High-affinity Nicotine Binding Sites Research Articles

Neurobiological Mechanisms of Nicotine Reward and Aversion.

Neuronal nicotinic acetylcholine receptors (nAChRs) regulate the rewarding actions of nicotine contained in tobacco that establish and maintain the smoking habit. nAChRs also regulate the aversive properties of nicotine, sensitivity to which decreases tobacco use and protects against tobacco use disorder. These opposing behavioral actions of nicotine reflect nAChR expression in brain reward and aversion circuits. nAChRs containing α4 and β2 subunits are responsible for the high-affinity nicotine binding sites in the brain and are densely expressed by reward-relevant neurons, most notably dopaminergic, GABAergic, and glutamatergic neurons in the ventral tegmental area. High-affinity nAChRs can incorporate additional subunits, including β3, α6, or α5 subunits, with the resulting nAChR subtypes playing discrete and dissociable roles in the stimulatory actions of nicotine on brain dopamine transmission. nAChRs in brain dopamine circuits also participate in aversive reactions to nicotine and the negative affective state experienced during nicotine withdrawal. nAChRs containing α3 and β4 subunits are responsible for the low-affinity nicotine binding sites in the brain and are enriched in brain sites involved in aversion, including the medial habenula, interpeduncular nucleus, and nucleus of the solitary tract, brain sites in which α5 nAChR subunits are also expressed. These aversion-related brain sites regulate nicotine avoidance behaviors, and genetic variation that modifies the function of nAChRs in these sites increases vulnerability to tobacco dependence and smoking-related diseases. Here, we review the molecular, cellular, and circuit-level mechanisms through which nicotine elicits reward and aversion and the adaptations in these processes that drive the development of nicotine dependence. SIGNIFICANCE STATEMENT: Tobacco use disorder in the form of habitual cigarette smoking or regular use of other tobacco-related products is a major cause of death and disease worldwide. This article reviews the actions of nicotine in the brain that contribute to tobacco use disorder.

Upregulation of Nicotinic Acetylcholine Receptor alph4+beta2 through a Ligand-Independent PI3Kbeta Mechanism That Is Enhanced by TNFalpha and the Jak2/p38Mapk Pathways.

High affinity nicotine-binding sites in the mammalian brain are neuronal nicotinic acetylcholine receptors (nAChR) assembled from at least alpha4 and beta2 subunits into pentameric ion channels. When exposed to ligands such as nicotine, these receptors respond by undergoing upregulation, a correlate of nicotine addiction. Upregulation can be measured using HEK293 (293) cells that stably express alpha4 and beta2 subunits using quantification of [3H]epibatidine ([3H]Eb) binding to measure mature receptors. Treatment of these cells with choline also produces upregulation through a hemicholinium3 (HC3)-sensitive (choline kinase) and an HC3-insensitive pathway which are both independent of the mechanism used by nicotine for upregulation. In both cases, upregulation is significantly enhanced by the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) which signals through its receptor Tnfr1 to activate p38Mapk. Here we report that the inhibition of class1 phosphoinositide 3-kinases isoform PI3Kbeta using the selective antagonist PI828 is alone sufficient to produce upregulation and enhance both nicotine and choline HC3-sensitive mediated upregulation. Further, these processes are impacted upon by an AG-490 sensitive Jak2-associated pathway. Both PI3Kbeta (negative) and Jak2 (positive) modulation of upregulation converge through p38Mapk and both overlap with TNFalpha enhancement of this process. Upregulation through the PI3Kbeta pathway did not require Akt. Collectively these findings support upregulation of endogenous alpha4beta2 as a balance among cellular signaling networks that are highly responsive to multiple environmental, inflammatory and metabolic agents. The findings also suggest how illness and metabolic stress could alter the expression of this important nicotinic receptor and novel avenues to intercede in modifying its expression.

