Impact of modulation of the α7 nicotinic acetylcholine receptor on nicotine reward in the mouse conditioned place preference test.

RATIONALE: The α7 nicotinic acetylcholine receptor (nAChR) has been implicated as a target in modulating nicotine reward. However, the effect of pharmacological agents that have been shown to alter the channel properties of the α7 nAChR is not well understood in nicotine reward. OBJECTIVES: This study aimed to investigate the impact of α7 nAChR pharmacological modulation on nicotine conditioned place preference (CPP) in mice by using positive allosteric modulators (PAMs) and a silent agonist. METHODS: The effect of the orthosteric α7 nAChR full agonist PNU282987 (1.3 and 9 mg/kg, s.c.), Type I α7 PAM NS1738 (1 and 10 mg/kg; i.p.), the Type II α7 PAM PNU120596 (0.3, 1, and 3 mg/kg, i.p.), and the α7 silent agonist NS6740 (1 and 3 mg/kg, i.p) on nicotine CPP was measured in mice. Mice were conditioned with either saline or nicotine (0.5 mg/kg) for 3 days in the CPP paradigm. RESULTS: The α7 full orthosteric agonist PNU282987 and the Type II α7 nAChR PAM PNU120596 reduced nicotine CPP, while the silent agonist NS6740 and Type I PAM NS1738 had no effect. The effects of PNU282987 and PNU120596 did not have an effect on morphine CPP. CONCLUSIONS: Taken together, our results suggest that modulation of the α7 nAChR can play important roles in nicotine CPP in mice. In addition, the Type II α7 nAChR PAM PNU120596 attenuated nicotine reward suggesting that endogenous acetylcholine/choline tone is sufficient to reduce nicotine CPP. These findings highlight a beneficial effect of using α7 nAChR PAMs in nicotine reward.

Imaging of cerebral α4ß2* nicotinic acetylcholine receptors with (-)-[(18)F]Flubatine PET: Implementation of bolus plus constant infusion and sensitivity to acetylcholine in human brain.

The positron emission tomography (PET) radioligand (-)-[(18)F]flubatine is specific to α4ß2(⁎) nicotinic acetylcholine receptors (nAChRs) and has promise for future investigation of the acetylcholine system in neuropathologies such as Alzheimer's disease, schizophrenia, and substance use disorders. The two goals of this work were to develop a simplified method for α4ß2(⁎) nAChR quantification with bolus plus constant infusion (B/I) (-)-[(18)F]flubatine administration, and to assess the radioligand's sensitivity to acetylcholine fluctuations in humans. Healthy human subjects were imaged following either bolus injection (n=8) or B/I (n=4) administration of (-)-[(18)F]flubatine. The metabolite-corrected input function in arterial blood was measured. Free-fraction corrected distribution volumes (VT/fP) were estimated with modeling and graphical analysis techniques. Next, sensitivity to acetylcholine was assessed in two ways: 1. A bolus injection paradigm with two scans (n=6), baseline (scan 1) and physostigmine challenge (scan 2; 1.5mg over 60min beginning 5min prior to radiotracer injection); 2. A single scan B/I paradigm (n=7) lasting up to 240min with 1.5mg physostigmine administered over 60min beginning at 125min of radiotracer infusion. Changes in VT/fP were measured. Baseline VT/fP values were 33.8±3.3mL/cm(3) in thalamus, 12.9±1.6mL/cm(3) in cerebellum, and ranged from 9.8 to 12.5mL/cm(3) in other gray matter regions. The B/I paradigm with equilibrium analysis at 120min yielded comparable VT/fP values with compartment modeling analysis of bolus data in extrathalamic gray matter regions (regional means <4% different). Changes in VT/fP following physostigmine administration were small and most pronounced in cortical regions, ranging from 0.8 to 4.6% in the two-scan paradigm and 2.8 to 6.5% with the B/I paradigm. These results demonstrate the use of B/I administration for accurate quantification of (-)-[(18)F]flubatine VT/fP in 120min, and suggest possible sensitivity of (-)-[(18)F]flubatine binding to physostigmine-induced changes in acetylcholine levels.

