The delayed rectifier K+ current (IK) in rabbit heart has long been thought to consist of only a single, rapidly activating, dofetilide-sensitive current, IKr. However, we find that IK of rabbit ventricular myocytes actually consists of both rapid and slow components, IKr and IKs, respectively, that can be isolated pharmacologically. Thus, after complete blockade of IKr with dofetilide, the remaining current, IKs, is homogeneous as judged by an envelope of tails test. IKs activates and deactivates slowly, continues to activate during sustained depolarizations, has a half-activation potential of 7.0 +/- 0.8 mV and slope factor of 11.0 +/- 0.7 mV, reverses at -77.2 +/- 1.3 mV (extracellular K+ concentration = 4 mM), is increased by removing extracellular K+, and is enhanced by isoproterenol and stocked by azimilide. Northern analysis demonstrates that the minK (IsK) gene, which encodes a subunit of the channel that underlies the IKs current, is expressed in rabbit heart. Expression of the rabbit protein in Xenopus oocytes elicits a slowly activating, voltage-dependent current, IsK, similar to those expressed previously from mouse, rat, guinea pig, and human genes. The results demonstrate that IKs is present in rabbit ventricle and therefore contributes to cardiac repolarization in this species.
The ␣7 nicotinic acetylcholine receptor (nAChR) is a promising target for treatment of cognitive dysfunction associated with Alzheimer's disease and schizophrenia. Here, we report the pharmacological properties of 5-morpholin-4-yl-pentanoic acid (4-pyridin-3-yl-phenyl)-amide [SEN12333 (WAY-317538)], a novel selective agonist of ␣7 nAChR. SEN12333 shows high affinity for the rat ␣7 receptor expressed in GH4C1 cells (K i ϭ 260 nM) and acts as full agonist in functional Ca 2ϩ flux studies (EC 50 ϭ 1.6 M). In whole-cell patch-clamp recordings, SEN12333 activated peak currents and maximal total charges similar to acetylcholine (EC 50 ϭ 12 M). The compound did not show agonist activity at other nicotinic receptors tested and acted as a weak antagonist at ␣3-containing receptors. SEN12333 treatment (3 mg/kg i.p.) improved episodic memory in a novel object recognition task in rats in conditions of spontaneous forgetting as well as cognitive disruptions induced via glutamatergic [5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate); MK-801] or cholinergic (scopolamine) mechanisms. This improvement was blocked by the ␣7-selective antagonist methyllycaconitine, indicating that it is mediated by ␣7 activation. SEN12333 also prevented a scopolamine-induced deficit in a passive avoidance task. In models targeting other cognitive domains, including attention and perceptual processing, SEN12333 normalized the apomorphine-induced deficit of prepulse inhibition. Neuroprotection of SEN12333 was demonstrated in quisqualate-lesioned animals in which treatment with SEN12333 (3 mg/kg/day i.p.) resulted in a significant protection of choline acetyltransferase-positive neurons in the lesioned hemisphere. Cumulatively, our results demonstrate that the novel ␣7 nAChR agonist SEN12333 has procognitive and neuroprotective properties, further demonstrating utility of ␣7 agonists for treatment of neurodegenerative and cognitive disorders.The family of nicotinic acetylcholine receptors, which comprises 16 different subunits in human (␣1-7, ␣9 -10, 1-4, ␦, ε, and ␥) that can form many functional homo-and heteropentameric receptor ion channel combinations, contributes to cholinergic neurotransmission in the nervous system and at the neuromuscular junction. The ␣7 nicotinic acetylcholine receptors (nAChRs) are rapidly desensitizing ligand-gated ion channels that are abundantly expressed in the cerebral cortex and the hippocampus, a limbic structure intimately linked to attention processing and memory formation (Gotti et al., 2006). In the hippocampus, ␣7 nAChRs are present in interneurons and glutamatergic pyramidal neurons, in which they are localized presynaptically in nerve terminals and postsynaptically in dendritic spines and soma. In line with their localization, ␣7 nAChRs modulate neurotransmitter release and are responsible for direct fast excitatory neuroArticle, publication date, and citation information can be found at
