P10-3 The sensitisation of TRPV4 in mechanical hyperalgesia is PKC pathways dependent

S160

Posters

P10-2 Edaravone attenuates the cerebral ischemic injury by inhibiting aquaporine-4

Conclusions: PKC and PKA enhance TRPV4 phosphorylation. AKAP79 may converge the effects of protein kinases on TRPV4.

K. Kikuchi1 , N. Miyagi1 , K. Kawahara2 , Y. Morimoto2 , N. Miura3 , T. Hashiguchi2 , I. Maruyama2 , H. Uchikado4 , T. Tokutomi4 , M. Shigemori4 1 Yame Public General Hospital, Japan, 2 Kagoshima University Graduate School of Medical and Dental Sciences, Japan, 3 Kagoshima University, Japan, 4 Kurume University, Japan

P10-5 Nerve excitability parameters are biomarkers that can differentiate between voltage-gated sodium (VGSC) blockers and may aid in drug development

Aquaporin-4 (AQP4) plays a role in post-ischemic edema formation. Pharmacological modulation of AQP4 function may thus provide a novel therapeutic strategy for the treatment of stroke, tumor-associated edema, and traumatic brain injury of the central nervous system (CNS) associated with altered brain water balance. Edaravone, a free-radical scavenger, significantly improved functional outcome compared with the placebo group in patients with acute ischemic stroke in Japan. In this study, edaravone (6 mg/kg i.v.) when administrated onset of ischemia/reperfusion, significantly reduced the infarct area and improved the neurological deficit scores at 24 hours after reperfusion in a rat transient focal ischemia model. Furthermore, edaravone markedly suppressed the AQP4-positive cells in cerebral infarction area. In light of observations that edaravone recommended for treatment of cerebral infarction specifically inhibited AQP4 in a rat transient focal ischemia model, we propose that edaravone might reduce cerebral edema through the inhibition of AQP4 in cerebral infarction. P10-3 The sensitisation of TRPV4 in mechanical hyperalgesia is PKC pathways dependent H.-C. Fan1,2 , X.-M. Zhang2 , J.-S. Chen1 , C.-C. Wang1 , P.A. McNaughton2 1 Department of Pediatrics, Tri-Service General Hospital and National Defense Medical Center, Taiwan, 2 Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom TRPV4, a member of the transient receptor family of ion channels, is thought to be a transducer of noxious hypo-osmotic and mechanical stimuli. It is interesting that combined action of multiple inflammatory mediators may activate TRPV4. In the present study, the role of TRPV4 in mechanical hyperalgesia induced by inflammatory mediators and the second-messenger pathways was investigated in detail. HEK293 cells, which were reported to lack TRPV4 this channel, were grown in DMEM containing serum, L-glutamine, penicillin and streptomycin at 37ºC. After transiently transfected with the human TRPV4 plasmid, cells were mounted in a perfusion chamber on a confocal microscope stage. Calcium imaging of there cells was performed with PKC agonists and inhibitors. We found that the activation of PKC by PMA cause more cells to be sensitized <37.94±3.15% > than the control group <5.59±1.40% >. Staurosporine <200nM>, a nonspecific PKC inhibitor, caused a decrease in the proportion of cells sensitized by PMA <7.78±2.37%>. PMA induced-sensitisation of TRPV4 was also blocked by BIM <11.90±3.01%>, a more specific inhibitor of PKC. Rottlerin was used to determine which PKC subtype was involved, and it largely abolished PMA induced sensitization of TRPV4 <1.90±1.48%>. PMA not only sensitized TRPV4, but also directly activated TRPV4 . The activation of TRPV4 by PMA was almost prevented by staurosporine <200nM><0.04±0.007>. Based on these data in HEK 293 cells, we showed that PMA has dual effects on TRPV4. It could sensitize repetitive TRPV4 to hypotonic challenge, and the sensitization of TRPV4 was possibly mediated by PKC d pathway. The direct activation of TRPV4 is also PKC dependent. PKC could possibly play a crucial role in inflammatory mediator-induced sensitization of TRPV4 for osmotic stimuli P10-4 Activation of the TRPV4 ion channel is enhanced by phosphorylation H.C. Fan1,2 , X. Zhang2 , P.A. McNaughton2 Department of Pediatrics, Tri-Service General Hospital and National Defense Medical Center, Taipei 11490, Taiwan, 2 Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, United Kingdom

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Objective: To investigate TRPV4 in mechanical hyperalgesia. Methods: Calcium imaging. Results: PKC and PKA enhance TRPV4 activation by phosphorylation which depends on assembly of them by AKAP79.

