Preproenkephalin mRNA and Methionine-Enkephalin Increase in Mouse Striatum After 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Treatment

Dopaminergic neurons that project to the striatum from the substantia nigra are thought to modulate methionine-enkephalin (Met-Enk) metabolism in the striatum. We administered a dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that produces a moderate depletion of dopamine in striatum, about 50%, without overt motor deficits, and found that Met-Enk-like immunoreactivity and preproenkephalin mRNA content increased in the tissue. Pretreatment with the monoamine oxidase B inhibitor deprenyl or the dopamine transport blocker nomifensine prevented these changes, suggesting that the changes were related to the partial loss of dopaminergic neurons rather than to MPTP. Moreover, administering GM1 ganglioside, which partially restores the MPTP-induced dopaminergic deficit, partially corrected the Met-Enk changes in the striatum as well. These findings are consistent with the hypothesis that dopaminergic input to the striatum, in part, modulates Met-Enk metabolism. Moreover, they show that moderate nigrostriatal lesions are sufficient to elevate Met-Enk and preproenkephalin mRNA contents and that restoration of dopaminergic function, as in our studies with GM1 ganglioside, restores the content of Met-Enk.     

Central injection of nicotine increases hepatic and splenic interleukin 6 (IL-6) mRNA expression and plasma IL-6 levels in mice: involvement of the peripheral sympathetic nervous system.

Accumulating evidence suggests that plasma levels of interleukin 6 (IL-6), a major cytokine stimulating the synthesis of acute-phase proteins, are intimately regulated by the central nervous system. Nicotine, one of the major drugs abused by humans, has been shown to affect immunological functions. In the present study, effects of intracerebroventricular (i.c.v.) injection of nicotine on plasma IL-6 levels were investigated in mice. Nicotine administered i.c.v. dose-dependently increased plasma IL-6 levels; the lowest effective dose was 0.3 ng/mouse and the maximal effect was attained with the dose of 105 ng/mouse. The nicotine (105 ng/mouse, i.c.v.)-induced plasma IL-6 levels peaked at 3 h and approached basal levels 6 h after injection. Mecamylamine, a nicotinic receptor antagonist, blocked nicotine-induced plasma IL-6 levels. Depletion of peripheral norepinephrine with 6-hydroxydopamine [100 mg/kg, intraperitoneal (i. p.)] inhibited the nicotine-induced plasma IL-6 levels by 57%, whereas central norepinephrine depletion with 6-hydroxydopamine (50 microgram/mouse, i.c.v.) had no effect. Pretreatment with prazosin (alpha1-adrenergic antagonist; 1 mg/kg, i.p.), yohimbine (alpha2-adrenergic antagonist; 1 mg/kg, i.p.), and ICI-118,551 (beta2-adrenergic antagonist; 2 mg/kg, i.p.), but not with betaxolol (beta1-adrenergic antagonist; 2 mg/kg, i.p.), inhibited nicotine-induced plasma IL-6 levels. Among the peripheral organs, including the pituitary, adrenals, heart, lung, liver, spleen, and lymph nodes, nicotine (105 ng/mouse, i.c.v.) increased IL-6 mRNA expression only in the liver and spleen, which was inhibited by peripheral norepinephrine depletion. These results suggest that stimulation of central nicotinic receptors induces plasma IL-6 levels and IL-6 mRNA expression in the liver and spleen via the peripheral sympathetic nervous system, alpha1-, alpha2-, and beta2-adrenoreceptors being involved.     

Acute nicotine administration increases somatostatin content and binding in the rat hypothalamus.

Within 4 minutes a single, intravenous injection of nicotine (0.3 mg/Kg) induced increases in somatostatin-like immunoreactivity concentrations in the rat hypothalamus but not in the striatum. These changes were associated with a significant increase in the specific binding of somatostatin to putative receptor sites in hypothalamic membranes, while no significant changes were found in striatum. The enhancement of somatostatin binding resulted from a rapid increase in the number of available receptors rather than a change in receptor affinity. This effect appears to be mediated by nicotinic cholinergic receptors, because pretreatment with a centrally active nicotinic receptor antagonist, mecamylamine (5.0 mg/Kg i.v.), prevented the nicotine-induced changes in somatostatin content and binding in the hypothalamus. Mecamylamine alone had no observable effect on the hypothalamic somatostatinergic system. These results suggest that the rat hypothalamic somatostatinergic system can be regulated by nicotine-like acetylcholine receptors.     

