cells/ml; C57bl/6: 11.43 ± 5.58 x 10 4 cells/mL). Treatment of Balb/c mice with etanercept during the challenge phase significantly inhibited AHR by 52% (12.5 mg/ml MCh: Penh reduced from 3.70 ± 0.34 to 2.49 ± 0.38, P < 0.05). Etanercept significantly (P < 0.05) altered the BALF cytokine profile, inhibiting IL-1b (52%), IL-5 (54%), IL-10 (36%), IL-12 (76%) and IFNc (68%). Etanercept had no significant effect on BALF neutrophilia or eosinophilia measured 48 h after allergen challenge. The sensitivity of components of this model to etanercept, a clinically used anti-TNF therapeutic, demonstrates the potential for utilising this model to investigate the mechanisms of persistent asthma and in the assessment of novel therapeutics.

Functional changes in background K + channels during the culture of pulmonary arteries B Manoury, S Etheridge, A Gurney University of Manchester, Manchester, UK The culture of blood vessels is gaining interest for use with transfection-based techniques (Gurney and Hunter, 2005) , but has been shown to alter the contractile properties of the vessels (Guibert et al., 2005) . The present study tested the effects of culture on the intrinsic tone of rat pulmonary arteries (PAs) and examined the function and expression of K + channels involved in regulating the resting membrane potential (E m ) and tone of pulmonary artery smooth muscle cells (PASMCs). Male Sprague-Dawley rats (250-300 g) were sacrificed according to Schedule I of the UK Scientific Procedures (Animals) Act 1986. Intrapulmonary arteries were isolated and cultured as previously described (Gurney et al., 2005) . Contractile responses of fresh and cultured PA to various drugs were measured using vessel myography. The E m of PASMC was recorded using the perforated patch technique and K + channel expression quantified using real time RT-PCR. The contractile response to 1 lM phenylephrine did not change during culture, but subsequent relaxation to 1lM carbachol was significantly reduced. Over 4 days in culture, contractile responses to 15 mM KCl, 1 mM 4-aminopyridine and 10 mM tetraethylammonium increased, while the vessels developed an uncharacteristic relaxation response to 1 lM nifedipine, 10 lM levcromakalim and Ca 2+ -free solution. These changes were associated with depolarisation of the PASMC E m and down regulation of Kv1.5, Kv2.1 and TASK-1 mRNA expression. Bubbling the culture medium with 95% O 2 partially reversed the changes observed. The changes in PA function that developed during culture are consistent with a progressive depolarisation of PASMC, probably resulting from reduced expression of background K + channels, especially TASK-1. O 2 enrichment appears to limit the changes induced by culture.

Chronic exposure to fibrin enhances agonist-induced calcium release in human pulmonary artery smooth muscle cells A Firth, J Yao, P Chiles, J Marsh, J Yuan University of California, San Diego, La Jolla, California, USA Acute pulmonary embolism occurs in more than 500 000 patients a year in the US. Chronic thromboembolic pulmonary hypertension (CTEPH) develops in approximately 4% of the patients (or~20 000 cases) with acute pulmonary embolism each year because of unresolved thromboemboli. CTEPH is a relatively common, progressive and potentially fatal disease process. One currently proposed theory for such poor resolution advocates that modification of fibrin in CTEPH patients causes resistance of emboli to fibrinolysis, which could lead to pulmonary vascular remodeling. Control of intracellular calcium is central to the regulation of cell migration, proliferation and contraction and contributes to the pathophysiological process of vascular remodeling. The current study investigates the regulation of intracellular calcium by chronic exposure of pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells to thrombin and fibrin. PASMC or PAEC were plated on control, fibrinogen (FNG), thrombin or fibrin coated cover slips for 72 h. Cells were loaded with a fluorescent calcium sensitive dye, Fura-2-AM, and changes in intracellular calcium in response to thrombin (5 nM) were recorded. Chronic exposure of PAEC to thrombin, an agonist of proteinase-activated receptors, significantly reduced the agonist-induced peak calcium transient (F/F 0 ratio) from 1.3 ± 0.07 (n = 27) to 0.97 ± 0.06 (n = 43), P < 0.001. However, in PASMC a substantial increase was observed. The peak calcium was significantly increased in PASMC chronically exposed to 4 lg/ml fibrin and the recovery rate of the agonist-induced calcium transients decelerated (P < 0.05). In PAEC, only higher FNG and fibrin concentrations (40 lg/ml) significantly affected the calcium transients; decreasing the peak calcium transient and the calcium transient peak width at 50% of the peak height (P < 0.05) when compared to control in PAEC. In conclusion, chronic exposure of cells to fibrinogen, fibrin and thrombin caused changes in intracellular calcium which could stimulate accelerated smooth muscle cell proliferation, migration and contraction. Prolonged exposure to thrombin had a potent effect reducing agonist-induced calcium transient in PAEC, suggesting a desensitization of receptors and intracellular calcium regulation in these cells.

