Cholecystokinin inhibits endocannabinoid-sensitive hippocampal IPSPs and stimulates others.

Cholecystokinin (CCK) is the most abundant neuropeptide in the central nervous system. In the hippocampal CA1 region, CCK is co-localized with GABA in a subset of interneurons that synapse on pyramidal cell somata and apical dendrites. CCK-containing interneurons also uniquely express a high level of the cannabinoid receptor, CB(1), and mediate the retrograde signaling process called DSI. Reported effects of CCK on inhibitory post-synaptic potentials (IPSPs) in hippocampus are inconsistent, and include both increases and decreases in activity. Hippocampal interneurons are very heterogeneous, and these results could be reconciled if CCK affected different interneurons in different ways. To test this prediction, we used sharp microelectrode recordings from pyramidal cells with ionotropic glutamate receptors blocked, and investigated the effects of CCK on pharmacologically distinct groups of IPSPs during long-term recordings. We find that CCK, acting via the CCK(2) receptor, increases some IPSPs and decreases others, and most significantly, that the affected IPSPs can be classified into two groups by their pharmacological properties. IPSPs that are increased by carbachol (CCh-sIPSPs), are depressed by CCK, omega-conotoxin GVIA, and endocannabinoids. IPSPs that are enhanced by CCK (CCK-sIPSPs) are blocked by omega-agatoxin IVA, and are unaffected by carbachol or endocannabinoids. Interestingly, a CCK(2) antagonist enhances CCh-sIPSPs, suggesting normally they may be partially suppressed by endogenous CCK. In summary, our data are compatible with the hypothesis that CCK has opposite actions on sIPSPs that originate from functionally distinct interneurons.

Conformational mobility of immobilized alpha3beta2, alpha3beta4, alpha4beta2, and alpha4beta4 nicotinic acetylcholine receptors.

Four affinity chromatography stationary phases have been developed based upon immobilized nicotinic acetylcholine receptor (nAChR) subtypes, the alpha3beta2, alpha3beta4, alpha4beta2, and alpha4beta4 nAChRs. The stationary phases were created using membranes from cell lines expressing the subtypes and an immobilized artificial membrane stationary phase. The immobilized nAChRs were characterized using frontal chromatography with the agonist epibatidine as the marker. The observed binding affinities for the agonists epibatidine, nicotine, and cytisine were consistent with reported values, indicating that the nAChRs retained their ability to bind agonists. The noncompetitive inhibitors (NCIs) of the nAChR (R)- and (S)-mecamylamine, phencylcidine, dextromethoprphan, and levomethorphan were also chromatographed on the columns using nonlinear chromatography techniques. The studies were carried out before and after exposure of the columns to epibatidine. The NCI retention times increased after exposure to epibtatidine as did the enantioselective separation of mecamylamine and methorphan. The results indicate that the immobilized nAChRs retained their ability to undergo agonist-induced conformational change from the resting to the desensitized states. The columns provide a unique ability to study the interactions of NCIs with both of these conformational states.

On-line screening of conformationally constrained nicotines and anabasines for agonist activity at the alpha3beta4- and alpha4beta2-nicotinic acetylcholine receptors using immobilized receptor-based liquid chromatographic stationary phases.

Liquid chromatography columns containing stationary phases based upon immobilized nicotinic acetylcholine receptors (nAChRs) were used to screen a series of conformationally constrained nicotine and anabasine derivatives for agonist activity. The alpha3beta4 nAChR and alpha4beta2 nAChR subtypes were used to prepare the chromatographic columns and [(3)H] epibatidine dihydrochloride ([(3)H] EB) was used as the marker ligand. Single displacement experiments were conducted with the test ligands and with nicotine and carbachol. Nicotine was used as an internal control for compounds with agonist activity and carbachol was used as an internal control for compounds with very weak agonistic activity (K(d) > 4700 nM for alpha3beta4). The displacement of [(3)H] EB by each of the test compounds and internal controls was calculated and expressed as Deltaml. Functional studies were then conducted using a stably transfected cell line that expresses the alpha3beta4 nAChR and EC(50) values were determined for the test compounds and the internal controls. A comparison of the Deltaml and EC(50) values indicated that 9/11 compounds had been correctly identified as agonists or non-agonists of the alpha3beta4 nAChR. A similar comparison could not be made for the alpha4beta2 nAChR, since the intact cell line was not available for testing. The results of the study suggest that the immobilized nAChR columns can be used for the rapid on-line screening of compounds for their relative affinities for the immobilized receptor and as an initial determination of qualitative functional activities.