Rare human nicotinic acetylcholine receptor α4 subunit (CHRNA4) variants affect expression and function of high-affinity nicotinic acetylcholine receptors.

Nicotine, the primary psychoactive component in tobacco smoke, produces its behavioral effects through interactions with neuronal nicotinic acetylcholine receptors (nAChRs). α4ß2 nAChRs are the most abundant in mammalian brain, and converging evidence shows that this subtype mediates the rewarding and reinforcing effects of nicotine. A number of rare variants in the CHRNA4 gene that encode the α4 nAChR subunit have been identified in human subjects and appear to be underrepresented in a cohort of smokers. We compared three of these variants (α4R336C, α4P451L, and α4R487Q) to the common variant to determine their effects on α4ß2 nAChR pharmacology. We examined [(3)H]epibatidine binding, interacting proteins, and phosphorylation of the α4 nAChR subunit with liquid chromatography and tandem mass spectrometry (LC-MS/MS) in HEK 293 cells and voltage-clamp electrophysiology in Xenopus laevis oocytes. We observed significant effects of the α4 variants on nAChR expression, subcellular distribution, and sensitivity to nicotine-induced receptor upregulation. Proteomic analysis of immunopurified α4ß2 nAChRs incorporating the rare variants identified considerable differences in the intracellular interactomes due to these single amino acid substitutions. Electrophysiological characterization in X. laevis oocytes revealed alterations in the functional parameters of activation by nAChR agonists conferred by these α4 rare variants, as well as shifts in receptor function after incubation with nicotine. Taken together, these experiments suggest that genetic variation at CHRNA4 alters the assembly and expression of human α4ß2 nAChRs, resulting in receptors that are more sensitive to nicotine exposure than those assembled with the common α4 variant. The changes in nAChR pharmacology could contribute to differences in responses to smoked nicotine in individuals harboring these rare variants.

TC-5619: an alpha7 neuronal nicotinic receptor-selective agonist that demonstrates efficacy in animal models of the positive and negative symptoms and cognitive dysfunction of schizophrenia.

A growing body of evidence suggests that the alpha7 neuronal nicotinic receptor (NNR) subtype is an important target for the development of novel therapies to treat schizophrenia, offering the possibility to address not only the positive but also the cognitive and negative symptoms associated with the disease. In order to probe the relationship of alpha7 function to relevant behavioral correlates we employed TC-5619, a novel selective agonist for the alpha7 NNR subtype. TC-5619 binds with very high affinity to the alpha7 subtype and is a potent full agonist. TC-5619 has little or no activity at other nicotinic receptors, including the alpha4beta2, ganglionic (alpha3beta4) and muscle subtypes. The transgenic th(tk-)/th(tk-) mouse model that reflects many of the developmental, anatomical, and multi-transmitter biochemical aspects of schizophrenia was used to assess the antipsychotic effects of TC-5619. In these mice TC-5619 acted both alone and synergistically with the antipsychotic clozapine to correct impaired pre-pulse inhibition (PPI) and social behavior which model positive and negative symptoms, respectively. Antipsychotic and cognitive effects of TC-5619 were also assessed in rats. Similar to the results in the transgenic mice, TC-5619 significantly reversed apomorphine-induced PPI deficits. In a novel object recognition paradigm in rats TC-5619 demonstrated long-lasting enhancement of memory over a wide dose range. These results suggest that alpha7-selective agonists such as TC-5619, either alone or in combination with antipsychotics, could offer a new approach to treating the constellation of symptoms associated with schizophrenia, including cognitive dysfunction.

Region-specific effects of N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide on nicotine-induced increase in extracellular dopamine in vivo.