Choline Promotes Nicotinic Receptor α4 + β2 Up-regulation

Neuronal nicotinic acetylcholine receptors (nAChR) composed of α4 + β2 subunits, the high affinity nicotine-binding site in the mammalian brain, up-regulate in response to chronic nicotine exposure. The identities of endogenous mediators of this process are unknown. We find that choline also up-regulates α4 + β2 nAChRs stably expressed by HEK293 cells as measured by increased [3H]epibatidine density. Choline-mediated up-regulation is dose-dependent and corresponds with an increase in β2 subunit protein expression. The choline kinase inhibitor hemicholinium-3 inhibits ∼60% of choline-mediated up-regulation revealing both an HC3-dependent and -independent pathway. Furthermore, choline-mediated up-regulation is not additive with up-regulation agents such as nicotine, but it is additive with weaker promoters of the up-regulation process. When co-applied with the pro-inflammatory cytokine tumor necrosis factor α, choline-mediated up-regulation is increased further through a mechanism that includes an increase in both α4 and β2 protein expression, and this is inhibited by the p38 MAPK inhibitor SB202190. These findings extend the view that up-regulation of α4 + β2 nAChRs is a normal physiological response to altered metabolic and inflammatory conditions.

Alpha4 and Beta2 Subunits of Neuronal Nicotinic Acetylcholine Receptor Genes Are Not Associated with Methamphetamine‐Use Disorder in the Japanese Population

The mesolimbic system is thought to be involved in the reinforcing action of many addictive drugs and the release of dopamine modulated by neuronal nicotine cholinergic receptors (nAChRs). Several investigations suggested that nAChRs on dopaminergic terminals play an important role in the development of some long-lasting adaptations associated with drug abuse. A majority of high-affinity nicotine binding sites in the brain have been showed in heteropentameric alpha4 (alpha4) and beta2 subunit (beta2) of nAChRs. Therefore, we conducted a genetic association analysis of the alpha4 gene (CHRNA4) and beta2 gene (CHRNB2) with methamphetamine (METH)-use disorder (191 cases and 753 controls). We first evaluated the linkage disequilibrium (LD) structure of these genes and selected 7 and 5 tagging SNPs (tag SNPs) on CHRNA4 and CHRNB2, respectively. Some tag SNPs were significantly associated with total METH-use disorder and METH-induced psychosis; however, these associations were no longer statistically significant after Bonferroni's correction for multiple testing. In conclusion, our results suggest that neither CHRNA4 nor CHRNB2 plays a major role in Japanese METH-use disorder.

Genetic association analysis of tagging SNPs in alpha4 and beta2 subunits of neuronal nicotinic acetylcholine receptor genes (CHRNA4 and CHRNB2) with schizophrenia in the Japanese population

Several lines of evidence suggest that nicotinic cholinergic dysfunction may contribute to the cognitive impairments in schizophrenia. The majority of high affinity nicotine binding sites in the human brain have been implicated in heteropentameric alpha4 and beta2 subunits of neuronal nicotinic acetylcholine receptors; therefore, these two neuronal nicotinic acetylcholine receptors genes (CHRNA4 and CHRNB2) are considered to be attractive candidate genes for the pathophysiology of schizophrenia. To represent these two genes in a gene-wide manner, we first evaluated the linkage disequilibrium structure using our own control samples. Thirteen SNPs (7 SNPs for CHRNA4 and 5 SNPs for CHRNB2) were selected as tagging SNPs. Using these tagging SNPs, we then conducted genetic association analysis of case-control samples (738 schizophrenia and 753 controls) in the Japanese population. No significant association was detected in the allele/genotype-wise or haplotype-wise analysis. Our results suggest that CHRNA4 and CHRNB2 do not play a major role in Japanese schizophrenia.

Up-regulation of Nicotinic Receptors by Nicotine Varies with Receptor Subtype

Recent evidence suggests that in addition to alpha4beta2 and alpha3-containing nicotinic receptors, alpha6-containing receptors are present in midbrain dopaminergic neurons and involved in the nicotine reward pathway. Using heterologous expression, we found that alpha6beta2, like alpha3beta2 and alpha4beta2 receptors, formed high affinity epibatidine binding complexes that are pentameric, trafficked to the cell surface, and produced acetylcholine-evoked currents. Chronic nicotine exposure up-regulated alpha6beta2 receptors with differences in up-regulation time course and concentration dependence compared with alpha4beta2 receptors, the predominant high affinity nicotine binding site in brain. The alpha6beta2 receptor up-regulation required higher nicotine concentrations than for alpha4beta2 but lower than for alpha3beta2 receptors. The alpha6beta2 up-regulation occurred 10-fold faster than for alpha4beta2 and slightly faster than for alpha3beta2. Our data suggest that nicotinic receptor up-regulation is subtype-specific such that alpha6-containing receptors up-regulate in response to transient, high nicotine exposures, whereas sustained, low nicotine exposures up-regulate alpha4beta2 receptors.

