Differential roles of prelimbic and infralimbic cholinergic neurotransmissions in control of cardiovascular responses to restraint stress in rats.

Previous studies showed a prominent role of the medial prefrontal cortex (mPFC), especially the prelimbic (PL) and infralimbic (IL) subregions, in behavioral and physiological responses to stressful stimuli. Nevertheless, the local neurochemical mechanisms involved are not completely understood. In this sense, previous studies identified cholinergic terminals within the mPFC, and stressful stimuli increased local acetylcholine release. Despite these pieces of evidence, the specific role of cholinergic neurotransmission in different subregions of the mPFC controlling the cardiovascular responses to stress has never been systematically evaluated. Therefore, the purpose of this study was to investigate the involvement of cholinergic neurotransmission present within PL and IL in cardiovascular responses to an acute session of restraint stress in rats. For this, rats received bilateral microinjection of the choline uptake inhibitor hemicholinium-3 before exposure to restraint stress. The arterial pressure and heart rate (HR) increases and the decrease in tail skin temperature as an indirect measurement of sympathetically-mediated cutaneous vasoconstriction were recorded throughout the restraint stress session. The results showed that the depletion of acetylcholine within the PL caused by local microinjection of hemicholinium-3 decreased the tachycardia to restraint stress, but without affecting the pressor response and the drop in tail skin temperature. Conversely, IL treatment with hemicholinium-3 decreased the restraint-evoked pressor response and the sympathetically-mediated cutaneous vasoconstriction without interfering with the HR response. Taken together, these results indicate functional differences of cholinergic neurotransmission within the PL and IL in control of cardiovascular and autonomic responses to stressful stimuli.

Molecular and Functional Analysis of Choline Transporters and Antitumor Effects of Choline Transporter-Like Protein 1 Inhibitors in Human Pancreatic Cancer Cells.

Choline, an organic cation, is one of the biofactors that play an important role in the structure and the function of biological membranes, and it is essential for the synthesis of phospholipids. Choline positron emission tomography-computed tomography (PET/CT) provides useful information for the imaging diagnosis of cancers, and increased choline accumulation has been identified in a variety of tumors. However, the molecular mechanisms of choline uptake and choline transporters in pancreatic cancer have not been elucidated. Here, we examined molecular and functional analyses of choline transporters in human pancreatic-cancer cell line MIA PaCa-2 and the elucidation of the action mechanism behind the antitumor effect of novel choline-transporter-like protein 1 (CTL1) inhibitors, Amb4269951 and its derivative Amb4269675. CTL1 and CTL2 mRNAs were highly expressed in MIA PaCa-2 cells, and CTL1 and CTL2 proteins were localized in the plasma membrane and the intracellular compartments, respectively. Choline uptake was characterized by Na+-independence, a single-uptake mechanism, and inhibition by choline-uptake inhibitor HC-3, similar to the function of CTL1. These results suggest that the uptake of extracellular choline in MIA PaCa-2 cells is mediated by CTL1. Choline deficiency and HC-3 treatment inhibited cell viability and increased caspase 3/7 activity, suggesting that the inhibition of CTL1 function, which is responsible for choline transport, leads to apoptosis-induced cell death. Both Amb4269951 and Amb4269675 inhibited choline uptake and cell viability and increased caspase-3/7 activity. Ceramide, which is increased by inhibiting choline uptake, also inhibited cell survival and increased caspase-3/7 activity. Lastly, both Amb4269951 and Amb4269675 significantly inhibited tumor growth in a mouse-xenograft model without any adverse effects such as weight loss. CTL1 is a target molecule for the treatment of pancreatic cancer, and its inhibitors Amb4269951 and Amb4269675 are novel lead compounds.

Paternal Δ9-Tetrahydrocannabinol Exposure Prior to Mating Elicits Deficits in Cholinergic Synaptic Function in the Offspring.

