nACh Receptor Antagonist Research Articles

N1H- and N1-Substituted Phenylguanidines as α7 Nicotinic Acetylcholine (nACh) Receptor Antagonists: Structure-Activity Relationship Studies.

We previously reported that N-(3-chlorophenyl)guanidine (1) represents a novel α7 nicotinic ACh (nACh) receptor antagonist chemotype. In the present study, a small series of compounds was synthesized with the intent to investigate the structure-activity relationship (SAR). Preliminary data suggested that the N-methyl analog of 1, 2, was several times more potent. Therefore, the chloro group at the aryl 3-position of 1 and its N1-methyl counterpart 2 were replaced with a number of substituents considering the electronic, lipophilic, and steric nature of the substituents. The potencies of the compounds to inhibit acetylcholine (ACh)-induced responses were obtained in Xenopus laevis oocytes expressing human α7 nicotinic ACh receptors (nAChRs) using a two-electrode voltage-clamp assay. We found that the nature of the 3-position substituents had relatively little (i.e., <10-fold) effect on potency, and the presence of an N1-isopropyl substituent was tolerated. Here, we report the first SAR investigation of this novel α7 nAChR antagonist chemotype.

Apigenin and Structurally Related Flavonoids Allosterically Potentiate the Function of Human α7-Nicotinic Acetylcholine Receptors Expressed in SH-EP1 Cells

Phytochemicals, such as monoterpenes, polyphenols, curcuminoids, and flavonoids, are known to have anti-inflammatory, antioxidant, neuroprotective, and procognitive effects. In this study, the effects of several polyhydroxy flavonoids, as derivatives of differently substituted 5,7-dihydroxy-4H-chromen-4-one including apigenin, genistein, luteolin, kaempferol, quercetin, gossypetin, and phloretin with different lipophilicities (cLogP), as well as topological polar surface area (TPSA), were tested for induction of Ca2+ transients by α7 human nicotinic acetylcholine (α7 nACh) receptors expressed in SH-EP1 cells. Apigenin (10 μM) caused a significant potentiation of ACh (30 μM)-induced Ca2+ transients, but did not affect Ca2+ transients induced by high K+ (60 mM) containing solutions. Co-application of apigenin with ACh was equally effective as apigenin preincubation. However, the effect of apigenin significantly diminished by increasing ACh concentrations. The flavonoids tested also potentiated α7 nACh mediated Ca2+ transients with descending potency (highest to lowest) by genistein, gossypetin, kaempferol, luteolin, phloretin, quercetin, and apigenin. The specific binding of α7 nACh receptor antagonist [125I]-bungarotoxin remained unchanged in the presence of any of the tested polyhydroxy flavonoids, suggesting that these compounds act as positive allosteric modulators of the α7-nACh receptor in SH-EP1 cells. These findings suggest a clinical potential for these phytochemicals in the treatment of various human diseases from pain to inflammation and neural disease.

Levetiracetam induction of theta frequency oscillations in rodent hippocampus in vitro.

Levetiracetam (LEV) has been demonstrated to improve cognitive function. Hippocampal theta rhythm (4-12 Hz) is associated with a variety of cognitively related behaviors, such as exploration in both humans and animal models. We investigated the effects of LEV on the theta rhythm in the rat hippocampal CA3 in hippocampal slices in vitro. We found that LEV increased the theta power in a dose-dependent manner. The increase in theta power can be blocked by GABAA receptor (GABAAR) or NMDA receptor (NMDAR) antagonists but not by AMPA receptor antagonist, indicating the involvement of GABAAR and NMDAR in the induction of theta activity. Interestingly, LEV enhancement of theta power can be also blocked by taurine or GABA-A agonist THIP, indicating that LEV induction of theta may be related to the indirect boosting of GABA action via reduction of extrasynaptic GABAAR activation. Furthermore, the increased theta power can be partially reduced by the mACh receptor (mAChR) antagonist atropine but not by nACh receptor antagonists, suggesting that mAChR activation provides excitatory input into local network responsible for LEV-induced theta. Our study demonstrated that LEV induced a novel theta oscillation in vitro, which may have implications in the treatment of the neuronal disorders with impaired theta oscillation and cognitive function.

