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1.  Modulation of TNF Release by Choline Requires α7 Subunit Nicotinic Acetylcholine Receptor-Mediated Signaling 
Molecular Medicine  2008;14(9-10):567-574.
The α7 subunit-containing nicotinic acetylcholine receptor (α7nAChR) is an essential component in the vagus nerve-based cholinergic anti-inflammatory pathway that regulates the levels of TNF, high mobility group box 1 (HMGB1), and other cytokines during inflammation. Choline is an essential nutrient, a cell membrane constituent, a precursor in the biosynthesis of acetylcholine, and a selective natural α7nAChR agonist. Here, we studied the anti-inflammatory potential of choline in murine endotoxemia and sepsis, and the role of the α7nAChR in mediating the suppressive effect of choline on TNF release. Choline (0.1–50 mM) dose-dependently suppressed TNF release from endotoxin-activated RAW macrophage-like cells, and this effect was associated with significant inhibition of NF-κB activation. Choline (50 mg/kg, intraperitoneally [i.p.]) treatment prior to endotoxin administration in mice significantly reduced systemic TNF levels. In contrast to its TNF suppressive effect in wild type mice, choline (50 mg/kg, i.p.) failed to inhibit systemic TNF levels in α7nAChR knockout mice during endotoxemia. Choline also failed to suppress TNF release from endotoxin-activated peritoneal macrophages isolated from α7nAChR knockout mice. Choline treatment prior to endotoxin resulted in a significantly improved survival rate as compared with saline-treated endotoxemic controls. Choline also suppressed HMGB1 release in vitro and in vivo, and choline treatment initiated 24 h after cecal ligation and puncture (CLP)-induced polymicrobial sepsis significantly improved survival in mice. In addition, choline suppressed TNF release from endotoxin-activated human whole blood and macrophages. Collectively, these data characterize the anti-inflammatory efficacy of choline and demonstrate that the modulation of TNF release by choline requires α7nAChR-mediated signaling.
PMCID: PMC2435495  PMID: 18584048
2.  Oral choline supplementation for postoperative pain 
BJA: British Journal of Anaesthesia  2013;111(2):249-255.
Activation of nicotinic receptors with nicotine has been shown to reduce post-surgical pain in clinical and preclinical studies. Choline is a selective agonist at α7-type nicotinic receptors that does not have addictive or sympathetic activating properties. It is anti-nociceptive in animal studies. We conducted a double-blind randomized trial of oral choline supplementation with lecithin to aid in the treatment of pain after gynaecological surgery.
Sixty women having open gynaecological surgery were randomly assigned to receive 20 g of lecithin before surgery or placebo. Plasma choline concentration and tumour necrosis factor (TNF) were measured. Pain report was the primary outcome measure.
We achieved a small but statistically significant increase in choline after surgery with oral supplementation. Plasma TNF was not decreased and pain report was not different between groups at rest or with movement. There were no adverse effects of treatment.
Oral supplementation with lecithin during the perioperative period resulted in very slow absorption and thus only a small increase in plasma choline was achieved. This concentration was inadequate to reduce TNF as has been shown in other studies. The absence of an anti-inflammatory effect was likely related to our failure to demonstrate efficacy in pain reduction.
PMCID: PMC3841409  PMID: 23568851
analgesia; anti-inflammatory agents; nutritional requirements; pain; pain measurement
Brain, behavior, and immunity  2010;24(6):959-967.
Nicotinic acetylcholine receptors (nAchR) are key receptors in the autonomic nervous system, but also are present on immune cells. The alpha seven subunit of nAchR (α7nAchR) suppresses pro-inflammation in peripheral monocytes by decreasing proinflammatory cytokine production. In spinal cord, α7nAchR are found on microglia, which are known to induce and maintain pain. We predicted that α7nAchR agonists might attenuate intrathecal HIV-1 gp120-induced, proinflammatory cytokine- and microglia-dependent mechanical allodynia. Choline, a precursor for acetylcholine and selective agonist for α7nAchR, was administered intrathecally either with, or 30 min after, intrathecal gp120. Choline significantly blocked and reversed gp120 induced mechanical allodynia for at least 4 hr after drug administration. In addition, intrathecal choline, delivered either with or 30 min after gp120, reduced gp120-induced IL-1β protein and pro-inflammatory cytokine mRNAs within the lumbar spinal cord. A second α7nAchR agonist, GTS-21, also significantly reversed gp120-induced mechanical allodynia and lumbar spinal cord levels of proinflammatory cytokine mRNAs and IL-1β protein. A role of microglia is suggested by the observation that intrathecal choline suppressed the gp120-induced expression of, cd11b, a macrophage/microglial activation marker. Taken together, the data support that α7nAchR may be a novel target for treating pain where microglia maintain the proinflammatory state within the spinal cord.
PMCID: PMC2902784  PMID: 20353818
pain; choline; intrathecal; rats; tumor necrosis factor
4.  Activation of the α7 nAChR Reduces Acid-Induced Acute Lung Injury in Mice and Rats 
New evidence indicates that neural mechanisms can down-regulate acute inflammation. In these studies, we tested the potential role of the α7 nicotinic acetylcholine receptor (α7 nAChR) in a rodent model of acid-induced acute lung injury. We first determined that the α7 nAChR was expressed by alveolar macrophages and lung epithelial cells. Then, using an acid-induced acute lung injury mouse model, we found that nicotine, choline, and PNU-282987 (a specific α7 nAChR agonist) decreased excess lung water and lung vascular permeability, and reduced protein concentration in the bronchoalveolar lavage (BAL). Deficiency of α7 nAChR resulted in a 2-fold increase in excess lung water and lung vascular permeability. The reduction of proinflammatory cytokines (macrophage inflammatory protein-2 and TNF-α) in the BAL with nicotine probably resulted from the suppression of NF-κB activation in alveolar macrophages. The beneficial effect of nicotine was also tested in rat model of acid-induced acute lung injury in which BAL protein and receptor for advanced glycation end products (RAGE), a marker of type I cell injury, were reduced by nicotine treatment. These results indicate that activation of α7 nAChR may provide a new therapeutic pathway for the treatment of acute lung injury.