BACKGROUND AND PURPOSE: Systemic administration of N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), an antagonist of nicotinic acetylcholine receptors (nAChRs) attenuated the nicotine-induced increase in dopamine levels in nucleus accumbens (NAcc). EXPERIMENTAL APPROACH: Using in vivo microdialysis, we investigated the effects of local perfusion of the novel nAChR antagonist bPiDDB into the NAcc or ventral tegmental area (VTA) on increased extracellular dopamine in NAcc, induced by systemic nicotine. We also examined the concentration-dependent effects of bPiDDB on the acetylcholine (ACh)-evoked response of specific recombinant neuronal nAChR subtypes expressed in Xenopus oocytes, using electrophysiological methods. KEY RESULTS: Nicotine (0.4 mg kg(-1), s.c.) increased extracellular dopamine in NAcc, which was attenuated by intra-VTA perfusion of mecamylamine (100 microM). Intra-VTA perfusion of bPiDDB (1 and 10 microM) reduced nicotine-induced increases in extracellular dopamine in NAcc. In contrast, intra-NAcc perfusion of bPiDDB (1 or 10 microM) failed to alter the nicotine-induced increase in dopamine in NAcc. Intra-VTA perfusion of bPiDDB alone did not alter basal dopamine levels, compared to control, nor the increased dopamine in NAcc following amphetamine (0.5 mg kg(-1), s.c.). Using Xenopus oocytes, bPiDDB (0.01-100 microM) inhibited the response to ACh on specific combinations of rat neuronal nAChR subunits, with highest potency at alpha3beta4beta3 and lowest potency at alpha6/3beta2beta3. CONCLUSIONS AND IMPLICATIONS: bPiDDB-Sensitive nAChRs involved in regulating nicotine-induced dopamine release are located in the VTA, rather than in the NAcc. As bPiDDB has properties different from the prototypical nAChR antagonist mecamylamine, further development may lead to novel nAChR antagonists for the treatment of tobacco dependence.

alpha7 Nicotinic receptor gene delivery into mouse hippocampal neurons leads to functional receptor expression, improved spatial memory-related performance, and tau hyperphosphorylation.

Brain alpha7 nicotinic receptors have become therapeutic targets for Alzheimer's disease (AD) based on their memory-enhancing and neuroprotective actions. This study investigated the feasibility of increasing neuronal alpha7 receptor functions using a gene delivery approach based on neuron-selective recombinant adeno-associated virus (rAAV)-derived vectors. In order to determine whether alpha7 receptor-mediated cytotoxicity was dependent on receptor density, rat alpha7 nicotinic receptors were expressed at high concentrations in GH4C1 cells as measured with nicotine-displaceable [3H]methyllycaconitine (MLA) binding. The potency of GTS-21 (an alpha7 receptor agonist) to induce cell loss was similar in these cells to that seen in pheochromocytoma (PC12) cells expressing nine-times-lower receptor levels, suggesting that cytotoxicity was more dependent on agonist concentration than receptor density. Hippocampal transduction with rat alpha7 nicotinic receptors increased [3H]MLA binding in this region in wild type and alpha7 receptor-knockout (KO) mice without apparent cytotoxicity. No difference was observed in Kd values for MLA binding between endogenous and transgenic receptors. Single cell recordings demonstrated that dentate granule cells that normally have no alpha7 receptor response did so following alpha7 receptor gene delivery in wild type mice. Recovery of alpha7 function was also observed in stratum oriens and stratum radiatum neurons of KO mice following gene delivery. Wild type mice exhibited improved acquisition performance in the Morris water task 1 month after bilateral hippocampal transductions with the rat alpha7 receptor gene compared with green fluorescent protein-transduced controls. However, both groups reached similar training levels and there was no difference in subsequent probe performance. Finally, this gene delivery approach was used to test whether alpha7 receptors affect tau-phosphorylation. Chronic (i.e. 2 month but not 2 week) expression of high levels of alpha7 receptors in hippocampus increased AT8 staining characteristic of hyperphosphorylated tau in that region, indicating that endogenous agonist-mediated receptor activation may be able to modulate this process.

Activity of alpha7-selective agonists at nicotinic and serotonin 5HT3 receptors expressed in Xenopus oocytes.