Activation of Toll-like receptor (TLR) 3 and TLR 8 lead to endothelin-1 release from pulmonary vascular smooth muscle: relevance to pulmonary hypertension R Badiger, A Liu, J Mitchell, P De Sousa, S Wort, M Paul-Clark Imperial College, London, UK Endothelin-1 (ET-1) is an important mediator in pulmonary hypertension. Usually released by the endothelium; it can be released by pulmonary vascular cells, and act in an autocrine manner (Wort et al., 2001) to cause vasoconstriction and smooth muscle hypertrophy. Pathogens, via pathogen-associated molecular patterns (PAMPS), can mediate profound vascular responses, e.g. the shock induced by lipopolysaccaride. These responses are mediated by Toll-like receptors (TLRs), (Mitchell et al., 2007) . Our aim was to determine if bacterial and viral PAMPS can lead to endothelin-1 release from pulmonary vascular smooth muscle, and thus whether pathogens can be triggers to pulmonary hypertension. Human pulmonary artery smooth muscle (HPASM) cells were grown in 96 well plates and were stimulated with a range of PAMPS. Supernatants were collected at 24 h. In addition, primary human endothelial (HE) cells were also stimulated with PAMPs. ET-1 was measured by ELISA. Our results showed that synthetic ligands for viral PAMPS, Poly I:C, (activates TLR 3), and LyoVec (ligand for TLR 8), cause ET-1 release from HPASM cells. Importantly, bacterial PAMPs did not cause ET-1 release in these cells. By contrast, activation of TLR8 or TLR3 did not elevate ET-1 in HE cells. These observations are the first to indicate a role for viral pathogen sensing in pulmonary hypertension. 

Antisense oligonucleotide against endothelin receptors modulate ET-1induced vasoconstriction of rat pulmonary arteries S Sauvageau, J Dupuis, E Thorin Montreal Heart Institute, Montreal/Quebec, Canada Most G protein-coupled receptors (GPCR) form homo and/or hetero-oligomeric complexes. This dimerization concept raises the problem of how two different receptors can influence the coupling of each other and determine the ultimate function of the complex. We previously reported an interaction between the ET A and ET B receptors (ET-R) in rat pulmonary arteries, which may modify endothelin-1-(ET-1) induced vasoconstriction (Sauvageau et al. 2007 ). To test this hypothesis we developed a novel non-pharmacological vessel culture technique combined with gene therapy. Small isolated pulmonary arteries (100-150 lM) from male wistar rats (200-250 g) were harvested and pooled for standard protein extraction. Heterodimerization of receptors was evaluated by immunoprecipitation of the ET B -R, followed by Western blotting for the expression of the ET A receptor in both denaturating and non-denaturating conditions. For gene therapy experiments, pulmonary arteries were incubated and exposed for 24 (ET A -R) or 72 h (ET B -R) to selective antisense (AS) oligonucleotides. A total of two different AS oligodeoxyribonucleotides (ON) phosphorothioate (PTO) sequences were used targeting either the rat ET A receptor (ET A -AS) or the rat ET B receptor (ET B -AS) mRNA sequences. The corresponding 2 scrambled (Sc) ON sequences were used as negative controls (Sc-ET A -AS; Sc-ET B -AS). Following the incubation period pulmonary arteries were mounted on a microvessel myograph to assess ET-1-induced constriction. Heterodimerization of both receptors was observed. Western blotting confirmed that each AS reduced protein expression of its targeted receptor. The use of ET B -AS significantly reduced the sensitivity to ET-1 ()7.90 ± 0.09; logEC 25 ± SEM, P < 0.05) compared to control vessel ()8.59 ± 0.17) without affecting the maximal vasoconstriction. In contrast, ET A -AS significantly increased the vascular sensitivity ()8.36 ± 0.04, P < 0.05) and the maximal vasoconstriction induced by ET-1 (100 ± 3, Emax ± SEM, P < 0.05) compared to controls ()7.83 ± 0.05, 89 ± 3%). In conclusion, suppression of ET B receptors reduced vascular sensitivity to ET-1 while suppression of ET A receptors increased the vascular sensitivity to this peptide. Hence, there is an interaction between the ET A and ET B receptors to induce the ET-1 response in rat pulmonary arteries. Furthermore, these results confirm the functional importance of ET B receptors in ET-1-induced vasoconstriction of small pulmonary arteries. Here, key transmembrane (TM) residues in TM 3, 5 and 7 have been mutated to provide structural insights into the nature of these two conformations. Mutations (D138A, D138S, S228A, S229A, S232A and N363A) were generated using the Strategene QuickChange mutagenesis kit. These constructs were then transfected into a CHO cell line stably expression a CRE-SPAP reporter gene. The cells were selected for 3 weeks using G418 for the receptor and hygromycin for the CRE-SPAP reporter. These stable mixed populations were then used in 3 H-CGP 12177 whole cell binding and CRE-SPAP reporter assays as previously described (Baker et al., 2003) . Agonist responses to isoprenaline and CGP 12177 had different sensitivities to b1-antagonists (e.g. CGP 20712A; log K D = )8.65 and )7.26 respectively). CGP 12177 acted as a high affinity antagonist (log K D = )9.18) at the 'catecholamine site' and as a lower affinity agonist at the 'CGP 12177 site' (log EC 50 = )8.12).

