microM Nicotine Research Articles

Expression of phosphorylated Akt in oral carcinogenesis and its induction by nicotine and alkaline stimulation

In Taiwan, it is well documented that cigarette smoking and areca nut chewing contribute to the risk of oral squamous cell carcinoma (OSCC). The role of phosphorylated Akt (p-Akt) in oral carcinogenesis induced by nicotine and alkaline environments was investigated. Immunohistochemistry (IHC) was used to detect p-Akt expression in cancerous (n = 30) precancerous (n = 30), and normal mucosa tissues (n = 10). Western blotting was used to detect time-dependent induction of p-Akt by 100 microM nicotine in normal human bronchial epithelial cell (NHBE), normal human oral keratinocytes (NHOK), immortalized human epithelial cells (HaCaT) and OEC-M1 cells, dose-dependent induction of p-Akt in OEC-M1 and HaCaT cells and pH effect of p-Akt in OEC-M1. The unpaired t-test and the Fisher's exact test were used to analyze the p-Akt immunoreactivity in various groups and its association with clinicopathological parameters. Higher p-Akt expression in cancerous group than in normal mucosa (P = 0.0002) and precancerous (P = 0.0049) groups was observed. A time-dependent increase in p-Akt in the NHBE, NHOK, HaCaT and OEC-M1 cell lines was observed with 100 microM nicotine treatment. The dose-dependent increase in p-Akt by nicotine treatment in HaCaT and OEC-M1 cells was obviously observed. Higher p-Akt expression in more alkaline environment (pH 8.0) was observed than at pH 7.4 in OEC-M1 cells. A potential role for increased p-Akt may relate to the dose and time of nicotine use. The potential role of an alkaline environment to enhance nicotine-related oral carcinogenesis may exist.

Nicotine Activates and Up-Regulates Nicotinic Acetylcholine Receptors in Bronchial Epithelial Cells

Prenatal nicotine exposure impairs normal lung development and leads to diminished pulmonary function after birth. Previous work from our laboratory has demonstrated that nicotine alters lung development by affecting a nonneuronal cholinergic autocrine loop that is expressed in lung. Bronchial epithelial cells (BECs) express choline acetyltransferase, the choline high-affinity transporter and nicotinic acetylcholine (ACh) receptor (nAChR) subunits. We now demonstrate through a combination of morphological and electrophysiological techniques that nicotine affects this autocrine loop by up-regulating and activating cholinergic signaling. RT-PCR showed the expression of alpha 3, alpha 4, alpha 7, alpha 9, alpha 10, beta2, and beta 4 nAChR mRNAs in rhesus monkey lung and cultured BECs. The expression of alpha 7, alpha 4, and beta2 nAChR was confirmed by immunofluorescence in the cultured BECs and lung. The electrophysiological characteristics of nAChR in BECs were determined using whole-cell patch-clamp on cultured BECs. Both ACh and nicotine evoked an inward current, with a rapid desensitizing current. Nicotine induced inward currents in a concentration-dependent manner, with an EC(50) of 26.7 microM. Nicotine-induced currents were reversibly blocked by the nicotinic antagonists, mecamylamine, dihydro-beta-erythroidine, and methyllcaconitine. Incubation of BECs with 1 microM nicotine for 48 hours enhanced nicotine-induced currents by roughly 26%. The protein tyrosine phosphorylation inhibitor, genistein, increased nicotine-induced currents by 58% and enhanced methyllcaconitine-sensitive currents (alpha 7 nAChR activities) 2.3-fold, whereas the protein tyrosine phosphatase inhibitor, pervanadate, decreased the effects of nicotine. These results demonstrate that chronic nicotine exposure up-regulates nAChR activity in developing lung, and that nAChR activity can be further modified by tyrosine phosphorylation.

Secondary motoneurons in juvenile and adult zebrafish: Axonal pathfinding errors caused by embryonic nicotine exposure