Little attention has been paid to the potential impact of paternal marijuana use on offspring brain development. We administered Δ9-tetrahydrocannabinol (THC, 0, 2, or 4 mg/kg/day) to male rats for 28 days. Two days after the last THC treatment, the males were mated to drug-naïve females. We then assessed the impact on development of acetylcholine (ACh) systems in the offspring, encompassing the period from the onset of adolescence (postnatal day 30) through middle age (postnatal day 150), and including brain regions encompassing the majority of ACh terminals and cell bodies. Δ9-Tetrahydrocannabinol produced a dose-dependent deficit in hemicholinium-3 binding, an index of presynaptic ACh activity, superimposed on regionally selective increases in choline acetyltransferase activity, a biomarker for numbers of ACh terminals. The combined effects produced a persistent decrement in the hemicholinium-3/choline acetyltransferase ratio, an index of impulse activity per nerve terminal. At the low THC dose, the decreased presynaptic activity was partially compensated by upregulation of nicotinic ACh receptors, whereas at the high dose, receptors were subnormal, an effect that would exacerbate the presynaptic defect. Superimposed on these effects, either dose of THC also accelerated the age-related decline in nicotinic ACh receptors. Our studies provide evidence for adverse effects of paternal THC administration on neurodevelopment in the offspring and further demonstrate that adverse impacts of drug exposure on brain development are not limited to effects mediated by the embryonic or fetal chemical environment, but rather that vulnerability is engendered by exposures occurring prior to conception, involving the father as well as the mother.

Acetylcholine receptors in the equatorial region of intrafusal muscle fibres modulate mouse muscle spindle sensitivity.

KEY POINTS: Acetylcholine receptors are aggregated in the central regions of intrafusal muscle fibres. Single unit muscle spindle afferent responses from isolated mouse extensor digitorum longus muscle were recorded in the absence of fusimotor input to ramp and hold stretches as well as to sinusoidal vibrations in the presence and absence of the acetylcholine receptor blockers d-tubocurarine and α-bungarotoxin. Proprioceptive afferent responses to both types of stretch were enhanced in the presence of either blocker. Blocking acetylcholine uptake and vesicular acetylcholine release by hemicholinium-3 also enhanced stretch-evoked responses. These results represent the first evidence that acetylcholine receptors negatively modulate muscle spindle responses to stretch. The data support the hypothesis that the sensory nerve terminal is able to release vesicles to fine-tune proprioceptive afferent sensitivity. ABSTRACT: Muscle spindles are complex stretch-sensitive mechanoreceptors. They consist of specialized skeletal muscle fibres, called intrafusal fibres, which are innervated in the central (equatorial) region by afferent sensory axons and in both polar regions by efferent γ-motoneurons. Previously it was shown that acetylcholine receptors (AChR) are concentrated in the equatorial region at the contact site between the sensory neuron and the intrafusal muscle fibre. To address the function of these AChRs, single unit sensory afferents were recorded from an isolated mouse extensor digitorum longus muscle in the absence of γ-motoneuron activity. Specifically, we investigated the responses of individual sensory neurons to ramp-and-hold stretches and sinusoidal vibrations before and after the addition of the competitive and non-competitive AChR blockers d-tubocurarine and α-bungarotoxin, respectively. The presence of either drug did not affect the resting action potential discharge frequency. However, the action potential frequencies in response to stretch were increased. In particular, frequencies of the dynamic peak and dynamic index to ramp-and-hold stretches were significantly higher in the presence of either drug. Treatment of muscle spindle afferents with the high-affinity choline transporter antagonist hemicholinium-3 similarly increased muscle spindle afferent firing frequencies during stretch. Moreover, the firing rate during sinusoidal vibration stimuli at low amplitudes was higher in the presence of α-bungarotoxin compared to control spindles also indicating an increased sensitivity to stretch. Collectively these data suggest a modulation of the muscle spindle afferent response to stretch by AChRs in the central region of intrafusal fibres possibly fine-tuning muscle spindle sensitivity.

Tetanic Facilitation of Neuromuscular Transmission by Adenosine A2A and Muscarinic M1 Receptors is Dependent on the Uptake of Choline via High-Affinity Transporters.