The ␣7 nicotinic acetylcholine receptor (nAChR) has been implicated in Alzheimer's disease and schizophrenia, leading to efforts targeted toward discovering agonists and positive allosteric modulators (PAMs) of this receptor. In a Ca 2ϩ flux fluorometric imaging plate reader assay, SB-206553 (3,5-dihydro -5 -methyl -N -3 -pyridinylbenzo [1, 2 -b : 4, 5 -bЈ] -di pyrrole-1(2H)-carboxamide), a compound known as a 5-hydroxytryptamine 2B/2C receptor antagonist, produced an 8-fold potentiation of the evoked calcium signal in the presence of an EC 20 concentration of nicotine and a corresponding EC 50 of 1.5 M for potentiation of EC 20 nicotine responses in GH4C1 cells expressing the ␣7 receptor. SB-206553 was devoid of direct ␣7 receptor agonist activity and selective against other nicotinic receptors. Confirmation of the PAM activity of SB-206553 on the ␣7 nAChR was obtained in patch-clamp electrophysiological experiments in GH4C1 cells, where it failed to evoke any detectable currents when applied alone, yet dramatically potentiated the currents evoked by an EC 20 (17 M) and EC 100 (124 M) of acetylcholine (ACh). Native nicotinic receptors in CA1 stratum radiatum interneurons of rat hippocampal slices could also be activated by ACh (200 M), an effect that was entirely blocked by the ␣7-selective antagonist methyllycaconitine (MLA). These ACh currents were potentiated by SB-206553, which increased the area of the current response significantly, resulting in a 40-fold enhancement at 100 M. In behavioral experiments in rats, SB-206553 reversed an MK-801 (dizocilpine maleate)-induced deficit in the prepulse inhibition of acoustic startle response, an effect attenuated in the presence of MLA. This latter observation provides further evidence in support of the potential therapeutic utility of ␣7 nAChR PAMs in schizophrenia.Nicotinic acetylcholine receptors (nAChRs) are formed of pentameric combinations of ␣ and non-␣ subunits with a high degree of complexity conferred by 10 different ␣ subunits (␣1-␣10) and seven different non-␣ subunits (1-4, ␥␦, ε)Article, publication date, and citation information can be found at
In this study, we describe the pharmacological characterization of novel aryl-ether, biaryl, and fluorene aspartic acid and diaminopropionic acid analogs as potent inhibitors of EAAT2, the predominant glutamate transporter in forebrain regions. The rank order of potency determined for the inhibition of human EAAT2 was(WAY-211686) (IC 50 ϭ 190 Ϯ 10 nM). WAY-213613 was the most selective of the compounds examined, with IC 50 values for inhibition of EAAT1 and EAAT3 of 5 and 3.8 M, respectively, corresponding to a 59-and 45-fold selectivity toward EAAT2. An identical rank order of potency [WAY-213613 (35 Ϯ 7 nM) Ͼ WAY-213394 (92 Ϯ 13 nM) ϭ WAY-212922 (95 Ϯ 8 nM) ϭ WAY-211686 (101 Ϯ 20 nM)] was observed for the inhibition of glutamate uptake in rat cortical synaptosomes, consistent with the predominant contribution of EAAT2 to this activity. Kinetic studies with each of the compounds in synaptosomes revealed a competitive mechanism of inhibition. All compounds were determined to be nonsubstrates by evaluating both the stimulation of currents in EAAT2-injected oocytes and the heteroexchange of D-[ 3 H]aspartate from cortical synaptosomes. WAY-213613 represents the most potent and selective inhibitor of EAAT2 identified to date. Taken in combination with its selectivity over ionotropic and metabotropic glutamate receptors, this compound represents a potential tool for the further elucidation of EAAT2 function.Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system. Glutamate transmission is mediated via interaction with the ligand-gated ion channel receptors, termed the ionotropic receptors, and the seven-transmembrane domain G-protein-coupled receptors, termed metabotropic glutamate receptors (Barnard, 1997;Schoepp et al., 1999). Activation of these receptors is responsible for the physiological actions of glutamate, whereas paradoxically, overstimulation of the ionotropic receptors contributes to the excitotoxic actions attributed to glutamate. Therefore, synaptic glutamate levels must be tightly regulated to maintain the integrity of synaptic transmission and to limit or prevent the pathophysiological activity of this excitatory neurotransmitter.A family of high-affinity Na ϩ -dependent glutamate transporters expressed in the plasma membranes of both neurons and astroglia is responsible for the clearance of extracellular glutamate by mediating the cellular uptake of glutamate in a Article, publication date, and citation information can be found at
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