215 Perinatal Nicotine Exposure Disrupts Breathing Rhythm by Interfering with O2 (NOT CO2) – Sensitive Respiratory Stabilizing Mechanisms.

There is a strong association between perinatal tobacco (especially nicotine) exposure and poor infant outcome, but the mechanisms linking the two are not well understood. Here we determined how perinatal nicotine exposure compromises neonatal respiratory control by comparing the effects of nicotine in normal (wild-type, WT) and knockout (KO) mice lacking nicotinic acetylcoline receptors (nAChRs) containing the b2 subunit. These nAChRs are a major component of high-affinity nicotine binding sites in the nervous system. Pregnant WT and KO mice were implanted with micro-osmotic minipumps on embryonic day 14. Pumps delivered either water or nicotine (6.0 mg.kg-1 day-1). Breathing was measured using dual-chamber plethysmograhy. Control WT and KO pups had comparable breathing rhythms at rest. Stress, however, unmasked important differences between genotypes. KO pups responded briskly to hypoxia, but breathing became highly irregular, with frequent pauses (apnoeas) during recovery from hypoxia. Irregular breathing and apnoeas were never seen in WT pups post-hypoxia. Control KO and WT pups responded similarly to CO2, with no evidence of breathing irregularity or apnoea in either genotype during recovery from bouts of intermittent hypercapnia. Nicotine-exposure augmented the response of the WT pups to hypoxia. WT pups also developed an irregular breathing rhythm due to frequent apnoeas between bouts of hypoxia. Nicotine-exposed WT pups consequently resembled the KO. Nicotine-exposure did not alter the hypoxic breathing pattern of KO pups. Moreover, perinatal nicotine exposure did not alter responses of either WT or KO pups to intermittent hypercapnia. Conclusion: Our findings suggest that nicotine increases the risk of neonatal apnoea by disrupting nAChR-dependent mechanisms that fine-tune breathing rhythm. These mechanisms are particularly sensitive to sudden changes in reflex drive from O2rather than CO2-sensitive chemoreflexes.

Exocytic Trafficking Is Required for Nicotine-induced Up-regulation of α4β2 Nicotinic Acetylcholine Receptors

The primary target for nicotine in the brain is the neuronal nicotinic acetylcholine receptor (nAChR). It has been well documented that nAChRs respond to chronic nicotine exposure by up-regulation of receptor numbers, which may underlie some aspects of nicotine addiction. In order to investigate the mechanism of nicotine-induced nAChR up-regulation, we have developed a cell culture system to assess membrane trafficking and nicotine-induced up-regulation of surface-expressed alpha(4)beta(2) nAChRs. Previous reports have implicated stabilization of the nAChRs at the plasma membrane as the potential mechanism of up-regulation. We have found that whereas nicotine exposure results in up-regulation of surface receptors in our system, it does not alter surface receptor internalization from the plasma membrane, postendocytic trafficking, or lysosomal degradation. Instead, we find that transport of nAChRs through the secretory pathway to the plasma membrane is required for nicotine-induced up-regulation of surface receptors. Therefore, nicotine appears to regulate surface receptor levels at a step prior to initial insertion in the plasma membrane rather than by altering their endocytic trafficking or degradation rates as had been previously suggested.

Time and brain region specific up-regulation of low affinity neuronal nicotinic receptors during chronic nicotine administration in mice

We studied the effects of chronic oral nicotine on brain low affinity nicotine binding sites. Mice received nicotine in the drinking water for 4 or 7 weeks. Receptor binding was measured at 24 or 48 h after cessation of nicotine administration with [3H]methyllycaconitine, an antagonist in α7 and α3/α6β2β3* nicotinic receptors in striatum, midbrain, hippocampus and cortex. Chronic nicotine for 4 weeks resulted in a significant increase in the [3H]methyllycaconitine binding in the striatum and cortex, whereas after 7 weeks the increase in binding could be found in the hippocampus but not in the other brain areas studied. For comparison, high affinity nicotine binding sites (mostly α4β2) were measured with [3H]epibatidine after 7-week chronic nicotine treatment. [3H]Epibatidine binding sites were increased in the hippocampus, midbrain and cortex, but not in the striatum. The up-regulation of [3H]methyllycaconitine binding was significant at 24 h but that of [3H]epibatidine binding sites was not observed until at 48 h after cessation of chronic nicotine. These results suggest that up-regulation of low affinity nicotine binding sites does occur during chronic nicotine administration. Furthermore, the low affinity and high affinity binding differ clearly as regards regions and duration suggesting that different nicotinic receptors respond differently to nicotine administration.