AE Succinimide, an Analogue of Methyllycaconitine, When Bound Generates a Nonconducting Conformation of the α4β2 Nicotinic Acetylcholine Receptor

Nicotinic acetylcholine (nACh) receptors are pentameric ligand-gated ion channels that mediate fast synaptic transmission. The α4β2 nACh receptor is highly expressed in the brain and exists in two functional stoichiometries: the (α4)2(β2)3 and (α4)3(β2)2 that differ by an ACh-binding site at the α4-α4 interface of (α4)3(β2)2 receptors. Methyllycaconitine (MLA) is an nACh receptor antagonist, and while potent at both α7 and α4β2 nACh receptors, it has a higher selectivity for the α7 nACh receptor. The anthranilate-succinimide ester side-chain is important for its activity and selectivity. Here we identify a simplified MLA analogue that contains only the A and E ring skeleton of MLA, AE succinimide, that binds close to the channel lumen to display insurmountable inhibition at α4β2 nACh receptors. Although inhibition by AE succinimide was found to be voltage-dependent indicating a possible pore channel blocker, substituted-cysteine accessibility experiments indicated it did not bind between 2'-16' region of the channel pore. Instead, we found that upon binding and in the presence of ACh, there is a conformational change to the channel membrane that was identified when the compound was assessed against (α4 V13'C)β2 nACh receptors. It was found that in the 3:2 stoichiometry the two adjacent α4 subunits containing 13' cysteine mutations formed a disulfide bond and occluded ion conductance. This was reversed by treatment with the reducing agent, dithiothreitol. Thus, AE succinimide has a different mechanism of inhibition to both MLA and other AE analogues, such as AE bicyclic alcohol, in that upon binding to an as yet unidentified site, AE succinimide in the presence of ACh induces a conformational change to the channel that generates a ligand-bound closed state.

Mechanism Underlying Organophosphate Paraoxon-Induced Kinetic Tremor.

Organophosphates (OPs) inhibit cholinesterase and hyperactivate the acetylcholinergic nervous system in the brain, causing motor disorders (e.g., tremor and seizures). Here, we performed behavioral and immunohistochemical studies in mice and rats to investigate the tremorgenic mechanism of paraoxon, an active metabolite of parathion. Treating animals with paraoxon (0.15-0.6mg/kg, i.p.) elicited kinetic tremor in a dose-dependent manner. Expressional analysis of Fos protein, a biomarker of neural excitation, revealed that a tremorgenic dose of paraoxon (0.6mg/kg) significantly and region-specifically elevated Fos expression in the cerebral cortex (e.g., sensory cortex), hippocampal CA1, globus pallidus, medial habenula, and inferior olive (IO) among 48 brain regions examined. A moderate increase in Fos expression was also observed in the dorsolateral striatum while the change was not statistically significant. Paraoxon-induced tremor was inhibited by the nicotinic acetylcholine (nACh) receptor antagonist mecamylamine (MEC), but not affected by the muscarinic acetylcholine receptor antagonist trihexyphenidyl (THP). In addition, paraoxon-induced Fos expression in the IO was also antagonized by MEC, but not by THP, and lesioning of the IO markedly suppressed tremorgenic action of paraoxon. The present results suggest that OPs elicit kinetic tremor at least partly by activating IO neurons via nACh receptors.

Propofol inhibits carbachol-induced chloride secretion by directly targeting the basolateral K+ channel in rat ileum epithelium.