Nicotine is a drug of abuse that has been reported to have many adverse effects on the developing nervous system. We previously demonstrated that embryonic exposure to nicotine alters axonal pathfinding of spinal secondary motoneurons in zebrafish. We hypothesize that these changes will persist into adulthood. The Tg(isl1:GFP) line of zebrafish, which expresses green fluorescent protein (GFP) in a subtype of spinal secondary motoneurons, was used to investigate potential long-term consequences of nicotine exposure on motoneuron development. Anatomical characterization of Tg(isl1:GFP) zebrafish ranging between 3 and 30 days postfertilization (dpf) was initially performed in fixed tissue to characterize axonal trajectories in larval and juvenile fish. Tg(isl1:GFP) embryos were transiently exposed to 5–30 μM nicotine. They were then rescued from nicotine and raised into later stages of life (3–30 dpf) and fixed for microscopic examination. Morphological analysis revealed that nicotine-induced abnormalities in secondary motoneuron anatomy were still evident in juvenile fish. Live imaging of Tg(isl1:GFP) zebrafish using fluorescent stereomicroscopy revealed that the nicotine-induced changes in motoneuron axonal pathfinding persisted into adulthood. We detected abnormalities in 37-dpf fish that were transiently exposed to nicotine as embryos. These fish were subsequently imaged over a 7-week period of time until they were ≈3 months of age. These pathfinding errors of spinal secondary motoneuron axons detected at 37 dpf persisted within the same fish until 86 dpf, the latest age analyzed. These findings indicate that exposure to nicotine during embryonic development can have permanent consequences for motoneuron anatomy in zebrafish. J. Comp. Neurol. 512:305–322, 2009. © 2008 Wiley-Liss, Inc.

Tobacco particulate matter is more potent than nicotine at upregulating nicotinic receptors on SH-SY5Y cells

The effect of total particulate matter (TPM) from cigarette smoke on the expression and binding properties of nicotinic acetylcholine receptors (nAChRs) was investigated using a human neuroblastoma cell line (SH-SY5Y). TPM but not nicotine on its own inhibited cell growth at nicotine concentrations above 5 microM. To examine effects on nAChR expression, intact cells were incubated with 3H-epibatidine, and a Bmax of 13 fmoles/10(5) cells (7.8 x 10(4) binding sites/cell) was measured in unexposed cells as well as in cells treated with 2 microM nicotine alone or with TPM containing 2 microM nicotine. Using Scatchard analysis, we measured a Kd of 0.3 nM for 3H-epibatidine binding to nAChRs. This Kd was increased to 1.3 nM by addition of nicotine or TPM extract, both at 2 microM nicotine. Bmax, however, was unaffected, suggesting competitive binding of nicotine to its receptor. Short-term and prolonged 3-day exposures of SH-SY5Y cells to either TPM or nicotine at nicotine concentrations ranging from 0.2 microM to 20 microM increased specific binding, suggesting upregulation of nAChR expression. Most significant, binding was consistently greater in cells pretreated with TPM than in cells pretreated with nicotine. We conclude that TPM contains compounds that are toxic to cells at high concentrations (cell growth inhibition) but that do not compete with nicotine for binding to nAChRs (Scatchard analysis). These non-nicotinic compounds are capable of increasing the expression of one or more of the nAChR subunits. Furthermore, our cell culture assay provides a useful in vitro model for assessing the relative addictiveness of different tobacco products, including that of non-nicotine components.

Nicotine-induced proliferation of isolated rat pancreatic acinar cells: effect on cell signalling and function.

The aim of the current study was to investigate whether nicotine treatment would induce the proliferation of isolated rat primary pancreatic acinar cells in culture by activating mitogen-activated protein kinase (MAPK) signalling and exocrine secretion. A nicotine dose- and time-response curve was initially developed to determine the optimal dose and time used for all subsequent studies. Proliferation studies were conducted by cell counting and confirmed further by bromodeoxyuridine (BrdU) incorporation and flow cytometry assays. MAPK signalling studies were conducted by Western blot analysis. Localization of ERK1/2 signals, with or without nicotine and the MAPK inhibitor, was visualized by immunofluorescence. Nicotine treatment caused dose-dependent activation of extracellular signal-regulated kinases (ERK1/2), the maxima occurring at 100 micro m and at 3 min after treatment; the response was suppressed by the ERK1/2 inhibitor. Maximal nicotine-induced cell proliferation occurred at 24 h, and UO126-treatment significantly reduced this response. Exposure of cells to 100 microm nicotine for 6 min significantly enhanced both baseline and cholecystokinin-stimulated cell function, and these effects were not affected by treatment with the inhibitor of ERK1/2 but were suppressed by mecamylamine, a nicotinic receptor antagonist. Our results suggest that nicotine treatment induced cell proliferation of isolated pancreatic acinar cells and that this is coupled with the activation of MAPK signalling with no effect on its function. Hence, in primary cells, the mechanism of induction and regulation of these two processes, cell proliferation and cell function, by nicotine treatment are independent of each other.