Activation of Tyrosine Hydroxylase by Nicotine in Rat Adrenal Gland

The administration of nicotine activates tyrosine hydroxylase in the rat adrenal gland. This activation is apparently maximal 25 min after a single subcutaneous injection of nicotine at 2.3 mg/kg. Repeated injections of nicotine (seven injections once every 30 min) are associated with a persistent activation of adrenal tyrosine hydroxylase for at least 3 h. The nicotinic receptor antagonist hexamethonium does not significantly inhibit the nicotine-mediated activation of tyrosine hydroxylase in innervated adrenal glands. However, hexamethonium completely blocks the activation of adrenal tyrosine hydroxylase by nicotine in denervated adrenal glands. Furthermore, even though a single injection of nicotine activates tyrosine hydroxylase in both innervated and denervated adrenal glands, repeated injections of nicotine do not activate tyrosine hydroxylase in denervated adrenal glands. Our results suggest that the systemic administration of nicotine activates adrenal tyrosine hydroxylase by two mechanisms: (1) via direct interaction with adrenal chromaffin cell nicotinic receptors; and (2) via stimulation of the CNS leading to the release from the splanchnic nerve of substances that interact with adrenal chromaffin cell receptors other than the nicotinic receptor.     

Parallel Decrease of Glutamic Acid Decarboxylase and Preproenkephalin mRNA in the Rat Striatum Following Chronic Treatment with a Dopaminergic D1 Antagonist and D2 Agonist

The levels of mRNA encoding glutamic acid decarboxylase (GAD) and preproenkephalin (PPE) were measured by Northern blot analysis, in the dorsal and the ventral part of the striatum, following long-term treatments with drugs acting selectively on D1 or D2 dopaminergic receptors. Chronic injection of the selective D1 antagonist SCH 23390 elicited a significant decrease in level of both GAD and PPE mRNA (-30%) in the dorsal striatum, whereas no significant change was observed in the ventral striatum. Chronic administration of both SCH 23390 and RU 24926, a D2 agonist, decreased the GAD and PPE mRNA levels in the dorsal (-38 and -57%, respectively) as well as in the ventral (-70 and -60%, respectively) striatum. In the ventral striatum the marked reduction of GAD mRNA levels was paralleled by a significant decrease of Vmax values of GAD enzymatic activity (-41%). These results suggest that the decrease in content of both GAD and PPE mRNA, promoted by the chronic blockade of D1 receptors, is mainly due to the action of dopamine acting on unaffected D2 receptors. Indeed, this decrease is further amplified when the D2 agonist and the D1 antagonist are administered together. Our results substantiate further the molecular mechanisms by which dopamine acts on different populations of GABAergic and enkephalinergic neurons in the two striatal regions examined.     

Genetic variation within the Chrna7 gene modulates nicotine reward‐like phenotypes in mice

Mortality from tobacco smoking remains the leading cause of preventable death in the world, yet current cessation therapies are only modestly successful, suggesting new molecular targets are needed. Genetic analysis of gene expression and behavior identified Chrna7 as potentially modulating nicotine place conditioning in the BXD panel of inbred mice. We used gene targeting and pharmacological tools to confirm the role of Chrna7 in nicotine conditioned place preference (CPP). To identify molecular events downstream of Chrna7 that may modulate nicotine preference, we performed microarray analysis of α7 knock‐out (KO) and wild‐type (WT) nucleus accumbens (NAc) tissue, followed by confirmation with quantitative polymerase chain reaction (PCR) and immunoblotting. In the BXD panel, we found a putative cis expression quantitative trait loci (eQTL) for Chrna7 in NAc that correlated inversely to nicotine CPP. We observed that gain‐of‐function α7 mice did not display nicotine preference at any dose tested, whereas conversely, α7 KO mice demonstrated nicotine place preference at a dose below that routinely required to produce preference. In B6 mice, the α7 nicotinic acetylcholine receptor (nAChR)‐selective agonist, PHA‐543613, dose‐dependently blocked nicotine CPP, which was restored using the α7 nAChR‐selective antagonist, methyllycaconitine citrate (MLA). Our genomic studies implicated a messenger RNA (mRNA) co‐expression network regulated by Chrna7 in NAc. Mice lacking Chrna7 demonstrate increased insulin signaling in the NAc, which may modulate nicotine place preference. Our studies provide novel targets for future work on development of more effective therapeutic approaches to counteract the rewarding properties of nicotine for smoking cessation .     