Nicotinic receptors containing alpha7 subunits are widely distributed in the central nervous system and are thought to be involved in a number of functions. However, it has been difficult to study alpha7-containing receptors in vivo because of a paucity of selective agonists. A new spirooxazolidinone compound, AR-R17779, was recently described as potent agonist at alpha7 receptors, but electrophysiological studies at other types of nicotinic receptors have not been carried out. We characterized the activity of AR-R17779 at alpha7, alpha4beta2, alpha3beta4, alpha3beta2, alpha3beta2alpha5 receptors expressed in Xenopus oocytes. In addition, since there is significant homology between nicotinic alpha7 and serotonin 5HT(3) receptors, the activity of AR-R17779 at expressed 5HT(3a) receptors was also examined. Finally, actions of tropisetron and ondansetron, two 5HT(3) antagonists, were explored. AR-R17779 was found to activate alpha7 receptors, but had no activity at other types of nicotinic receptors, and also had no activity at 5HT(3a) receptors. Tropisetron activated, while ondansetron acted as an antagonist, at alpha7 nicotinic receptors. The two 5HT(3) antagonists also acted as antagonists at alpha4beta2 and alpha3beta4 nicotinic receptors. Thus, AR-R17779 was confirmed to be a selective nicotinic alpha7 receptor agonist and to be without activity at 5HT(3) receptors. In contrast, the actions of tropisetron and ondansetron on nicotinic receptors were complex.

2-(2-Piperidyl)- and 2-(2-pyrrolidyl)chromans as nicotine agonists: synthesis and preliminary pharmacological characterization.

As part of an effort to develop a new class of subtype selective nicotine agonists, we have synthesized and tested a group of 12 hydroxylated 2-(2-piperidyl)- and 2-(2-pyrrolidyl)chromans. In rat brain membranes, all 12 compounds displayed poor affinity for [(125)I]-alpha-bunagarotoxin binding sites. In contrast, three compounds, 17c, 24, and 26, displayed moderate to high affinity for [(3)H]cytisine binding sites, while three (17b, 18b,c) and six (17a,d,e and 18a,d,e) compounds showed weak and poor affinity, respectively, for these same sites. In subsequent studies, compounds 17a and 17c were found to stimulate the efflux of (86)Rb(+) from rat cortical synaptosomes, an indication of agonist activity. Further, both 17c and 26 displayed high intrinsic activity in stimulating the release of [(3)H]dopamine from striatal synaptosomes; however, only 17c was effective at stimulating the release of [(3)H]acetylcholine from cortical synaptosomes, suggesting differential selectivity. In cloned human nicotinic acetylcholine receptors (nAChR) expressed in Xenopus oocytes, both 17c and 26 activated alpha7 and alpha3beta2 receptor subtypes in a dose-dependent manner, but 26 was clearly the more potent agonist. Last, neither compound displayed dose-dependent activation of alpha4beta2 nAChRs. We conclude that 2-(2-azacyclic)chromans appear to be a promising new class of nicotine agonists.

Inhibition of wild-type and mutant neuronal nicotinic acetylcholine receptors by local anesthetics.

Inhibition of neuronal nicotinic receptors can be regulated by the presence of specific amino acids in the beta subunit second transmembrane domain (TM2) domain. We show that the incorporation of a mutant beta4 subunit, which contains sequence from the muscle beta subunit at the TM2 6' and 10' positions of the neuronal beta4 subunit, greatly reduces the sensitivity of receptors to the local anesthetic [2-(triethylamino)-N-(2,6-dimethylphenyl)acetamide] (QX-314). Although differing in potency, the inhibition of both wild-type alpha3beta4 receptors and alpha3beta4(6'F10'T) receptors by QX-314 is voltage-dependent and noncompetitive. Interestingly, the potency of the local anesthetic tetracaine for the inhibition of alpha3beta4 and alpha3beta4(6'F10'T) receptors seems unchanged when measured at -50 mV. However, whereas the onset of inhibition of wild-type alpha3beta4 receptors is voltage-dependent and noncompetitive, the onset of inhibition of alpha3beta4(6'F10'T) receptors by tetracaine is unaffected by membrane voltage, and at concentrations < or = 30 microM seems to be competitive with acetylcholine. This may be due to either direct effects of tetracaine at the acetylcholine binding site or preferential block of closed rather than open channels in the mutant receptors. Further analysis of receptors containing the 6' mutation alone suggests that although the 6' mutation is adequate to alter the voltage dependence of tetracaine inhibition, both point mutations are required to produce the apparent competitive effects.

