Prenatal Hypoxia Affects Nicotine Consumption and Withdrawal in Adult Rats via Impairment of the Glutamate System in the Brain.

The role of damaging factors in the prenatal period as a basis for drug addiction in offspring is of great interest. In this study, we aim at deciphering the effects and possible mechanisms of prenatal severe hypoxia (PSH) on predisposition to nicotine addiction in adult rats. In PSH rats, we found an increasing tendency to nicotine consumption in the two-bottle choice test. After 2 weeks of chronic treatment with nicotine via osmotic minipump (9 mg/kg per day), we assessed the symptoms of withdrawal in the conditioned place aversion test after mecamylamine (an antagonist of nicotinic acetylcholine receptors, nAChR) treatment. We showed that the mecamylamine-precipitated withdrawal aversion was stronger in the PSH group than in the control group. This suggests that PSH acts as a predisposing factor for developing nicotine addiction in adulthood. PSH rats also demonstrated an increased level of phosphorylated DARPP-32 protein (known as the relay for dopamine and glutamate signaling) at 34 threonine (pThr34DARPP-32) in relation to its total amount in the nucleus accumbens of the striatum (NAc). Meanwhile, no changes in both the content of dopamine in the mesolimbic pathway and the first type of dopamine receptors (DAR1) in NAc were found. The increased rate of DARPP-32 phosphorylation in adult PSH rats might result from excessive glutamatergic stimulation of the dopaminergic (DA) neurons of the ventral tegmental area (VTA) caused by activation of presynaptic nAChR by nicotine. This hypothesis is supported by the observed increase in VGluT2-positive terminals to Nurr1-positive neuronal bodies in VTA in PSH animals. Thus, the altered glutamate signaling phenotype might play a significant role in the development of PSH-related nicotine addiction.

Acetylcholine suppresses LPS-induced endothelial cell activation by inhibiting the MAPK and NF-κB pathways.

BACKGROUND AND OBJECTIVE: Endothelial cell activation plays a critical role in leukocyte recruitment during inflammation and infection. We previously found that cholinergic stimulation (via vagus nerve stimulation) attenuates vascular endothelial impairment and reduces the inflammatory profile in ovariectomized rats. However, the specific molecular mechanism is unclear. This study was designed to explore the effects and molecular mechanisms of cholinergic agonists (acetylcholine [ACh]) on lipopolysaccharide (LPS)-induced endothelial cell activation in vitro. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of LPS (10/100/1000 ng/mL) to activate endothelial cells. HUVECs were untreated, treated with ACh (10-5 M) alone, treated with 100 ng/mL LPS alone, or treated with different concentrations of ACh (10-9/10-8/10-7/10-6/10-5 M) before LPS stimulation. HUVECs were also pre-treated with 10-6 M ACh with or without mecamylamine (an nAChR blocker) (10 µΜ) and methyllycaconitine (a specific α7 nAChR blocker) (10 µΜ) and incubated with or without LPS. ELISA, western blotting, cell immunofluorescence, and cell adhesion assays were used to examine inflammatory cytokine production, adhesion molecule expression, monocyte-endothelial cell adhesion and activation of the MAPK/NF-κB pathways. RESULTS: LPS (at 10 ng/mL, 100 ng/mL and 1,000 ng/mL) increased VCAM-1 expression in HUVECs in a dose-dependent manner (with no significant difference between LPS at 100 ng/mL and 1,000 ng/mL). ACh (10-9 M-10-5 M) blocked adhesion molecule expression (VCAM-1, ICAM-1, and E-selectin) and inflammatory cytokine production (TNF-α, IL-6, MCP-1, IL-8) in response to LPS in a dose-dependent manner (with no significant difference between 10-5 and 10-6 M Ach). LPS was also shown to significantly enhance monocyte-endothelial cell adhesion, which was largely abrogated by treatment with ACh (10-6M). VCAM-1 expression was blocked by mecamylamine rather than methyllycaconitine. Lastly, ACh (10-6 M) significantly reduced LPS-induced phosphorylation of NF-κB/p65, IκBα, ERK, JNK and p38 MAPK in HUVECs, which was blocked by mecamylamine. CONCLUSIONS: ACh protects against LPS-induced endothelial cell activation by inhibiting the MAPK and NF-κB pathways, which are mediated by nAChR, rather than α7 nAChR. Our results may provide novel insight into the anti-inflammatory effects and mechanisms of ACh.