Mutations to D138 abolished all 3 H-CGP 12177 binding and all functional responses. N363 mutations abolished all CGP 12177 binding and responses and markedly reduced responses to isoprenaline and cimaterol. Each of the TM5 serine residues (S228A, S229A, S232A) were found to contribute to catecholamine binding, but none was singularly responsible. Cimaterol binding (a non-catechol agonist) however was minimally affected. The S228A and S229A mutations reduced both the affinity of CGP 12177 for the catecholamine site (25.5 and 7.1-fold) and also its agonist potency (15.5 and 6.8-fold) at the 'CGP 12177 site'. Thus D138 and N363 are essential for binding and function of both conformations of the b1-adrenoceptor and each of the TM5 serine mutants retained the pharmacology of the two conformations of the b1-adrenoceptor. In the present study we addressed the following questions: 1) Is the local diffusion of the receptor in the cell membrane restricted to small domains?, 2) if the long distance diffusion of the receptor in the membrane is not macroscopically bounded in local domains, can local diffusion explain this large scale diffusion quantitatively?

, 3) what is the nature of the compartmental connectivity in the cell in terms of receptor trafficking, when the cellular distribution of receptor is in the steadystate?. We used HEK293 cells permanently transfected with human b2 adrenoceptor fused to the N-terminal of the dendra protein (a GFP-like fluorescent protein that undergoes an irreversible spectral conversion from {Ex. 488, Em.507 nm} to {Ex. 558, Em.575 nm} upon subsecond irradiation with 488 nm~1 W/cm-sq argon laser) as a model system. This construct allowed us to locally (and instantaneously) label the receptors in a small region of the membranes in living cells. We used a confocal microscope (Leica) equipped with appropriate lasers, thermostatic baths and software for data collection. Solutions of two-dimensional diffusion equation (analytical or numerical) for appropriate boundary and initial conditions were used for quantitative evaluation of the data. We found that 1) functional integrity of the dendra-tagged receptor remains intact (as assessed by agonist stimulated cyclase activation or radioligand binding experiments), 2) inward or outward flux of the receptor to, or from a small membrane patch (~4 micron-sq) can be symmetrically explained by the same simple diffusion process with a diffusion coefficient of~0.1 micron-sq/s (with an average mobile fraction of 85%), 3) this process is found to be independent of the activity state of the receptor, as assessed by using constitutively active mutants of the receptor, 4) only a part of the large scale movement of the receptor in the membrane can be explained by the same local diffusion process, implying the presence of large-scales diffusion barriers in the membrane, 5) cell-wide re-distribution of the receptor protein in the membrane is governed by the same local diffusion process (i.e. the entire cell membrane is apparently available to the receptor molecules, and 6) all the visible compartments (within the spatial resolution of the microscope) in the cell are interconnected within the time frame of hours. These are the first experiments that demonstrate these points.

Role of key residues in TM2 and TM6 in the two agonist conformations of the human b1-adrenoceptor J Baker, R Proudman, N Hawley, S Hill University of Nottingham, Nottingham, UK Functional studies with CGP 12177 at the human b1-adrenoceptor have provided evidence for two active conformations that have markedly different pharmacological properties (Baker et al., 2003) . In the b-2 adrenoceptor Asp79 in TM2 and residues in TM6 (Asn 293 and Phe290) have been implicated in receptor activation mediated by agonists (Kobilka, 2007) . Here, the equivalent residues in the b-1 adrenoceptor have been mutated to investigate the contribution of these residues to the two agonist conformations of the b1-adrenoceptor. Mutations (wild-type, (WT), D104A, D104N, N341A and F343A) were generated using the Strategene QuickChange mutagenesis kit. These constructs were then transfected into a CHO cell line stably expression a CRE-SPAP reporter gene. The cells were selected for 3 weeks using G418 for the receptor and hygromycin for the CRE-SPAP reporter. These stable mixed populations were then used in 3 H-CGP 12177 whole cell binding and CRE-SPAP reporter assays as previously described (Baker et al., 2003) . Responses to isoprenaline and CGP 12177 were inhibited in the WT receptor by CGP 20712A to give a log K D values of )8.65 and )7.26 respectively, indicative of the two conformations of the b1-adrenoceptor. Mutations of Asp104 had very little effect on the affinity of ligands however functional responses including that to CGP 12177 were greatly reduced. Mutations in TM6 (N341A and F341A) slightly reduced the binding affinity of isoprenaline and CGP 12177 but had little effect on their agonist actions. Antagonism by CGP 20712A of these two responses was similar ()log K D values = )7.6 and )7.6 for F341A; )8.1 and )8.2 for N344A with isoprenaline and CGP 1277 as agonist respectively). This suggests that CGP 20712A was no longer able to discriminate between the two agonist conformations of the receptor. These studies suggest that F341 and N344 may have important roles in defining the two conformations of the b1-adrenoceptor. 