Nicotine and Biochanin A, but Not Cigarette Smoke, Induce Anti-Inflammatory Effects on Keratinocytes and Endothelial Cells in Patients with Behçet's Disease

During periods of smoking, patients with Behçet's disease have less oral aphthae than in abstinence. To elucidate this observation, human keratinocytes and dermal microvascular endothelial cells (HMEC-1) were incubated with serum of 20 patients with Behçet's disease and 20 healthy controls for 4 hours. Maximum non-toxic concentrations were determined and the cells were further treated with 6 microM nicotine, 3.3% cigarette smoke extract (CES), 100 microM biochanin A, and 6.25/12.5 microM pyrrolidine dithiocarbamate alone and in combinations for 24 hours. Serum IL-8 levels of patients were significantly lower than those of controls. However, after 4 hours incubation with patients' sera, IL-8 release by both cell types was markedly increased when compared with the corresponding serum levels. The levels of IL-6 and vascular endothelial growth factor (VEGF) release were after 4 hours similar with the corresponding levels in serum. IL-1 was not detected. Nicotine significantly decreased IL-8 and -6 release by HMEC-1 maintained in both patients' and controls' sera, but only IL-6 release by keratinocytes maintained in patients' sera. VEGF release by both cells was markedly increased after nicotine treatment in either serum. CES significantly decreased IL-8 release and increased production of VEGF in keratinocytes maintained in patients' serum. The phytoestrogen biochanin A alone and in combination with nicotine further decreased the secretion of IL-8, -6, and VEGF in all experimental settings. Our data support a specific anti-inflammatory effect of nicotine on keratinocytes and endothelial cells maintained in the serum of patients with Behçet's disease. Moreover, biochanin A is likely to exhibit similar and even more profound results than nicotine.

Nicotine upregulates the expression of P2Y12 on vascular cells and megakaryoblasts

P2Y12 is the major platelet receptor that mediates ADP-induced aggregation. P2Y12 is also expressed by vascular cells. The factors that regulate P2Y12 expression have not been determined. Since nicotine (NIC) has effects on platelet activation and vascular function, and because nicotinic and purinerigic receptors may interact, we determined whether nicotine altered P2Y12 expression. Four cell lines (human coronary artery endothelial cells, HCAEC; human umbilical vein endothelial cells, HUVEC; human aortic smooth muscle cells, HASMC; and human megakaryoblastic cells, MEG-01) were cultured in the absence or presence of nicotine. Immunoblotting for P2Y12, P2Y2, and actin was performed. Nicotine, at concentrations of 0.1-1.0 microM, induced P2Y12 (but not P2Y2) expression in all the four cell lines. HASMC exhibited the greatest induction with a sixfold mean increase in P2Y12 expression in response to 0.25 microM nicotine. The induction was inhibited by nicotinic acetylcholine receptor antagonists. Healthy smokers were observed to have higher P2Y12 expression in platelet lysates compared to non-smokers. Nicotine induces the expression of P2Y12 in vascular cells and megakaryoblasts, and is mediated by nicotinic acetylcholine receptors. Smokers exhibit higher platelet P2Y12, possibly mediated via nicotine. These results may contribute to a better understanding of the effects of cigarette smoking on platelet activation and the vessel wall. The factors that regulate the expression of P2Y12, the platelet ADP receptor, have not been determined. Four cell lines (human coronary artery endothelial cells, HCAEC; human umbilical vein endothelial cells, HUVEC; human aortic smooth muscle cells, HASMC; and human megakaryoblastic cells, MEG-01) were cultured in the absence or presence of nicotine. Nicotine, at concentrations of 0.1-1.0 microM, induced P2Y12 expression in all the four cell lines. HASMC exhibited the greatest induction with a sixfold mean increase in P2Y12 expression in response to 0.25 microM nicotine. The induction was inhibited by nicotinic acetylcholine receptor antagonists. Healthy smokers were observed to have higher P2Y12 expression in platelet lysates compared to non-smokers. These results may contribute to a better understanding of the effects of cigarette smoking on platelet activation and the vessel wall.

Effect of acetylcholinesterase inhibitors on the binding of nicotinic α4β2 receptor PET radiotracer, 18F‐nifene: A measure of acetylcholine competition