Propofol is a widely used intravenous general anesthetic. Acetylcholine (ACh) is critical in controlling epithelial ion transport. This study was to investigate the effects of propofol on ACh-evoked secretion in rat ileum epithelium. The Ussing chamber technique was used to investigate the effects of propofol on carbachol (CCh)-evoked short-circuit currents (Isc). Propofol (10-2 -10-6 mol/L) attenuated CCh-evoked Isc of rat ileum mucosa in a dose-dependent manner. The inhibitory effect of propofol was only evident after application to the serosal side. Pretreatment with tetrodotoxin (TTX, 0.3μmol/L, n=5) had no effect on propofol-induced inhibitory effect, whereas serosal application of K+ channel inhibitor, glibenclamide, but not, an ATP-sensitive K+ channel inhibitor, largely reduced the inhibitory effect of propofol. In addition, pretreatment with either hexamethonium bromide (HB, nicotinic nACh receptor antagonist) or Cl- channel blockers niflumic acid and cystic fibrosis transmembrane conductance regulator (inh)-172 did not produce any effect on the propofol-induced inhibitory effect. Propofol inhibits CCh-induced intestinal secretion by directly targeting basolateral K+ channels.

Nicotine evokes kinetic tremor by activating the inferior olive via α7 nicotinic acetylcholine receptors

Nicotinic acetylcholine (nACh) receptors are implicated in the pathogenesis of movement disorders (e.g., tremor) and epilepsy. Here, we performed behavioral and immunohistochemical studies using mice and rats to elucidate the mechanisms underlying nicotine-induced tremor. Treatments of animals with nicotine (0.5–2mg/kg, i.p.) elicited kinetic tremor, which was completely suppressed by the nACh receptor antagonist mecamylamine (MEC). The specific α7 nACh receptor antagonist methyllycaconitine (MLA) also inhibited nicotine-induced tremor, whereas the α4β2 nACh antagonist dihydro-β-erythroidine (DHβE) or the peripheral α3β4 nACh antagonist hexamethonium showed no effects. Mapping analysis of Fos protein expression, a biological marker of neural excitation, revealed that a tremorgenic dose (1mg/kg) of nicotine region-specifically elevated Fos expression in the piriform cortex (PirC), medial habenula, solitary nucleus and inferior olive (IO) among 44 brain regions examined. In addition, similarly to the tremor responses, nicotine-induced Fos expression in the PirC and IO was selectively antagonized by MLA, but not by DHβE. Furthermore, an electrical lesioning of the IO, but not the PirC, significantly suppressed the induction of nicotine tremor. The present results suggest that nicotine elicits kinetic tremor in rodents by activating the IO neurons via α7 nACh receptors.

Microinjection of acetylcholine into cerebellar fastigial nucleus induces blood depressor response in anesthetized rats

It is well known that the cerebellar fastigial nucleus (FN) is involved in cardiovascular modulation, and has direct evidence of cholinergic activity; however, whether and how acetylcholine (ACh) in the FN modulates blood pressure has not been investigated. In this study, we analyzed mean arterial pressure, maximal change in mean arterial pressure, and the reaction time of blood pressure changes after microinjection of cholinergic reagents into the FN in anesthetized rats. The results showed that ACh evoked a concentration-dependent (10, 30 and 100mM) effect on blood pressure down-regulation. The muscarinic ACh (mACh) receptor antagonist atropine, but not the nicotinic ACh (nACh) receptor antagonist mecamylamine, blocked the ACh-mediated depressor response. The mACh receptor agonist oxotremorine M, rather than nACh receptor agonist nicotine, mimicked the ACh-mediated blood pressure decrease in a dose-dependent manner (10, 30 and 100mM). These results indicate that cholinergic input in the cerebellar FN exerts a depressor effect on systemic blood pressure regulation, and such effects are substantially contributed by mACh rather than nACh receptors, although the precise mechanism concerning the role of mACh receptor in FN-mediated blood pressure modulation remains to be elucidated.

Methamphetamine-like discriminative stimulus effects of bupropion and its two hydroxy metabolites in male rhesus monkeys.