Better antidepressant efficacy of mecamylamine in combination with L-NAME than with L-arginine.

Aff ;ective disorders, including anxiety and mood disorders, are a constellation of psychiatric diseases that aff ;ect over 10 % of the world's population. It has been proposed that drugs that change nicotinic acetylcholine receptor (nAChR) activity can affect mood- and anxiety-related behaviors. Also, neuronal nitric oxide synthase (nNOS) is closely associated with the pathophysiology of these disorders. To limit the potential adverse effects of alteration in cholinergic and nitric oxide (NO) systems, we investigated the combined efficacy of subthreshold doses of nAChR antagonist mecamylamine and NO ligands (L-arginine as agonist and l-NAME as an antagonist) on depression- and anxiety-related behaviors in male NMRI mice. Depression-related behaviors using the forced swim test (FST) and anxiety-like activity using the hole-board test were assessed. In our results, mecamylamine (3 mg/kg) showed antidepressant-like properties, and it also tended to have anxiolytic-like effects, though not significant. Concomitant treatment of subthreshold doses of mecamylamine (1 mg/kg) and l-arginine (25 mg/kg), l-NAME (1 mg/kg), or l-arginine/L-NAME were antidepressive. In contrast, l-arginine/L-NAME alone or in associated with mecamylamine showed anxiogenic-like efficacy. Isobolographic analysis exhibited an additive antidepressant effect of the combined subthreshold doses of mecamylamine and l-arginine, and a synergistic antidepressant effect of the combined subthreshold doses of mecamylamine and l-NAME. It should be noted that mecamylamine (3 mg/kg) elicited hypolocomotion. Our results suggest that mecamylamine produces a better antidepressant efficacy in combination with l-NAME than with l-arginine.

Antinociceptive effects of novel epibatidine analogs through activation of α4ß2 nicotinic receptors.

The study of α4ß2 nicotinic receptors has provided new indications in the treatment of pain. Efforts have been made to explore new α4ß2 nicotinic receptor agonists, including TC-2559, as antinociceptive drugs. In this study, we discovered a set of novel epibatidine analogs with strong binding affinities to the α4ß2 nicotinic receptors. Among these compounds, C-159, C-163, and C-9515 attenuated formalin-induced nociceptive responses in mice; C-9515 caused the most potent analgesic effect, which was blocked by mecamylamine, a non-selective nicotinic receptor antagonist. Furthermore, C-9515 potently inhibited chronic constriction injury (CCI)-induced neuropathic pain in rats, which was sensitive to DHßE, a selective α4ß2 subtype antagonist, indicating that its analgesic effect was mediated by the activation of the α4ß2 nicotinic receptors. In conclusion, the epibatidine analog C-9515 was found to be a potent α4ß2 nicotinic receptor agonist with potent analgesic function, which demonstrated potential for the further exploration of its druggability.

Nicotine-Induced Effects on Nicotinic Acetylcholine Receptors (nAChRs), Ca2+ and Brain-Derived Neurotrophic Factor (BDNF) in STC-1 Cells.