Analysis of mecamylamine stereoisomers on human nicotinic receptor subtypes.

Because mecamylamine, a nicotinic receptor antagonist, is used so often in nicotine research and because mecamylamine may have important therapeutic properties clinically, it is important to fully explore and understand its pharmacology. In the present study, the efficacy and potency of mecamylamine and its stereoisomers were evaluated as inhibitors of human alpha 3 beta 4, alpha 3 beta 2, alpha 7, and alpha 4 beta 2 nicotinic acetylcholine receptors (nAChRs), as well as mouse adult type muscle nAChRs and rat N-methyl-D-aspartate (NMDA) receptors expressed in Xenopus oocytes. The selectivity of mecamylamine for neuronal nAChR was manifested primarily in terms of slow recovery rates from mecamylamine-induced inhibition. Neuronal receptors showed a prolonged inhibition after exposure to low micromolar concentrations of mecamylamine. Muscle-type receptors showed a transient inhibition by similar concentrations of mecamylamine, and NMDA receptors were only transiently inhibited by higher micromolar concentrations. Mecamylamine inhibition of neuronal nAChR was noncompetitive and voltage dependent. Although there was little difference between S-(+)-mecamylamine and R-(-)-mecamylamine in terms of 50% inhibition concentration values for a given receptor subtype, there appeared to be significant differences in the off-rates for the mecamylamine isomers from the receptors. Specifically, S-(+)-mecamylamine appeared to dissociate more slowly from alpha 4 beta 2 and alpha 3 beta 4 receptors than did R-(-)-mecamylamine. In addition, it was found that muscle-type receptors appeared to be somewhat more sensitive to R-(-)-mecamylamine than to S-(+)-mecamylamine. Together, these findings suggest that in chronic (i.e., therapeutic) application, S-(+)-mecamylamine might be preferable to R-(-)-mecamylamine in terms of equilibrium inactivation of neuronal receptors with decreased side effects associated with muscle-type receptors.

The activation and inhibition of human nicotinic acetylcholine receptor by RJR-2403 indicate a selectivity for the alpha4beta2 receptor subtype.

Human nicotinic acetylcholine (ACh) receptor subtypes expressed in Xenopus oocytes were characterized in terms of their activation by the experimental agonist RJR-2403. Responses to RJR-2403 were compared with those evoked by ACh and nicotine. These agonists were also characterized in terms of whether application of the drugs had the effect of producing a residual inhibition that was manifest as a decrease in subsequent control responses to ACh measured 5 min after the washout of the drug. For the activation of alpha4beta2 receptors, RJR-2403 had an efficacy equivalent to that of ACh and was more potent than ACh. RJR-2403 was less efficacious than ACh for other human receptor subtypes, suggesting that it is a partial agonist for all these receptors. Nicotine activated peak currents in human alpha4beta2 and alpha3beta2 receptors that were 85 and 50% of the respective ACh maximum responses. Nicotine was an efficacious activator of human alpha7 receptors, with a potency similar to ACh, whereas RJR-2403 had very low potency and efficacy for these receptors. At concentrations of <1 mM, RJR-2403 did not produce any residual inhibition of subsequent ACh responses for any receptor subtype. In contrast, nicotine produced profound residual inhibition of human alpha4beta2, alpha3beta2, and alpha7 receptors with IC(50) values of 150, 200, and 150 microM, respectively. Co-expression of the human alpha5 subunit with alpha3 and beta2 subunits had the effect of producing protracted responses to ACh and increasing residual inhibition by ACh and nicotine but not RJR-2403. In conclusion, our results, presented in the context of the complex pharmacology of nicotine for both activating and inhibiting neuronal nicotinic receptor subtypes, suggest that RJR-2403 will be a potent and relatively selective activator of human alpha4beta2 receptors.

Subtype-selective inhibition of neuronal nicotinic acetylcholine receptors by cocaine is determined by the alpha4 and beta4 subunits.

