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Background and Purpose An increasing body of evidence suggests that the purinergic receptor P2X, ligand-gated ion channel, 7 (P2X7) in the CNS may play a key role in neuropsychiatry, neurodegeneration and chronic pain. In this study, we characterized JNJ-47965567, a centrally permeable, high-affinity, selective P2X7 antagonist. Experimental Approach We have used a combination of in vitro assays (calcium flux, radioligand binding, electrophysiology, IL-1β release) in both recombinant and native systems. Target engagement of JNJ-47965567 was demonstrated by ex vivo receptor binding autoradiography and in vivo blockade of Bz-ATP induced IL-1β release in the rat brain. Finally, the efficacy of JNJ-47965567 was tested in standard models of depression, mania and neuropathic pain. Key Results JNJ-47965567 is potent high affinity (pKi 7.9 ± 0.07), selective human P2X7 antagonist, with no significant observed speciation. In native systems, the potency of the compound to attenuate IL-1β release was 6.7 ± 0.07 (human blood), 7.5 ± 0.07 (human monocytes) and 7.1 ± 0.1 (rat microglia). JNJ-47965567 exhibited target engagement in rat brain, with a brain EC50 of 78 ± 19 ng·mL−1 (P2X7 receptor autoradiography) and functional block of Bz-ATP induced IL-1β release. JNJ-47965567 (30 mg·kg−1) attenuated amphetamine-induced hyperactivity and exhibited modest, yet significant efficacy in the rat model of neuropathic pain. No efficacy was observed in forced swim test. Conclusion and Implications JNJ-47965567 is centrally permeable, high affinity P2X7 antagonist that can be used to probe the role of central P2X7 in rodent models of CNS pathophysiology.

TRPA1 activation by respiratory irritants and pungent foods causes neurogenic inflammation, possibly contributing to wheezing, cough and dyspnea in humans. In this study we characterized JNJ-414776...

Heteromeric KCNQ5/Q3 channels were stably expressed in Chinese Hamster ovary cells and characterized using the whole cell voltage-clamp technique. KCNQ5/Q3 channels were activated by the novel anticonvulsant, retigabine (EC50 1.4 μM) by a mechanism that involved drug-induced, leftward shifts in the voltage-dependence of channel activation (−31.8 mV by 30 μM retigabine). KCNQ5/Q3 channels were inhibited by linopirdine (IC50 7.7 μM) and barium (IC50 0.46 mM), at concentrations similar to those required to inhibit native M-currents. These findings identify KCNQ5/Q3 channels as a molecular target for retigabine and raise the possibility that activation of KCNQ5/Q3 channels may be responsible for some of the anti-convulsant activity of this agent. Furthermore, the sensitivity of KCNQ5/Q3 channels to linopirdine supports the possibility that potassium channels comprised of KCNQ5 and KCNQ3 may make a contribution to native M-currents. British Journal of Pharmacology (2001) 132, 381–384; doi:10.1038/sj.bjp.0703861

1. Cardiac hypertrophy and prolongation of the cardiac action potential are hallmark features of heart disease. We examined the molecular mechanisms and the functional consequences of this action potential prolongation on calcium handling in right ventricular myocytes obtained from rats 8 weeks following ligation of the left anterior descending coronary artery (post-myocardial infarction (MI) myocytes). 2. Compared with myocytes from sham-operated rats (sham myocytes), post-MI myocytes showed significant reductions in transient outward K+ current (Ito) density (sham 19.7 +/- 1.1 pA pF-1 versus post-MI 11.0 +/- 1.3 pA pF-1; means +/- s.e.m.), inward rectifier K+ current density (sham -13.7 +/- 0.6 pA pF-1 versus post-MI -10.3 +/- 0.9 pA pF-1) and resting membrane potential (sham -84.4 +/- 1.3 mV versus post-MI -74.1 +/- 2.6 mV). Depressed Ito amplitude correlated with significant reductions in Kv4.2 and Kv4.3 mRNA and Kv4.2 protein levels. Kv1.4 mRNA and protein levels were increased and coincided with the appearance of a slow component of recovery from inactivation for Ito. 3. In current-clamp recordings, post-MI myocytes showed a significant increase in [Ca2+]i transient amplitude compared with sham myocytes. Using voltage-clamp depolarizations, no intrinsic differences in Ca2+ handling by the sarcoplasmic reticulum or in L-type Ca2+ channel density (ICa,L) were detected between the groups. 4. Stimulation of post-MI myocytes with an action potential derived from a sham myocyte reduced the [Ca2+] transient amplitude to the sham level and vice versa. 5. The net Ca2+ influx per beat via ICa,L was increased about 2-fold in myocytes stimulated with post-MI action potentials compared with sham action potentials. 6. Our findings demonstrate that reductions in K+ channel expression in post-MI myocytes prolong action potential duration resulting in elevated Ca2+ influx and [Ca2+]i transients.