Regulation of G-protein-coupled receptor kinase 2 (GRK2) by calmodulin and protein kinase C J Brockmann a , K Lorenz a , MJ Lohse a , C Krasel b a Department of Pharmacology, University of Wuerzburg, Wuerzburg, Germany; b School of Pharmacy, University of Reading, Reading, Berkshire, UK G-protein-coupled receptor kinases (GRKs) mediate the first step of homologous desensitisation of G-protein-coupled receptors, namely phosphorylation of agonistactivated receptors. It has been previously shown that one of the GRKs, GRK2 is subject to inhibition by calmodulin. We have previously postulated that this inhibition can be relieved by protein kinase C which phosphorylates GRK2 on Ser-29, within a putative calmodulin-binding site. We have now investigated this regulatory mechanism further. To directly assess GRK2-calmodulin interaction, we covalently labelled calmodulin with dansyl chloride and investigated its interaction with various concentrations of GRK2, GRK2(1-53) and GRK2(552-689) by measuring dansyl fluorescence at 486 nm. Fluorescence increases in a saturable manner when calmodulin interacts with increasing concentrations of a binding partner. GRK2 activity was assessed in the presence and absence of calmodulin by measuring phosphorylation of rhodopsin (membrane-bound substrate) or tubulin (soluble; a kind gift from Jens Mü ller, DESY, Hamburg, Germany) using 32 P-ATP.

To mimick phosphorylation of GRK2 at Ser-29, we constructed a Ser29Asp mutant (S29D). GRK2 interacted with calmodulin in the presence of 5 mM EGTA and the interaction increased at 1 mM Ca 2+ . Calmodulin was able to interact with both GRK2(1-53) and GRK2(552-689), in agreement with predictions from the calmodulin target database (http://calcium.uhnres.utoronto.ca/ctdb/), but interaction with the N-terminus occurred only in the presence of 1 mM Ca 2+ whereas interaction with the C-terminus was evident in the presence of 5 mM EGTA. GRK2(1-53) S29D showed reduced interaction with calmodulin compared to GRK2(1-53). Calmodulin inhibited GRK2 in a strictly calcium-dependent manner. Rhodopsin phosphorylation was inhibited with higher potency (IC 50~2 lM) than GRK2-mediated tubulin phosphorylation (IC 50~1 00 lM). In a different set of experiments, calmodulin inhibited GRK2 S29D activity approximately 10-fold less potently than GRK2 activity. These experiments support the hypothesis that calmodulin is able to inhibit GRK2 interaction with membrane-bound substrates by interacting with the GRK2 N-terminus. This interaction (and thus the inhibition) is relieved if Ser-29 within the calmodulin binding site is phosphorylated by protein kinase C.

Molecular pharmacology (14.30-15.30)

Structural determinants regulating P2Y12 purinergic internalization into a distinct population of clathrin-coated pits C Harding, S Nisar, S Mundell University of Bristol, Bristol, UK Upon activation many G protein-coupled receptors (GPCRs) internalise by clathrinmediated endocytosis and are subsequently sorted to undergo recycling or lysosomal degradation. Recent research from our laboratory (Mundell et al., 2006) and others (Puthenveedu and von Zastrow 2006) has suggested that there may be multiple populations of clathrin-coated pits (CCPs) available to selectively sort GPCR cargo. In this present study we examined the structural determinants present in the COOH tail of the P2Y 12 receptor that regulate their internalization into CCPs. These studies were undertaken either in CHO cells stably transfected with receptor constructs or in HEK293 cells. N-terminal tagged (either HA or FLAG) receptor constructs were co-transfected into cells using Lipofectamine. Surface receptor surface loss was measured by ELISA and cellular distribution of HA-tagged or FLAG-tagged receptor examined by immunofluorescence microscopy as previously described (Mundell et al., 2006) . Our initial studies confirmed that deletion of the last four amino acids (E 339 stop), a PDZ motif (ETPM), of the P2Y 12 receptor attenuated receptor internalization. Interestingly mutation of P341 to alanine in this motif also attenuated receptor internalization. Subsequent immunofluorescent studies revealed that both P341A and E 339 stop trafficked to clathrin-coated pits but did not colocalize with full length P2Y 12 receptor at these sites. In addition colocalization with arrestin, which regulates P2Y 12 receptor entry into CCPs was not evident for E 339 stop. Subsequent experiments revealed that following internalization both E 339 stop and P341A did not traffic normally back to the cell surface but were retained in an as yet uncharacterised endosomal compartment. This intracellular retention attenuated receptor resensitization. In conclusion this study demonstrates that the presence and integrity of a PDZ motif on the P2Y 12 receptor is essential for correct targeting to distinct populations of CCPs and subsequent receptor internalization and traffic. 