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels of the central and peripheral nervous system that regulate synaptic activity from both pre- and postsynaptic sites. Nicotine binding to brain nAChRs is thought to underlie the induction of behavioral addiction to nicotine, probably as a result of desensitizing/inhibitory effects. Here, another commonly abused drug, cocaine, is shown to selectively inhibit particular nAChR subtypes with a potency in the low micromolar range by interacting with separate sites associated with the alpha4 and beta4 nAChR subunits. Chimeric receptor subunits and site-directed mutants were used to localize sequence determinants of cocaine affinity to: 1) a region of alpha4 located between residues 128 and 267 and 2) a site within the pore-lining M2 domain of beta4 that includes the 13' phenylalanine residue. The voltage dependence for inhibition associated with each site is consistent with these results. Analysis of the effects of incorporation of mutant and chimeric subunits also permitted identification of sequence elements important in receptor activation. For alpha3-containing receptors, a region or regions contained within the N-terminal extracellular domain of neuronal beta subunits influence the time course of responses to acetylcholine. Conversely, the 13' residue of the beta4 subunit M2 region is important in determining acetylcholine potency, indicating a role for this residue in agonist binding/gating processes. In summary, the present work describes sequence elements critical in both cocaine inhibition and acetylcholine activation of nAChRs and indicates that nAChRs may provide a site of interaction for the effects of nicotine and cocaine in the nervous system.

alpha7 receptor-selective agonists and modes of alpha7 receptor activation.

The alpha7-selective agonists 3-(2, 4-dimethoxybenzylidene)-anabaseine (GTS-21), also known as DMXB, and 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21) produce a variety of behavioral and cytoprotective effects that may be related to the activation of either large transient currents at high concentrations or small sustained currents at lower agonist concentrations. We are using acutely dissociated hypothalamic neurons, which express a central nervous system (CNS) alpha7-type receptor, to test a model for the concentration-dependent desensitization of alpha7-mediated responses. Our results confirm that 4OH-GTS-21 is a potent activator of neuronal alpha7 nicotinic-acetylcholine receptor. The rapid application of agonist leads to a brief period of maximal receptor-activation followed by desensitization. Rise rates, decay rates, and the degree to which current was desensitized were all concentration-dependent. Following the initial peak response to a 300-microM 4OH-GTS-21 application, current is reduced to baseline values within about 100 ms. Application of 30 microM 4OH-GTS-21 produced both a transient peak current and a sustained current that decayed only slowly after the removal of agonist. In the case of a 300-microM 4OH-GTS-21 application, after agonist was removed, we saw a rebound response up to the level of the 30-microM sustained current. The data, therefore, suggest that a sufficient level of agonist occupation can be retained on the receptor to promote activation for up to several hundred milliseconds.

Antagonist activities of mecamylamine and nicotine show reciprocal dependence on beta subunit sequence in the second transmembrane domain.

We show that a portion of the TM2 domain regulates the sensitivity of beta subunit-containing rat neuronal nicotinic AChR to the ganglionic blocker mecamylamine, such that the substitution of 4 amino acids of the muscle beta subunit sequence into the neuronal beta4 sequence decreases the potency of mecamylamine by a factor of 200 and eliminates any long-term effects of this drug on receptor function. The same exchange of sequence that decreases inhibition by mecamylamine produces a comparable potentiation of long-term inhibition by nicotine. Inhibition by mecamylamine is voltage-dependent, suggesting a direct interaction of mecamylamine with sequence elements within the membrane field. We have previously shown that sensitivity to TMP (tetramethylpiperidine) inhibitors is controlled by the same sequence elements that determine mecamylamine sensitivity. However, inhibition by bis-TMP compounds is independent of voltage. Our experiments did not show any influence of voltage on the inhibition of chimeric receptors by nicotine, suggesting that the inhibitory effects of nicotine are mediated by binding to a site outside the membrane's electric field. An analysis of point mutations indicates that the residues at the 6' position within the beta subunit TM2 domain may be important for determining the effects of both mecamylamine and nicotine in a reciprocal manner. Single mutations at the 10' position are not sufficient to produce effects, but 6' 10' double mutants show more effect than do the 6' single mutants.

Characterization of the neuroprotective and toxic effects of alpha7 nicotinic receptor activation in PC12 cells.

