Allosteric modulation of α4ß2* nicotinic acetylcholine receptors: Desformylflustrabromine potentiates antiallodynic response of nicotine in a mouse model of neuropathic pain.

BACKGROUND: Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels. The α4ß2 subtype of nAChRs plays an important role in the mediation of pain and several nicotine-evoked responses. Agonists and partial agonists of α4ß2 nAChRs show efficacy in animal pain models. In addition, the antinociceptive properties of nicotine, a non-selective nAChR agonist with a high affinity for α4ß2 nAChRs, is well-known. There is a growing body of evidence pointing to allosteric modulation of nAChRs as an alternative treatment strategy in experimental pain. Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) at α4ß2 nAChRs that enhances agonist responses without activating receptors. We hypothesized that dFBr may enhance nicotine-induced antinociception. METHODS: The present study investigated whether dFBr could attenuate mouse chronic constriction injury (CCI)-induced neuropathic pain by increasing endogenous cholinergic tone or potentiating the nicotine-evoked antiallodynic response. RESULTS: We found that subcutaneous administration of dFBr failed to reduce pain behaviour on its own. However, the combination of dFBr with nicotine significantly reversed neuropathic pain behaviour dose- and time-dependently without motor impairment. Our data revealed that this effect was mediated by the α4ß2 nAChRs by using competitive α4ß2 antagonist dihydro-ß-erythroidine. In addition, dFBr failed to potentiate the antiallodynic effect of morphine, which shows the effect of dFBr is unique to α4ß2 nAChRs. CONCLUSIONS: The present results suggest that allosteric modulation of α4ß2 nAChR may provide new strategies in chronic neuropathic pain. SIGNIFICANCE: α4ß2 nAChRs are involved in pain modulation. dFBr, a PAM at α4ß2 nAChRs, potentiates the nicotine response dose-dependently in neuropathic pain. Thus, the present results suggest that allosteric modulation of α4ß2* nAChR may provide new strategies in chronic neuropathic pain.

A vertical flow chamber for Xenopus oocyte electrophysiology and automated drug screening.

Xenopus laevis oocytes are used extensively in the study of ion channel coupled receptors. Efficient use of oocytes for ion channel characterization requires a system that is inherently stable, reproducible, minimizes drug volumes, and maximizes oocyte longevity. We have constructed a vertical flow oocyte recording chamber to address the aforesaid issues, where the oocyte is placed in a funnel-shaped chamber and perfused from the bottom of the funnel. The vertical rather than horizontal flow of perfusate results in an unusually stable environment for oocyte recording. Two-electrode voltage clamp recordings from a single oocyte are acquired easily and routinely over several hours while maintaining stable baseline currents and reproducible response profiles. Chamber characteristics were tested using a serotonin ligand-gated ion channel receptor (5-HT3R). Data obtained from this system corresponds well with published data. To further test the stability and reliability of this perfusion chamber, we constructed an automated oocyte perfusion system utilizing a commonly available HPLC autosampler. We were able to obtain dose-response curves for various 5-HT3AR ligands using the automated perfusion system with minimal user intervention. Such a system can easily satisfy need for automated oocyte electrophysiology in academic settings, especially small to medium sized laboratories.

5-HT(3)R binding of lerisetron: an interdisciplinary approach to drug-Receptor interactions.

The design, synthesis, and use of lerisetron-based molecular probes to investigate the 5-HT(3)R binding site are described. A SAR study, which involved distance and electronic parameter modifications of lerisetron's N-benzyl group, resulted in the discovery of a partial agonist.

Structural features of the ligand-binding domain of the serotonin 5HT3 receptor.

The nicotinic acetylcholine receptor (AChR) and the serotonin type 3 receptor (5HT3R) are members of the ligand-gated ion channel gene family. Both receptors are inhibited by nanomolar concentrations of d-tubocurarine (curare) in a competitive fashion. Chemical labeling studies on the AChR have identified tryptophan residues on the gamma (gammaTrp-55) and delta (deltaTrp-57) subunits that interact with curare. Comparison of the sequences of these two subunits with the 5HT3R shows that a tryptophan residue is found in the homologous position in the 5HT3R (Trp-89), suggesting that this residue may be involved in curare-5HT3R interactions. Site-directed mutagenesis at position Trp-89 markedly reduces the affinity of the 5HT3R for the antagonists curare and granisetron but has little effect on the affinity for the agonist serotonin. To further examine the role of this region of the receptor in ligand-receptor interactions, alanine-scanning mutagenesis analysis of the region centered on Trp-89 (Thr-85 to Trp-94) was carried out, and the ligand binding properties of the mutant receptors were determined. Within this region of the receptor, curare affinity is reduced by substitution only at Trp-89, whereas serotonin affinity is reduced only by substitution at Arg-91. On the other hand, granisetron affinity is reduced by substitutions at Trp-89, Arg-91, and Tyr-93. This differential effect of substitutions on ligand affinity suggests that different ligands may have different points of interaction within the ligand-binding pocket. In addition, the every-other-residue periodicity of the effects on granisetron affinity strongly suggests that this region of the ligand-binding site of the 5HT3R (and by inference, other members of the ligand-gated ion channel family) is in a beta-strand conformation.