The analysis of group effects (AGE) on the binding of drugs to receptors: what you can do with a library of values of log.K (pA 2 ) R Barlow Retired, Cumbria, UK Published values of log.K (pA 2 ) for 496 competitive antagonists acting at muscarinic (M 3 ) receptors in isolated guinea-pig ileum at 37°C, collected in the package DIXDATA, have been used to calculate the 'group effect' (DlogK) for large numbers (24-89) of pairs with and without a particular group. Values of group effects, analyzed from their cumulative frequency curves, in some instances fit a single population but most can be resolved into at least two components, indicating different areas of binding. Groups can have positive effects in some pairs of compounds but negative effects in others suggesting there may be a limit to the size of the group which can be accommodated. For hydroxyl groups and others which have only small apparent molal volumes it is likely to involve interactions with water. Cumulative frequency curves for the effects of temperature on affinity for these muscarinic receptors show that an appreciable fraction of the group effect on binding is associated with changes in entropy, even though the compounds are paired. From the geometry of the drugs it is possible to construct crude models showing areas (or pockets) in the receptor with which the drugs may interact but these need to be checked against models derived from aminoacid sequences. The validity of these models likewise needs to be checked by comparing values of log.K which they may provide against the experimental ones. Comparisons can be made between receptor types: the effect of replacing hydrogen by an aromatic ring in antagonists acting at nicotinic receptors in the frog rectus abdominis is smaller than for muscarinic receptors in ileum. This paper shows what you can do with a library of values of log.K (pA 2 ). It would be particularly interesting to compare group effects for muscarinic receptors with those for histamine H1 receptors in ileum.

Effects of rosiglitazone and sumatriptan in human isolated small and large coronary arteries K Bagot, B Sheldrick Asterand UK Ltd, Royston, UK Recently, the FDA expressed concerns over a potential increased risk for heart attacks in relation to rosiglitazone (www.fda.gov/bbs/topics/NEWS/2007/ NEW01636.html) (PPARc agonist used in diabetes management). Coronary vasoconstriction, a known side effect of some drugs such as triptans, has been demonstrated in human isolated tissues (MaassenVanDenBrink, A et al., 1998) . Therefore, in the current study, coronary constrictor potential of rosiglitazone was compared to sumatriptan and 5-HT in human isolated small and large coronary arteries (HCA). HCA were taken from three different non-diseased hearts obtained from ethically approved organ procurement organisations. Small HCA (SHCA, 0.3-0.5 mm internal diameter (i.d.)) and large HCA (LHCA, Rosiglitazone -8.3 ± 9.8 -)21.6 ± 15.3 -)9.8 ± 3.8 Sumatriptan 6.4 ± 0.4 42.7 ± 13.9 6.5 ± 0.1 45.8 ± 17.4 6.2 ± 0.1 31.6 ± 9.9 5-HT 7.4 (7.1-7.6) a 95.7 (79-112) a 6.9 ± 0.1 89.6 ± 13.3 6.9 (6.5-7.3) a 85.2 ± 23.5 Vehicle not tested not tested -)1.9 ± 1.6 -)18.1 ± 13.4

