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Relationship between ATP-induced membrane potential and contraction in smooth muscle of chicken anterior mesenteric artery
Abstracts
3 mg/kg (group 1, n = 4) or lidocaine 5, 10, 20 and 30 mg/kg (group 2, n = 4) iv. (*p < 0.05 vs predose). In group 1, surface QRS duration increased by 0.8, 2.5, 3.8 and 5.8 ms*, H–V was prolonged by 2.5, 3.8, 6* and 7* ms at 0.5,1, 2 and 3 mg/kg. Intraventricular conduction (V12–VHis) increased by 2, 3.5, 6.5* and 8.7* ms with 400 ms CL pacing, 1.3, 3.8, 7* and 9.7* ms with 300 ms, 0.8, 6.5, 9* and 13.3 ms* with 200 ms CL, meanwhile, intracardiac QRS duration prolonged by 7.5, 13*, 19.8* and 25.6%* under 400 ms CL, 6.7, 11.4*, 20.4* and 26.1* under 300 ms pacing, 9.5%, 12.2%*, 25.2%* and 32.4%* under 200 ms CL. In group 2, surface QRS duration increased by 1.2, 1.7, 2.2 and 2.7 ms, while H–V was prolonged by 1.3, 3.3, 3.5 and 7.3* with 5,10, 20 and 30 mg/kg. V12–VHis increased by 1, 2, 2 and 3 ms with 400 ms CL,1.5,1.8, 3.3 and 3.3 ms under 300 ms, 3.3, 2.3, 5.3* and 5.5 ms* under 200 ms; QRS duration prolonged by 4.8, 5.7, 8 and 12.2%* with 400 ms pacing, 4.8, 6.2, 10.2* and 14.3* with 300 ms pacing, 7.6, 8.9, 13.7* and 20.4%* under 200 ms pacing. HBE recordings may be more sensitive than surface QRS for evaluating sodium channel inhibition. Combined QRS with HBE recordings provide a sensitive platform to assess safety with respect to sodium channel.
doi:10.1016/j.vascn.2011.03.034
Poster No: 29 N-cadherin monoclonal antibody 12E3 blocks action potential conduction in human isolated Purkinje fibers Julie V. Selkirk a, Aileen D. McHarg a, Dusko Trajkovik a, Patrick D. Lappin a, Sherry LaPorte b, Jeanette Dilley b, Guy Page c, Shanon Stous c, Ky Truong c, Paul Miller c, Antonio Guia c, Andrea Ghetti c
e9
Poster No: 30 Relationship between ATP-induced membrane potential and contraction in smooth muscle of chicken anterior mesenteric artery Marwan Draid a, Marwan Draid a, AbuBakr El-Mahmoudy b, Takahiko Shiina c, Yasutake Shimizu c, Tadashi Takewaki c a Department of Pharmacology, Toxicology & Forensic Medicine, Faculty of Veterinary Medicine, Al-Fateh University, Tripoli, Libya b Department of Pharmacology, Faculty of Veterinary Medicine, Benha University, 13736 Moshtohor, Qalioubeya, Egypt c Department of Basic Veterinary Science, The United Graduate School, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan The relationship between ATP-induced membrane potential and contraction was investigated in the smooth muscle of chicken anterior mesenteric artery using microelectrode and tension recording techniques respectively. Application of ATP to endotheliumdenuded arterial preparations developed concentration-dependent, slow depolarization which started only at concentrations not less than 10 μM. On the other hand the tension recording experiment revealed that ATP produced concentration-dependent contractions, which were evident at concentrations lower than those evoked depolarizing response to ATP (as lower 100 nM). Both depolarizing & contracting were abolished by the P2X receptor blocker, PPADS (50 μM) but not in the presence of P2Y receptor blocker CBF3GA (100 μM); indicating that the excitatory responses of ATP are mediated via activation of P2X receptor. These findings show that ATP-mediated contraction is not dependent on membrane depolarization, at least at low concentration in the first order branches of chicken anterior mesenteric artery.
a
Pfizer, Inc., San Diego, CA, USA Pfizer Rinat, South San Francisco, CA, USA c AVIVA Biosciences Inc., San Diego, CA, USA b
N-cadherin is a member of the cadherin gene family which encodes proteins that mediate calcium ion-dependent cell–cell adhesion. Conditional knockout of N-cadherin in mice resulted in cardiovascular-related death. The N-cadherin protein consists of five extracellular cadherin repeats, a transmembrane domain and a highly conserved cytoplasmic tail. As such antibodies may be derived that will specifically block the function of N-cadherin for cell adhesion, but perhaps not impact formation of gap junctions and cardiac function. 12E3, a mouse monoclonal IgG was developed with this strategy in mind. Dosing of 12E3 to monkeys however, revealed significant cardiotoxicity. The aim of the current study was to investigate the effect of 12E3 when applied to ex vivo human isolated Purkinje fibers. 12E3, at concentrations similar to exposures seen in the monkey, caused disturbances in the propagation of action potentials in Purkinje fibers. Effects of 12E3 were incremental relative to antibody concentration, stimulation frequency, and time post antibody perfusion, as such complete ablation of action potential propagation was evident with 0.1 mg/ml, 0.5 Hz, after 15 min exposure and with 0.2 mg/ml, 1 Hz after 2 min exposure. 12E3mediated blockade of action potentials could be restored if the voltage was increased, even up to 15 min after initial perfusion. We have demonstrated using immunohistochemistry that 12E3 penetrated the Purkinje fiber tissue and thus conclude that blockade of Ncadherin caused disruption to the gap junctions sufficient to block action potential propagation.