In addition to the T2R bitter taste receptors, neuronal nicotinic acetylcholine receptors (nAChRs) have recently been shown to be involved in the bitter taste transduction of nicotine, acetylcholine and ethanol. However, at present it is not clear if nAChRs are expressed in enteroendocrine cells other than beta cells of the pancreas and enterochromaffin cells, and if they play a role in the synthesis and release of neurohumoral peptides. Accordingly, we investigated the expression and functional role of nAChRs in enteroendocrine STC-1 cells. Our studies using RT-PCR, qRT-PCR, immunohistochemical and Western blotting techniques demonstrate that STC-1 cells express several α and ß nAChR subunits. Exposing STC-1 cells to nicotine acutely (24h) or chronically (4 days) induced a differential increase in the expression of nAChR subunit mRNA and protein in a dose- and time-dependent fashion. Mecamylamine, a non-selective antagonist of nAChRs, inhibited the nicotine-induced increase in mRNA expression of nAChRs. Exposing STC-1 cells to nicotine increased intracellular Ca2+ in a dose-dependent manner that was inhibited in the presence of mecamylamine or dihydro-ß-erythroidine, a α4ß2 nAChR antagonist. Brain-derived neurotrophic factor (BDNF) mRNA and protein were detected in STC-1 cells using RT-PCR, specific BDNF antibody, and enzyme-linked immunosorbent assay. Acute nicotine exposure (30 min) decreased the cellular content of BDNF in STC-1 cells. The nicotine-induced decrease in BDNF was inhibited in the presence of mecamylamine. We also detected α3 and ß4 mRNA in intestinal mucosal cells and α3 protein expression in intestinal enteroendocrine cells. We conclude that STC-1 cells and intestinal enteroendocrine cells express nAChRs. In STC-1 cells nAChR expression is modulated by exposure to nicotine in a dose- and time-dependent manner. Nicotine interacts with nAChRs and inhibits BDNF expression in STC-1 cells.

Non-noxious skin stimulation activates the nucleus basalis of Meynert and promotes NGF secretion in the parietal cortex via nicotinic ACh receptors.

The effects of non-noxious skin stimulation on nerve growth factor (NGF) secretion in the parietal cortex were examined in anesthetized rats. Innocuous skin stimulation was delivered to the left hindlimb with a soft-hair brush. Extracellular NGF in the right parietal cortex was collected by microdialysis methods using a protein-permeable probe and was measured using an enzyme-linked immune-sorbent assay. Brushing produced a significant increase in extracellular NGF levels. This NGF response was not observed in rats pretreated with a nicotinic ACh receptor (nAChR) antagonist mecamylamine. We further examined whether brushing could activate the basal forebrain nucleus (nucleus basalis of Meynert, NBM), which is the main source of cholinergic fibers in the cerebral cortex, by means of functional MRI. The blood oxygen level-dependent signal in the right NBM was significantly higher during brushing compared to baseline. The results suggest that non-noxious skin stimulation activates NBM and promotes NGF secretion in the parietal cortex via nAChRs.

Molecular interactions between mecamylamine enantiomers and the transmembrane domain of the human α4ß2 nicotinic receptor.

To characterize the binding sites of mecamylamine enantiomers on the transmembrane domain (TMD) of human (h) (α4)3(ß2)2 and (α4)2(ß2)3 nicotinic acetylcholine receptors (AChRs), we used nuclear magnetic resonance (NMR), molecular docking, and radioligand binding approaches. The interactions of (S)-(+)- and (R)-(-)-mecamylamine with several residues, determined by high-resolution NMR, within the hα4ß2-TMD indicate different modes of binding at several luminal (L) and nonluminal (NL) sites. In general, the residues sensitive to each mecamylamine enantiomer are similar at both receptor stoichiometries. However, some differences were observed. The molecular docking experiments were crucial for delineating the location and orientation of each enantiomer in its binding site. In the (α4)2(ß2)3-TMD, (S)-(+)-mecamylamine interacts with the L1 (i.e., between positions -3' and -5') and L2 (i.e., between positions 16' and 20') sites, whereas the ß2-intersubunit (i.e., cytoplasmic end of two ß2-TMDs) and α4/ß2-intersubunit (i.e., cytoplasmic end of α4-TM1 and ß2-TM3) sites are shared by both enantiomers. In the (α4)3(ß2)2-TMD, both enantiomers bind with different orientations to the L1' (closer to ring 2') and α4-intrasubunit (i.e., at the cytoplasmic ends of α4-TM1 and α4-TM2) sites, but only (R)-(-)-mecamylamine interacts with the L2' (i.e., closer to ring 20') and α4-TM3-intrasubunit sites. Our findings are important because they provide, for the first time, a structural understanding of the allosteric modulation elicited by mecamylamine enantiomers at each hα4ß2 stoichiometry. This advancement could be beneficial for the development of novel therapies for the treatment of several neurological disorders.