Effect of Chronic Nicotine Treatment on Nicotinic Autoreceptor Function and N-[3H]Methylcarbamylcholine Binding Sites in the Rat Brain

It has been reported that N-methylcarbamylcholine (MCC), a nicotinic agonist, binds to central nicotinic receptors and causes an increase of acetylcholine (ACh) release from certain central cholinergic nerve terminals. The present experiments determine whether these two phenomena change in response to the chronic administration of nicotine, a procedure known to result in an increase in nicotinic binding sites. Chronic nicotine caused a brain region-specific up-regulation of [3H]MCC sites; binding increased in the frontal cortex, parietal cortex, striatum, and hippocampus, but not in the occipital cortex or cerebellum. The effect of nicotine was selective to nicotinic binding sites, because muscarinic sites, both M1 ([ 3H]pirenzepine) and M2 ([3H]ACh), were unaffected by chronic nicotine treatment. MCC increased the release of ACh from the frontal cortex and hippocampus by a calcium-dependent mechanism; MCC did not alter ACh release from striatum or occipital cortex of control animals. The MCC-induced increase in ACh release was not apparent in those animals which had been treated with nicotine. There was a partial recovery of nicotinic autoreceptor function when animals were allowed to recover (4 days) following chronic nicotine treatment, but the density of binding sites remained increased compared to control. Chronic nicotine did not change the potassium-evoked release of ACh from the frontal cortex or hippocampus, but decreased this measure from striatum. It also decreased the ACh content of the striatum, but not that of the cortex or the hippocampus; the activity of choline acetyltransferase was not altered in any of the regions tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presynaptic Nicotinic Modulation of Dopamine Release in the Three Ascending Pathways Studied by In Vivo Microdialysis: Comparison of Naive and Chronic Nicotine-Treated Rats

Abstract: The modulation of dopamine release by presynaptic nicotinic receptors in vitro is well established, but the significance of this effect in vivo is unclear. We have characterised the effect of nicotine, locally applied via a microdialysis probe, on dopamine release from the terminal regions of three ascending dopaminergic pathways in conscious, freely moving rats. Nicotine caused a dose-dependent increase in dopamine release in the striatum, the nucleus accumbens, and, to a lesser extent, the frontal cortex. Metabolite levels were unaltered by any concentration of nicotine. Prior administration of mecamylamine via the probe abolished the nicotine-evoked increase in dopamine release, confirming the mediation of nicotinic receptors. The dose dependence of mecamylamine-sensitive, nicotine-evoked dopamine release was similar in all three brain regions. However, 10⁻⁵ M tetrodotoxin totally blocked nicotine-stimulated dopamine release in the striatum and the accumbens but not the cortex. Daily subcutaneous injections of nicotine (0.4 mg kg⁻¹ for 7 days) increased the response to a subsequent local application of nicotine in the striatum, and a similar trend was found in the other brain areas. The same daily dose of nicotine given as a continuous infusion had no effect, whereas infusion of 4 mg kg⁻¹ day⁻¹ increased the response to a subsequent nicotine challenge. The localisation and regulation of nicotinic receptors in the terminal fields of dopaminergic pathways are discussed.     