The alpha7 nicotinic receptor partial agonist DMXB protected differentiated PC12 cells from NGF+ serum deprivation over a concentration range (1-10 microM) that correlated with activation of protein kinase C. Increased toxicity was observed at a higher concentration of DMXB (30 microM) that did not elevate protein kinase C activity, but did increase tyrosine protein kinase activity. Neuroprotection was blocked with the protein kinase C-inhibitor bis-indolemaleimide, while toxicity was attenuated with the tyrosine protein kinase-antagonists herbimycin and genistein. The alpha7-selective antagonist methyllyconitine attenuated both the protective and toxic actions of DMXB, but in temporally distinct manners. Methyllyconitine (1 microM) attenuated toxicity when added 10 s before, but not 10 s after, 30 microM DMXB. In contrast, it blocked neuroprotection when added 10 min post-agonist addition. This temporal difference in receptor-activation that was necessary for protection vs. toxicity reflected the time courses for agonist-induced desensitization of the receptor expressed in Xenopus oocytes. These results indicate that alpha7 nicotinic receptors act through different intracellular transduction processes to protect or kill cells. Further, they suggest that the transduction processes may be differentially activated depending on the amplitude and duration of calcium signals.

Subtype-specific effects of lithium on glutamate receptor function.

We report that substitution of sodium with lithium (Li+) in the extracellular solution causes subtype-specific changes in the inward and outward currents of glutamate receptors (GluRs), without a shift in reversal potential. Li+ produces an increase of inward and outward currents of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors and decreases in the currents of kainate (KA) and N-methyl-D-aspartate receptors. The greatest effect of Li+ was observed with GluR3. A concentration-response curve for GluR3 reveals that the potentiation caused by Li+ is greatest at saturating agonist concentrations. GluR1, which shows no potentiation by Li+ at 100 microM KA, shows a small but significant potentiation at saturating KA and glutamate concentrations. The effects of Li+ on outward current, where Li+ is not the primary charge carrier, and the lack of reversal potential shift argue for a mechanism of potentiation not associated with Li+ permeation. This potentiation of current is specific for Li+ because rubidium, although causing an increase of inward current, shifted the reversal potential and did not increase outward current. The effects of Li+ are different for KA, a weak desensitizing agonist, and glutamate, a strong desensitizing agonist, suggesting that Li+ might interact with a mechanism of desensitization. By using cyclothiazide (CTZ) to reduce desensitization of GluR3, we find that for low concentrations of KA and glutamate potentiation of the response by a combination of CTZ and Li+ is no greater than by CTZ or Li+ alone. However, at high concentrations of agonist, the potentiation of the response by a combination of CTZ and Li+ is significantly greater than by CTZ or Li+ alone. This potentiation was additive for glutamate but not for KA. At high agonist concentration in the presence of CTZ, the intrinsically lower desensitization produced with KA-evoked responses may preclude Li+ from potentiating the current to the same degree as it can potentiate glutamate-evoked responses. The additive effects of CTZ and Li+ were unique to the flop variant of GluR3.

Lithium modulates desensitization of the glutamate receptor subtype gluR3 in Xenopus oocytes.

Analysis of splice variants and site-directed mutants of the AMPA receptor GluR3 expressed in Xenopus oocytes has shown that lithium produces a large potentiation of the GluR3 flop splice variant and suggested that lithium might inhibit rapid desensitization, which is characteristic of this receptor (Karkanias, N. and Papke, R., Subtype-specific effects of lithium on glutamate receptor function. J. Neurophysiol., 81 (1999) 1506-1512). We now show that mutation of the 769R/ G desensitization site (Lomeli, H.M.J., Melcher, T., Hoger, T., Geiger, J.R., Kuner, T., Monyer, H., Higuchi, M.B.A. and Seeburg, P.H, Control of kinetic properties of AMPA receptor channels by nuclear RNA editing. Science, 9(266) (1994) 1709-1713) greatly attenuates the lithium-induced potentiation of GluR3. Additionally, experiments with the non-desensitizing site-directed mutant GluR3(L507Y) (Stern-Bach, Y., Russo, S., Neuman, M. and Rosenmund, C., A point mutation in the glutamate binding site blocks desensitization of AMPA receptors. Neuron, 21 (1998) 907-918) further confirms that lithium enhances GluR3 responses by reducing desensitization, since lithium's effects are reversed in this mutant. Lithium's effects on GluR3 desensitization are distinct from the effects of aniracetam on desensitization. Specifically, aniracetam, which potentiates wild-type AMPA receptors, is ineffective on the non-desensitizing GluR3(L507Y) mutant, but has synergistic effects with lithium on wild-type receptors.