Gestational exposure of mice to secondhand cigarette smoke causes bronchopulmonary dysplasia blocked by the nicotinic receptor antagonist mecamylamine.

BACKGROUND: Cigarette smoke (CS) exposure during gestation may increase the risk of bronchopulmonary dysplasia (BPD)-a developmental lung condition primarily seen in neonates that is characterized by hypoalveolarization, decreased angiogenesis, and diminished surfactant protein production and may increase the risk of chronic obstructive pulmonary disease. OBJECTIVE: We investigated whether gestational exposure to secondhand CS (SS) induced BPD and sought to ascertain the role of nicotinic acetylcholine receptors (nAChRs) in this response. METHODS: We exposed BALB/c and C57BL/6 mice to filtered air (control) or SS throughout the gestation period or postnatally up to 10 weeks. Lungs were examined at 7 days, 10 weeks, and 8 months after birth. RESULTS: Gestational but not postnatal exposure to SS caused a typical BPD-like condition: suppressed angiogenesis [decreased vascular endothelial growth factor (VEGF), VEGF receptor, and CD34/CD31 (hematopoietic progenitor cell marker/endothelial cell marker)], irreversible hypoalveolarization, and significantly decreased levels of Clara cells, Clara cell secretory protein, and surfactant proteins B and C, without affecting airway ciliated cells. Importantly, concomitant exposure to SS and the nAChR antagonist mecamylamine during gestation blocked the development of BPD. CONCLUSIONS: Gestational exposure to SS irreversibly disrupts lung development leading to a BPD-like condition with hypoalveolarization, decreased angiogenesis, and diminished lung secretory function. Nicotinic receptors are critical in the induction of gestational SS-induced BPD, and the use of nAChR antagonists during pregnancy may block CS-induced BPD.

Analysis of nicotine-induced DNA damage in cells of the human respiratory tract.

Epithelium of the upper and lower airways is a common origin of tobacco-related cancer. The main tobacco alkaloid nicotine may be associated with tumor progression. The potential of nicotine in inducing DNA mutations as a step towards cancer initiation is still controversially discussed. Different subtypes of nicotinic acetylcholine receptors (nAChR) are expressed in human nasal mucosa and a human bronchial cell line representing respiratory mucosa as a possible target for receptor-mediated pathways. In the present study, both cell systems were investigated with respect to DNA damage induced by nicotine and its mechanisms. Specimens of human nasal mucosa were harvested during surgery of the nasal air passage. After enzymatic digestion over night, single cells were exposed to an increasing nicotine concentration between 0.001 mM and 4.0mM. In a second step co-incubation was performed using the antioxidant N-acetylcysteine (NAC) and the nAChR antagonist mecamylamine. DNA damage was assessed using the alkali version of the comet assay. Dose finding experiments for mecamylamine to evaluate the maximal inhibitory effect were performed in the human bronchial cell line BEAS-2B with an increasing mecamylamine concentration and a constant nicotine concentration. The influence of nicotine in the apoptotic pathway was evaluated in BEAS-2B cells with the TUNEL assay combined with flow cytometry. After 1h of nicotine exposure with 0.001, 0.01, 0.1, 1.0 and 4.0mM, significant DNA damage was determined at 1.0mM. Further co-incubation experiments with mecamylamine and NAC were performed using 1.0mM of nicotine. The strongest inhibitory effect was measured at 1.0mM mecamylamine and this concentration was used for co-incubation. Both, the antioxidant NAC at a concentration of 1.0mM, based on the literature, as well as the receptor antagonist were capable of complete inhibition of the nicotine-induced DNA migration in the comet assay. A nicotine-induced increase or decrease in apoptosis as assessed by the TUNEL assay in BEAS-2B could not be detected. These results support the hypothesis that oxidative stress is responsible for nicotine-induced DNA damage. Similar results exist for other antioxidants in different cell systems. The decrease in DNA damage after co-incubation with a nAChR antagonist indicates a receptor-dependent pathway of induction for oxidative stress. Further investigations concerning pathways of receptor-mediated DNA damage via nAChR, the role of reactive oxygen species and apoptosis in this cell system will elucidate underlying mechanisms.

