Effects of methoxsalen, a CYP2A5/6 inhibitor, on nicotine dependence behaviors in mice

Metabolism of nicotine to inactive cotinine by hepatic enzyme CYP2A6 is the principal pathway by which active nicotine is removed from circulation. We therefore hypothesized that inhibition of mouse CYP2A5, the ortolog of human CYP2A6, by methoxsalen (8-methoxypsoralen) alter dependence-related behaviors of nicotine in the mouse. Conditioned place preference (CPP) test was used to assess the appetitive reward-like properties and precipitated nicotine withdrawal to assess physical (somatic and hyperalgesia) and affective (anxiety-related behaviors) measures. The nicotine plasma levels were also measured with or without methoxsalen pretreatment. Methoxsalen (15 and 30 mg/kg, intraperitoneally) pretreatment enhanced nicotine-induced preference in mice (p < 0.05). However, there was a lack of enhancement of nicotine in the CPP test after the highest dose of the CYP-2A5 inhibitor. Similarly to the CPP results, repeated administration of methoxsalen increased the intensity of mecamylamine-precipitated withdrawal signs. The potentiation of nicotine preference and withdrawal intensity by methoxsalen was accompanied by significant increase in nicotine plasma levels in mice (p < 0.05). Finally, methoxsalen enhanced the ability of a very low dose of nicotine (0.05 mg/kg) to reverse withdrawal signs in mice undergoing spontaneous withdrawal after chronic nicotine infusion (p < 0.05). In conclusion, inhibition of nicotine metabolism by methoxsalen alters the behavioral effects of nicotine in the mouse. Combining CYP2A6 inhibitors with low dose nicotine replacement therapies may have a beneficial role in smoking cessation because it will decrease the drug elimination rate and maintain plasma and brain nicotine levels.United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (DA-05274)United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (R01DA032246) (P30DA033934) (P50DA005274

Functional interaction between Lypd6 and nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) affect multiple physiological functions in the brain and their functions are modulated by regulatory proteins of the Lynx family. Here, we report for the first time a direct interaction of the Lynx protein LY6/PLAUR domain‐containing 6 (Lypd6) with nAChRs in human brain extracts, identifying Lypd6 as a novel regulator of nAChR function. Using protein cross‐linking and affinity purification from human temporal cortical extracts, we demonstrate that Lypd6 is a synaptically enriched membrane‐bound protein that binds to multiple nAChR subtypes in the human brain. Additionally, soluble recombinant Lypd6 protein attenuates nicotine‐induced hippocampal inward currents in rat brain slices and decreases nicotine‐induced extracellular signal‐regulated kinase phosphorylation in PC12 cells, suggesting that binding of Lypd6 is sufficient to inhibit nAChR‐mediated intracellular signaling. We further show that perinatal nicotine exposure in rats (4 mg/kg/day through minipumps to dams from embryonic day 7 to post‐natal day 21) significantly increases Lypd6 protein levels in the hippocampus in adulthood, which did not occur after exposure to nicotine in adulthood only. Our findings suggest that Lypd6 is a versatile inhibitor of cholinergic signaling in the brain, and that Lypd6 is dysregulated by nicotine exposure during early development. [Image: see text] Regulatory proteins of the Lynx family modulate the function of nicotinic receptors (nAChRs). We report for the first time that the Lynx protein Lypd6 binds to nAChRs in human brain extracts, and that recombinant Lypd6 decreases nicotine‐induced ERK phosphorylation and attenuates nicotine‐induced hippocampal inward currents. Our findings suggest that Lypd6 is a versatile inhibitor of cholinergic signaling in the brain

Effects of Menthol on Nicotine Pharmacokinetic, Pharmacology and Dependence in Mice.

Although menthol, a common flavoring additive to cigarettes, has been found to impact the addictive properties of nicotine cigarettes in smokers little is known about its pharmacological and molecular actions in the brain. Studies were undertaken to examine whether the systemic administration of menthol would modulate nicotine pharmacokinetics, acute pharmacological effects (antinociception and hypothermia) and withdrawal in male ICR mice. In addition, we examined changes in the brain levels of nicotinic receptors of rodents exposed to nicotine and menthol. Administration of i.p. menthol significantly decreased nicotine's clearance (2-fold decrease) and increased its AUC compared to i.p. vehicle treatment. In addition, menthol pretreatment prolonged the duration of nicotine-induced antinociception and hypothermia (2.5 mg/kg, s.c.) for periods up to 180 min post-nicotine administration. Repeated administration of menthol with nicotine increased the intensity of mecamylamine-precipitated withdrawal signs in mice exposed chronically to nicotine. The potentiation of withdrawal intensity by menthol was accompanied by a significant increase in nicotine plasma levels in these mice. Western blot analyses of α4 and β2 nAChR subunit expression suggests that chronic menthol impacts the levels and distribution of these nicotinic subunits in various brain regions. In particular, co-administration of menthol and nicotine appears to promote significant increase in β2 and α4 nAChR subunit expression in the hippocampus, prefrontal cortex and striatum of mice. Surprisingly, chronic injections of menthol alone to mice caused an upregulation of β2 and α4 nAChR subunit levels in these brain regions. Because the addition of menthol to tobacco products has been suggested to augment their addictive potential, the current findings reveal several new pharmacological molecular adaptations that may contribute to its unique addictive profile