Acetylcholinesterase inhibitors (AChEI's) are used to treat Alzheimer's disease (AD), and the putative mode of action is to increase acetylcholine (ACh) levels. Our goal is to evaluate competition of ACh with nicotinic alpha4beta2 receptor PET agonist radiotracer, 2-[(18)F]fluoro-3-[2-((S)-3-pyrrolinyl)methoxy]pyridine ((18)F-nifene). This ability to measure ACh-(18)F-nifene competition may have potential to assess efficacy of AChEI's in vivo. In vitro studies in rat brain slices used two AChEI's, physostigmine (PHY) and galanthamine (GAL). Brain slices were incubated with (18)F-nifene and various concentrations of PHY (0.2-20 microM) or GAL (0.4-4 microM) prior to (18)F-nifene treatment. For ACh competition, slices were also incubated with PHY + 100 nM ACh or GAL + 100 nM ACh or 100 nM ACh alone. Nonspecific binding of (18)F-nifene was determined using 300 microM nicotine. In the in vitro rat brain homogenate binding assay, ACh inhibited (3)H-cytisine binding to alpha4beta2 receptors with K(i) values of 19.2 nM (with PHY) and 34.7 microM (no PHY) indicating approximately 1.8 x 10(3) weaker binding of ACh in the absence of AChEI. Binding of (18)F-nifene was not affected by PHY (0.2-20 microM) or ACh 100 nM alone but decreased substantially by PHY + ACh 100 nM in all brain regions (down by >40% of control in thalamus). Similarly, for GAL (4 microM) no effect on (18)F-nifene binding occurred but GAL (0.4-4 microM) + ACh 100 nM showed a reduction of (18)F-nifene binding in all brain regions (down by approximately 15%). The reduction in both cases is a result of ACh competition with (18)F-nifene in the presence of AChEI. These preliminary in vitro results suggest that ACh is able to compete with (18)F-nifene at the alpha4beta2 receptors in the presence of PHY or GAL. The effect is AChEI-concentration dependent and is greater for PHY than GAL. Thus (18)F-nifene has promise for assessing ACh levels and AChEI effects in vivo.

Mitogen-Activated Protein Kinase Pathway Was Significantly Activated in Human Bronchial Epithelial Cells by Nicotine

Nicotine is potentially associated with the onset of chronic obstructive pulmonary disease (COPD) and lung cancer. To gain insights into the molecular mechanism underlying such nicotine-induced conditions, microarray- bioinformatics analysis was carried out in the present study to explore the gene expression profiles in human bronchial epithelial cells (HBECs) treated with 5 microM nicotine for 4, 8, and 10 h. Of 1,800 assessed genes overall, 260 (14.4%) were upregulated and 17 (0.9%) down regulated significantly. Gene ontology analysis demonstrated that most of the differentially expressed genes belonged to the category of molecular function, especially to the subcategories of enzyme activity. The integration of obtained information with bioinformatics tools in DAVID and KEGG databases indicated that the greatest number of overexpressed genes was involved in mitogen-activated protein kinase (MAPK) pathway. Membrane array analysis subsequently suggested that both extracellular signal-regulated kinase (ERK) 1/2 and c-Jun-NH(2)-terminal kinase (JNK) signalings but not p38 MAPK signaling were activated in response to nicotine. Pretreatment of HBECs with specific inhibitors against ERK 1/2 and JNK but not p38 could significantly inhibit nicotine-induced interleukin- 8 production. These results suggest that MAPK pathway may mediate the effect of nicotine through ERK 1/2 and JNK but not p38 in HBECs treated with nicotine.

Nicotinic α4β2 receptor imaging agents

The alpha4beta2 nicotinic acetylcholine receptor (nAChR) has been implicated in various neurodegenerative diseases. Optimal positron emission tomography (PET) imaging agents are therefore highly desired for this receptor. We report here the development and initial evaluation of 2-fluoro-3-[2-((S)-3-pyrrolinyl)methoxy]pyridine (nifene). In vitro binding affinity of nifene in rat brain homogenate using 3H-cytisine exhibited a K(i) = 0.50 nM for the alpha4beta2 sites. The radiosynthesis of 2-18F-fluoro-3-[2-((S)-3-pyrrolinyl)methoxy]pyridine (18F-nifene) was accomplished in 2.5 h with an overall radiochemical yield of 40-50%, decay corrected. The specific activity was estimated to be approx. 37-185 GBq/micromol. In vitro autoradiography in rat brain slices indicated selective binding of 18F-nifene to anteroventral thalamic (AVT) nucleus, thalamus, subiculum, striata, cortex and other regions consistent with alpha4beta2 receptor distribution. Rat cerebellum showed some binding, whereas regions in the hippocampus had the lowest binding. The highest ratio of >13 between AVT and cerebellum was measured for 18F-nifene in rat brain slices. The specific binding was reduced (>95%) by 300 microM nicotine in these brain regions. Positron emission tomography imaging study of 18F-nifene (130 MBq) in anesthetized rhesus monkey was carried out using an ECAT EXACT HR+ scanner. PET study showed selective maximal uptake in the regions of the anterior medial thalamus, ventro-lateral thalamus, lateral geniculate, cingulate gyrus, temporal cortex including the subiculum. The cerebellum in the monkeys showed lower binding than the other regions. Thalamus-to-cerebellum ratio peaked at 30-35 min postinjection to a value of 2.2 and subsequently reduced. The faster binding profile of 18F-nifene indicates promise as a PET imaging agent and thus needs further evaluation.