The dopamine transporter (DAT) inhibitor and nicotinic acetylcholine (nACh) receptor antagonist bupropion is being investigated as a candidate 'agonist' medication for methamphetamine addiction. In addition to its complex pharmacology, bupropion also has two distinct pharmacologically active metabolites. However, the mechanism by which bupropion produces methamphetamine-like 'agonist' effects remains unknown. The aim of the present study was to determine the role of DAT inhibition, nACh receptor antagonism, and the hydroxybupropion metabolites in the methamphetamine-like discriminative stimulus effects of bupropion in rhesus monkeys. In addition, varenicline, a partial agonist at the nACh receptor, and risperidone, a dopamine antagonist, were tested as controls. Monkeys (n=4) were trained to discriminate 0.18 mg/kg intramuscular methamphetamine from saline in a two-key food-reinforced discrimination procedure. The potency and time course of methamphetamine-like discriminative stimulus effects were determined for all compounds. Bupropion, methylphenidate, and 2S,3S-hydroxybupropion produced full, at least 90%, methamphetamine-like effects. 2R,3R-Hydroxybupropion, mecamylamine, and nicotine also produced full methamphetamine-like effects, but drug potency was more variable between monkeys. Varenicline produced partial methamphetamine-like effects, whereas risperidone did not. Overall, these results suggest DAT inhibition as the major mechanism of the methamphetamine-like 'agonist' effects of bupropion, although nACh receptor antagonism appeared, at least partially, to contribute. Furthermore, the contribution of the 2S,3S-hydroxybupropion metabolite could not be completely ruled out.

Stimulation of α7 nicotinic acetylcholine receptor regulates glutamate transporter GLAST via basic fibroblast growth factor production in cultured cortical microglia

The α7 nicotinic acetylcholine (nACh) receptor expressed in microglia has a crucial role in neuroprotection. Simulation of α7 nACh receptor leads to increased expression of glutamate/aspartate transporter (GLAST), which in turn decreases synaptic glutamate levels. However, the upregulation of GLAST in cultured rat cortical microglia appears long after (over 18h) stimulation of the α7 nACh receptor with nicotine. Thus, the current study elucidated the pathway responsible for the induction of GLAST expression in cultured cortical microglia. Nicotine-induced GLAST mRNA expression was significantly inhibited by cycloheximide pretreatment, indicating that a protein intermediary, such as a growth factor, is required for GLAST expression. The expression of fibroblast growth factor-2 (FGF-2) mRNA in cortical microglia was significantly increased 6 and 12h after treatment with nicotine, and this increase was potently inhibited by pretreatment with methyllycaconitine, a selective α7 nACh receptor antagonist. The treatment with nicotine also significantly increased FGF-2 protein expression. Furthermore, treatment with recombinant FGF-2 increased GLAST mRNA, protein expression and 14C-glutamate uptake, a functional measurement of GLAST activity. Conversely, pretreatment with PD173074, an inhibitor of FGF receptor (FGFR) tyrosine kinase, significantly prevented the nicotine-induced expression of GLAST mRNA, its protein and 14C-glutamate uptake. Reverse transcription polymerase chain reaction confirmed FGFR1 mRNA expression was confined to cultured cortical microglia. Together, the current findings demonstrate that the neuroprotective effect of activation of microglial α7 nACh receptors could be due to the expression of FGF-2, which in turn increases GLAST expression, thereby clearing glutamate from synapse and decreasing glutamate neurotransmission.

The α3β4* nicotinic ACh receptor subtype mediates physical dependence to morphine: mouse and human studies.

Recent data have indicated that α3β4* neuronal nicotinic (n) ACh receptors may play a role in morphine dependence. Here we investigated if nACh receptors modulate morphine physical withdrawal. To assess the role of α3β4* nACh receptors in morphine withdrawal, we used a genetic correlation approach using publically available datasets within the GeneNetwork web resource, genetic knockout and pharmacological tools. Male and female European-American (n = 2772) and African-American (n = 1309) subjects from the Study of Addiction: Genetics and Environment dataset were assessed for possible associations of polymorphisms in the 15q25 gene cluster and opioid dependence. BXD recombinant mouse lines demonstrated an increased expression of α3, β4 and α5 nACh receptor mRNA in the forebrain and midbrain, which significantly correlated with increased defecation in mice undergoing morphine withdrawal. Mice overexpressing the gene cluster CHRNA5/A3/B4 exhibited increased somatic signs of withdrawal. Furthermore, α5 and β4 nACh receptor knockout mice expressed decreased somatic withdrawal signs compared with their wild-type counterparts. Moreover, selective α3β4* nACh receptor antagonists, α-conotoxin AuIB and AT-1001, attenuated somatic signs of morphine withdrawal in a dose-related manner. In addition, two human datasets revealed a protective role for variants in the CHRNA3 gene, which codes for the α3 nACh receptor subunit, in opioid dependence and withdrawal. In contrast, we found that the α4β2* nACh receptor subtype is not involved in morphine somatic withdrawal signs. Overall, our findings suggest an important role for the α3β4* nACh receptor subtype in morphine physical dependence.