Activation of p-ERK1/2 by nicotine in pancreatic tumor cell line AR42J: effects on proliferation and secretion

The objectives of the present study were to determine the effect of nicotine on MAPK signaling and on the proliferation of AR42J cells as well as to assess the relationship between MAPK activation and exocrine secretion in these cells. AR42J cells were incubated with nicotine and analyzed for the activation of MAPK by Western blot analysis using their respective antibodies and confirmed by immunohistochemistry. The effect of nicotine on cell proliferation was determined by the spectrophotometric method, and cell function was assessed by cholecystokinin (CCK)-stimulated amylase release into the culture medium. Nicotine at a dose of 100 microM induced phospho-ERK1/2 activation maximally in 3 min compared with untreated cells. Furthermore, immunofluorescence study confirmed the nicotine-induced increase in translocation of phospho-ERK1/2 to the nucleus. Activation of phospho-ERK1/2 was inhibited by an ERK1/2 pathway inhibitor but not by a nicotine receptor antagonist. At the same dose, there was significantly enhanced proliferation of AR42J cells until 72 h without toxic effect, as the percentage of lactate dehydrogenase release remained unchanged. Other MAPKs, c-Jun NH2-terminal kinase 1/2 and p38 MAPK, were not affected by nicotine treatment. At a nicotine dose of 100 microM, the CCK-stimulated release of amylase was maximal at 6 min, and, although a nicotinic receptor antagonist inhibited this response, it was not inhibited by the ERK1/2 pathway inhibitor. We conclude that nicotine treatment induced activation of ERK1/2 and increased the proliferation of AR42J cells. The data further indicate that MAPK signaling by nicotine is independent of the secretory response.     

The alpha4beta2alpha5 nicotinic cholinergic receptor in rat brain is resistant to up-regulation by nicotine in vivo

We used immunoprecipitation with subunit-specific antibodies to examine the distribution of heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) that contain the α5 subunit in the adult rat brain. Among the regions of brain we surveyed, the α5 subunit is associated in ∼37% of the nAChRs in the hippocampus, ∼24% of the nAChRs in striatum, and 11–16% of the receptors in the cerebral cortex, thalamus, and superior colliculus. Sequential immunoprecipitation assays demonstrate that the α5 subunit is associated with α4β2* nAChRs exclusively. Importantly, in contrast to α4β2 nAChRs, which are increased by 37–85% after chronic administration of nicotine, the α4β2α5 receptors are not increased by nicotine treatment. These data thus indicate that the α4β2α5 nAChRs in rat brain are resistant to up-regulation by nicotine in vivo , which suggests an important regulatory role for the α5 subunit. To the extent that nicotine-induced up-regulation of α4β2 nAChRs is involved in nicotine addiction, the resistance of the α4β2α5 subtype to up-regulation may have important implications for nicotine addiction.     

Genetic correlations between nicotine reinforcement‐related behaviors and propensity toward high or low alcohol preference in two replicate mouse lines

Common genetic factors may contribute to the high comorbidity between tobacco smoking and alcohol use disorder. Here, we assessed behavioral and biological effects of nicotine in replicate mouse lines selectively bred for high (HAP2/3) or low alcohol preference (LAP2/3). In Experiment 1, free‐choice (FC) oral nicotine and quinine intake were assessed in HAP2/3 and LAP2/3 mice. Effects of nicotinic acetylcholine receptor blockade by mecamylamine on nicotine intake in HAP2 mice were also examined. In Experiment 2, HAP2/3 and LAP2/3 mice were tested for differences in sensitivity to nicotine‐induced taste conditioning. In Experiment 3, the effects of a single nicotine injection on nucleus accumbens (NAc) and dorsal striatum monoamine levels in HAP2/3 and LAP2/3 mice were tested. In Experiment 1, HAP2/3 mice showed greater nicotine intake and intake ratio than LAP2/3 mice. There were no line differences in quinine intake. Mecamylamine reduced nicotine intake and intake ratio in HAP2 mice. In Experiment 2, HAP2/3 mice showed weaker nicotine‐induced conditioned taste aversion (CTA) compared with LAP2/3 mice. In Experiment 3, nicotine treatment increased NAc dopamine turnover across both HAP2/3 and LAP2/3 mouse lines. These results show that there is a positive genetic correlation between oral alcohol intake (high alcohol intake/preference selection phenotype) and oral nicotine intake and a negative genetic correlation between oral alcohol intake and sensitivity to nicotine‐induced CTA.     