In vitro regulation of serotonin transporter activity by protein kinase A and nicotinic acetylcholine receptors in the prefrontal cortex of rats

We investigated the effect of in vitro exposure to nicotinic acetylcholine receptors (nAChRs), agonists, antagonists, and protein kinase A (PKA) modulators on the activity of the serotonin transporter (SERT) in prefrontocortical (PFC) synaptosomes. The plasma membrane SERT is an active transport mechanism specific for serotonin. Receptors and second messengers capable of altering transporter activity would be expected to have profound effects on serotonergic neurotransmission and on functions involving serotonergic input, such as cognition, anxiety, and mood. Our data suggest that activation of nAChRs, quite likely via PKA, increase the activity of the SERT in the PFC and, thereby, can alter 5-HT levels in a region important in the behavioral effects of nicotine and 5-HT. Nicotine at 4 microM increased [(3)H]5-HT uptake by 75%. Because the nAChR antagonists mecamylamine and dihydro-beta-erythrodine (DHbetaE) both decreased [(3)H]5-HT uptake into synaptosomes, it appeared that the SERT might be tonically activated by acetylcholine present within our synaptosomal preparations. Blocking PKA significantly decreased [(3)H]5-HT, while stimulation of PKA activity significantly increased the uptake. A 66% decrease compared with control was produced by 100 microM Rp-cAMP, and a 41% increase in 5-HT uptake over control was observed with 30 microM Sp-cAMPs. Furthermore, the enhancement in uptake produced by 4 microM nicotine was inhibited in a time-dependent fashion by preincubation with 10 microM Rp-cAMP. A better understanding of the influence of the cholinergic system and the receptors involved in the trafficking of SERT would help clarify the important relationship between the cholinergic and serotonergic systems and the role these systems play in behavior.

Activation and desensitization of neuronal nicotinic receptors modulate glutamatergic transmission on neonatal rat hypoglossal motoneurons

In the neonate the muscles of the tongue, which are exclusively innervated by the XII cranial nerves originating from the brainstem nucleus hypoglossus, must contract rhythmically in coincidence with breathing, suckling and swallowing. These motor commands are generated by hypoglossal motoneurons excited by glutamatergic inputs. Because in forebrain areas the efficiency of glutamatergic transmission is modulated by neuronal nicotinic receptors (nAChRs), the role and identity of nAChRs within the nucleus hypoglossus of the neonatal rat were explored using an in vitro brainstem slice preparation. This area expressed immunoreactivity for alpha4, alpha7 and beta2 nAChR subunits. Whole-cell patch-clamp recording from hypoglossal motoneurons showed lack of spontaneous cholinergic events mediated by nAChRs even in the presence of a cholinesterase inhibitor. However, pharmacological antagonism of alpha7- or beta2-containing receptors depressed glutamatergic currents arising either spontaneously or by electrical stimulation of the reticular formation. Hypoglossal motoneurons expressed functional nAChRs with characteristics of alpha4beta2 and alpha7 receptor subunits. Such receptors underwent fast desensitization (time constant of 200 ms) with full recovery within 1 min. Low (0.5 microm) concentration of nicotine first facilitated glutamatergic transmission on motoneurons and later depressed it through receptor desensitization. When 0.1 microm nicotine was used, only depression of synaptic transmission occurred, in keeping with the suggestion that nAChRs can be desensitized without prior activation. These results highlight the role of tonic nAChR activity in shaping excitatory inputs to hypoglossal motoneurons, and suggest that nAChR desensitization by ambient nicotine could contribute to disorders of tongue muscle movements.

CYP2A6 and CYP2B6 are involved in nornicotine formation from nicotine in humans: Interindividual differences in these contributions