Beta2-containing nicotinic receptors contribute to the organization of sleep and regulate putative micro-arousals in mice.

The cholinergic system is involved in arousal and in rapid eye movement sleep (REMS). To evaluate the contribution of nicotinic acetylcholine receptors (nAChRs) to these functions, we studied with polygraphic recordings the regulation of sleep in mice lacking the beta2 subunit gene of the nAChRs, a major component of high-affinity nicotine binding sites in the brain. Nicotine (1-2 mg/kg, i.p.) increased wakefulness in wild-type but not knock-out animals, indicating that beta2-containing nAChRs mediate the arousing properties of nicotine. Under normal conditions, the beta2-/- mice displayed the same amounts of waking, non-REM sleep (NREMS) and REMS as their wild-type counterparts. However, they exhibited longer REMS episodes and a reduced fragmentation of NREMS by events characterized notably by a transient drop in EEG power and frequently associated with EMG activation, tentatively referred to as micro-arousals. Respiration monitoring showed that these events were accompanied with, but not caused by, breathing irregularities. Sleep deprivation of beta2-/- mice resulted in a normal increase in REMS episode duration and NREMS delta power but yielded a reduction of the number of micro-arousals in NREMS. In contrast, in beta2-/- mice, a 1 hr immobilization stress failed to produce the normal rebound in REMS in the following 12 hr and, instead, was associated with increased NREMS fragmentation and sustained corticosterone levels. Our results show that the beta2-containing nAChRs contribute to the organization of sleep by regulating the transient phasic activity in NREMS, the REMS onset and duration, and the REMS-promoting effect of stress.

Functional and molecular characterization of neuronal nicotinic ACh receptors in rat hippocampal interneurons

Publisher Summary This chapter elucidates the molecular makeup of functional neuronal nicotinic acetylcholine receptors (nAChRs) in various regions of the brain and nervous system, particularly in the rat hippocampal interneurons. nAChRs are ligand-gated ion channels that are widely expressed in the central and peripheral nervous system, where they can mediate fast excitatory synaptic transmission. The subunit composition of nAChRs determines their functional properties. A variety of functional and molecular studies have suggested that the two major subtypes of nAChRs found in the hippocampus are composed of α7 (the majority of the α-bungarotoxin binding sites) and α4β2 (the high affinity nicotine binding sites) subunits. Intrinsic cholinergic interneurons are shown to pattern network activity via nAChRs in rat hippocampal slices, and nAChR activation increases theta activity in freely moving rabbits. Thus, the activation of nAChRs via cholinergic input activates inhibitory interneurons, which in turn contributes to the synchronization of the principal cell firing. These mechanisms help in understanding the link between the activation of these receptors and cognition.

Characterization of human alpha 4 beta 2-nicotinic acetylcholine receptors stably and heterologously expressed in native nicotinic receptor-null SH-EP1 human epithelial cells.