PMCID: PMC1976545  PMID: 17431097
α7 nicotinic acetylcholine receptor; acute lung injury; alveolar macrophage; nicotine; proinflammatory cytokines
5.  Peripheral nerve injury alters spinal nicotinic acetylcholine receptor pharmacology 
European journal of pharmacology  2008;590(1-3):163-169.
Nicotinic acetylcholine receptors are widely expressed in the rat spinal cord and modulate innocuous and nociceptive transmission. The present studies were designed to investigate the plasticity of spinal nicotinic acetylcholine receptors modulating mechanosensitive information following spinal nerve ligation. A tonic inhibitory cholinergic tone mediated by dihydro-β-erythroidine- (DHβE) and methyllycaconitine- (MLA) sensitive nicotinic acetylcholine receptors was identified in the normal rat spinal cord and cholinergic tone at both populations of nicotinic acetylcholine receptors was lost ipsilateral to spinal nerve ligation. The administration of intrathecal nicotinic acetylcholine receptor agonists reduced mechanical paw pressure thresholds with a potency of epibatidine = A-85380 >> nicotine > choline in the normal rat. Following spinal nerve ligation, intrathecal epibatidine and nicotine produced an ipsilateral antinociception, but intrathecal A-85380 and choline did not. The antinociceptive response to intrathecal nicotine was blocked with the α7* and α9α10*-selective nicotinic acetylcholine receptor antagonist, MLA, and the αβ heteromeric nicotinic acetylcholine receptor antagonist, DHβE. The antinociceptive effects of both intrathecal nicotine and epibatidine were mediated by GABAA receptors. Spinal [3H]epibatidine saturation binding was unchanged in spinal nerve-ligated rats, but spinal nerve ligation did increase the ability of nicotine to displace [3H]epibatidine from spinal cord membranes. Spinal nerve ligation altered the expression of nicotinic acetylcholine receptor subunits ipsilaterally, with a large increase in the modulatory α5 subunit. Taken together these results suggest that pro- and antinociceptive populations of spinal nicotinic acetylcholine receptors modulate the transmission of mechanosensitive information and that spinal nerve ligation-induced changes in spinal nicotinic acetylcholine receptors likely result from a change in subunit composition rather than overt loss of nicotinic acetylcholine receptor subtypes.
PMCID: PMC2569862  PMID: 18573248
neuropathic pain; antinociception; nicotine; nociception
6.  Cytidine 5′-Diphosphocholine (CDP-Choline) in Stroke and Other CNS Disorders 
Neurochemical research  2005;30(1):15-23.
Brain phosphatidylcholine (PC) levels are regulated by a balance between synthesis and hydrolysis. Pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1α/β) activate phospholipase A2 (PLA2) and PC-phospholipase C (PC-PLC) to hydrolyze PC. PC hydrolysis by PLA2 releases free fatty acids including arachidonic acid, and lyso-PC, an inhibitor of CTP-phosphocholine cytidylyltransferase (CCT). Arachidonic acid metabolism by cyclooxygenases/lipoxygenases is a significant source of reactive oxygen species. CDP-choline might increase the PC levels by attenuating PLA2 stimulation and loss of CCT activity. TNF-α also stimulates proteolysis of CCT. TNF-α and IL-1β are induced in brain ischemia and may disrupt PC homeostasis by increasing its hydrolysis (increase PLA2 and PC-PLC activities) and inhibiting its synthesis (decrease CCT activity). The beneficial effects of CDP-choline may result by counteracting TNF-α and/or IL-1 mediated events, integrating cytokine biology and lipid metabolism. Re-evaluation of CDP-choline phase III stroke clinical trial data is encouraging and future trails are warranted. CDP-choline is non-xenobiotic, safe, well tolerated, and can be considered as one of the agents in multi-drug treatment of stroke.
PMCID: PMC1934404  PMID: 15756928
Cerebral ischemia; citicoline; clinical trials; interleukin-1β; phospholipases; phospholipids; reactive oxygen species; tumor necrosis factor; lipidomics
7.  An Autoradiographic Analysis Of Rat Brain Nicotinic Receptor Plasticity Following Dietary Choline Modification 
Choline is known to be involved with numerous physiological functions of the nervous system and also acts as a direct acting agonist of α7 nicotinic acetylcholine receptors (nAChRs). The purpose of this study was to conduct a brain region-specific evaluation of changes in nAChR subtype expression following dietary choline modification. In addition, we assessed changes in body weight, food/water intake, as well as changes spatial learning (Morris Water Maze) in response to dietary choline modification. Male Sprague Dawley rats were exposed to standard, choline supplemented or choline deficient diets for periods of 14 or 28 days. Choline supplemented animals gained significantly less weight over the course of the experiment, in spite of the fact that there were minimal differences in food consumption between the dietary regimens. Spatial memory did not differ between animals maintained on a standard rat diet, and the choline supplemented food. Brains of the animals kept on the diets for 14 and 28 days were used for quantitative autoradiographic analysis of nicotinic receptor subtypes using 125I-Bungarotoxin (α7) and 125I-Epibatidine (non-α7). There were no significant differences in nicotinic receptor binding or physiologic parameters measured between animals fed standard and choline deficient diets. However 2 weeks of dietary choline supplementation caused significant up-regulation of α7 receptors without significant effect on the density of non-α7 nAChRs. Increases in BTX binding predominantly occurred in cortical and hippocampal brain regions and ranged between 14 and 30 percent depending on the brain region. The results of our study suggest that choline acts as a selective agonist at α7 nicotinic cholinergic receptors in the rat central nervous system.
PMCID: PMC1976431  PMID: 16753203
ℑ7 nAChR; Cholinergic; Acetylcholine; Morris Water Maze; Up-regulation; Bungarotoxin; Epibatidine
8.  The Effects of Perinatal Choline Supplementation on Hippocampal Cholinergic Development in Rats Exposed to Alcohol During the Brain Growth Spurt 
Hippocampus  2012;22(8):1750-1757.