BACKGROUND/AIMS: In this study, we evaluated the functional impact of facilitatory presynaptic adenosine A2A and muscarinic M1 receptors in the recovery of neuromuscular tetanic depression caused by the blockage of high-affinity choline transporter (HChT) by hemicholinium-3 (HC-3), a condition that mimics a myasthenia-like condition. METHODS: Rat diaphragm preparations were indirectly stimulated via the phrenic nerve trunk with 50-Hz frequency trains, each consisting of 500-750 supramaximal intensity pulses. The tension at the beginning (A) and at the end (B) of the tetanus was recorded and the ratio (R) B/A calculated. RESULTS: Activation of A2A and M1 receptors with CGS21680 (CGS; 2 nmol/L) and McN-A-343c (McN; 3 µmol/L) increased R values. Similar facilitatory effects were obtained with forskolin (FSK; 3 µmol/L) and phorbol 12-myristate 13-acetate (PMA; 10 µmol/L), which activate adenylate cyclase and protein kinase C respectively. HC-3 (4 µmol/L) decreased transmitter exocytosis measured by real-time videomicroscopy with the FM4-64 fluorescent dye and prevented the facilitation of neuromuscular transmission caused by CGS, McN, and FSK, with a minor effect on PMA. The acetylcholinesterase inhibitor, neostigmine (NEO; 0.5 µmol/L), also decreased transmitter exocytosis. The paradoxical neuromuscular tetanic fade caused by NEO (0.5 µmol/L) was also prevented by HC-3 (4 µmol/L) and might result from the rundown of the positive feedback mechanism operated by neuronal nicotinic receptors (blocked by hexamethonium, 120 µmol/L). CONCLUSION: Data suggest that the recovery of tetanic neuromuscular facilitation by adenosine A2A and M1 receptors is highly dependent on HChT activity and may be weakened in myasthenic patients when HChT is inoperative.

Assessment of Radiation Resistance and Therapeutic Targeting of Cancer Stem Cells: A Raman Spectroscopic Study of Glioblastoma.

Radiation is the standard therapy used for treating Glioblastoma (GBM), a grade IV brain cancer. Glioma Stem-like Cells (GSCs), an integral part of GBM, enforces resistance to radiation therapy of GBM. Studying the differential biomolecular composition of GSCs with varying levels of radiation sensitivity can aid in identifying the molecules and their associated pathways which impose resistance to cells thereby unraveling new targets which would serve as potential adjuvant therapy. Raman spectroscopy being a noninvasive, label free technique can determine the biomolecular constituent of cells under live conditions. In this study, we have deduced Raman spectral signatures to predict the radiosensitivity of any GSC accurately using the inherent and radiation induced biomolecular composition. Our study identified the differential regulation of several biomolecules which can be potential targets for adjuvant therapy. We radiosensitized the resistant GSCs using small molecule inhibitors specific to the metabolic pathways of these biomolecules. Efficient antitumor therapy can be attained with lower dosage of radiation along with these inhibitors and thus improving the survival rate of GBM patients with reduced side-effects from radiation.

Regulation of synaptic acetylcholine concentrations by acetylcholine transport in rat striatal cholinergic transmission.

In addition to hydrolysis by acetylcholine esterase (AChE), acetylcholine (ACh) is also directly taken up into brain tissues. In this study, we examined whether the uptake of ACh is involved in the regulation of synaptic ACh concentrations. Superfusion experiments with rat striatal segments pre-incubated with [3 H]choline were performed using an ultra-mini superfusion vessel, which was developed to minimize superfusate retention within the vessel. Hemicholinium-3 (HC-3) at concentrations less than 1 µM, selectively inhibited the uptake of [3 H]choline by the high affinity-choline transporter 1 and had no effect on basal and electrically evoked [3 H]efflux in superfusion experiments. In contrast, HC-3 at higher concentrations, as well as tetraethylammonium (>10 µM), which inhibited the uptake of both [3 H]choline and [3 H]ACh, increased basal [3 H]overflow and potentiated electrically evoked [3 H]efflux. These effects of HC-3 and tetraethylammonium were also observed under conditions where tissue AChE was irreversibly inactivated by diisopropylfluorophosphate. Specifically, the potentiation of evoked [3 H]efflux was significantly higher in AChE-inactivated preparations and was attenuated by atropine. On the other hand, striatal segments pre-incubated with [3 H]ACh failed to increase [3 H]overflow in response to electrical stimulation. These results show that synaptic ACh concentrations are significantly regulated by the postsynaptic uptake of ACh, as well as by AChE hydrolysis and modulation of ACh release mediated through presynaptic muscarinic ACh receptors. In addition, these data suggest that the recycling of ACh-derived choline may be minor in cholinergic terminals. This study reveals a new mechanism of cholinergic transmission in the central nervous system.