Central amygdala nicotinic and 5-HT1A receptors mediate the reversal effect of nicotine and MDMA on morphine-induced amnesia

The present study was designed to investigate possible involvement of the central amygdala (CeA) nicotinic acetylcholine (nACh) and 5-hydroxytryptamine 1A (5-HT1A) receptors in the reversal effect of nicotine and 3,4-methylenedioxy-N-methylamphetamine (MDMA or ecstasy) on morphine-induced amnesia. Two guide cannulas were stereotaxically implanted in the CeA regions and a step-through passive avoidance task was used for the assessment of memory retrieval in adult male Wistar rats. Our results indicated that post-training s.c. administration of morphine (3–7-mg/kg) impaired memory retrieval. Pre-test administration of nicotine (0.3- and 0.5-mg/kg, s.c.) reversed morphine-induced amnesia. In addition, pre-test intra-CeA injection of MDMA (1–2-μg/rat) with an ineffective dose of nicotine (0.1-mg/kg, s.c.) improved memory retrieval, suggesting the interactive effect of the drugs on memory formation. It should be noted that that pre-test intra-CeA injection of 2-μg/rat of MDMA by itself produced amnesia. Interestingly, pre-test intra-CeA injection of mecamylamine, a nACh receptor antagonist (1–2-μg/rat) or (S)-WAY 100135 (0.25–1-μg/rat), a selective 5-HT1A receptor antagonist inhibited the improvement of morphine-induced amnesia which was produced by pre-test co-injection of nicotine and MDMA. Pre-test intra-CeA injection of the same doses of MDMA, mecamylamine or (S)-WAY 100135 by itself had no effect on morphine-induced amnesia. Moreover, pre-test injection of the same doses of mecamylamine or (S)-WAY 100135 into the CeA alone could not change memory retrieval. Taken together, it can be concluded that there is a functional interaction between morphine, nicotine and MDMA via the CeA nicotinic and serotonergic receptor mechanisms in passive avoidance memory retrieval. Moreover, cross state-dependent memory retrieval may have been induced between the drugs and this probably depends on the rewarding effects of the drugs.

Effects of the nicotinic acetylcholine receptor antagonist mecamylamine on the discriminative stimulus effects of cocaine in male rhesus monkeys.

Preclinical drug discrimination procedures have been useful in understanding the pharmacological mechanisms of the subjective-like effects of abused drugs. Converging lines of evidence from neurochemical and behavioral studies implicate a potential role of nicotinic acetylcholine (nACh) receptors in the abuse-related effects of cocaine. The aim of the present study was to determine the effects of the nACh receptor antagonist mecamylamine on the discriminative stimulus effects of cocaine in nonhuman primates. The effects of mecamylamine on the cocaine-like discriminative stimulus effects of nicotine were also examined. Male rhesus monkeys (n = 5) were trained to discriminate 0.32 mg/kg, IM cocaine from saline in a 2-key, food-reinforced discrimination procedure. Initially, potency and time course of cocaine-like discriminative stimulus effects were determined for nicotine and mecamylamine alone. Test sessions were then conducted examining the effects of mecamylamine on cocaine or the cocaine-like discriminative stimulus effects of nicotine. Curiously, mecamylamine produced partial cocaine-like discriminative stimulus effects. Mecamylamine did not significantly alter the discriminative stimulus effects of cocaine up to doses that significantly decreased rates of operant responding. Mecamylamine and nicotine combinations were not different than saline. These results confirm previous nonhuman primate studies of partial substitution with nicotine and extend these findings with mecamylamine. Furthermore, these results extend previous results in rats suggesting cocaine may have nACh receptor antagonist properties.

