trans-metanicotine and cytisine

Dopaminergic (DAergic) neuronal activity in the ventral tegmental area (VTA) is thought to contribute generally to pleasure, reward, and drug reinforcement and has been implicated in nicotine dependence. nAChRs expressed in the VTA exhibit diverse subunit compositions, but the functional and pharmacological properties are largely unknown. Here, using patch-clamp recordings in single DAergic neurons freshly dissociated from rat VTA, we clarified three functional subtypes of nAChRs (termed ID, IID and IIID receptors) based on whole-cell current kinetics and pharmacology. Kinetic analysis demonstrated that comparing to ID, IID receptor-mediated current had faster activation and decay constant and IIID receptor-mediated current had larger current density. Pharmacologically, ID receptor-mediated current was sensitive to the alpha4beta2-nAChR agonist RJR-2403 and antagonist dihydro-beta-erythroidine (DHbetaE); IID receptor-mediated current was sensitive to the selective alpha7-nAChR agonist choline and antagonist methyllycaconitine (MLA); while IIID receptor-mediated current was sensitive to the beta4-containing nAChR agonist cytisine and antagonist mecamylamine (MEC). The agonist concentration-response relationships demonstrated that IID receptor-mediated current exhibited the highest EC(50) value compared to ID and IIID receptors, suggesting a relatively low agonist affinity of type IID receptors. These results suggest that the type ID, IID and IIID nAChR-mediated currents are predominately mediated by activation of alpha4beta2-nAChR, alpha7-nAChR and a novel nAChR subtype(s), respectively. Collectively, these findings indicate that the VTA DAergic neurons express diversity and multiplicity of functional nAChR subtypes. Interestingly, each DAergic neuron predominantly expresses only one particularly functional nAChR subtype, which may have distinct but important roles in regulation of VTA DA neuronal function, DA transmission and nicotine dependence.

Topics: Acetylcholine; Aconitine; Action Potentials; Alkaloids; Animals; Azocines; Brain; Choline; Dihydro-beta-Erythroidine; Dopamine; Dose-Response Relationship, Drug; Gene Expression; Kinetics; Mecamylamine; Neurons; Nicotine; Nicotinic Agonists; Nicotinic Antagonists; Patch-Clamp Techniques; Protein Subunits; Quinolizines; Rats; Rats, Wistar; Receptors, Nicotinic; Reverse Transcriptase Polymerase Chain Reaction; Tyrosine 3-Monooxygenase; Ventral Tegmental Area

We have investigated a series of 7-azaindoles as potential partial agonists of the alpha4beta2 nicotinic acetylcholine receptor (nAChR). Three series of 7-azaindole derivatives have been synthesized and evaluated for rat brain neuronal nicotinic receptor affinity and functional activity. Compound (+)-51 exhibited the most potent nAChR binding (Ki = 10 nM). Compound 30A demonstrated both moderate binding affinity and partial agonist potency, thus representing a promising lead for the indications of cognition and smoking cessation.

Topics: Alkaloids; Animals; Aza Compounds; Azocines; Brain; Indoles; Kinetics; Neurons; Nicotinic Agonists; Quinolizines; Radioligand Assay; Rats; Receptors, Nicotinic

To develop a homogeneous high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel alpha4beta2 nicotinic acetylcholine receptor (nAChR) modulators.. Membrane preparation of HEK293 cells expressing alpha4beta2 nAChR, [(3)H]cytisine and wheat germ agglutinin (WGA)-coupled microbeads were used to develop an HTS assay based on SPA technology. This method was validated against a conventional filter binding approach and applied to large-scale screening of a library containing 32 000 synthetic compounds. Intracellular calcium measurement was carried out to verify the bioactivities of the hits found by the SPA assay.. IC(50) values of 2 reference compounds (epibatidine and RJR 2403) determined by SPA and filter binding methods were comparable and consistent with those reported elsewhere. A total of 54 compounds, showing more than 60% competitive inhibition on [(3)H]cytisine binding to alpha4beta2 nAChR, were identified initially following an HTS campaign. Secondary screening confirmed that 17 compounds with novel chemical structures possessed relatively high binding affinity to alpha4beta2 nAChR (K(i)<2 micromol/L). Eight compounds displayed antagonistic effects with >50% inhibition on ABT-594-induced calcium mobilization while none showed any agonist activity.. This homogeneous binding assay is a highly efficient, amenable to automation and robust tool to screen potential alpha4beta2 nAChR modulators in an HTS setting. Its application may be expanded to other membrane receptors and ion channels.

Topics: Alkaloids; Azetidines; Azocines; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cell Line; Drug Evaluation, Preclinical; Embryo, Mammalian; Humans; Kidney; Nicotine; Nicotinic Agonists; Nicotinic Antagonists; Protein Binding; Pyridines; Quinolizines; Receptors, Nicotinic; Scintillation Counting

The GABA/glycine-mediated inhibitory activity in the substantia gelatinosa (SG) of the spinal cord is critical in the control of nociceptive transmission. We examined whether and how SG inhibitory activity might be regulated by neuronal nicotinic receptors (nAChRs). Patch-clamp recordings were performed in SG neurons of spinal slice preparations from adult rats. We provided electrophysiological evidence that inhibitory presynaptic terminals in the SG expressed nAChRs and their activation resulted in large increases in the frequency of spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) in over 90% SG neurons tested. The enhancement of inhibitory activity was mediated by increases in the release of GABA/glycine, and direct Ca(2+) entry through SG presynaptic nAChRs appeared to be involved. Miniature IPSC frequency could be enhanced by the nAChR agonists nicotine or cytisine. Nicotine could still elicit large increases in mIPSC frequency in the presence of the alpha4beta2 nAChR antagonist dihydro-beta-erythroidine (5 microM) and the alpha7 nAChR-selective antagonist methyllycaconitine (40 nM). However, nicotine did not produce a significant enhancement of mIPSC frequency in the presence of the broad spectrum nAChR antagonist mecamylamine (5 microM). Nicotinic agonist-evoked whole-cell currents from SG neurons and the antagonist profiles also indicated the presence of a subtype of nAChRs, which were different from the major central nervous system nAChR subtypes, i.e. alpha4beta2* or alpha7 nAChRs. Together, our results suggest that a subtype of nAChR, possibly alpha3beta4* nAChR or a new nAChR type, is highly expressed at the inhibitory presynaptic terminals in SG of adult rats and play a role in the control of inhibitory activity in SG.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Age Factors; Alkaloids; Animals; Azocines; Choline; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; Glycine; Neural Inhibition; Nicotine; Nicotinic Agonists; Nootropic Agents; Patch-Clamp Techniques; Quinolizines; Rats; Rats, Sprague-Dawley; Receptors, Nicotinic; Substantia Gelatinosa; Synaptic Transmission