Data are mean ± s.e.mean (n = 3-6 rings, 3 donors), or a range (n = 2 rings, 2 donors). Emax = % KCl 100 mM Molecular pharmacology (16.00-16.45) C040 L-carnitine protects neuroblastoma (SH-SY5Y) cells from oxidative stress by mitochondria, stress response proteins and GADD genes J Ye, B Ding, C Yan, C Wang Qingdao University, Medical College, Qingdao, China Oxidative stress plays an important role in neurodegenerative disorders and H 2 O 2induced oxidative toxicity is a well-described model of oxidative stress-induced neurodegeneration. L-carnitine (LC) is an endogenous mitochondrial membrane compound and some studies have reported that LC could effectively protect various functions of mitochondria and cells against oxidative injury both in vitro and in vivo. However, the exact molecular mechanism of LC on oxidative stress in neurodegeneration is unclear. In the present study we used the human neuroblastoma SH-SY5Y cell line as an in vitro model and assessed the effect of L-carnitine on hydrogen peroxide (H 2 O 2 )-mediated oxidative stress and neurotoxicity. Cells in culture were treated for 24 h with 100, 200, 300, 400, 500 lM H 2 O 2 alone or pretreated with 0.1, 1, 10, 100, 300 and 1000 lM L-carnitine. H 2 O 2 produced a dose-related decrease in cell viability as measured by Trypan blue exclusion assays, a reduction in the mitochondrial metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) and an increase in DNA fragmentation analysis. Pretreatment with LC 2 h inhibited H 2 O 2 -induced cell death in a concentration-dependent manner. And analysis of DNA fragmentation showed that LC could prevent H 2 O 2 -induced DNA damage and apoptosis. Western blot analysis showed that LC could inhibit the release of of cytochrome c from mitochondria and upregulated the expression of heat shock proteins (HSP27, HSP 70 and HSP 90) levels compared with untreated control cells. Meanwhile, the expression of two endogenous anti-oxidant defense components, growth arrest and DNA damage-inducible (GADD) mRNA, GADD45 and GADD153, were observed at the early phase during cell death in 400 lM H 2 O 2 -treated control groups and LC could elevate the expression of GADD genes to protected DNA from oxidative damage. Taken together, these results demonstrate that LC exerts protective effects against oxidative stress in part by protect mitochondria and up-regulating the levels of endogenous anti-oxidant defense components and stress proteins, GADD genes and HSPs. This evidences support the pharmacological potential of LC in the management of oxidative stress, neurotoxicity and neurodegenerative diseases. Previous work has demonstrated that the treatment of diabetes with PPARc agonists is associated with a reduced risk of some cardiovascular complications. We have, therefore, examined the effects of PPARc agonists on platelet function. Washed platelets were stimulated with PPARc ligands and collagen-induced aggregation was measured using optical aggregometry. Calcium levels were measured by spectrofluorimetry in Fura-2AM loaded platelets. Tyrosine phosphorylation levels of early signalling components of the GPVI signalling pathway were measured using imunoblot analysis. The role of PPARc agonists in thrombus formation was assessed using an in vitro flow system, where fluorescently labelled whole blood was perfused through a collagen coated capillary at a shear rate of 1000/s in the presence or absence of PPARc agonists. Thrombus volume was quantified by confocal microscope using Leica Sp2 software. In this study, we report that PPARc ligand rosiglitazone inhibits collagen-stimulated platelet aggregation. This was accompanied by a reduction in collagen-stimulated intracellular calcium mobilization and reduction of thrombus formation on immobilised collagen under arterial flow conditions in a concentration-dependent manner. This was accompanied by inhibition of collagen-stimulated tyrosine phosphorylation of phospholipase Cc2. We propose that the PPARc ligands may have beneficial clinical actions through inhibition of platelet activation. Furthermore our results demonstrate a novel non-genomic mode for nuclear receptor action, and functional cross-talk between the collagen receptor GPVI and nuclear receptor signalling families in platelets.

Identification of the anti-apoptosis activity of nerve growth factor on cardiac myocytes A Caporali, G Sala-Newby, C Emanueli Bristol Heart Institute, University of Bristol, Bristol, UK Neurotrophins (NTs) control the survival and regeneration of neurons. Recent research showed that NTs posses cardiovascular actions. In this study, we investigated the hypothesis that the NT nerve growth factor (NGF) prevents cardiomyocyte apoptosis. We demonstrated that cultured rat neonatal cardiomyocytes (RNCMs) produce NGF and express its trkA receptor. RNCMs given a neutralizing antibody for NGF or the trkA inhibitor K252a underwent apoptosis, thus suggesting that NGF is an endogenous pro-survival factor for cardiomyocytes. Recombinant NGF induced trkA phosphorylation, followed by Ser473-phosphorylation and nuclear translocation of Akt in RNCMs. In response to Akt activation, Forkhead transcription factors Foxo-3a and Foxo-1 were phosphorylated and excluded from the nucleus. Adenovirus (Ad)-mediated NGF over-expression RNCMs protected RNCMs apoptosis induced by either hypoxia/reoxygenation or angiotensin II. Inhibitory approaches using K252a, LY294002 (a pan-phosphatidyl inositol 3-kinase -PI3K-inhibitor), and adenoviruses carrying a dominant negative mutant form of Akt (Ad.DN.Akt) or an Akt-resistant Foxo-3a (Ad.AAA-Foxo-3a) demonstrated that the pathway encompassing trkA, PI3K-Akt, and Foxo is essential for the pro-survival effect of NGF. The anti-apoptosis action of NGF was confirmed in adult myocytes extracted from the mouse heart, which were submitted to the angiotensin II apoptosis test in the presence of recombinant NGF. Finally, intramyocardial NGF gene transfer prevented cardiomyocyte apoptosis in a murine model of myocardial infarction.