Primary cultures of rat cortical microglia treated with nicotine increases in the expression of excitatory amino acid transporter 1 (GLAST) via the activation of the α7 nicotinic acetylcholine receptor

Although the clearance of glutamate from the synapse under physiological conditions is performed by astrocytic glutamate transporters, their expression might be diminished under pathological conditions. Microglia glutamate transporters, however, might serve as a back-up system when astrocytic glutamate uptake is impaired, and could have a prominent neuroprotective function under pathological conditions. In the current study, the effect of nicotine, well known as a neuroprotective molecule, on the function of glutamate transporters in cultured rat cortical microglia was examined. Reverse transcription polymerase chain reaction and pharmacological approaches demonstrated that, glutamate/aspartate transporter (GLAST), not glutamate transporter 1 (GLT-1), is the major functional glutamate transporter in cultured cortical microglia. Furthermore, the α7 subunit was demonstrated to be the key subunit comprising nicotinic acetylcholine (nACh) receptors in these cells. Treatment of cortical microglia with nicotine led to a significant increase of GLAST mRNA expression and 14C-glutamate uptake in a concentration- and time-dependent manner, which were markedly inhibited by pretreatment with methyllycaconitine, a selective α7 nACh receptor antagonist. The nicotine-induced expression of GLAST mRNA and protein is mediated through an inositol trisphosphate (IP3) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) depend intracellular pathway, since pretreatment with either xestospongin C, an IP3 receptor antagonist, or KN-93, a CaMKII inhibitor, blocked GLAST expression. Together, these findings indicate that activation of nACh receptors, specifically those expressing the α7 subunit, on cortical microglia could be a key mechanism of the neuroprotective effect of nACh receptor ligands such as nicotine.

Serotonergic involvement in the amelioration of behavioral abnormalities in dopamine transporter knockout mice by nicotine

Dopamine transporter knockout (DAT KO) mice exhibit elevated extracellular dopamine levels in brain regions that include the striatum and the nucleus accumbens, but not the prefrontal cortex. DAT KO mice model some aspects of psychiatric disorders, including schizophrenia. Smoking is more common in patients with schizophrenia, suggesting that nicotine might ameliorate aspects of the behavioral abnormalities and/or treatment side effects seen in these individuals. We report nicotine-induced normalization of effects on locomotion and prepulse inhibition of acoustic startle (PPI) in DAT KO mice that require intact serotonin 5-HT1A systems. First, we observed that the marked hyperactivity displayed by DAT KO mice was reduced by administration of nicotine. This nicotine effect was blocked by pretreatment with the non-specific nicotinic acetylcholine (nACh) receptor antagonist mecamylamine, or the 5-HT1A antagonist WAY100635. Secondly, we examined the effects of nicotine on PPI in DAT KO mice. Treatment with nicotine significantly ameliorated the PPI deficits observed in DAT KO mice. The ameliorating action of nicotine on PPI deficits in DAT KO mice was blocked by mecamylamine, the α7 nACh receptor antagonist methyllycaconitine or WAY100635, while the α4β2 nACh receptor antagonist dihydro-β-erythroidinehydrobromide (DHβE) produced only a non-significant trend toward attenuation of nicotine effects. Finally, we observed that administration of the 5-HT1A receptor agonist 8-OH-DPAT also ameliorated the deficit in PPI observed in DAT KO mice. This amelioration was antagonized by pretreatment with WAY100635. These data support the idea that nicotine might ameliorate some of the cognitive dysfunctions found in schizophrenia in a 5-HT1A-dependent fashion.This article is part of a Special Issue entitled ‘Cognitive Enhancers’.