Synthesis of oxadiazolidinedione derivatives as quisqualic acid analogues and their evaluation at a quisqualate-sensitized site in the rat hippocampus.

The ability of quisqualic acid (1) to sensitize neurons to depolarization by omega-phosphono alpha-amino acid analogues of excitatory amino acids is a highly specific phenomenon and is termed the QUIS effect. In an attempt to elucidate the structure-activity relationships for this sensitization, analogues 2-6 of quisqualic acid have been synthesized. Compounds 4, 5, and 6 showed no quisqualate sensitization with respect to L-2-amino-6-phosphonohexanoic acid (L-AP6), while compounds 2 and 3 were 1/10 and 1/1000, respectively, as active as quisqualic acid in sensitizing neurons toward L-AP6.

Immunocytochemical evidence that quisqualate is selectively internalized into a subset of hippocampal neurons.

Quisqualic acid (QUIS) has been shown to interact with several glutamate receptor subtypes and uptake sites. We have previously demonstrated that a brief exposure of hippocampal cells to QUIS sensitizes them to depolarization by the alpha-amino-omega-phosphonate analogues of glutamate, AP4, AP5, and AP6. This QUIS-induced sensitization is accompanied by the active uptake of QUIS into hippocampal slices. In order to localize the sites of QUIS uptake into rat hippocampal slices, a polyclonal antibody against QUIS was raised in rabbits. Utilizing immunocytochemical techniques, we have identified immunoreactive axons and dendrites after brief exposure times to QUIS, and perikarya after longer exposure times to QUIS. The intensity of the QUIS immunoreactivity increased as the exposure time to QUIS increased. QUIS immunoreactivity was primarily found in stratum oriens and stratum radiatum, of regions CA1, CA2, and CA3 of the hippocampus as well as in the hilus and molecular layer of the dentate gyrus. The distribution and morphology of QUIS immunoreactive cells appeared to be similar to those of GABAergic interneurons. Glial fibrillary acidic protein (GFAP) did not co-localize with the QUIS-internalizing cells suggesting that they are not glia. Ultrastructural analysis revealed QUIS immunoreactive profiles within the stratum radiatum. Immunostained profiles at both the light and EM levels appeared, in many cases, to be swollen and showed signs of degeneration. Such changes were only evident in tissue exposed to QUIS. These data demonstrate that QUIS is taken up by a select group of neurons in the rat hippocampus.

Utilization of the resolved L-isomer of 2-amino-6-phosphonohexanoic acid (L-AP6) as a selective agonist for a quisqualate-sensitized site in hippocampal CA1 pyramidal neurons.

Brief exposure of rat hippocampal slices to quisqualic acid (QUIS) sensitizes neurons to depolarization by the alpha-amino-omega-phosphonate excitatory amino acid (EAA) analogues AP4, AP5 and AP6. These phosphonates interact with a novel QUIS-sensitized site. Whereas L-AP4 and D-AP5 cross-react with other EAA receptors, DL-AP6 has been shown to be relatively selective for the QUIS-sensitized site. This specificity of DL-AP6, in conjunction with the apparent preference of this site for L-isomers, suggested that the hitherto unavailable L-isomer of AP6 would be a potent and specific agonist. We report the resolution of the D- and L-enantiomers of AP6 by fractional crystallization of the L-lysine salt of DL-AP6. We also report the pharmacological responses of kainate/AMPA, NMDA, lateral perforant path L-AP4 receptors and the CA1 QUIS-sensitized site to D- and L-AP6, and compare these responses to the D- and L-isomers of AP3, AP4, AP5 and AP7. The D-isomers of AP4, AP5 and AP6 were 5-, 3- and 14-fold less potent for the QUIS-sensitized site than their respective L-isomers. While L-AP4 and L-AP5 cross-reacted with NMDA and L-AP4 receptors, L-AP6 was found to be highly potent and specific for the QUIS-sensitized site (IC50 = 40 microM). Its IC50 values for kainate/AMPA, NMDA and L-AP4 receptors were > 10, 3 and 0.8 mM, respectively. As with AP4 and AP5, sensitization to L-AP6 was reversed by L-alpha-aminoadipate.