Autolyse the cell in order to save it? Inducing, then blocking, autolysis as a strategy for delaying cell death in the probiotic Lactobacillus reuteri.

OBJECTIVE: To examine whether choline and its derivatives can be used to preserve viable cells of Lactobacillus reuteri in autolytic models. RESULTS: A phosphate-induced autolytic model in de Man, Rogosa and Sharpe medium (MRS) was used. Viable cell counts were determined by plated on MRS-agar. Choline and hemicholinium-3 (HC-3) significantly blocked autolysis of L. reuteri at 360 mM and 4 mM, respectively. Viable cell counts corroborated these observations. Importantly, autolytically induced cells treated with choline and hemicholinium-3 were significantly more viable then even non-induced cells. Over-production of a known autolytic protein, spirosin, was not attenuated in the presence of choline and hemicholinium-3. CONCLUSION: Inducing autolysis and then blocking it with choline and its analogs is a promising approach for retaining the viability of L. reuteri cells.

Hemicholinium-3 sensitive choline transport in human T lymphocytes: Evidence for use as a proxy for brain choline transporter (CHT) capacity.

The synaptic uptake of choline via the high-affinity, hemicholinium-3-dependent choline transporter (CHT) strongly influences the capacity of cholinergic neurons to sustain acetylcholine (ACh) synthesis and release. To advance research on the impact of CHT capacity in humans, we established the presence of the neuronal CHT protein in human T lymphocytes. Next, we demonstrated CHT-mediated choline transport in human T cells. To address the validity of T cell-based choline uptake as a proxy for brain CHT capacity, we isolated T cells from the spleen, and synaptosomes from cortex and striatum, of wild type and CHT-overexpressing mice (CHT-OXP). Choline uptake capacity in T cells from CHT-OXP mice was two-fold higher than in wild type mice, mirroring the impact of CHT over-expression on synaptosomal CHT-mediated choline uptake. Monitoring T lymphocyte CHT protein and activity may be useful for estimating human CNS cholinergic capacity and for testing hypotheses concerning the contribution of CHT and, more generally, ACh signaling in cognition, neuroinflammation and disease.

Inhibition of the high affinity choline transporter enhances hyperalgesia in a rat model of chronic pancreatitis.

BACKGROUND: The mechanisms underlying chronic and persistent pain associated with chronic pancreatitis (CP) are not completely understood. The cholinergic system is one of the major neural pathways of the pancreas. Meanwhile, this system plays an important role in chronic pain. We hypothesized that the high affinity choline transporter CHT1, which is a main determinant of cholinergic signaling capacity, is involved in regulating pain associated with CP. METHODS: CP was induced by intraductal injection of 2% trinitrobenzene sulfonic acid (TNBS) in Sprague-Dawley rats. Pathological examination was used to evaluate the inflammation of pancreas and hyperalgesia was assessed by measuring the number of withdrawal events evoked by application of the von Frey filaments. CHT1 expression in pancreas-specific dorsal root ganglia (DRGs) was assessed through immunohistochemistry and western blotting. We also intraperitoneally injected the rats with hemicholinium-3 (HC-3, a specific inhibitor of CHT1). Then we observed its effects on the visceral hyperalgesia induced by CP, and on the acetylcholine (ACh) levels in the DRGs through using an acetylcholine/acetylcholinesterase assay kit. RESULTS: Signs of CP were observed 21 days after TNBS injection. Rats subjected to TNBS infusions had increased sensitivity to mechanical stimulation of the abdomen. CHT1-immunoreactive cells were increased in the DRGs from rats with CP compared to naive or sham rats. Western blots indicated that CHT1 expression was significantly up-regulated in TNBS-treated rats when compared to naive or sham-operated rats at all time points following surgery. In the TNBS group, CHT1 expression was higher on day 28 than on day 7 or day 14, but there was no statistical difference in CHT1 expression on day 28 vs. day 21. Treatment with HC-3 (60 µg/kg, 80 µg/kg, or 100 µg/kg) markedly enhanced the mechanical hyperalgesia and reduced ACh levels in a dose-dependent manner in rats with CP. CONCLUSION: We report for the first time that CHT1 may be involved in pain modulation in CP, as it plays an important role in pain inhibition. Increased CHT1 activity or the up-regulation of its expression may be used to treat pain in patients with CP.