Naturally expressed nicotinic acetylcholine receptors composed of alpha4 and beta2 subunits (alpha4beta2-nAChR) are the predominant form of high affinity nicotine binding site in the brain implicated in nicotine reward, mediation of nicotinic cholinergic transmission, modulation of signaling through other chemical messages, and a number of neuropsychiatric disorders. To develop a model system for studies of human alpha4beta2-nAChR allowing protein chemical, functional, pharmacological, and regulation of expression studies, human alpha4 and beta2 subunits were stably introduced into the native nAChR-null human epithelial cell line SHEP1. Heterologously expressed alpha4beta2-nAChR engage in high-affinity, specific binding of 3H-labeled epibatidine (H-EBDN; macroscopic KD = 10 pM; kon = 0.74/min/nM, koff = 0.013/min). Immunofluorescence studies show alpha4 and beta2 subunit protein expression in virtually every transfected cell, and microautoradiographic studies show expression of 125I-labeled iodo-deschloroepibatidine binding sites in most cells. H-EBDN binding competition studies reveal high affinity for nicotinic agonists and lower affinity for nicotinic antagonists. Heterologously expressed alpha4beta2-nAChR functional studies using 86Rb+ efflux assays indicate full efficacy of epibatidine, nicotine, and acetylcholine; partial efficacy for 1,1-dimethyl-4-phenyl-piperazinium, cytisine, and suberyldicholine; competitive antagonism by dihydro-beta-erythroidine, decamethonium, and methyllycaconitine; noncompetitive antagonism by mecamylamine and eserine; and mixed antagonism by pancuronium, hexamethonium, and d-tubocurarine. These results demonstrate utility of transfected SH-EP1 cells as models for studies of human alpha4beta2-nAChR, and they also reveal complex relationships between apparent affinities of drugs for radioligand binding and functional sites on human alpha4beta2-nAChR.

Melatonin modulation of presynaptic nicotinic acetylcholine receptors located on short noradrenergic neurons of the rat vas deferens: a pharmacological characterization.

Melatonin, the pineal hormone produced during the dark phase of the light-dark cycle, modulates neuronal acetylcholine receptors located presynaptically on nerve terminals of the rat vas deferens. Recently we showed the presence of high affinity nicotine-binding sites during the light phase, and low and high affinity binding sites during the dark phase. The appearance of the low affinity binding sites was due to the nocturnal melatonin surge and could be mimicked by exposure to melatonin in vitro. The aim of the present research was to identify the receptor subtypes responsible for the functional response during the light and the dark phase. The rank order of potency of agonists was dimethylphenylpiperazinium (DMPP) = cytisine > nicotine > carbachol and DMPP = nicotine = cytisine > carbachol, during the light and dark phase, respectively, due to an increase in apparent affinity for nicotine. Mecamylamine similarly blocked the DMPP response during the light and the dark phase, while the response to nicotine was more efficiently blocked during the light phase. In contrast, methyllycaconitine inhibited the nicotine-induced response only at 21:00 h. Since alpha7 nicotinic acetylcholine receptors (nAChRs) have low affinity for nicotine in binding assays, we suggest that a mixed population composed of alpha3ss4 - plus alpha7-bearing nAChR subtypes is present at night. This plasticity in receptor subtypes is probably driven by melatonin since nicotine-induced contraction in organs from animals sacrificed at 15:00 h and incubated with melatonin (100 pg/ml, 4 h) is not totally blocked by mecamylamine. Thus melatonin, by acting directly on the short adrenergic neurons that innervate the rat vas deferens, induces the appearance of the low affinity binding site, probably an alpha7 nAChR subtype.

Expression of the α3 nicotinic receptor subunit mRNA in aging and Alzheimer's disease

Changes in the number of high-affinity nicotine binding sites have been widely reported in specific regions of the human brain during aging and in degenerative neurological diseases associated with aging, such as Alzheimer's disease. Nicotinic receptors are highly diverse and a description of the molecular subtypes affected in such conditions has not been achieved to date. To investigate the status of the α3 subunit-containing subtypes in such conditions, we assessed by in situ hybridisation the α3 mRNA density in the hippocampus, entorhinal cortex and thalamus of Alzheimer's patients and age-matched controls. No significant difference in the expression of the α3 mRNA, either qualitative or quantitative, was found between Alzheimer's individuals and controls in any of the analysed areas. This result suggests that the nicotine binding changes occurring in these areas in Alzheimer's patients are not correlated to a variation of the α3 mRNA in the same regions. Nevertheless, a negative correlation between the α3 mRNA density and the age was observed in the entorhinal cortex of both the Alzheimer's and the normal subjects, suggesting a potentially extensive decay of the α3-expressing neurons or loss of α3-containing receptors in intact neurons of the entorhinal cortex in the late elderly.

Cortical cholinergic dysfunction after human head injury.