1. Noradrenaline sensitivity and relaxation to acetylcholine were investigated in the isolated perfused mesentery and in aortic rings of control and streptozotocin (STZ)-induced (50 mg kg-1) diabetic Charles River rats. 2. In addition, noradrenaline sensitivity and acetylcholine relaxation were similarly assessed in streptozotocin-induced diabetic rats treated from the time of onset of diabetes with the aldose reductase inhibitor, ponalrestat (100 mg kg-1 day-1). 3. The untreated diabetic rats (2-10 weeks after injection of STZ) demonstrated enhanced vascular sensitivity to noradrenaline in the perfused mesenteric arterial tree, compared with age matched controls (pEC50 [-log concentration (M)]: diabetic 5.62 +/- 0.09, n = 18, versus control 5.23 +/- 0.07, n = 16, P < 0.01). 4. Acetylcholine-induced relaxation was significantly impaired in the perfused mesentery of the diabetic animals compared to controls (pED50 [-log dose (mol)]: diabetic 9.87 +/- 0.10, n = 20, versus controls, 10.29 +/- 0.09, n = 20, P < 0.05). 5. In contrast, the aortic ring preparations demonstrated no significant functional differences between the diabetic and control groups in response to either noradrenaline (pEC50: diabetic 7.66 +/- 0.08, n = 15, versus controls 7.55 +/- 0.06, n = 15, NS), or acetylcholine (pEC50: diabetics 7.30 +/- 0.06, n = 15, versus controls 7.40 +/- 0.09, n = 15, NS). 6. Treatment with the aldose reductase inhibitor, ponalrestat, did not affect the increased vascular reactivity to noradrenaline, or impaired relaxation to acetylcholine in the perfused mesentery.

TRPA1 is a nociceptor that is activated by allicin, cinnamaldehyde, allyl isothiocynate (AITC), gingerol, carvacrol, farnesyl thiosalicylic acid (FTS), disulfiram, chlordantoin, and 15d-PGJ2. Forma...

This unit provides an overview of the principal electrophysiological techniques commonly used for the study of ionic currents and the ion channels that mediate them. These techniques include electroencephalograms (EEGs), electrocardiograms (ECGs), single- and multiunit extracellular recording, multielectrode arrays, transepithelial recording, impedance measurements, and current-clamp, voltage-clamp, patch-clamp, and lipid bilayer recording. The unit also discusses recent advances in high-throughput, automated electrophysiological techniques for drug discovery and the use of stem cells as a tissue source.

1. Inhibition constant (Ki) were determined for a range of opioid standards using two binding assays; [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin ([3H]-GLYOL) binding to guinea-pig brain membranes in HEPES buffer and [3H]-naloxone binding to rat whole brain membranes in Krebs/HEPES buffer. 2. These values were compared with affinity measurements determined by antagonism of GLYOL on the rat isolated vas deferens preparation and by the receptor occlusion technique of Furchgott on the guinea-pig ileum longitudinal muscle, myenteric plexus preparation. 3. Agonists demonstrated markedly reduced binding affinity in the [3H]-naloxone binding assay where binding was conducted in the presence of sodium. 4. A strong correlation was obtained between Ki values from the [3H]-naloxone binding assay and affinity values determined in both isolated tissue preparations. Ki values obtained from [3H]-GLYOL binding did not correlate well with affinity data determined by isolated tissue techniques. 5. These findings suggest that functionally relevant receptors exhibit low agonist affinity.