Cell signalling (14.30-15.15) C043 Communicative methods of teaching: A structuralistic model of the construction of knowledge in problem based learning P Papaioannidou Department of Pharmacology, Medical School, Aristotle University, Thessaloniki, Greece Although methodology of teaching has been developed extensively the last years, teachers of science and biological sciences usually use a methodology of teaching, which is about 70-years-old. During the last few years, an effort has been made in medical, to use contemporary communicative methods of teaching, like problem based teaching. Nevertheless, it is obvious that lack of theoretical knowledge on the procedure of learning and on methodology of teaching restricts the application of communicative methods of teaching. The aim of this work is to present the theories of constructivism in learning, and to develop a new structuralistic model of the construction of knowledge in integrated methods of teaching, which may serve as the theoretical basis for problem based learning (PBL) and problem based teaching (PBT). The psychological constructivism of Piaget, the social constructivism of Durkheim and the radical constructivism of von Glaserfeld have given the basis for the development of communicative methods of teaching. Neo-Piazetian theories have developed models of learning process and have suggested new strategies for solving problems in teaching and learning. Understanding memory and its organization can make teaching and learning easier. Factors influencing memory and learning are: motivated attention, grade of familiarity with new information, the way of classifying and relating new information with old data, chunking of perceived information, repetition, and knowledge of rules of memory processing and data storing. Problem based teaching is a communicative, student oriented method of teaching, which offers strong motivation for learning and memorizing. In this work, as well as presenting the theoretical background, models of the construction of knowledge are presented briefly, as well as the new structuralistic model of the construction of knowledge in PBL, which has been developed by the author. All the data are presented in a simple, interactive and friendly to the audience manner, with a lot of images and in a way that provides strong motivation for participation of the audience. Structuralistic theories of cognitive development have given new tools in methodology of teaching, and lead to the development of communicative and very effective methods of teaching and learning, like PBL and PBT. 

Lipopolysaccharide augments the contractions of rat prostatic vas deferens via mechanisms that do not involve the endothelin receptors S Ertac-Serdar, AB Iskit, MO Guc Department of Pharmacology, Faculty of Medicine, Hacettepe University, Ankara, Turkey Lipopolysaccharide (LPS)-induced hyporesponsiveness of isolated smooth muscles to vasoconstristors is a hall-mark of experimental sepsis. Contrarily, alpha adrenoceptor-induced contractions of mesenteric arteries from LPS-treated mice were reported to be enhanced via the activation of Rho-kinase pathway which involves also the endothelin peptides (Buyukafsar et al., 2004) .Thus, we investigated the effect of LPS on the contractile responsiveness of rat prostatic vas deferens with special emphasis on the role of endothelin peptides. Wistar albino rats (250-350 g) pretreated with LPS (4 mg/kg, ip) was then given bosentan (30 mg/kg, ip twice at 2nd and 12th hour after LPS). Parallel controls received saline (0.9% NaCl, ip). At 24th hour, prostatic sections of vas deferens were isolated into organ baths containing Krebs-Henseleit solution at 37°C and contracted by electrical field stimulation (EFS, 0.1-100 Hz, supramaximal voltage, 2 min duration for 10 s) or by cummulatively added phenylephrine (0.1 lM-0.1 mM). All data were expressed as means ± SEM of number (n) of observations. Ordinary one-way ANOVA or twoway ANOVA for repeated measures were used where appropriate and statistical significance was accepted when P < 0.05. LPS significantly augmented the contractile responses to EFS (e.g. mg contraction to 3 Hz, control: 3.6 ± 0.4; LPS: 5.1 ± 0.4, P = 0.0076, n = 15) and to phenylephrine (i.e. two-way ANOVA for repeated measures applied to curves obtained from control versus LPS-treated animals revealed P = 0.0486). However, bosentan had no significant effect on neither EFS nor phenylephrine-induced contractions. Therefore, we conclude that LPS augments the EFS-or alpha adrenoceptor-mediated contractions of rat prostatic vas deferens via mechanisms that do not involve the endothelin receptors.

Buyukafsar et al., Eur J Pharmacol. 2004; 498: 211-217 .