doi:10.1016/j.vascn.2011.03.035
doi:10.1016/j.vascn.2011.03.036
Poster No: 31 Guinea pig Langendorff perfused heart model for evaluating the effect of drug candidates on cardiac function JianYong Qian, HaiMing Tang, Martin Sanders, Liang Guo Hoffmann-La Roche Inc. Nutley NJ 07110, USA The adverse effect of drug candidates on cardiac function is one of the major concerns throughout drug development. To identify such liability in early preclinical settings, a guinea pig Langendorff perfused heart model was validated by known inotropic modulators. Under retrograde perfusion at constant pressure (85 mm Hg), ouabain and digoxin at 0.1–0.3 μM exhibited quite similar effects in increasing the left ventricular developed pressure (LVDP) by ~34%, ±dP/dt max by ~ 53% and shortening QT/QTcf by ~ 21% significantly comparing with the time-matched vehicle group. Both manifested toxic effect (sustained ventricular tachycardia and/or ventricular fibrillation) at 0.3–1.0 μM. Digoxigenin caused a little less effect even at 10-fold higher concentrations. Propranolol (0.3, 1.0 and 3.0 μM) dose-dependently decreased LVDP by ~31%, ±dP/dt by ~ 38%. At 3.0 μM, it also reduced sinus heart rate (15%), and increased PR interval (13%) and QRS duration (45%). Nifedipine (0.003, 0.01 and 0.03 μM) dose-dependently reduced LVDP by ~40%, ±dP/dt by ~43% and QT/QTcf by ~ 12%. Furthermore it increased coronary perfusion volume by ~ 14%. The actions of both positive (digitalis) and negative (β-adrenergic receptor and calcium channel blockers) inotropic drugs and their proarrhythmic properties of digitalis were detected at clinical relevant concentrations in this Langendorff perfused heart
3 mg/kg (group 1, n = 4) or lidocaine 5, 10, 20 and 30 mg/kg (group 2, n = 4) iv. (*p < 0.05 vs predose). In group 1, surface QRS duration increased by 0.8, 2.5, 3.8 and 5.8 ms*, H–V was prolonged by 2.5, 3.8, 6* and 7* ms at 0.5,1, 2 and 3 mg/kg. Intraventricular conduction (V12–VHis) increased by 2, 3.5, 6.5* and 8.7* ms with 400 ms CL pacing, 1.3, 3.8, 7* and 9.7* ms with 300 ms, 0.8, 6.5, 9* and 13.3 ms* with 200 ms CL, meanwhile, intracardiac QRS duration prolonged by 7.5, 13*, 19.8* and 25.6%* under 400 ms CL, 6.7, 11.4*, 20.4* and 26.1* under 300 ms pacing, 9.5%, 12.2%*, 25.2%* and 32.4%* under 200 ms CL. In group 2, surface QRS duration increased by 1.2, 1.7, 2.2 and 2.7 ms, while H–V was prolonged by 1.3, 3.3, 3.5 and 7.3* with 5,10, 20 and 30 mg/kg. V12–VHis increased by 1, 2, 2 and 3 ms with 400 ms CL,1.5,1.8, 3.3 and 3.3 ms under 300 ms, 3.3, 2.3, 5.3* and 5.5 ms* under 200 ms; QRS duration prolonged by 4.8, 5.7, 8 and 12.2%* with 400 ms pacing, 4.8, 6.2, 10.2* and 14.3* with 300 ms pacing, 7.6, 8.9, 13.7* and 20.4%* under 200 ms pacing. HBE recordings may be more sensitive than surface QRS for evaluating sodium channel inhibition. Combined QRS with HBE recordings provide a sensitive platform to assess safety with respect to sodium channel.