Nicotinic alpha5 subunit deletion locally reduces high-affinity agonist activation without altering nicotinic receptor numbers

Neuronal nicotinic acetylcholine receptor subunit alpha5 mRNA is widely expressed in the CNS. An alpha5 gene polymorphism has been implicated in behavioral differences between mouse strains, and alpha5-null mutation induces profound changes in mouse acute responses to nicotine. In this study, we have examined the distribution and prevalence of alpha5* nicotinic acetylcholine receptor in mouse brain, and quantified the effects of alpha5-null mutation on pre-synaptic nicotinic acetylcholine receptor function (measured using synaptosomal (86)Rb(+) efflux) and overall [(125)I]epibatidine binding site expression. alpha5* nicotinic acetylcholine receptor expression was found in nine of fifteen regions examined, although < 20% of the total nicotinic acetylcholine receptor population in any region contained alpha5. Deletion of the alpha5 subunit gene resulted in localized loss of function (thalamus, striatum), which was itself confined to the DHbetaE-sensitive receptor population. No changes in receptor expression were seen. Consequently, functional changes must occur as a result of altered function per unit of receptor. The selective depletion of high agonist activation affinity sites results in overall nicotinic function being reduced, and increases the overall agonist activation affinity. Together, these results describe the receptor-level changes underlying altered behavioral responses to nicotine in nicotinic acetylcholine receptor alpha5 subunit-null mutants.     

Effect of tianeptine and fluoxetine on the levels of Met-enkephalin and mRNA encoding proenkephalin in the rat.

The purpose of the present study was to evaluate the effects of acute and repeated treatment with two antidepressant drugs (ADs) of opposite pharmacological profile, i.e. tianpetine (TIA, serotonin reuptake enhancer) and fluoxetine (FLU, serotonin reuptake inhibitor) on the levels of Met-Enkephalin, (Met-Enk, a member ofopioid peptide family, which has been suggested to play a role in the mechanism of action ADs) as well as on mRNA coding for proenkephalin (mRNA PENK) in various regions of the rat brain, pituitary, adrenal glands and plasma. Male Wistar rats were treated acutely or repeatedly (10 mg/kg p.o., twice daily for 14 days) with TIA or FLU. Tissue for biochemical experiments was taken 2 h after last dose of appropriate drug. The levels of Met-Enk were estimated by radioimmunoassay, mRNA PENK was measured using in situ hybridization. From the results obtained in the present study it may be concluded that repeated administration of TIA or FLU induced similar changes in the levels of Met-Enk in the rat hippocampus, striatum, hypothalamus and neurointermediate lobe of pituitary. Such an effect is interesting, especially if one takes into account the differences in pharmacological profile between these two antidepressant drugs. It may be suggested that serotonin level might not be crucial for inducing the alterations in the content of Met-Enk. Since we did not observe any changes in the levels of PENK mRNA in the studied rat brain regions after repeated administration of TIA or FLU, it seems that the observed changes in the levels of Met-Enk do not result from effects of these antidepressants on biosynthesis of PENK, but rather from alterations in the peptide release. Another interesting finding of the present study was that in the anterior lobe of pituitary, adrenal glands and plasma, repeated administration of TIA induced alterations in the contents of Met-Enk, while repeated administration of FLU remained without any effect. It is tempting to speculate that such a differentiation between the effects of these two antidepressants might be linked to the well known feature of TIA (but not FLU) which has been shown to reduce both basal and stress-evoked activity of the hypothalamic-pituitary-adrenal (HPA) axis.     

Interactive effects of nicotine and MPTP on striatal tetrahydrobiopterin in mice

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin known to cause dopamine (DA) neuron degeneration, while the psychoactive compound nicotine is known to excite DA neurons. Tetrahydrobiopterin is the cofactor for tyrosine hydroxylase (TOH) in the regulation of DA biosynthesis. The present study investigated the interactions between nicotine and MPTP on striatal biopterin, DA and TOH activity in BALB/c mice. The results indicated that both acute and chronic nicotine administrations at various concentrations significantly increased biopterin and DA levels in the striatum, while MPTP markedly decreased these measures. Pretreatment with nicotine at a dose having no significant effect alone, partially protected against MPTP's toxicity on biopterin and DA. Increasing the dose of nicotine did not have a further protective action. The toxicity of MPTP on TOH was also prevented by nicotine. Further, the above effects of nicotine were probably mediated through the cholinergic nicotinic receptors since mecamylamine reversed the effects of nicotine. These results suggest that nicotine interacts with the dopaminergic system probably at the level of DA biosynthesis through activating TOH and its coenzyme tetrahydrobiopterin.     