Prenatal alcohol exposure leads to long-lasting cognitive and attention deficits, as well as hyperactivity. Using a rat model, we have previously shown that perinatal supplementation with the essential nutrient, choline, can reduce the severity of some fetal alcohol effects, including hyperactivity and deficits in learning and memory. In fact, choline can mitigate alcohol-related learning deficits even when administered after developmental alcohol exposure, during the postnatal period. However, it is not yet known how choline is able to mitigate alcohol-related behavioral alterations. Choline may act by altering cholinergic signaling in the hippocampus. This study examined the effects of developmental alcohol exposure and perinatal choline supplementation on hippocampal M1 and M2/4 muscarinic receptors. Sprague-Dawley rat pups were orally intubated with ethanol (5.25 mg/kg/day) from postnatal days (PD) 4-9, a period of brain development equivalent to the human 3rd trimester; control subjects received sham intubations. From PD 4-30, subjects were injected s.c. with choline chloride (100 mg/kg/day) or saline vehicle. Open field activity was assessed from PD 30-33 and brain tissue was collected on PD 35 for autoradiographic analysis. Ethanol-exposed subjects were more active compared to controls during the first two days of testing, an effect attenuated with choline supplementation. Developmental alcohol exposure significantly decreased the density of muscarinic M1 receptors in the dorsal hippocampus, an effect that was not altered by choline supplementation. In contrast, developmental alcohol exposure significantly increased M2/4 receptor density, an effect mitigated by choline supplementation. In fact, M2/4 receptor density of subjects exposed to alcohol and treated with choline did not differ significantly from that of controls. These data suggest that developmental alcohol exposure can cause long-lasting changes in the hippocampal cholinergic system and that perinatal choline supplementation may attenuate alcohol-related behavioral changes by influencing cholinergic systems.
PMCID: PMC3382021  PMID: 22431326
muscarinic; fetal alcohol; treatment; hyperactivity; nutrition
9.  Upregulating Nonneuronal Cholinergic Activity Decreases TNF Release from Lipopolysaccharide-Stimulated RAW264.7 Cells 
Mediators of Inflammation  2014;2014:873728.
Nonneuronal cholinergic system plays a primary role in maintaining homeostasis. It has been proved that endogenous neuronal acetylcholine (ACh) could play an anti-inflammatory role, and exogenous cholinergic agonists could weaken macrophages inflammatory response to lipopolysaccharide (LPS) stimulation through activation of α7 subunit-containing nicotinic acetylcholine receptor (α7nAChR). We assumed that nonneuronal cholinergic system existing in macrophages could modulate inflammation through autocrine ACh and expressed α7nAChR on the cells. Therefore, we explored whether LPS continuous stimulation could upregulate the nonneuronal cholinergic activity in macrophages and whether increasing autocrine ACh could decrease TNF release from the macrophages. The results showed that, in RAW264.7 cells incubated with LPS for 20 hours, the secretion of ACh was significantly decreased at 4 h and then gradually increased, accompanied with the enhancement of α7nAChR expression level. The release of TNF was greatly increased from RAW264.7 cells at 4 h and 8 h exposure to LPS; however, it was suppressed at 20 h. Upregulating choline acetyltransferase (ChAT) expression through ChAT gene transfection could enhance ACh secretion and reduce TNF release from the infected RAW264. 7cells. The results indicated that LPS stimulation could modulate the activity of nonneuronal cholinergic system of RAW264.7 cells. Enhancing autocrine ACh production could attenuate TNF release from RAW264.7 cells.
PMCID: PMC3964895  PMID: 24733966
10.  Neuronal stimulation with 5-hydroxytryptamine 4 receptor induces anti-inflammatory actions via α7nACh receptors on muscularis macrophages associated with postoperative ileus 
Gut  2010;60(5):638-647.
The main symptom of postoperative ileus (POI) is an intestinal motility disorder in which monocytes/macrophages and neutrophils play crucial roles. Prokinetic 5-hydroxytryptamine 4 receptor (5-HT4R) agonists and dopamine receptor antagonists are potential therapeutic agents for directly ameliorating the motility disorder associated with POI.
To determine the effects of the 5-HT4R agonists mosapride citrate (MOS) and CJ-033466 on intestinal smooth muscle contractility relative to immune reactions after POI.
Intestinal manipulation (IM) was applied to the rat distal ileum. Both MOS (0.3 and 1 mg/kg, s.c.) and CJ-033466 (1 mg/kg, s.c.) were administered to the animals before and after IM. At 24 h after IM, isolated intestinal smooth muscle contractile activity in vitro, gastrointestinal transit in vivo, inflammatory mediator expression and leucocyte infiltration were measured.
After IM, ileal circular muscle contractility in vitro and gastrointestinal transit in vivo were reduced and the number of macrophages and neutrophils increased in the inflamed muscle layer, resulting in the induction of inflammatory mediators such as interleukin 1 β (IL-1β), IL-6, tumour necrosis factor α (TNFα), monocyte chemoattractant protein 1 (MCP-1) and inducible nitric oxide synthase (iNOS). Both MOS and CJ-033466 significantly attenuated not only the intestinal motility dysfunction but also the leucocyte infiltration and inflammatory mediator expression after IM. The autonomic ganglionic blocker hexamethonium (1 mg/kg, i.p.) and the α7-nicotinic acetylcholine receptor (α7nAChR) antagonist methyl lycaconitine citrate (0.087 mg/kg, i.p.) blocked MOS-mediated ameliorative actions. Immunohistochemically, α7nAChR is expressed by monocytes/macrophages but not by neutrophils in the inflamed intestine.
Stimulating the 5-HT4R accelerates acetyl choline (ACh) release from cholinergic myenteric neurons, which subsequently activates α7nAChR on activated monocytes/macrophages to inhibit their inflammatory reactions in the muscle layer. In addition to their gastroprokinetic action, 5-HT4R agonists might serve as novel therapeutic agents for POI characterised by anti-inflammatory potency.