doi:10.1016/j.vascn.2011.03.034
Poster No: 29 N-cadherin monoclonal antibody 12E3 blocks action potential conduction in human isolated Purkinje fibers Julie V. Selkirk a, Aileen D. McHarg a, Dusko Trajkovik a, Patrick D. Lappin a, Sherry LaPorte b, Jeanette Dilley b, Guy Page c, Shanon Stous c, Ky Truong c, Paul Miller c, Antonio Guia c, Andrea Ghetti c
e9
Poster No: 30 Relationship between ATP-induced membrane potential and contraction in smooth muscle of chicken anterior mesenteric artery Marwan Draid a, Marwan Draid a, AbuBakr El-Mahmoudy b, Takahiko Shiina c, Yasutake Shimizu c, Tadashi Takewaki c a Department of Pharmacology, Toxicology & Forensic Medicine, Faculty of Veterinary Medicine, Al-Fateh University, Tripoli, Libya b Department of Pharmacology, Faculty of Veterinary Medicine, Benha University, 13736 Moshtohor, Qalioubeya, Egypt c Department of Basic Veterinary Science, The United Graduate School, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan The relationship between ATP-induced membrane potential and contraction was investigated in the smooth muscle of chicken anterior mesenteric artery using microelectrode and tension recording techniques respectively. Application of ATP to endotheliumdenuded arterial preparations developed concentration-dependent, slow depolarization which started only at concentrations not less than 10 μM. On the other hand the tension recording experiment revealed that ATP produced concentration-dependent contractions, which were evident at concentrations lower than those evoked depolarizing response to ATP (as lower 100 nM). Both depolarizing & contracting were abolished by the P2X receptor blocker, PPADS (50 μM) but not in the presence of P2Y receptor blocker CBF3GA (100 μM); indicating that the excitatory responses of ATP are mediated via activation of P2X receptor. These findings show that ATP-mediated contraction is not dependent on membrane depolarization, at least at low concentration in the first order branches of chicken anterior mesenteric artery.
a
Pfizer, Inc., San Diego, CA, USA Pfizer Rinat, South San Francisco, CA, USA c AVIVA Biosciences Inc., San Diego, CA, USA b
N-cadherin is a member of the cadherin gene family which encodes proteins that mediate calcium ion-dependent cell–cell adhesion. Conditional knockout of N-cadherin in mice resulted in cardiovascular-related death. The N-cadherin protein consists of five extracellular cadherin repeats, a transmembrane domain and a highly conserved cytoplasmic tail. As such antibodies may be derived that will specifically block the function of N-cadherin for cell adhesion, but perhaps not impact formation of gap junctions and cardiac function. 12E3, a mouse monoclonal IgG was developed with this strategy in mind. Dosing of 12E3 to monkeys however, revealed significant cardiotoxicity. The aim of the current study was to investigate the effect of 12E3 when applied to ex vivo human isolated Purkinje fibers. 12E3, at concentrations similar to exposures seen in the monkey, caused disturbances in the propagation of action potentials in Purkinje fibers. Effects of 12E3 were incremental relative to antibody concentration, stimulation frequency, and time post antibody perfusion, as such complete ablation of action potential propagation was evident with 0.1 mg/ml, 0.5 Hz, after 15 min exposure and with 0.2 mg/ml, 1 Hz after 2 min exposure. 12E3mediated blockade of action potentials could be restored if the voltage was increased, even up to 15 min after initial perfusion. We have demonstrated using immunohistochemistry that 12E3 penetrated the Purkinje fiber tissue and thus conclude that blockade of Ncadherin caused disruption to the gap junctions sufficient to block action potential propagation.
doi:10.1016/j.vascn.2011.03.035
doi:10.1016/j.vascn.2011.03.036
Poster No: 31 Guinea pig Langendorff perfused heart model for evaluating the effect of drug candidates on cardiac function JianYong Qian, HaiMing Tang, Martin Sanders, Liang Guo Hoffmann-La Roche Inc. Nutley NJ 07110, USA The adverse effect of drug candidates on cardiac function is one of the major concerns throughout drug development. To identify such liability in early preclinical settings, a guinea pig Langendorff perfused heart model was validated by known inotropic modulators. Under retrograde perfusion at constant pressure (85 mm Hg), ouabain and digoxin at 0.1–0.3 μM exhibited quite similar effects in increasing the left ventricular developed pressure (LVDP) by ~34%, ±dP/dt max by ~ 53% and shortening QT/QTcf by ~ 21% significantly comparing with the time-matched vehicle group. Both manifested toxic effect (sustained ventricular tachycardia and/or ventricular fibrillation) at 0.3–1.0 μM. Digoxigenin caused a little less effect even at 10-fold higher concentrations. Propranolol (0.3, 1.0 and 3.0 μM) dose-dependently decreased LVDP by ~31%, ±dP/dt by ~ 38%. At 3.0 μM, it also reduced sinus heart rate (15%), and increased PR interval (13%) and QRS duration (45%). Nifedipine (0.003, 0.01 and 0.03 μM) dose-dependently reduced LVDP by ~40%, ±dP/dt by ~43% and QT/QTcf by ~ 12%. Furthermore it increased coronary perfusion volume by ~ 14%. The actions of both positive (digitalis) and negative (β-adrenergic receptor and calcium channel blockers) inotropic drugs and their proarrhythmic properties of digitalis were detected at clinical relevant concentrations in this Langendorff perfused heart