Choline induces opposite changes in pyramidal neuron excitability and synaptic transmission through a nicotinic receptor-independent process in hippocampal slices.

Choline is present at cholinergic synapses as a product of acetylcholine degradation. In addition, it is considered a selective agonist for α5 and α7 nicotinic acetylcholine receptors (nAChRs). In this study, we determined how choline affects action potentials and excitatory synaptic transmission using extracellular and intracellular recording techniques in CA1 area of hippocampal slices obtained from both mice and rats. Choline caused a reversible depression of evoked field excitatory postsynaptic potentials (fEPSPs) in a concentration-dependent manner that was not affected by α7 nAChR antagonists. Moreover, this choline-induced effect was not mimicked by either selective agonists or allosteric modulators of α7 nAChRs. Additionally, this choline-mediated effect was not prevented by either selective antagonists of GABA receptors or hemicholinium, a choline uptake inhibitor. The paired pulse facilitation paradigm, which detects whether a substance affects presynaptic release of glutamate, was not modified by choline. On the other hand, choline induced a robust increase of population spike evoked by orthodromic stimulation but did not modify that evoked by antidromic stimulation. We also found that choline impaired recurrent inhibition recorded in the pyramidal cell layer through a mechanism independent of α7 nAChR activation. These choline-mediated effects on fEPSP and population spike observed in rat slices were completely reproduced in slices obtained from α7 nAChR knockout mice, which reinforces our conclusion that choline modulates synaptic transmission and neuronal excitability by a mechanism independent of nicotinic receptor activation.

Choline on the Move: Perspectives on the Molecular Physiology and Pharmacology of the Presynaptic Choline Transporter.

Genetic, biochemical, physiological, and pharmacological approaches have advanced our understanding of cholinergic biology for over 100 years. High-affinity choline uptake (HACU) was one of the last features of cholinergic signaling to be defined at a molecular level, achieved through the cloning of the choline transporter (CHT, SLC5A7). In retrospect, the molecular era of CHT studies initiated with the identification of hemicholinium-3 (HC-3), a potent, competitive CHT antagonist, though it would take another 30 years before HC-3, in radiolabeled form, was used by Joseph Coyle's laboratory to identify and monitor the dynamics of CHT proteins. Though HC-3 studies provided important insights into CHT distribution and regulation, another 15 years would pass before the structure of CHT genes and proteins were identified, a full decade after the cloning of most other neurotransmitter-associated transporters. The availability of CHT gene and protein probes propelled the development of cell and animal models as well as efforts to gain insights into how human CHT gene variation affects the risk for brain and neuromuscular disorders. Most recently, our group has pursued a broadening of CHT pharmacology, elucidating novel chemical structures that may serve to advance cholinergic diagnostics and medication development. Here we provide a short review of the transformation that has occurred in HACU research and how such advances may promote the development of novel therapeutics.

Choline kinase alpha-Putting the ChoK-hold on tumor metabolism.

It is well established that lipid metabolism is drastically altered during tumor development and response to therapy. Choline kinase alpha (ChoKα) is a key mediator of these changes, as it represents the first committed step in the Kennedy pathway of phosphatidylcholine biosynthesis and ChoKα expression is upregulated in many human cancers. ChoKα activity is associated with drug resistant, metastatic, and malignant phenotypes, and represents a robust biomarker and therapeutic target in cancer. Effective ChoKα inhibitors have been developed and have recently entered clinical trials. ChoKα's clinical relevance was, until recently, attributed solely to its production of second messenger intermediates of phospholipid synthesis. The recent discovery of a non-catalytic scaffolding function of ChoKα may link growth receptor signaling to lipid biogenesis and requires a reinterpretation of the design and validation of ChoKα inhibitors. Advances in positron emission tomography, magnetic resonance spectroscopy, and optical imaging methods now allow for a comprehensive understanding of ChoKα expression and activity in vivo. We will review the current understanding of ChoKα metabolism, its role in tumor biology and the development and validation of targeted therapies and companion diagnostics for this important regulatory enzyme. This comes at a critical time as ChoKα-targeting programs receive more clinical interest.

Molecular and functional characterization of choline transporter in the human trophoblastic cell line JEG-3 cells.