Effects of acetylcholine on sodium-dependent high-affinity glutamate transport in rat striatal homogenates

Incubation of rat striatal tissue in the presence of acetylcholine, carbachol, oxotremorine, or nicotine results in a significant decrease in the sodium-dependent high-affinity glutamate uptake (HAGU). The cholinergic inhibitory effect on glutamate transport is no more detectable in the presence of atropine, a cholinergic receptor antagonist. These data support the hypothesis that glutamatergic nerve ending activity in the striatum is modulated by cholinergic neurons. The effects would involve both muscarinic and nicotinic presynaptic receptors located on the corticostriatal glutamatergic terminals.     

二甲基苯蒽对小鼠皮肤表皮鸟氨酸脱羧酶和转化生长因子βmRNA水平的影响

本文利用Ｎｏｒｔｈｅｒｎ印迹分析技术研究了完全致癌物二甲基苯蒽（ＤＭＢＡ）对小鼠表皮鸟氨酸脱羧酶（ＯＤＣ）ｍＲＮＡ和转化生长因子β（ＴＧＦ－β）ｍＲＮＡ水平的影响。ＤＭＢＡ在１５μｇ和９００μｇ剂量下一次涂用于小鼠皮肤,可使表皮ＯＤＣｍＲＮＡ和ＴＧＦ－βｍＲＮＡ表达增加。ＯＤＣｍＲＮＡ为单一的２．０ｋｂ大小的条带,其表达在在３６ｈ时最为明显。而ＴＧＦ－βｍＲＮＡ大小为２．５ｋｂ和１．９ｋｂ,其表达在３６ｈ时最高,到４８ｈ几乎降至正常,但在７２ｈ又有增加。ＯＤＣ和ＴＧＦ－β可能在ＤＭＢＡ诱癌过程中起重要作用。     

Binding and functional activity of nicotinic cholinergic receptors in selected rat brain regions are increased following long-term but not short-term nicotine treatment

Chronic nicotine exposure up-regulates neuronal nicotinic receptors, but the functional consequences for these receptors is less well understood. Following 2 weeks of nicotine or saline treatment by osmotic minipump, the functional activity of nicotinic receptors was measured by concentration-response curves for epibatidine-stimulated (86)Rb efflux. Nicotine-treated animals had a significantly higher maximal efflux in cerebral cortex and superior colliculus, but not in thalamus or interpeduncular nucleus plus medial habenula. This increase was confirmed in a separate experiment with stimulation by single concentrations of epibatidine (cortex, superior colliculus) or nicotine (cortex only). Chronic nicotine did not alter (86)Rb efflux stimulated by cytisine, an alpha3beta4-selective agonist, or by potassium chloride, in any region. Short-term (16 h) nicotine exposure caused no changes in either (86)Rb efflux or receptor binding measured with [(3)H]epibatidine. Binding was significantly increased after 2 weeks nicotine exposure in cortex, superior colliculus and thalamus, but not in interpeduncular nucleus plus medial habenula. The increases in epibatidine-stimulated (86)Rb efflux in the four regions tested was linearly correlated with the increases in [(3)H]epibatidine binding in these regions (R(2) = 0.91), suggesting that rat brain receptors up-regulated by chronic nicotine are active. These results have important consequences for understanding nicotinic receptor neurobiology in smokers and users of nicotine replacement therapy.     

Acute reduction of brain substance P induced by nicotine

Ten minutes after a single injection of 0.8 mg/kg nicotine SC (free base) the level of substance P-like immunoreactivity (SPLI) was reduced by 61–73% in rat caudate-putamen, nucleus accumbens, and olfactory tubercle, with smaller and not significant reductions in the frontal cortex, substantia nigra, and ventral tegmental area. The nicotinic receptor antagonist mecamylamine (1.0 mg/kg IP) prevented the reductions in SPLI. The rapidity and the degree of the changes in SPLI after nicotine exceed those previously reported for other agents and implicate substance P neurotransmission as a major component of nicotinic action.Preliminary data were presented at the 17th annual meeting of the American Society for Neurochemistry, Montreal, 1986 (1).