PMCID: PMC3071096  PMID: 21115544
Prokinetic agents; motility disorder; abdominal surgery; macrophages; inflammation; macrophages; motility disorders; prokinetic agent
11.  N,N′-Alkane-diyl-bis-3-picoliniums as Nicotinic Receptor Antagonists: Inhibition of Nicotine-induced Dopamine Release and Hyperactivity 
The current study evaluated a new series of N,N′-alkane-diyl-bis-3-picolinium (bAPi) analogs with C6–C12 methylene linkers as nicotinic receptor (nAChR) antagonists, for nicotine-evoked [3H]dopamine (DA) overflow, for blood-brain barrier choline transporter affinity and for attenuation of discriminative stimulus and locomotor stimulant effects of nicotine. bAPi analogs exhibited little affinity forα4β2* andα7* high affinity ligand binding sites, nor for nAChRs modulating DA transporter function. With the exception of C6, all analogs inhibited nicotine-evoked [3H]DA overflow (IC50=2 nM–6μM; Imax=54–64%), with N,N′-dodecane-1,12-diyl-bis-3-picolinium dibromide (C12, bPiDDB) being most potent. bPiDDB did not inhibit electrically-evoked [3H]DA overflow, suggesting specific nAChR inhibitory effects and a lack of toxicity to DA neurons. Schild analysis suggested that bPiDDB interacts in an orthosteric manner at nAChRs mediating nicotine-evoked [3H]DA overflow. To determine if bPiDDB interacts with α-conotoxin MII-sensitive α6β2-containing nAChRs, slices were exposed concomitantly to maximally-effective concentrations of bPiDDB (10 nM) and α-conotoxin MII (1 nM). Inhibition of nicotine-evoked [3H]DA overflow was not different with the combination compared with either antagonist alone, suggesting that bPiDDB interacts with α6β2-containing nAChRs. C7, C8, C10 and C12 analogs exhibited high affinity for the blood-brain barrier choline transporter in vivo, suggesting brain bioavailability. Although, none of the analogs altered the discriminative stimulus effect of nicotine, C8, C9, C10 and C12 analogs decreased nicotine-induced hyperactivity in nicotine-sensitized rats, without reducing spontaneous activity. Further development of nAChR antagonists that inhibit nicotine-evoked DA release and penetrate brain to antagonize DA-mediated locomotor stimulant effects of nicotine as novel treatments for nicotine addiction is warranted.
PMCID: PMC3089982  PMID: 18460644
12.  Influence of δ-Opioid Receptors in the Behavioral Effects of Nicotine 
Neuropsychopharmacology  2012;37(10):2332-2344.
Multiple studies in animal models and humans suggest that the endogenous opioid system is an important neurobiological substrate for nicotine addictive properties. In this study, we evaluated the participation of δ-opioid receptors in different behavioral responses of nicotine by using δ-opioid receptor knockout mice. Acute nicotine administration induced hypolocomotion and antinociception in wild-type mice, which were similar in knockout animals. The development of tolerance to nicotine-induced antinociception was also similar in both genotypes. In agreement, the expression and functional activity of δ-opioid receptors were not modified in the different layers of the spinal cord and brain areas evaluated after chronic nicotine treatment. The somatic manifestation of the nicotine withdrawal syndrome precipitated by mecamylamine was also similar in wild-type and δ-opioid receptor knockout mice. In contrast, nicotine induced a conditioned place preference in wild-type animals that was abolished in knockout mice. Moreover, a lower percentage of acquisition of intravenous nicotine self-administration was observed in mice lacking δ-opioid receptors as well as in wild-type mice treated with the selective δ-opioid receptor antagonist naltrindole. Accordingly, in-vivo microdialysis studies revealed that the enhancement in dopamine extracellular levels induced by nicotine in the nucleus accumbens was reduced in mutant mice. In summary, the present results show that δ-opioid receptors are involved in the modulation of nicotine rewarding effects. However, this opioid receptor does not participate either in several acute effects of nicotine or in the development of tolerance and physical dependence induced by chronic nicotine administration.
PMCID: PMC3422497  PMID: 22669166
dependence; tolerance; reward; knockout; microdialysis; self-administration; behavioral science; addiction & substance abuse; animal models; psychopharmacology; dependence; tolerance; reward; knockout; microdialysis
13.  Cardiopulmonary Arrest and Resuscitation Disrupts Cholinergic Anti-Inflammatory Processes: A Role for Cholinergic α7 Nicotinic Receptors 
The Journal of Neuroscience  2011;31(9):3446-3452.
Cardiac arrest is a leading cause of death worldwide. While survival rates following sudden cardiac arrest remain relatively low, recent advancements in patient care have begun to increase the proportion of individuals who survive cardiac arrest. However, many of these individuals subsequently develop physiological and psychiatric conditions that likely result from ongoing neuroinflammation and neuronal death. The present study was conducted to better understand the pathophysiological effects of cardiac arrest on neuronal cell death and inflammation, and their modulation by the cholinergic system. Using a well validated model of cardiac arrest, here we show that global cerebral ischemia increases microglial activation, proinflammatory cytokine mRNA expression (interleukin-1β, interleukin-6, tumor necrosis factor-α), and neuronal damage. Cardiac arrest also induces alterations in numerous cellular components of central cholinergic signaling, including a reduction in choline acetyltransferase enzymatic activity and the number of choline acetyltransferase-positive neurons, as well as, reduced acetylcholinesterase and vesicular acetylcholine transporter mRNA. However, treatment with a selective agonist of the α7 nicotinic acetylcholine receptor, the primary receptor mediating the cholinergic anti-inflammatory pathway, significantly decreases the neuroinflammation and neuronal damage resulting from cardiac arrest. These data suggest that global cerebral ischemia results in significant declines in central cholinergic signaling, which may in turn diminish the capacity of the cholinergic anti-inflammatory pathway to control inflammation. Furthermore, we provide evidence that pharmacological activation of α7 nicotinic acetylcholine receptors provide significant protection against ischemia-related cell death and inflammation within a clinically relevant time frame.
PMCID: PMC3758544  PMID: 21368056
14.  Rivastigmine Alleviates Experimentally Induced Colitis in Mice and Rats by Acting at Central and Peripheral Sites to Modulate Immune Responses 
PLoS ONE  2013;8(2):e57668.