A potential inhibitory role of hydrogen sulphide on carrageenan-induced acute arthritis in rats E Ekundi-Valentim, A Barreto, S Teixeira, M Muscará, S Costa University São Paulo, Department of Pharmacology, São Paulo, Brazil Hydrogen sulphide (H 2 S), a well known environment pollutant, has been proven to be produced endogenously in mammalian tissues and now seems to play an emerging role in physiological and pathophysiological conditions (Szabó, 2007) . This study was undertaken to evaluate the relevance of H 2 S in carrageenan (CGN)-induced experimental acute arthritis. Male Wistar rats (180-220 g) were subjected, under halothane anaesthesia, to intra articular injection (i.art. injection) of 3% CGN or saline (50 ll; control group). Sixty min before CGN injection, either an inhibitor of H 2 S formation, DL-propargylglycine (PAG; 53 mmol/knee joint), or an H 2 S donor, Lawson's reagent (3.6 lmol/knee joint), was injected i.art. in the ipsilateral (IPSI) knee joint. Following 4 h CGN injection, functional assays revealed that the IPSI knee of vehicle-treated rats exhibited a potent oedema associated with pain scored behaviour and high contents of myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) activity in the synovial fluid. Treatment with Lawson's reagent significantly attenuated the CGN-induced oedema (3.32 ± 0.1 and 2.47 ± 0.2 mm** for control and treated, respectively; n = 7-8), pain score (1.7 ± 0.2 and 0.87 ± 0.2* for control and treated) and MPO activity (281 ± 41.6 and 74 ± 9.6** U/cavity for control and treated), but failed to reduce increased iNOS activity (0.44 ± 0.2 and 2.4 ± 0.3 pmol/min/mg protein for control and treated). In contrast, the treatment of rats with PAG had no effect on CGN-induced arthritic signs and further potentiated synovial iNOS activity (4.45 ± 0.24*** pmol/min/mg protein; n = 4). We show for the first time that exogenous supply of H 2 S in the knee joint allowed a significant reduction of CGN-induced acute arthritis signs and symptoms, although did not prevent the up-regulation of iNOS, which may contribute to the pathogenesis of arthritis. FAPESP and CNPq for financial support. Ekundi is a recipient of a grant from University Agostinho Neto, Angola. Reference: Szabó C. Nat Rev Drug Discov. 2007; 6(11): 917-35.

Up-regulation of histamine H1R receptor and TNF-a gene expression by exogenous administration of histamine in the rat paw D Molyva, K Kalokasidis, H Dedi, V Kokkas, V Mirtsou, A Goulas Department of Pharmacology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece In addition to provoking acute symptoms of inflammation, histamine may be involved in late phase allergic responses, as implied by reports of H1R up-regulation in the nasal mucosa of patients with allergic rhinitis, as well as in animal models thereof (Dinh et al., 2005; Murata et al., 2004) . In following recent reports indicating that histamine can induce H1R expression in various types of cells in culture (Das et al., 2007) , we recorded the time course of H1R gene expression, as well as that of TNF-a, following intradermal administration of histamine in the rat paw. Male Wistar rats (250-300 g) were used in this study. Acute reaction to local subcutaneous histamine administration (100 ll, 5 mM) was followed by measuring paw oedema formation, with the use of a plethysmometer. H1R and TNF-a gene expression at various time-points following histamine administration was determined semi-quantitatively by conventional, end-point, RT-PCR on RNA isolated from subcutaneous tissue excised from rat paws. Histamine induced a significant increase in steady state mRNA levels of both H1R and TNF-a in the rat paw. The time course of this up-regulation was specific for the two transcripts. H1R expression was relatively low during the first 3 h, peaked at hours four to five, and then fell again at hour six, post-histamine. TNF-a gene expression responded in biphasic manner, with a steady increase during the first 3 h, followed by a reduction at hour four and a new increase, but at a lower rate, over hours five and six following the injection of histamine into the paw. This complex pattern of H1R and TNF-a gene expression is suggestive of histamine's ability to elicit late phase allergic responses. FAPESP and CNPq for financial support. Ekundi is a recipient of a grant from University Agostinho Neto, Angola. Pain and inflammation (14.30-15.15) 

After 15 weeks, we determined body weight, glycemia, blood pressure, insulin, glucose and insulin resistance, lipid profile, uric acid, malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and glutathione peroxidase (GPx) in blood and in liver homogenate. A histopathological exam of liver, kidney and aorta was performed. For statistical analysis we used analysis of variance (ANOVA one way) followed by Tukey's multiple comparison tests. Compared to control group, group III exhibited statistical significant increased levels of uric acid (2.35 ± 0.84 vs. 3.71 ± 0.85 mg/dL, P = 0.017), total cholesterol, low density lipoprotein cholesterol, triglycerides and oxidative stress parameters. The group IV presented statistical significant reduced levels of triglycerides (161.33 ± 46.51 vs. 215.83 ± 55.84 mg/dL

Px4 EBs than H7 control EBs (n = 3) at the mid-to late-stages of differentiation. Following FACS, the Zn 2+ positive cells were found to be positive for INS expression by RT-PCR and Q-PCR, they also contained c-peptide protein (77 ± 7 pg/10 4 cells; n = 3) and secreted c-peptide in response to stimulation with the insulin-secretagogue, tolbutamide (100 lM

These studies describe for the first time the enhancement of beta-cell differentiation in hESC by constitutive expression of PAX4, and also a novel method to separate differentiating insulin-secreting cells from undifferentiated precursors