Nicotinic ligands modulate ethanol-induced dopamine function in mice.

The aim of the present study was to examine the effects of two neuronal nicotinic acetylcholine receptor ligands on acute ethanol-induced dopamine (DA) function in the C57BL/6J mouse ventral striatumusing an ex vivo assay and high-performance liquid chromatography coupled with electrochemicaldetection. Acute systemic injection of ethanol (2.5 g/kg) significantly increased the DA and dihydroxyphenylacetic acid (DOPAC) content in the ventral striatum. Pretreatment with lobeline (1 or 10 mg/kg) inhibited the ethanol-induced increase in the tissue DA and DOPAC content in the ventral striatum. Similarly, pretreatment with cytisine (0.5 or 3 mg/kg) also reduced the ethanol-induced increase in the tissue DA and DOPAC content in the ventral striatum. However, when given alone lobeline or cytisine did not produce significant effect on the DA or DOPAC content in the ventral striatum compared with controls. These findings provide evidence that lobeline and cytisine modulate ethanol-induced DA function by targeting nicotinic acetylcholine receptors in the ventral striatum, a reward-relevant brain region implicated in ethanol dependence.

Tricyclic antidepressants and mecamylamine bind to different sites in the human alpha4beta2 nicotinic receptor ion channel.

The interaction of tricyclic antidepressants with the human (h) alpha4beta2 nicotinic acetylcholine receptor in different conformational states was compared with that for the noncompetitive antagonist mecamylamine by using functional and structural approaches. The results established that: (a) [(3)H]imipramine binds to halpha4beta2 receptors with relatively high affinity (K(d)=0.83+/-0.08 microM), but imipramine does not differentiate between the desensitized and resting states, (b) although tricyclic antidepressants inhibit (+/-)-epibatidine-induced Ca(2+) influx in HEK293-halpha4beta2 cells with potencies that are in the same concentration range as that for (+/-)-mecamylamine, tricyclic antidepressants inhibit [(3)H]imipramine binding to halpha4beta2 receptors with affinities >100-fold higher than that for (+/-)-mecamylamine. This can be explained by our docking results where imipramine interacts with the leucine (position 9') and valine (position 13') rings by van der Waals contacts, whereas mecamylamine interacts electrostatically with the outer ring (position 20'), (c) van der Waals interactions are in agreement with the thermodynamic results, indicating that imipramine interacts with the desensitized and resting receptors by a combination of enthalpic and entropic components. However, the entropic component is more important in the desensitized state, suggesting local conformational changes. In conclusion, our data indicate that tricyclic antidepressants and mecamylamine efficiently inhibit the ion channel by interacting at different luminal sites. The high proportion of protonated mecamylamine calculated at physiological pH suggests that this drug can be attracted to the channel mouth before binding deeper within the receptor ion channel finally blocking ion flux.

Levamisole and ryanodine receptors. II: An electrophysiological study in Ascaris suum.