The cholinergic anti-inflammatory system and α7 nicotinic receptors in macrophages have been proposed to play a role in neuroimmunomodulation and in the etiology of ulcerative colitis. We investigated the ability of a cholinesterase (ChE) inhibitor rivastigmine, to improve the pathology of ulcerative colitis by increasing the concentration of extracellular acetylcholine in the brain and periphery. In combination with carbachol (10 µM), rivastigmine (1 µM) significantly decreased the release of nitric oxide, TNF-α, IL-1β and IL-6 from lipopolysaccharide-activated RAW 264.7 macrophages and this effect was abolished by α7 nicotinic receptor blockade by bungarotoxin. Rivastigmine (1 mg/kg) but not (0.5 mg/kg), injected subcutaneously once daily in BALB/c mice with colitis induced by 4% dextran sodium sulphate (DSS), reduced the disease activity index (DAI) by 60% and damage to colon structure. Rivastigmine (1 mg/kg) also reduced myeloperoxidase activity and IL-6 by >60%, and the infiltration of CD11b expressing cells by 80%. These effects were accompanied by significantly greater ChE inhibition in cortex, brain stem, plasma and colon than that after 0.5 mg/kg. Co-administration of rivastigmine (1 mg/kg) with the muscarinic antagonist scopolamine significantly increased the number of CD11b expressing cells in the colon but did not change DAI compared to those treated with rivastigmine alone. Rivastigmine 1 and 2 mg given rectally to rats with colitis induced by rectal administration of 30 mg dintrobezene sulfonic acid (DNBS) also caused a dose related reduction in ChE activity in blood and colon, the number of ulcers and area of ulceration, levels of TNF-α and in MPO activity. The study revealed that the ChE inhibitor rivastigmine is able to reduce gastro-intestinal inflammation by actions at various sites at which it preserves ACh. These include ACh released from vagal nerve endings that activates alpha7 nicotinic receptors on circulating macrophages and in brainstem neurons.
PMCID: PMC3585220  PMID: 23469045
15.  Targeting alpha-7 nicotinic neurotransmission in schizophrenia: A novel agonist strategy 
Schizophrenia research  2013;148(0):138-144.
Alpha7 nicotinic acetylcholine receptor (α7 nAChR) agonists may be valuable treatments for negative symptoms and cognitive impairment in schizophrenia. Unfortunately, chronic exposure to an agonist may reduce the receptor’s sensitivity. Therefore, we combined CDP-choline, a dietary source of the direct agonist choline, with galantamine, a positive allosteric modulator (PAM) of nicotinic acetylcholine receptors, to improve the efficiency of transducing the choline signal and, possibly, preserve the receptor in a sensitive state. We conducted a single-site, double-blind randomized clinical trial comparing galantamine/CDP-choline to placebos in schizophrenia patients with negative symptoms who were receiving second generation antipsychotics. Forty-three subjects received galantamine and CDP-choline or matching placebos for 16 weeks. The primary outcome measure was the 5-item Marder negative-symptoms factor of the Positive and Negative Syndrome Scale (PANSS). Cognition and functioning were also assessed. Trial completion was high; 79%. There was no significant treatment effect on negative symptoms, other PANSS symptom factors, or the MATRICS Cognitive Consensus Battery. There were significant treatment effects in overall functioning and a test of free verbal recall. Three subjects discontinued treatment in the active treatment group for gastro-intestinal adverse events (AE). The most common AE for galantamine/CDP-choline was abdominal pain; for placebo it was headache and sweating. Although there was no significant treatment effect on negative symptoms, the direction of effect mirrored the effects on a cognitive measure and overall functioning. Further study of α7 nAChR agonist/PAMs are warranted in larger studies that will have greater power.
PMCID: PMC3732552  PMID: 23768813
acetylcholine; alpha7 nicotinic receptor; schizophrenia; positive allosteric modulator; negative symptoms; neurocognition
16.  Activation of α7 nicotinic acetylcholine receptor by nicotine selectively up-regulates cyclooxygenase-2 and prostaglandin E2 in rat microglial cultures 
Nicotinic acetylcholine (Ach) receptors are ligand-gated pentameric ion channels whose main function is to transmit signals for the neurotransmitter Ach in peripheral and central nervous system. However, the α7 nicotinic receptor has been recently found in several non-neuronal cells and described as an important regulator of cellular function. Nicotine and ACh have been recently reported to inhibit tumor necrosis factor-α (TNF-α) production in human macrophages as well as in mouse microglial cultures. In the present study, we investigated whether the stimulation of α7 nicotinic receptor by the specific agonist nicotine could affect the functional state of activated microglia by promoting and/or inhibiting the release of other important pro-inflammatory and lipid mediator such as prostaglandin E2.
Expression of α7 nicotinic receptor in rat microglial cell was examined by RT-PCR, immunofluorescence staining and Western blot. The functional effects of α7 receptor activation were analyzed in resting or lipopolysaccharide (LPS) stimulated microglial cells pre-treated with nicotine. Culture media were assayed for the levels of tumor necrosis factor, interleukin-1β, nitric oxide, interleukin-10 and prostaglandin E2. Total RNA was assayed by RT-PCR for the expression of COX-2 mRNA.
Rat microglial cells express α7 nicotinic receptor, and its activation by nicotine dose-dependently reduces the LPS-induced release of TNF-α, but has little or no effect on nitric oxide, interleukin-10 and interleukin-1β. By contrast, nicotine enhances the expression of cyclooxygenase-2 and the synthesis of one of its major products, prostaglandin E2.
Since prostaglandin E2 modulates several macrophage and lymphocyte functions, which are instrumental for inflammatory resolution, our study further supports the existence of a brain cholinergic anti-inflammatory pathway mediated by α7 nicotinic receptor that could be potentially exploited for novel treatments of several neuropathologies in which local inflammation, sustained by activated microglia, plays a crucial role.