Nornicotine is an N-demethylated metabolite of nicotine. In the present study, human cytochrome P450 (P450) isoform(s) involved in nicotine N-demethylation were identified. The Eadie-Hofstee plot of nicotine N-demethylation in human liver microsomes was biphasic with high-affinity (apparent K(m) = 173 +/- 70 microM, V(max) = 57 +/- 17 pmol/min/mg) and low-affinity (apparent K(m) = 619 +/- 68 microM, V(max) = 137 +/- 6 pmol/min/mg) components. Among 13 recombinant human P450s expressed in baculovirus-infected insect cells (Supersomes), CYP2B6 exhibited the highest nicotine N-demethylase activity, followed by CYP2A6. The apparent K(m) values of CYP2A6 (49 +/- 12 microM) and CYP2B6 (550 +/- 46 microM) were close to those of high- and low-affinity components in human liver microsomes, respectively. The intrinsic clearances of CYP2A6 and CYP2B6 Supersomes were 5.1 and 12.5 nl/min/pmol P450, respectively. In addition, the intrinsic clearance of CYP2A13 expressed in Escherichia coli (44.9 nl/min/pmol P450) was higher than that of CYP2A6 expressed in E. coli (2.6 nl/min/pmol P450). Since CYP2A13 is hardly expressed in human livers, the contribution of CYP2A13 to the nicotine N-demethylation in human liver microsomes would be negligible. The nicotine N-demethylase activity in microsomes from 15 human livers at 20 microM nicotine was significantly correlated with the CYP2A6 contents (r = 0.578, p < 0.05), coumarin 7-hydroxylase activity (r = 0.802, p < 0.001), and S-mephenytoin N-demethylase activity (r = 0.694, p < 0.005). The nicotine N-demethylase activity at 100 microM nicotine was significantly correlated with the CYP2B6 contents (r = 0.677, p < 0.05) and S-mephenytoin N-demethylase activities (r = 0.740, p < 0.005). These results as well as the inhibition analyses suggested that CYP2A6 and CYP2B6 would significantly contribute to the nicotine N-demethylation at low and high substrate concentrations, respectively. The contributions of CYP2A6 and CYP2B6 would be dependent on the expression levels of these isoforms in any human liver.

Locally Administered Low Nicotine-Induced Neurotransmitter Changes in Areas of Cognitive Function

The present study examined the effect of a low-dose of nicotine; below that one expects to be achieved from a single cigarette, on brain regional heterogeneity and sensitivity of catecholaminergic responses. 1 microM nicotine was infused into six brain areas via a microdialysis probe: the dorsal and ventral hippocampus, the medial temporal and prefrontal cortex, the basolateral amygdala, and the ventral tegmental area (VTA). The nicotine concentration in the brain tissue near the probe site was approximately 0.1 microM. Nicotine-induced increases and decreases could be noted in dopamine (DA), norepinephrine (NE), and serotonin (5HT) levels. In particular, DA and 5HT decreased in both hippocampal areas, while NE increased in the dorsal and decreased in the ventral hippocampus. In the cortical areas, DA and NE increased and 5HT was not significantly altered. In the amygdala all three neurotransmitters increased and in the VTA, all three decreased. Many of the nicotine-induced changes in neurotransmitter concentrations were reversed in the presence of atropine. Where nicotine induced decreases in DA and 5HT in the VTA, increases were observed in the presence of atropine. A similar reversal was seen with NE in the VTA and ventral hippocampus. In contrast, the increases in DA observed in the cortex and amygdala and the increases in NE observed in the cortex, amygdala and dorsal hippocampus were inhibited by the presence of atropine. 5HT was also significantly decreased in the amygdala and both cortical areas in the presence of atropine, where nicotine alone had no significant effect. We conclude, that at low doses, nicotine significantly alters the release of DA, NE, and 5HT--in some areas increasing, in others decreasing endogenous neurotransmitter levels. This data, in conjunction with previous experiments, indicates that the effects of nicotine are regionally heterogeneous and arise from both direct and indirect actions on various receptors and neurotransmitter systems and nicotine's effects at low doses differ from that at higher doses. The changes in effects in the presence of atropine suggest that muscarinic acetylcholine receptors play a major role in nicotine's actions on neurotransmitter systems.

Cellular responses to nicotinic receptor activation are decreased after prolonged exposure to galantamine in human neuroblastoma cells

In this study, we have examined cellular responses of neuroblastoma SH-SY5Y cells after chronic treatment with galantamine, a drug used to treat Alzheimer's disease that has a dual mechanism of action: inhibition of acetylcholinesterase and allosteric potentiation of nicotinic acetylcholine receptors (nAChR). Acute experiments confirmed that maximum potentiation of nicotinic responses occurs at 1 microM galantamine; hence this concentration was chosen for chronic treatment. Exposure to 1 microM galantamine for 4 days decreased Ca(2+) responses (by 19.8+/-3.6%) or [(3)H]noradrenaline ([(3)H]NA) release (by 23.9+/-3.3%) elicited by acute application of nicotine. KCl-evoked increases in intracellular Ca(2+) were also inhibited by 10.0+/-1.9% after 4 days' treatment with galantamine. These diminished responses are consistent with the downregulation of downstream cellular processes. Ca(2+) responses evoked by activation of muscarinic acetylcholine receptors were unaffected by chronic galantamine treatment. Exposure to the more potent acetylcholinesterase inhibitor rivastigmine (1 microM) for 4 days failed to alter nicotine-, KCl-, or muscarinic receptor-evoked increases in intracellular Ca(2+). These observations support the hypothesis that chronic galantamine exerts its effects through interaction with nAChR in this cell line. Exposure to 10 microM nicotine for 4 days produced decreases in acute nicotine- (18.0+/-3.5%) and KCl-evoked Ca(2+) responses (10.6+/-2.5%) and nicotine-evoked [(3)H]NA release (26.0+/-3.3%) that are comparable to the effects of a corresponding exposure to galantamine. Treatment with 1 microM galantamine did not alter numbers of [(3)H]epibatidine-binding sites in SH-SY5Y cells, in contrast to 62% upregulation of these sites in response to 10 microM nicotine. Thus, chronic galantamine acts at nAChR to decrease subsequent functional responses to acute stimulation with nicotine or KCl. This effect appears to be independent of the upregulation of nAChR-binding sites.