Resistance to antinematodal drugs like levamisole has increased and there is a need to understand what factors affect the responses to these anthelmintics. In our previous study, we examined the role of ryanodine receptors in muscle contraction pathways. Here we have examined interactions of levamisole receptors, ryanodine receptors (RyRs), the excitatory neuropeptide AF2, and coupling to electrophysiological responses. We examined the effects of a brief application of levamisole on Ascaris suum body muscle under current-clamp. The levamisole responses were characterized as an initial primary depolarization, followed by a slow secondary depolarizing response. We examined the effects of AF2 (KHEYLRFamide), 1 microM applied for 2 min. We found that AF2 potentiated the secondary response to levamisole and had no significant effect on the primary depolarization. Further, the reversal potentials observed during the secondary response suggested that more than one ion was involved in producing this potential. AF2 potentiated the secondary response in the presence of 30 microM mecamylamine suggesting the effect was independent of levamisole sensitive acetylcholine receptors. The secondary response, potentiated by AF2, appeared to be dependent on cytoplasmic events triggered by the primary depolarization. Ion-substitution experiments showed that the AF2 potentiated secondary response was dependent on extracellular calcium and chloride suggesting a role for the calcium-activated anion channel. Caffeine mimicked the AF2 potentiated secondary response and 0.1 microM ryanodine inhibited it. 1.0 microM ryanodine increased spiking showing that it affected membrane excitability. A model is proposed showing ryanodine receptors mediating effects of AF2 on levamisole responses.

Original nootropic drug noopept prevents memory deficit in rats with muscarinic and nicotinic receptor blockade.

Antiamnesic activity of Noopept was studied on the original three-way model of conditioned passive avoidance response, which allows studying spatial component of memory. Cholinoceptor antagonists of both types (scopolamine and mecamylamine) decreased entry latency and reduced the probability for selection of the safe compartment. Noopept abolished the antiamnesic effect of cholinoceptor antagonists and improved spatial preference.

In vivo effects of the anatoxin-a on striatal dopamine release.

Anatoxin-a is an important neurotoxin that acts a potent nicotinic acetylcholine receptor agonist. This characteristic makes anatoxin-a an important tool for the study of nicotinic receptors. Anatoxin-a has been used extensively in vitro experiments, however anatoxin-a has never been studied by in vivo microdialysis studies. This study test the effect of anatoxin-a on striatal in vivo dopamine release by microdialysis.The results of this work show that anatoxin-a evoked dopamine release in a concentration-dependent way. Atropine had not any effect on dopamine release evoked by 3.5 mM anatoxin-a. However, perfusion of nicotinic antagonists mecamylamine and alpha-bungarotoxin induced a total inhibition of the striatal dopamine release. Perfusion of alpha7*-receptors antagonists, metillycaconitine or alpha-bungarotoxin, partially inhibits the release of dopamine stimulated by anatoxin-a. These results show that anatoxin-a can be used as an important nicotinic agonist in the study of nicotinic receptor by in vivo microdialysis technique and also support further in vivo evidences that alpha7*nicotinic AChRs are implicated in the regulation of striatal dopamine release.

Nicotine stimulates adhesion molecular expression via calcium influx and mitogen-activated protein kinases in human endothelial cells.