PMCID: PMC548670  PMID: 15670336
Brain macrophages; inflammation; TNF; IL-10; Prostaglandin E2
17.  Block of Muscle Nicotinic Receptors by Choline Suggests that the Activation and Desensitization Gates Act as Distinct Molecular Entities 
The Journal of General Physiology  2006;127(6):703-717.
Ion channel block in muscle acetylcholine nicotinic receptors (AChRs) is an extensively reported phenomenon. Yet, the mechanisms underlying the interruption of ion flow or the interaction of the blocker with the channel's gates remain incompletely characterized. In this paper, we studied fast channel block by choline, a quaternary-ammonium cation that is also an endogenous weak agonist of this receptor, and a valuable tool in structure–function studies. Analysis of the single-channel current amplitude as a function of both choline concentration and voltage revealed that extracellular choline binds to the open-channel pore with millimolar apparent affinity (KB ≅ 12 mM in the presence of ∼155 mM monovalent and 3.5 mM divalent, inorganic cations), and that it permeates the channel faster than acetylcholine. This, together with its relatively small size (∼5.5 Å along its longest axis), suggests that the pore-blocking choline binding site is the selectivity filter itself, and that current blockages simply reflect the longer-lived sojourns of choline at this site. Kinetic analysis of single-channel traces indicated that increasing occupancy of the pore-blocking site by choline (as judged from the reduction of the single-channel current amplitude) is accompanied by the lengthening of (apparent) open interval durations. Consideration of a number of possible mechanisms firmly suggests that this prolongation results from the local effect of choline interfering with the operation of the activation gate (closure of blocked receptors is slower than that of unblocked receptors by a factor of ∼13), whereas closure of the desensitization gate remains unaffected. Thus, we suggest that these two gates act as distinct molecular entities. Also, the detailed understanding gained here on how choline distorts the observed open-time durations can be used to compensate for this artifact during activation assays. This correction is necessary if we are to understand how choline binds to and gates the AChR.
PMCID: PMC2151541  PMID: 16735755
18.  Short-term nutritional folate deficiency in rats has a greater effect on choline and acetylcholine metabolism in the peripheral nervous system than in the brain, and this effect escalates with age 
Nutrition research (New York, N.Y.)  2010;30(10):722-730.
The hypothesis of this study is that a folate-deficient diet (FD) has a greater effect on cholinergic system in the peripheral nervous system than in the brain, and that this effect escalates with age. It was tested by comparing choline and acetylcholine levels in male Sprague Dawley rats fed either control or folate-deficient diets for 10 weeks, starting at age 4 weeks (the young group) or 9 months (the adult group). FD consumption resulted in depletion of plasma folate in both age groups. In young folate-deficient rats, liver and lung choline levels were significantly lower than those in the respective controls. No other significant effects of FD on choline and acetylcholine metabolism were found in young rats. In adult rats, FD consumption markedly decreased choline levels in the liver, kidneys, and heart; furthermore, choline levels in the cortex and striatum were moderately elevated, although hippocampal choline levels were not affected. Acetylcholine levels were higher in the heart, cortex, and striatum but lower in the hippocampus in adult folate-deficient rats, as compared to controls. Higher acetylcholine levels in the striatum in adult folate-deficient rats were also associated with higher dopamine release in the striatal slices. Thus, both age groups showed higher cholinergic metabolic sensitivity to FD in the peripheral nervous system than in the brain. However, compensatory abilities appeared to be better in the young group, implicating the adult group as a preferred model for further investigation of folate-choline-acetylcholine interactions and their role in brain plasticity and cognitive functions.
PMCID: PMC3000554  PMID: 21056288
Folic acid; choline; neurotransmitters; liver; hippocampal choline; rats
19.  Specificity and Control of Choline–O-Sulfate Transport in Filamentous Fungi 
Journal of Bacteriology  1968;96(5):1574-1585.
Choline-O-sulfate uptake by Penicillium notatum showed the following characteristics. (i) Transport was mediated by a permease which is highly specific for choline-O-sulfate. No significant inhibition of transport was caused by choline, choline-O-phosphate, acetylcholine, ethanolamine-O-phosphate, ethanolamine-O-sulfate, methanesulfonyl choline, 2-aminoethane thiosulfate, or the monomethyl or dimethyl analogues of choline-O-sulfate. Similarly, no significant inhibition was caused by any common sulfur amino acid or inorganic sulfur compound. Mutants lacking the inorganic sulfate permease possessed the choline-O-sulfate permease at wild-type levels. (ii) Choline-O-sulfate transport obeyed saturation kinetics (Km = 10−4 to 3 × 10−4m; Vmax = 1 to 6 μmoles per g per min). The kinetics of transport between 10−9 and 10−1m external choline-O-sulfate showed that only one saturable mechanism is present. (iii) Transport was sensitive to 2,4-dinitrophenol, azide, N-ethylmaleimide, p-chloromercuribenzoate, and cyanide. Ouabain, phloridzin, and eserine had no effect. (iv) Transport was pH-dependent with an optimum at pH 6. Variations in the ionic strength of the incubation medium had no effect. (v) Transport was temperature-dependent with a Q10 of greater than 2 between 3 and 40 C. Transport decreased rapidly above 40 C. (vi) Ethylenediaminetetraacetate (sodium salts, pH 6) had no effect, nor was there any stimulation by metal or nonmetal ions. Cu++, Ag+, and Hg++ were inhibitory. (vii) The initial rate at which the ester is transported was independent of intracellular hydrolysis. After long periods of incubation (> 10 min), a significant proportion of the transported choline-O-sulfate was hydrolyzed intracellulary. In the presence of 5 × 10−3m external choline-O-sulfate, the mycelia accumulated choline-O-sulfate to an apparent intracellular concentration of 0.075 m by 3 hr. Transport was unidirectional. No efflux or exchange of 35S-choline-O-sulfate was observed when preloaded mycelia were suspended in buffer alone or in buffer containing a large excess of unlabeled choline-O-sulfate. (viii) The specific transport activity of the mycelium depended on the sulfur source used for growth. (ix) Sulfur starvation of sulfur-sufficient mycelium resulted in an increase in the specific transport activity of the mycelium. This increase was prevented by cycloheximide, occurred only when a metabolizable carbon source was present, and resulted from an increase in the Vmax of the permease, rather than from a decrease in Km. The increase could be partially reversed by refeeding the mycelia with unlabeled choline-O-sulfate, sulfide, sulfite, l-homocysteine, l-cysteine, or compounds easily converted to cysteine. The results strongly suggested that the choline-O-sulfate permease is regulated primarily by repression-derepression, but that intracellular choline-O-sulfate and cysteine can act as feedback inhibitors.