Modulation of human α4β2 nicotinic acetylcholine receptors by brominated and halogen-free flame retardants as a measure for in vitro neurotoxicity

Brominated flame retardants (BFRs) are abundant persistent organic pollutants with well-studied toxicity. The toxicological and ecological concern associated with BFRs argues for replacement by safer alternatives. However, the (neuro)toxic potential of alternative halogen-free flame retardants (HFFRs) is unknown. Previous research identified the nervous system as a sensitive target organ for BFRs, with modulation of excitatory nicotinic acetylcholine (nACh) receptors as one of the modes of action. Since it is essential to assess the (neuro)toxic potential of HFFRs before large scale use, we measured the effects of three BFRs and 13 HFFRs on the function of human α4β2 nACh receptors, expressed in Xenopus oocytes, using the two-electrode voltage-clamp technique. The results demonstrate that some BFRs (TBBPA and to a lesser extent BDE-209) and HFFRs (TPP, Alpi, APP, MMT and to a lesser extent ATH, ATO, MHO, MPP, RDP and ZHS) act as nACh receptor antagonists. Contrary, BPS, BDP, DOPO and ZS were unable to modulate nACh receptors. Despite the lack of toxicological data on HFFRs and the need for additional studies to perform a full (neuro)toxic risk assessment, the current data on antagonistic effects on nACh receptors could be an important step in prioritizing viable HFFRs for substitution of BFRs.

Regulation of Glutamate Release by α7 Nicotinic Receptors: Differential Role in Methamphetamine-Induced Damage to Dopaminergic and Serotonergic Terminals

Regulation of glutamate release is an important underlying mechanism in mediating excitotoxic events such as damage to dopamine (DA) and serotonin (5-HT) neurons observed after exposure to methamphetamine (Meth). One way to regulate glutamate release may be through the modulation of α7 nicotinic acetylcholine (nACh) receptors. Meth administration is known to increase acetylcholine release; however, it is unknown whether Meth increases glutamate release and causes long-term damage to both DA and 5-HT terminals through the activation of α7 nACh receptors. To test this hypothesis, the α7 nACh receptor antagonist, methyllycaconitine (MLA), was administered before the administration of repeated doses of Meth while simultaneously monitoring extracellular striatal glutamate with in vivo microdialysis. In addition, the subsequent long-term decreases in markers of dopaminergic and serotonergic terminals, including DA reuptake transporter (DAT), serotonin reuptake transporter (SERT), vesicular monoamine transporter-2, vesicular DA, and vesicular 5-HT content in the rat striatum, were measured. The results show that MLA pretreatment prevented Meth-induced increases in striatal glutamate and protected against the subsequent long-term decreases in striatal DAT and vesicular DA content without affecting the hyperthermia produced by Meth. In contrast, the Meth-induced decreases in striatal SERT immunoreactivity and vesicular 5-HT content were not affected by MLA. This suggests that the α7 nACh receptor differentially mediates glutamate-dependent damage to DA but not 5-HT terminals in a manner that is independent of hyperthermia. Furthermore, antagonism of α7 nACh receptors may be a possible therapeutic strategy for decreasing extracellular glutamate and preventing the excitotoxic damage observed in other DA-related neurodegenerative disorders.

360 POSTER Ror2 in renal cell carcinoma: evaluating its role in RCC tumorigenesis

The α7 nicotinic acetylcholine (nACh) receptor expressed in microglia has a crucial role in neuroprotection. Simulation of α7 nACh receptor leads to increased expression of glutamate/aspartate transporter (GLAST), which in turn decreases synaptic glutamate levels. However, the upregulation of GLAST in cultured rat cortical microglia appears long after (over 18 h) stimulation of the α7 nACh receptor with nicotine. Thus, the current study elucidated the pathway responsible for the induction of GLAST expression in cultured cortical microglia. Nicotine-induced GLAST mRNA expression was significantly inhibited by cycloheximide pretreatment, indicating that a protein intermediary, such as a growth factor, is required for GLAST expression. The expression of fibroblast growth factor-2 (FGF-2) mRNA in cortical microglia was significantly increased 6 and 12 h after treatment with nicotine, and this increase was potently inhibited by pretreatment with methyllycaconitine, a selective α7 nACh receptor antagonist. The treatment with nicotine also significantly increased FGF-2 protein expression. Furthermore, treatment with recombinant FGF-2 increased GLAST mRNA, protein expression and 14C-glutamate uptake, a functional measurement of GLAST activity. Conversely, pretreatment with PD173074, an inhibitor of FGF receptor (FGFR) tyrosine kinase, significantly prevented the nicotine-induced expression of GLAST mRNA, its protein and 14C-glutamate uptake. Reverse transcription polymerase chain reaction confirmed FGFR1 mRNA expression was confined to cultured cortical microglia. Together, the current findings demonstrate that the neuroprotective effect of activation of microglial α7 nACh receptors could be due to the expression of FGF-2, which in turn increases GLAST expression, thereby clearing glutamate from synapse and decreasing glutamate neurotransmission.