Analysis of 3-(4-hydroxy, 2-Methoxybenzylidene)anabaseine selectivity and activity at human and rat alpha-7 nicotinic receptors.

3-(2,4-dimethoxybenzylidene)anabaseine (GTS-21) is a selective partial agonist for rat alpha-7 nicotine receptors with reportedly much lower efficacy for human alpha-7 receptors. Because this drug improves memory-related performance in nonhuman primates, and is presently in a clinical trial for Alzheimer's disease, we investigated the potential effects of its primary human metabolite, 3-(4-hydroxy, 2-methoxy-benzylidene)anabaseine) on human as well as rat nicotinic acetylcholine receptor. 4OH-GTS-21 exhibited a similar level of efficacy for both rat and human alpha-7 receptors expressed in Xenopus oocytes. It displaced high affinity [125I]alpha-bungarotoxin binding to human SK-N-SH cell-membranes (Ki 0.17 microM) and rat PC12 cell-membranes (Ki 0.45 microM). GTS-21 also displaced [125I]alpha-bungarotoxin binding to PC12 cell membranes with high potency (Ki 0.31 microM), but was much less potent in this regard in SK-N-SH cells (23 microM). 4OH-GTS-21 produced less residual inhibition of either the human or rat AChR subtypes than GTS-21 did. To compare the neuroprotective efficacies of GTS-21 and 4OH-GTS-21 in both species, an amyloid-toxicity model (Abeta 25-35) was used. 4OH-GTS-21 was protective in both human and rat cell lines, although GTS-21 was effective only in the latter. These studies suggest that the efficacy of GTS-21 in primates may depend on a pro-drug function.

The correction of alpha7 nicotinic acetylcholine receptor concentration-response relationships in Xenopus oocytes.

The rapid desensitization of alpha7 nicotinic acetylcholine receptors (nAChR) has presented a serious problem for the characterization of this receptor subtype, potentially confounding the interpretation of concentration-response relationships. However, the consistency of cell geometry and solution flow in oocyte recordings permits estimations of instantaneous concentrations to be made in this system. Results interpreted with predicted instantaneous concentrations suggest that estimates of EC50 derived from conventional analysis may overestimate the actual EC50 values by a factor of 10 and underestimate Hill slopes by a factor of 2-3. If the limiting desensitization process of alpha7 receptors is driven by the agonist concentration itself rather than by time-dependent processes, then similar dependencies may exist between the response and instantaneous agonist concentration in other systems.

Lithium homeostasis in Xenopus oocytes: implications for the study of signal transduction.

The Xenopus oocyte has been shown to be a useful model for the study of signal transduction pathways. The present study investigated whether or not the oocyte could be used to study the effects of lithium on signal transduction mechanisms by comparing the dynamics of lithium homeostasis in the oocyte and a human immortalized hippocampal cell line using Flame Atomic Emission Spectroscopy (FAES). A biphasic pattern of lithium uptake was observed in the oocyte in the presence of 5 mM extracellular lithium. The late phase of lithium uptake, which started after 30 minutes of incubation time, was sensitive to phloretin, an inhibitor of Na+/Li+ counter-transport. Differences in lithium efflux kinetics further characterized the two observed phases of accumulation and also suggested that lithium might be distributed in different pools within the oocyte, including one sequestered in organelles or associated with cytosolic proteins. An analogous sequestered pool was not, however, observed in the hippocampal cell line indicating that lithium is distributed differently in these cell types. This suggests that the Xenopus oocyte might not be a suitable model for evaluating the effects of lithium on signal transduction pathways because of the unknown contribution of the sequestered pool on predicting relevant physiological effects.