Porcine CYP2A Polymorphisms and Activity

CYP2A6 in man catalyzes the oxidation of nicotine-forming cotinine and 7-hydroxylation of coumarin, which is used as test substrate for CYP2A6 in man. Large interindividual differences are found in man and some are due to genetic polymorphism. The 7-hydroxylation of coumarin is present in pigs, and an inter-individual variation has been found that might be due to polymorphisms. To enable the finding of polymorphism in pigs, the minipig cDNA was sequenced. Two cDNAs were found and translated to a 494 and a 487 amino acid long protein, both cDNAs were found in all but one pig. The 494 a.a. protein showed high homology to the human and 100% homology to the conventional pig CYP2A19 protein. In the wild type protein, all 6 substrate recognition sites were found, whereas the short protein only contained the first 5 substrate recognition sites. SSCP analysis revealed 3 polymorphisms. In order to study the effect of these polymorphisms on enzyme activity, microsomes were incubated with nicotine and coumarin. The polymorphisms appeared to have no effect on either enzyme activity as the specific enzyme activity towards nicotine and coumarin were approximately the same for all pigs. The specificity of pig CYP2A was investigated and it was found that the formation of cotinine correlated with the immunochemical level of CYP2A as did the coumarin hydroxylation. Anti-human CYP2A inhibitory antibody inhibited coumarin 7-hydroxylation by about 90% and formation of cotinine by 44--60% and 85--100% at substrate concentrations of 500 microM and 50 microM respectively, showing that coumarin and nicotine (50 microM) are very specific substrates for CYP2A in pigs, whereas the CYP2A only is responsible for about 50% of the cotinine formation at a 500 microM nicotine incubation concentration. These results show that the large interindividual differences in porcine CYP2A activity are not caused by polymorphisms but transcriptional regulation and the coumarin 7-hydroxylation is as specific a reaction for porcine CYP2A as for human CYP2A6.

Local perfusion of nicotine differentially modulates somatodendritic dopamine release in the rat ventral tegmental area after nicotine preexposure.

We examined the effects of nicotine perfusion into the ventral tegmental area (VTA) on extracellular dopamine (DA) levels in rats using in vivo microdialysis. Local perfusion with nicotine for 80 min (10-100 microM) modestly increased (approximately 105-131% of basal) the extracellular DA levels in the VTA of rats that had been pretreated with saline for 5 days. In animals that had been pretreated with nicotine for 5 days (0.3 mg/kg, s.c.), perfusion with nicotine for 80 min (10-100 microM) dose-dependently increased the extracellular DA levels in the VTA of rats and did so to a greater extent than in saline-pretreated animals (125-171% of basal). Co-perfusion through the dialysis probe with 100 microM mecamylamine, a nonselective nicotinic acetylcholine receptor (nAChR) antagonist, or 100 microM dihydro-beta-erythroidine, a high affinity and competitive nAChR antagonist, attenuated the enhancement of extracellular DA levels produced by 100 microM nicotine alone. These results suggest that local nicotine challenge potentiated the somatodendritic DA release after nicotine preexposure by stimulation of high-affinity nAChRs in the VTA.