To evaluate the effect of nicotine on endothelium dysfunction and development of vascular diseases, we investigated the influence on adhesion molecular expression mediated by nicotine and the mechanism of this effect in human umbilical vein endothelial cells (HUVECs). The result showed that nicotine could induce surface/soluble vascular cell adhesion molecule (VCAM-1) and endothelial selectin (E-selectin) expression in a time-response decline manner and the peak appeared at 15 min. This action could be mediated by mitogen-activated protein kinase/extracellular signal regulated kinase 1/2 (MAPK/ERK1/2) and MAPK/p38 because their activation could be distinctly blocked by MAPK inhibitors, PD098059 or SB203580. Mecamylamine (non-selective nicotinic receptor inhibitor), alpha-bungarotoxin (alpha7 nicotinic receptor inhibitor) could block Ca2+ accumulation, and then, prevented the phosphorylation on ERK1/2 and p38. They also inhibited the surface/soluble VCAM-1, E-selectin production of HUVECs modulated by nicotine. Therefore, we concluded that: (i) nicotine obviously up-regulates VCAM-1 and E-selectin expression at 15 min in HUVECs, (ii) nicotine activates HUVECs triggered by the ERK1/2 and p38 phosphorylation with an involvement of intracellular calcium mobilization chiefly mediated by alpha7 nicotinic receptor, (iii) intracellular Ca2+ activates a sequential pathway from alpha7 nicotinic receptor to the phosphorylation of ERK1/2, p38. These elucidate that nicotine activates HUVECs through fast signal transduction pathway and arguments their capacity of adhesion molecular production. Further more nicotine may contribute its influence to the progression of vascular disease such as atherosclerotic lesion.

Evaluation in rats and primates of [11C]-mecamylamine, a potential nicotinic acetylcholine receptor radioligand for positron emission tomography.

Mecamylamine is a well-described non specific antagonist of nicotinic acetylcholine receptors (nAChRs), used in therapy and in psychopharmacological studies. [(11)C]-Mecamylamine was prepared and evaluated as a putative radioligand for positron emission tomography to study nicotinic acetylcholine receptors. The radiosynthesis consisted in the [(11)C]-methylation of the desmethyl precursor within 40 min with 30-40% radiochemical yield decay corrected. Biodistribution studies in rats showed that radioligand crossed the blood-brain barrier (0.39% ID at 30 min) and only unmetabolized tracer was recovered from brain at 45 min. Ex vivo autoradiography studies in rats did not indicate preferential uptake, and pre-treatment mecamylamine or with chlorisondamine, an nicotinic receptor inhibitor, did not demonstrate a significant specific binding. To investigate possible specie differences and effects of anesthesia, in vivo positron emission tomography (PET) studies were carried out on anaesthetized baboons and conscious macaques. The regional brain distribution of [(11)C]-mecamylamine in the two species of primates exhibited similar kinetics as did the rat with steady state reached about 45-50 min after radiotracer administration. Uptake values were two-fold higher in brain of conscious macaque than in anaesthetized baboon (thalamus: 0.258% ID/(kg mL) in conscious macaques and 0.129% ID/(kg mL) in baboons). PET images showed a radioactivity distribution which was quite homogeneous throughout the brain but with somewhat higher uptake in grey matter than in white. Brain distribution was unaltered by saturation or displacement studies. Possible explanation for the failure to establish specific binding in vivo could be long-lived structural modifications of the ionotropic channel by the unlabeled ligand administered before the tracer. In conclusion, [(11)C]-mecamylamine did not satisfy the requirements for a PET tracer of nicotinic acetylcholine receptors.

Second hand smoke stimulates tumor angiogenesis and growth.

Exposure to second hand smoke (SHS) is believed to cause lung cancer. Pathological angiogenesis is a requisite for tumor growth. Lewis lung cancer cells were injected subcutaneously into mice, which were then exposed to sidestream smoke (SHS) or clean room air and administered vehicle, cerivastatin, or mecamylamine. SHS significantly increased tumor size, weight, capillary density, VEGF and MCP-1 levels, and circulating endothelial progenitor cells (EPC). Cerivastatin (an inhibitor of HMG-coA reductase) or mecamylamine (an inhibitor of nicotinic acetylcholine receptors) suppressed the effect of SHS to increase tumor size and capillary density. Cerivastatin reduced MCP-1 levels, whereas mecamylamine reduced VEGF levels and EPC. These studies reveal that SHS promotes tumor angiogenesis and growth. These effects of SHS are associated with increases in plasma VEGF and MCP-1 levels, and EPC, mediated in part by isoprenylation and nicotinic acetylcholine receptors.

Spinal gabapentin and antinociception: mechanisms of action.