COMPUTER SIMULATION OF COTRANSMISSION BY EXCITATORY AMINO ACIDS AND ACETYLCHOLINE IN THE ENTERIC NERVOUS SYSTEM

The role of cotransmission by α-amino-3-hydroxy-5-methyl-4-isoxalose propionic acid (AMPA), L-aspartate, N-methyl-D-aspartate (NMDA), and acetylcholine (ACh) as well as the coexpression of AMPA, NMDA, and nicotinic ACh (nACh) receptors on the electrophysiological activity of the primary sensory (AH) and motor (S) neurons of the enteric nervous system are numerically assessed. Results of computer simulations showed that AMPA and L-Asp alone can induce fast action potentials of short duration on AH and S neurons. Costimulation of nACh and AMPA receptors on the soma of the S neuron resulted in periodic spiking activity. A characteristic biphasic response was recorded from the AH neuron after coactivation of AMPA and NMDA receptors. Glutamate alone acting on NMDA receptors caused prolonged depolarization of the AH neuron and failed to depolarize the S neuron. Cojoint stimulation of the AMPA or nACh receptors was required to produce the effect of glutamate. The overall electrical response of neurons to the activation of NMDA receptors was long-term depolarization. Acetylcholine, AMPA, and glutamate acting alone or cojointly enhanced phasic contraction of the longitudinal smooth muscle. Treatment of neurons with AMPA, NMDA, and nACh receptor antagonists revealed intricate properties of the AH and S neurons. Application of MK-801, D-AP5, and CPP reduced the excitability of the AH neuron and totally abolished electrical activity in the S neuron. The information gained into the cotransmission by excitatory amino acids and acetylcholine in the enteric nervous system may be beneficial in the development of novel effective therapeutics to treat diseases associated with altered visceral nociception, i.e. irritable bowel syndrome.

Heteromeric Nicotinic Acetylcholine–Dopamine Autoreceptor Complexes Modulate Striatal Dopamine Release

In the striatum, dopamine and acetylcholine (ACh) modulate dopamine release by acting, respectively, on dopamine D(2) autoreceptors and nicotinic ACh (nACh) heteroreceptors localized on dopaminergic nerve terminals. The possibility that functional interactions exist between striatal D(2) autoreceptors and nACh receptors was studied with in vivo microdialysis in freely moving rats. Local perfusion of nicotine in the ventral striatum (shell of the nucleus accumbens) produced a marked increase in the extracellular levels of dopamine, which was completely counteracted by co-perfusion with either the non-alpha(7) nACh receptor antagonist dihydro-beta-erythroidine or the D(2-3) receptor agonist quinpirole. Local perfusion of the D(2-3) receptor antagonist raclopride produced an increase in the extracellular levels of dopamine, which was partially, but significantly, counteracted by coperfusion with dihydro-beta-erythroidine. These findings demonstrate a potent crosstalk between G protein-coupled receptors and ligand-gated ion channels in dopaminergic nerve terminals, with the D(2) autoreceptor modulating the efficacy of non-alpha(7) nACh receptor-mediated modulation of dopamine release. We further demonstrate physical interactions between beta(2) subunits of non-alpha(7) nicotinic acetylcholine receptors and D(2) autoreceptors in co-immunoprecipitation experiments with membrane preparations from co-transfected mammalian cells and rat striatum. These results reveal that striatal non-alpha(7) nicotinic acetylcholine receptors form part of heteromeric dopamine autoreceptor complexes that modulate dopamine release.