PMCID: PMC315213  PMID: 5726299
20.  Antinociceptive and Anti-Inflammatory Effects of Octacosanol from the Leaves of Sabicea grisea var. grisea in Mice 
Sabicea species are used in the Amazon for treatment of fever and malaria, which suggests that its chemical constituents may have some effect on pain and inflammation. Phytochemical analysis of the hexane fraction obtained from the crude ethanol extract from Sabicea grisea var. grisea Cham. & Schltdl (Rubiaceae), an endemic plant in Brazil, resulted in the isolation of octacosanol. This study investigated the antinociceptive and anti-inflammatory effects of the octacosanol in different experimental models. The crude ethanolic extract and hexane fraction obtained from the leaves of S. grisea produced an inhibition of acetic acid-induced pain. Moreover, octacosanol isolated from the hexane fraction produced a significant inhibition of pain response elicited by acetic acid. Pre-treatment with yohimbine, an alpha 2-adrenergic receptor antagonist, notably reversed the antinociceptive activity induced by octacosanol in the abdominal constriction test. Furthermore, mice treated with octacosanol did not exhibit any behavioral alteration during the hot plate and rota-rod tests, indicating non-participation of the supraspinal components in the modulation of pain by octacosanol with no motor abnormality. In the formalin test, octacosanol did not inhibit the licking time in first phase (neurogenic pain), but significantly inhibited the licking time in second phase (inflammatory pain) of mice. The anti-inflammatory effect of octacosanol was evaluated using carrageenan-induced pleurisy. The octacosanol significantly reduced the total leukocyte count and neutrophils influx, as well as TNF-α levels in the carrageenan-induced pleurisy. This study revealed that the mechanism responsible for the antinociceptive and anti-inflammatory effects of the octacosanol appears to be partly associated with an inhibition of alpha 2-adrenergic transmission and an inhibition of pathways dependent on pro-inflammatory cytokines. Finally, these results demonstrated that the octacosanol from the leaves of S. grisea possesses antinociceptive and anti-inflammatory activities, which could be of relevance for the pharmacological control of pain and inflammatory processes.
PMCID: PMC3291979  PMID: 22408410
octacosanol; anti-inflammatory; antinociception; TNF-α; Sabicea grisea
21.  Lipopolysaccharide Up-regulates Alpha7 Acetylcholine Receptors: Stimulation with GTS-21 Mitigates Growth Arrest of Macrophages and Improves Survival in Burned Mice 
Shock (Augusta, Ga.)  2012;38(2):213-219.
Nicotinic stimulation of the alpha7 acetylcholine receptors (α7AChRs) mitigates the lipopolysaccharide (LPS)-induced TNF-α and other cytokines release in macrophages. This effect is blocked by α7AChR antagonist, α-bungarotoxin (BTX). We tested and confirmed the hypotheses that LPS up-regulates α7AChRs and the prototypical α7AChR antagonists, vecuronium, and BTX do not block the effects of GTS-21, a specific α7AChR agonist, on TNF-α release. With the knockdown of α7AChR expression by siRNA, GTS-21 effects on inhibition of TNF-α release were not demonstrable. In addition, GTS-21 mitigated the LPS-induced growth arrest of macrophages in vitro in J774A.1 cells and ex vivo in peritoneal macrophages obtained from mice at three days after burn. Moreover, GTS-21 reduced mortality after burn injury in mice. These results indicate that (i) LPS up-regulates α7AChRs; (ii) the therapeutic beneficial effects of GTS-21 on cytokine release are specifically mediated via α7AChRs and are preserved even when co-treated with prototypical antagonist, BTX, or clinically used muscle nicotinic antagonist, vecuronium; (iii) activation of α7AChRs by GTS-21 partially reverses the LPS-induced proliferation arrest and (iv) GTS-21 reduces mortality in mice with burn injury. The in vivo beneficial effects of GTS-21 in burn injury warrant further studies.
PMCID: PMC3399057  PMID: 22683726
alpha7 acetylcholine receptor agonist; alpha7 acetylcholine receptor antagonists; α-bungarotoxin; tumor necrosis factor-α; up-regulation; vecuronium
22.  Nicotinic acetylcholine receptors control acetylcholine and noradrenaline release in the rodent habenulo-interpeduncular complex 
British journal of pharmacology  2014;171(23):5209-5224.
Nicotinic acetylcholine receptors (nACh receptors) play a central role in the habenulo-interpeduncular system. We studied nicotine-induced release of NA and ACh in the habenula and interpeduncular nucleus (IPN).
The habenula and IPN were loaded with [3H]-choline or [3H]-NA and placed in superfusion chambers. [3H]-ACh release was also stimulated using nicotinic agonists, electrical pulses and elevated [KCl]o in hippocampal and cortical slices from rats, wild-type mice and mice lacking α5, α7, β2, or β4 nACh receptor subunits. Finally, we analysed nACh receptor subtypes in the IPN using immunoprecipitation.
Nicotine induced release of [3H]-ACh in the IPN of rats and mice. This release was calcium-dependent but not blocked by tetrodotoxin (TTX); moreover, [3H]-ACh release was abolished in β4-knockout mice but was unaffected in β2- and α5-knockout mice. In contrast, nicotine-induced release of [3H]-NA in the IPN and habenula was blocked by TTX and reduced in both β2-knockout and β4-knockout mice, and dose–response curves were right-shifted in α5-knockout mice. Although electrical stimuli triggered the release of both transmitters, [3H]-ACh release required more pulses delivered at a higher frequency.