Assembly of α4β2 Nicotinic Acetylcholine Receptors Assessed with Functional Fluorescently Labeled Subunits: Effects of Localization, Trafficking, and Nicotine-Induced Upregulation in Clonal Mammalian Cells and in Cultured Midbrain Neurons

Fura-2 recording of Ca2+ influx was used to show that incubation in 1 microm nicotine (2-6 d) upregulates several pharmacological components of acetylcholine (ACh) responses in ventral midbrain cultures, including a MLA-resistant, DHbetaE-sensitive component that presumably corresponds to alpha4beta2 receptors. To study changes in alpha4beta2 receptor levels and assembly during this upregulation, we incorporated yellow and cyan fluorescent proteins (YFPs and CFPs) into the alpha4 or beta2 M3-M4 intracellular loops, and these subunits were coexpressed in human embryonic kidney (HEK) 293T cells and cultured ventral midbrain neurons. The fluorescent receptors resembled wild-type receptors in maximal responses to ACh, dose-response relations, ACh-induced Ca2+ influx, and somatic and dendritic distribution. Transfected midbrain neurons that were exposed to nicotine (1 d) displayed greater levels of fluorescent alpha4 and beta2 nicotinic ACh receptor (nAChR) subunits. As expected from the hetero-multimeric nature of alpha4beta2 receptors, coexpression of the alpha4-YFP and beta2-CFP subunits resulted in robust fluorescence resonance energy transfer (FRET), with a FRET efficiency of 22%. In midbrain neurons, dendritic alpha4beta2 nAChRs displayed greater FRET than receptors inside the soma, and in HEK293T cells, a similar increase was noted for receptors that were translocated to the surface during PKC stimulation. When cultured transfected midbrain neurons were incubated in 1 microm nicotine, there was increased FRET in the cell body, denoting increased assembly of alpha4beta2 receptors. Thus, changes in alpha4beta2 receptor assembly play a role in the regulation of alpha4beta2 levels and responses in both clonal cell lines and midbrain neurons, and the regulation may result from Ca2+-stimulated pathways.

Apoptosis of spinal cord neurons by preventing depletion nicotine attenuates arachidonic acid-induced of neurotrophic factors.

Increased levels of free fatty acids and, in particular, arachidonic acid can lead to induction of apoptosis of spinal cord neurons. Because of the importance of neurotrophic factors in cell survival and death, mRNA and protein levels of brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (FGF-2) were studied in cultured spinal cord neurons treated with arachidonic acid. In addition, the present study focused on the effects of nicotine and neuronal nicotinic acetylcholine receptors (nAChRs) on these processes. A 2-h exposure to arachidonic acid markedly diminished expression of BDNF and FGF-2. These effects were fully prevented by pretreatment with 10 microM nicotine. Mecamylamine (a non-specific antagonist of nAChRs) and alpha-bungarotoxin (a specific antagonist of the nAChRalpha7) completely inhibited nicotine-mediated protection against arachidonic acid-induced alterations of BDNF and FGF-2. In addition, nicotine, BDNF and FGF-2 fully protected against arachidonic acid-induced apoptosis of spinal cord neurons. BDNF and FGF-2 were effective in prevention of apoptotic cell death even when applied 2 h after the beginning of arachidonic acid treatment. These results suggest that arachidonic acid can induce apoptosis of spinal cord neurons by depletion of neurotrophic factors and that nicotine can protect against these effects through the nAChRalpha7-mediated pathway.

Nicotine stimulates osteoclast resorption in a porcine marrow cell model.

Combustible tobacco use is generally linked with accelerated periodontal bone loss and diminished post-surgical wound healing; however, the pathogenesis of this process at the cellular level remains unclear. Nicotine is known to affect human gingival fibroblast orientation, attachment, and beta1 integrin expression, yet little is known about its effects on osteoclasts, the cells most responsible for bone resorption. The purpose of this study was to determine the effects of physiologically relevant nicotine levels on porcine osteoclast function as measured by resorption of calcium phosphate. Pure nicotine was diluted in medium to the following concentrations: 0.03 microM, 0.15 microM, 0.30 microM, 0.60 microM, and 1.50 microM. Porcine osteoclasts were seeded onto calcium phosphate multi-test slides and incubated at 37 degrees C with half media changes every 24 hours. Cells received 0, 0.15, 0.30, 0.60, and 1.50 microM nicotine, or 25 nM parathyroid hormone (PTH). Osteoclast resorption was quantified by measuring the resorbed surface area of the calcium phosphate substrate. Osteoclast cultures resorbed bone slices and calcium phosphate substrate. All nicotine concentrations and PTH resulted in statistically significantly greater mean percent resorptions than the control group (P < 0.05). However, no statistical difference was found between the various nicotine doses or PTH. The number of osteoclasts increased in a linear relationship to the increasing nicotine concentrations; however, no correlation was found between osteoclast number and the amount of resorption. Nicotine is non-toxic to osteoclasts at the clinically relevant levels tested. Nicotine appears to stimulate osteoclast differentiation and resorption of calcium phosphate, the major component of bone. Nicotine-modulated osteoclast stimulation may, in part, explain the increased rapidity of periodontal bone loss and refractory disease incidence in smokers.