K.G. Shields1,2 , M. Koltzenburg1,2 Neural Plasticity Unit, UCL Institute of Child Health, London, United Kingdom, 2 Department of Clinical Neurophysiology, The National Hospital for Neurology and Neurosurgery, Queen Square, London, United Kingdom

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Objective: To validate nerve excitability parameters as a biomarker for blockade of voltage-gated sodium channel (VGSC) function using an invitro rat cutaneous sensory nerve preparation. Methods: The saphenous skin-nerve preparation was placed in a tissue bath perfused with synthetic interstitial fluid (SIF). Strength duration time constant (SD), rheobase, threshold electrotonus, recovery cycle and a current/voltage relationship were recorded using non-polarizable electrodes and an automated threshold tracking programme. VGSC blockers lidocaine, carbamazepine (CBZ) and lacosamide were dissolved in SIF at concentrations 1, 3, 10, 30 and 100 uM. Nerve excitability measurements were performed before and after perfusion for 20 minutes. Each dose response was calculated and compared with baseline values using students paired and unpaired t-tests (P = 0.05). Results: Lidocaine had reversible dose-dependent effects on all excitability parameters. SD, depolarising threshold electrotonus (TEd) and superexcitability were all decreased and the I/V curve was shifted towards outward rectification. TEd was the most sensitive parameter; effects being seen at 1uM (N = 8 df = 7, t = 2.4, P = 0.05), indicating use-dependent blockade of VGSCs. Significant differences in SD (N = 9, df = 8 t = 3.66, P = 0.006) and superexcitability (N = 8, df = 7, t = 3.48, P = 0.01) were seen at 30 uM. CBZ had qualitatively similar effects though with less efficacy. Lacosamide followed the same trend except for superexcitability which dose dependently increased up to 30 uM (N = 5, df = 4, t = 4.12, P = 0.01). Comparison of efficacy (TEd) revealed lidocaine > carbamazepine > lacosamide. Conclusions: This model is a sensitive biomarker for drug activity at VGSCs, capable of discriminating quantitative and qualitative differences between VGSC blockers. It may be used to screen for activity and provide comparative information on drug dosing. P10-6 Use of changes in specific nerve excitability parameters as a biomarker for the blockade of HCN/Ih by Org 34167 M. Koltzenburg1 , J. Udo de Haes2 , A. Androulidakis3 , V. Low3 , T. Mant3 , G. Ruigt2 1 UCL Institute of Neurology, United Kingdom, 2 Merck Research Laboratories, USA, 3 GDRU London, UK Objective: Blockers of voltage-gated sodium channels (VGSC) have previously been shown to modulate a number of nerve excitability parameters in human peripheral nerve. Here we investigated the effects of an inhibitor of the hyperpolarization-activated cyclic nucleotide gated (HCN) channels. Methods: Over a period of 10 days, eight male volunteers received an ascending oral dose (10 to 40 mg) of the non-selective HCN inhibitor Org 34167. Four times daily we measured strength duration time constant (SDTC), rheobase, depolarising (TEd) and hyperpolarising (TEh) threshold electrotonus, recovery cycle (RC) and a current-voltage (IV) relationship of motor axons of the median nerve at the wrist using a computerized threshold tracking programme (QTRAC). Results: Org34167 had a dose-dependent effect on TEh and IV, but baseline threshold, rheobase, STDC, TEd and RC remained unchanged. The higher intensity level of the hyperpolarizing conditioning stimulus the more pronounced the effects were. At 300 ms and 100% conditioning stimulus the nadir of TEh changed from about 350% to 650%. At the same time S3 decreased from a baseline of approximately 70% to 0%. Using the MEMFit modelling function of QTracP the effects could entirely be explained by a dose dependent blockade of HCN. A more than 50% reduction of HCN could be achieved before subjects reported treatment emergent adverse events. All nerve excitability parameters normalized after washout.