Effects of menthol pretreatment on nicotine-induced antinociception and hypothermia dose-response curves in mice.

<p>Vehicle or menthol (100 mg/kg, ip) was administered 30 min before various doses of nicotine (0.5, 1.5, 2, and 2.5 mg/kg s.c.) and mice were tested in <b>(A)</b> the tail-flick test, <b>(B)</b> the hot-plate test, and <b>(C)</b> hypothermia. Each point represents the mean ± SE of 8–12 mice.</p

Nicotine Enhances the Hypnotic and Hypothermic Effects of Alcohol in the Mouse

BACKGROUND: Ethanol (EtOH) and nicotine abuse are 2 leading causes of preventable mortality in the world, but little is known about the pharmacological mechanisms mediating co-abuse. Few studies have examined the interaction of the acute effects of EtOH and nicotine. Here, we examine the effects of nicotine administration on the duration of EtOH-induced loss of righting reflex (LORR) and characterize the nature of their pharmacological interactions in C57BL/6J mice. METHODS: We assessed the effects of EtOH and nicotine and the nature of their interaction in the LORR test using isobolographic analysis after acute injection in C57BL/6J male mice. Next, we examined the importance of receptor efficacy using nicotinic partial agonists varenicline and sazetidine. We evaluated the involvement of major nicotinic acetylcholine receptor (nAChR) subtypes using nicotinic antagonist mecamylamine and nicotinic α4- and α7-knockout mice. The selectivity of nicotine\u27s actions on EtOH-induced LORR was examined by testing nicotine\u27s effects on the hypnotic properties of ketamine and pentobarbital. We also assessed the development of tolerance after repeated nicotine exposure. Last, we assessed whether the effects of nicotine on EtOH-induced LORR extend to hypothermia and EtOH intake in the drinking in the dark (DID) paradigm. RESULTS: We found that acute nicotine injection enhances EtOH\u27s hypnotic effects in a synergistic manner and that receptor efficacy plays an important role in this interaction. Furthermore, tolerance developed to the enhancement of EtOH\u27s hypnotic effects by nicotine after repeated exposure of the drug. α4* and α7 nAChRs seem to play an important role in nicotine-EtOH interaction in the LORR test. In addition, the magnitude of EtOH-induced LORR enhancement by nicotine was more pronounced in C57BL/6J than DBA/2J mice. Furthermore, acute nicotine enhanced ketamine and pentobarbital hypnotic effects in the mouse. Finally, nicotine enhanced EtOH-induced hypothermia but decreased EtOH intake in the DID test. CONCLUSIONS: Our results demonstrate that nicotine synergistically enhances EtOH-induced LORR in the mouse

Summary of the potency of nicotine in male and female mice in different behavioral tests.

<p>ED₅₀ values (±CL) were calculated from the dose-response curve of the different groups and expressed as mg/kg.</p><p>Animals were pretreated with either vehicle or menthol (100 mg/kg, i.p.) followed by nicotine (2.5 mg/kg s.c.) at different doses and then tested 5 min (tail-flick and hot-plate tests) or 20 min later (hypothermia). Each point represents the mean ± SE of 6 to 8 mice.</p

Nicotine plasma levels in adult male ICR mouse after pre-treatment with vehicle or menthol in the withdrawal testing.

<p>*P<0.05 compared to vehicle/nicotine group.</p><p>Mice received nicotine (12 mg/kg/day) for 7 days. On day 8, mice were sacrificed and blood samples were drawn. Values are shown as mean ± S.E.M. (n = 6-8/group)</p

Time course of nicotine plasma concentrations in mice pretreated with menthol.

<p>Nicotine was administered (2.5 mg/kg s.c.), 30 min after pretreatment with vehicle or menthol (100 mg/kg i.p.). Each time point represents the mean ± SEM of 7 to 10 animals. For the vehicle pretreatment, values for nicotine plasma levels at 2 h were below the limits of detection. Each point represents the mean ± SE of 8–12 mice. Nic = nicotine.</p