Quisqualic acid induced sensitization and the active uptake of L-quisqualic acid by hippocampal slices.

Hippocampal CA1 pyramidal cell neurons are sensitized to depolarization by L-2-amino-4-phosphonobutanoic acid (L-AP4) following exposure to L-quisqualic acid (QUIS). It has been proposed that induction of this 'QUIS-effect' involves uptake of L-QUIS by hippocampal cells. We have used o-phthaldialdehyde (OPA) derivatization and high performance liquid chromatographic (HPLC) separation of extracts from hippocampal slices which have been exposed to varied concentrations of L-QUIS to investigate L-QUIS uptake into hippocampal slices. We observe uptake rates such that the internal concentration of L-QUIS exceeds the bath concentration within 7 min. The fact that this uptake is concentrative indicates that it is mediated by an active transport system. In addition, uptake of L-QUIS may be linked to the induction of the QUIS-effect. At low concentrations of L-QUIS (< 4 microM), the QUIS-effect is only partially induced within the 4 min incubation time which maximally induces the effect when 16 microM L-QUIS is used. However, repeated 4 min exposure periods of slices to low L-QUIS concentrations will eventually induce the QUIS-effect even when each exposure is separated by extensive washout periods. Hence induction is dependent on both concentration and total exposure time. We also examined the effects of L-alpha-aminoadipic acid and L-serine-O-sulfate on the rate of L-QUIS uptake. Exposure of slices to these compounds prior to treatment with L-QUIS will block the physiological effects of L-QUIS. We found that these 'pre-blocking' compounds did not decrease the rate of L-QUIS uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-function relationships for analogues of L-2-amino-4-phosphonobutanoic acid on the quisqualic acid-sensitive AP4 receptor of the rat hippocampus.

Hippocampal CA1 pyramidal cell neurons are sensitized to depolarization by L-2-amino-4-phosphonobutanoic acid (L-AP4) following exposure to L-quisqualic acid (QUIS). We have examined the interaction of 43 structural analogues of L-AP4 with both the 'induction' site and the QUIS-sensitive AP4 site in rat hippocampus. The synthesis of cis- and trans-4-phosphonoxy-L-proline, 3-(RS)-amino-5-phosphonopentanoic acid and 2(RS)-amino-5-phenyl-4(RS)-phosphonopentanoic acid (gamma-benzyl AP4) are described. None of the test compounds interact with the induction site; thus L-QUIS remains the only compound known to induce this effect. However, one compound (L-2-amino-3-(5-tetrazolyl)-propanoic acid (L-aspartate tetrazole) 'pre-blocked' and reversed the effects of QUIS. In addition, the potency of 16 analogues increased more than 4-fold following exposure of slices to L-QUIS. Among these, L-AP4, L-AP5, 2-amino-4-(methylphosphino)butanoic acid (AMPB), and E-1(RS)-amino-3(RS)-phosphonocyclopentanecarboxylic acid (E-cyclopentyl AP4) displayed IC50 values of less than 0.100 mM after QUIS. The results presented here suggest that the QUIS-sensitive AP4 site requires a spatial configuration of functional groups similar to that present in E-cyclopentyl AP4. The presence of a primary amino group and a phosphorus-containing group (either monoanionic or dianionic) appear to be required, however, a carboxyl group is not essential for interaction. The pharmacology of the QUIS-sensitive AP4 site suggests that it is distinct from other known binding sites for L-AP4 in the central nervous system (CNS).

Structure-function relationships for kynurenic acid analogues at excitatory pathways in the rat hippocampal slice.

Eight kynurenic acid analogues were bath-applied to rat hippocampal slices while recording extracellular synaptic field potentials and the potencies of these analogues for inhibition of these responses were compared to that of kynurenic acid. Quinaldic acid, 4-hydroxyquinoline, 4-hydroxypicolinic acid, L-kynurenine and picolinic acid inhibited evoked field potentials, but were at least 15-fold less potent than kynurenic acid in all pathways tested. Xanthurenic acid was inactive in the pathways tested. Quinolinic acid and dipicolinic acid showed signs of agonist activity with IC50's of approx. 400 microM and 2500 microM, respectively. These studies show that the 2-carboxy group and the 4-hydroxy moiety are essential for the antagonist activity exhibited by kynurenate. They also show that the unsubstituted second aromatic ring greatly enhances the affinity of kynurenate for these receptors and that substitution in at least one position on this aromatic ring abolishes activity.