Spinal gabapentin has been known to show the antinociceptive effect. Although several assumptions have been suggested, mechanisms of action of gabapentin have not been clearly established. The present study was undertaken to examine the action mechanisms of gabapentin at the spinal level. Male SD rats were prepared for intrathecal catheterization. The effect of gabapentin was assessed in the formalin test. After pretreatment with many classes of drugs, changes of effect of gabapentin were examined. General behaviors were also observed. Intrathecal gabapentin produced a suppression of the phase 2 flinching, but not phase 1 in the formalin test. The antinociceptive action of intrathecal gabapentin was reversed by intrathecal NMDA, AMPA, D-serine, CGS 15943, atropine, and naloxone. No antagonism was seen following administration of bicuculline, saclofen, prazosin, yohimbine, mecamylamine, L-leucine, dihydroergocristine, or thapsigargin. Taken together, intrathecal gabapentin attenuated only the facilitated state. At the spinal level, NMDA receptor, AMPA receptor, nonstrychnine site of NMDA receptor, adenosine receptor, muscarinic receptor, and opioid receptor may be involved in the antinociception of gabapentin, but GABA receptor, L-amino acid transporter, adrenergic receptor, nicotinic receptor, serotonin receptor, or calcium may not be involved.

Spinal Gabapentin and Antinociception: Mechanisms of Action

Spinal gabapentin has been known to show the antinociceptive effect. Although several assumptions have been suggested, mechanisms of action of gabapentin have not been clearly established. The present study was undertaken to examine the action mechanisms of gabapentin at the spinal level. Male SD rats were prepared for intrathecal catheterization. The effect of gabapentin was assessed in the formalin test. After pretreatment with many classes of drugs, changes of effect of gabapentin were examined. General behaviors were also observed. Intrathecal gabapentin produced a suppression of the phase 2 flinching, but not phase 1 in the formalin test. The antinociceptive action of intrathecal gabapentin was reversed by intrathecal NMDA, AMPA, D-serine, CGS 15943, atropine, and naloxone. No antagonism was seen following administration of bicuculline, saclofen, prazosin, yohimbine, mecamylamine, L-leucine, dihydroergocristine, or thapsigargin. Taken together, intrathecal gabapentin attenuated only the facilitated state. At the spinal level, NMDA receptor, AMPA receptor, nonstrychnine site of NMDA receptor, adenosine receptor, muscarinic receptor, and opioid receptor may be involved in the antinociception of gabapentin, but GABA receptor, L-amino acid transporter, adrenergic receptor, nicotinic receptor, serotonin receptor, or calcium may not be involved.

[3H]benzylpempidine, a new radioligand for probing a putative channel site on nicotinic cholinergic receptors.

A study was undertaken to assess the receptor binding characteristics of [3H]4-benzylpempidine to an allosteric site on calf brain membranes associated with nicotinic cholinergic receptors and to compare the binding affinity of novel arylpempidine analogs with their ability to antagonize the behavioral effects of nicotine in mice. Scatchard analysis of the binding yielded a K(d) of 20 nM and a B(max) of 330 fmols/mg membrane protein. [3H]4-benzylpempidine appears to be a more satisfactory ligand than [3H]mecamylamine, since it possessed a 50-fold greater affinity and its binding was far less sensitive to inorganic ions and Tris. Among the arylpempidine analogs 4-m-chlorobenzylidenepempidine and 4-benzylidenepempidine had the lowest K(i) values (1.4 nM and 5.0 nM, respectively) and were the most potent in antagonizing nicotine-induced seizures in mice. Although the K(i) values for pempidine and mecamylamine were 1-2 orders of magnitude greater than any of the arylpempidines, the dose required to antagonize nicotine-induced seizures in mice was comparable to the arylpempidines. One explanation for this apparent discrepancy in the correlation of binding affinity and nicotine antagonism is the lower brain penetration of arylpempidines compared to mecamylamine, following their systemic administration to mice.