Our results confirm previous findings that β4-containing nACh receptors are critical for [3H]-ACh release in the mouse IPN. Experiments using α5-knockout mice also revealed that unlike in the hippocampus, nicotine-induced [3H]-NA release in the habenulo-interpeduncular system is altered in this knockout model. As α5-containing nACh receptors play a key role in nicotine intake, our results add NA to the list of transmitters involved in this mechanism.
PMCID: PMC4244193  PMID: 25041479
23.  Nicotinic acetylcholine receptors control acetylcholine and noradrenaline release in the rodent habenulo-interpeduncular complex 
British Journal of Pharmacology  2014;171(23):5209-5224.
Background and purpose
Nicotinic acetylcholine receptors (nACh receptors) play a central role in the habenulo-interpeduncular system. We studied nicotine-induced release of NA and ACh in the habenula and interpeduncular nucleus (IPN).
Experimental approach
The habenula and IPN were loaded with [3H]-choline or [3H]-NA and placed in superfusion chambers. [3H]-ACh release was also stimulated using nicotinic agonists, electrical pulses and elevated [KCl]o in hippocampal and cortical slices from rats, wild-type mice and mice lacking α5, α7, β2, or β4 nACh receptor subunits. Finally, we analysed nACh receptor subtypes in the IPN using immunoprecipitation.
Key results
Nicotine induced release of [3H]-ACh in the IPN of rats and mice. This release was calcium-dependent but not blocked by tetrodotoxin (TTX); moreover, [3H]-ACh release was abolished in β4-knockout mice but was unaffected in β2- and α5-knockout mice. In contrast, nicotine-induced release of [3H]-NA in the IPN and habenula was blocked by TTX and reduced in both β2-knockout and β4-knockout mice, and dose–response curves were right-shifted in α5-knockout mice. Although electrical stimuli triggered the release of both transmitters, [3H]-ACh release required more pulses delivered at a higher frequency.
Conclusions and implications
Our results confirm previous findings that β4-containing nACh receptors are critical for [3H]-ACh release in the mouse IPN. Experiments using α5-knockout mice also revealed that unlike in the hippocampus, nicotine-induced [3H]-NA release in the habenulo-interpeduncular system is altered in this knockout model. As α5-containing nACh receptors play a key role in nicotine intake, our results add NA to the list of transmitters involved in this mechanism.
PMCID: PMC4244193  PMID: 25041479
24.  Perinatal choline effects on neonatal pathophysiology related to later schizophrenia risk 
The American journal of psychiatry  2013;170(3):290-298.
Deficient cerebral inhibition is a pathophysiological brain deficit related to poor sensory gating and attention in schizophrenia and other disorders. Cerebral inhibition develops perinatally, influenced by genetic and in utero factors. Amniotic choline activates fetal α7-nicotinic acetylcholine receptors and facilitates development of cerebral inhibition. Increasing this activation may protect infants from future illness by promoting normal brain development.
A randomized placebo-controlled clinical trial of dietary phosphatidylcholine supplementation was conducted with 100 healthy pregnant women, who consented to the study at second trimester. Supplementation to twice normal dietary levels for mother or newborn continued through the third postnatal month. All women received dietary advice regardless of treatment. Infants’ electroencephalographic recordings of inhibition of the P50 component of the cerebral evoked response to paired sounds were analyzed. Criterion for inhibition was suppression of the amplitude of the second P50 response by at least half, compared to the first response.
No adverse effects of choline were observed in maternal health and delivery, birth, or infant development. More choline-treated infants (76%) suppressed the P50 response, compared to placebo-treated infants (43%) at the fifth postnatal week (effect size 0.7). There was no difference at the 13th week. A CHRNA7 genotype associated with schizophrenia diminished P50 inhibition in the placebo-treated infants, but not in the choline-treated infants.
Neonatal developmental delay in inhibition is associated with attentional problems as the child matures. Perinatal choline activates timely development of cerebral inhibition, even in the presence of gene mutations that otherwise delay it.
PMCID: PMC3640283  PMID: 23318559
25.  A New IRAK-M-Mediated Mechanism Implicated in the Anti-Inflammatory Effect of Nicotine via α7 Nicotinic Receptors in Human Macrophages 
PLoS ONE  2014;9(9):e108397.
Nicotine stimulation of α7 nicotinic acetylcholine receptor (α7 nAChR) powerfully inhibits pro-inflammatory cytokine production in lipopolysaccharide (LPS)-stimulated macrophages and in experimental models of endotoxemia. A signaling pathway downstream from the α7 nAChRs, which involves the collaboration of JAK2/STAT3 and NF-κB to interfere with signaling by Toll-like receptors (TLRs), has been implicated in this anti-inflammatory effect of nicotine. Here, we identifiy an alternative mechanism involving interleukin-1 receptor-associated kinase M (IRAK-M), a negative regulator of innate TLR-mediated immune responses. Our data show that nicotine up-regulates IRAK-M expression at the mRNA and protein level in human macrophages, and that this effect is secondary to α7 nAChR activation. By using selective inhibitors of different signaling molecules downstream from the receptor, we provide evidence that activation of STAT3, via either JAK2 and/or PI3K, through a single (JAK2/PI3K/STAT3) or two convergent cascades (JAK2/STAT3 and PI3K/STAT3), is necessary for nicotine-induced IRAK-M expression. Moreover, down-regulation of this expression by small interfering RNAs specific to the IRAK-M gene significantly reverses the anti-inflammatory effect of nicotine on LPS-induced TNF-α production. Interestingly, macrophages pre-exposed to nicotine exhibit higher IRAK-M levels and reduced TNF-α response to an additional LPS challenge, a behavior reminiscent of the ‘endotoxin tolerant’ phenotype identified in monocytes either pre-exposed to LPS or from immunocompromised septic patients. Since nicotine is a major component of tobacco smoke and increased IRAK-M expression has been considered one of the molecular determinants for the induction of the tolerant phenotype, our findings showing IRAK-M overexpression could partially explain the known influence of smoking on the onset and progression of inflammatory and infectious diseases.
PMCID: PMC4178160  PMID: 25259522

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