Loss of cholinergic neurotransmission is implicated in memory impairment and cognitive dysfunction after head injury. The aim of the present study was to investigate presynaptic markers, particularly in relation to cholinergic neurotransmission in human postmortem brain from patients who died following a head injury and age-matched controls. Choline acetyltransferase activity and high-affinity nicotinic receptor binding sites were assayed in the inferior temporal gyrus, cingulate gyrus, and superior parietal cortex of 16 head-injured patients and 8 controls. Synaptophysin immunoreactivity was determined in the left cingulate gyrus from the same patient groups. In the head-injured group, choline acetyltransferase activity was consistently reduced in each cortical region compared to control subjects. The presence of a subdural haematoma and a prolonged survival period after head injury tended to be associated with lower choline acetyltransferase activity. In contrast to the marked reduction in choline acetyltransferase activity, nicotine receptor binding was unchanged in head-injured compared to control patients. Synaptophysin immunoreactivity in the cingulate gyrus was reduced by approximately 30% (p < 0.05) in the head-injured group compared to controls. Correlation of choline acetyltransferase activity with synaptophysin immunoreactivity indicated there is a deficit of cholinergic presynaptic terminals in postmortem human brain following head injury.

Mice Deficient in the α7 Neuronal Nicotinic Acetylcholine Receptor Lack α-Bungarotoxin Binding Sites and Hippocampal Fast Nicotinic Currents

The alpha7 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) is abundantly expressed in hippocampus and is implicated in modulating neurotransmitter release and in binding alpha-bungarotoxin (alpha-BGT). A null mutation for the alpha7 subunit was prepared by deleting the last three exons of the gene. Mice homozygous for the null mutation lack detectable mRNA, but the mice are viable and anatomically normal. Neuropathological examination of the brain revealed normal structure and cell layering, including normal cortical barrel fields; histochemical assessment of the hippocampus was also normal. Autoradiography with [3H]nicotine revealed no detectable abnormalities of high-affinity nicotine binding sites, but there was an absence of high-affinity [125I]alpha-BGT sites. Null mice also lack rapidly desensitizing, methyllycaconitine-sensitive, nicotinic currents that are present in hippocampal neurons. The results of this study indicate that the alpha-BGT binding sites are equivalent to the alpha7-containing nAChRs that mediate fast, desensitizing nicotinic currents in the hippocampus. These mice demonstrate that the alpha7 subunit is not essential for normal development or for apparently normal neurological function, but the mice may prove to have subtle phenotypic abnormalities and will be valuable in defining the functional role of this gene product in vivo.

Synthesis of UB-165: A novel nicotinic ligand and anatoxin-a/epibatidine hybrid

Abstract UB-165 (5), a hybrid corresponding to natural anatoxin-a and epibatidine, has been synthesised and shows significant potency at the high affinity nicotine binding site in rat brain. Ent-(5) shows a much lower level of activity which parallels the sense of enantiospecificity associated with anatoxin-a.

Presence of Muscarinic Acetylcholine Receptors in the Cattle Tick Boophilus microplus and in Epithelial Tissue Culture Cells of Chironomus tentans

A muscarinic acetylcholine receptor (mAChR) has been demonstrated and partially characterized in larvae of the cattle tick Boophilus microplus. Its properties are compared with mAChR from an epithelial cell line from the dipteran insect Chironomus tentans. Competition studies with cholinergic ligands of different specificity revealed the muscarinic nature of the cholinergic receptors investigated in both species. In homogenates from tick larvae, specific binding sites for [3H]quinuclidinyl benzilate (QNB) with high affinity (1·2±(0·13) nM; Bmax 22·5 pmol mg protein−1) were detected that do not bind nicotinic compounds specifically. The estimated IC50 values for nicotine, imidacloprid and α-bungarotoxin were all in the mM range. Additionally, with tick larvae, high-affinity nicotinic binding sites were detected with [3H]nicotine which could be displaced by high concentrations of imidacloprid or QNB. The estimated IC50 values for nicotine, α-bungarotoxin, imidacloprid and QNB were 43(±8) nM, 0·8(±0·2) μM, 2·8(±0·6) μM and 78(±1·9) μM, respectively. With homogenates of the non-neuronal insect cell line from C. tentans, only high-affinity binding sites for [3H]QNB were found. Muscarinic antagonists selectively displaced [3H]quinuclidinyl benzilate (QNB) binding to tick larvae homogenates. The mAChR of B. microplus preferred pirenzepine (IC50 2·13(±1·02) μM) among different subtype-specific mAChR antagonists (4-DAMP had IC50 49·9(±9·13) μM and methoctramine had IC50 121(±14·2) μM) indicating a type of binding site similar to the vertebrate M1 mAChR subtype. The tick muscarinic receptor seems to be a G-protein-coupled receptor, as concluded from the 4·8-fold reduction in receptor affinity for binding of the muscarinic agonist oxotremorine M upon treatment with the non-hydrolysable GTP-analogue γ-S-GTP. Binding data for the agonists oxotremorine M (IC50 71·3(±19·6) μM) and carbachol (IC50 253(±87·1) μM) parallel the biological efficacy of these compounds, in that, while oxotremorine M showed some activity against ticks, carbachol was ineffective.

Presence of Muscarinic Acetylcholine Receptors in the Cattle TickBoophilus microplusand in Epithelial Tissue Culture Cells ofChironomus tentans

A muscarinic acetylcholine receptor (mAChR) has been demonstrated and partially characterized in larvae of the cattle tick Boophilus microplus. Its properties are compared with mAChR from an epithelial cell line from the dipteran insect Chironomus tentans. Competition studies with cholinergic ligands of different specificity revealed the muscarinic nature of the cholinergic receptors investigated in both species. In homogenates from tick larvae, specific binding sites for [ 3 H]quinuclidinyl benzilate (QNB) with high affinity (1.2 ± (0.13) nM ; B max 22.5 pmol mg protein -1 ) were detected that do not bind nicotinic compounds specifically. The estimated IC 50 values for nicotine, imidacloprid and α-bungarotoxin were all in the mM range. Additionally, with tick larvae, high-affinity nicotinic binding sites were detected with [ 3 H]nicotine which could be displaced by high concentrations of imidacloprid or QNB. The estimated IC 50 values for nicotine, α-bungarotoxin, imidacloprid and QNB were 43(±8) nM, 0.8(±0.2) μM, 2.8(±0.6) μM and 78(±1.9) μM, respectively. With homogenates of the non-neuronal insect cell line from C. tentans, only high-affinity binding sites for [ 3 H]QNB were found. Muscarinic antagonists selectively displaced [ 3 H]quinuclidinyl benzilate (QNB) binding to tick larvae homogenates. The mAChR of B. microplus preferred pirenzepine (IC 50 2.13(±1.02) μM) among different subtype-specific mAChR antagonists (4-DAMP had IC 50 49.9(±9.13) μM and methoctramine had IC 50 121(±14.2) μM) indicating a type of binding site similar to the vertebrate M1 mAChR subtype. The tick muscarinic receptor seems to be a G-protein-coupled receptor, as concluded from the 4.8-fold reduction in receptor affinity for binding of the muscarinic agonist oxotremorine M upon treatment with the non-hydrolysable GTP-analogue γ-S-GTP. Binding data for the agonists oxotremorine M (IC 50 71.3(±19.6) μM) and carbachol (IC 50 253(±87.1) μM) parallel the biological efficacy of these compounds, in that, while oxotremorine M showed some activity against ticks, carbachol was ineffective.

Long-term ethanol and nicotine treatment elicit tolerance to ethanol.

Several previous studies have shown that 1 to 2 weeks of treatment with ethanol elicits tolerance to several effects produced by ethanol and cross-tolerance to nicotine-induced hypothermia. Similarly, short-term, high-dose nicotine treatment produces tolerance to nicotine and cross-tolerance to ethanol-induced hypothermia. In the studies reported here, C57BL/6 mice were force-fed ethanol, nicotine, or an ethanol/nicotine combination in the drinking water for 6 months. All of the chronic drug-treated mice developed tolerance to ethanol as measured by open-field activity, body temperature, and sleep-time tests. Ethanol tolerance is due, in part, to enhanced metabolism and reduced CNS sensitivity in the two ethanol-treated groups but only to reduced CNS sensitivity in the nicotine-treated group. Similar levels of tolerance to nicotine developed in those two groups that were nicotine-treated, but no tolerance to nicotine was seen in those animals treated with ethanol alone. The tolerance to nicotine may be related to an upregulation of brain (cortex, hippocampus, and hypothalamus) [3H]-nicotine binding, but ethanol tolerance is not readily explained by changes in the number of the brain high affinity nicotine binding sites.