INTRODUCTION: Choline is essential for the synthesis of the major membrane phospholipid phosphatidylcholine (PC), the methyl donor betaine and the neurotransmitter acetylcholine (ACh), which is involved in several vital biological functions that play key roles in fetal development. In this study, we examined the molecular and functional characteristics of choline uptake in the human trophoblastic cell line JEG-3. METHODS: We examined [(3)H]choline uptake in the human trophoblastic cell line JEG-3. The expression of CTL1 and CTL2 was evaluated by quantitative real-time PCR, western blotting and immunocytochemistry. RESULTS: We demonstrated that JEG-3 cells take up [(3)H] choline by a saturable process that is mediated by a Na(+)-independent and pH-dependent transport system. The cells have two different [(3)H] choline transport systems, high- and low-affinity, with Km values of 28.4 ± 5.0 µM and 210.6 ± 55.1 µM, respectively. Cationic compounds and hemicholinium-3 (HC-3) inhibited choline uptake. Choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein were highly expressed in JEG-3 cells and were localized to the plasma membrane. DISCUSSION: The present results suggest that choline is mainly transported via a high-affinity choline transport system (CTL1) and a low-affinity choline transport system (CTL2) in human trophoblastic JEG-3 cells. These transporters play an important role in the growth of the fetus.

Identification and characterization of ML352: a novel, noncompetitive inhibitor of the presynaptic choline transporter.

The high-affinity choline transporter (CHT) is the rate-limiting determinant of acetylcholine (ACh) synthesis, yet the transporter remains a largely undeveloped target for the detection and manipulation of synaptic cholinergic signaling. To expand CHT pharmacology, we pursued a high-throughput screen for novel CHT-targeted small molecules based on the electrogenic properties of transporter-mediated choline transport. In this effort, we identified five novel, structural classes of CHT-specific inhibitors. Chemical diversification and functional analysis of one of these classes identified ML352 as a high-affinity (Ki = 92 nM) and selective CHT inhibitor. At concentrations that fully antagonized CHT in transfected cells and nerve terminal preparations, ML352 exhibited no inhibition of acetylcholinesterase (AChE) or cholineacetyltransferase (ChAT) and also lacked activity at dopamine, serotonin, and norepinephrine transporters, as well as many receptors and ion channels. ML352 exhibited noncompetitive choline uptake inhibition in intact cells and synaptosomes and reduced the apparent density of hemicholinium-3 (HC-3) binding sites in membrane assays, suggesting allosteric transporter interactions. Pharmacokinetic studies revealed limited in vitro metabolism and significant CNS penetration, with features predicting rapid clearance. ML352 represents a novel, potent, and specific tool for the manipulation of CHT, providing a possible platform for the development of cholinergic imaging and therapeutic agents.

No evidence for role of extracellular choline-acetyltransferase in generation of gamma oscillations in rat hippocampal slices in vitro.

Acetylcholine (ACh) is well known to induce persistent γ-oscillations in the hippocampus when applied together with physostigmine, an inhibitor of the ACh degrading enzyme acetylcholinesterase (AChE). Here we report that physostigmine alone can also dose-dependently induce γ-oscillations in rat hippocampal slices. We hypothesized that this effect was due to the presence of choline in the extracellular space and that this choline is taken up into cholinergic fibers where it is converted to ACh by the enzyme choline-acetyltransferase (ChAT). Release of ACh from cholinergic fibers in turn may then induce γ-oscillations. We therefore tested the effects of the choline uptake inhibitor hemicholinium-3 (HC-3) on persistent γ-oscillations either induced by physostigmine alone or by co-application of ACh and physostigmine. We found that HC-3 itself did not induce γ-oscillations and also did not prevent physostigmine-induced γ-oscillation while washout of physostigmine and ACh-induced γ-oscillations was accelerated. It was recently reported that ChAT might also be present in the extracellular space (Vijayaraghavan et al., 2013). Here we show that the effect of physostigmine was prevented by the ChAT inhibitor (2-benzoylethyl)-trimethylammonium iodide (BETA) which could indicate extracellular synthesis of ACh. However, when we tested for effects of extracellularly applied acetyl-CoA, a substrate of ChAT for synthesis of ACh, physostigmine-induced γ-oscillations were attenuated. Together, these findings do not support the idea that ACh can be synthesized by an extracellularly located ChAT.

Prenatal nicotine alters the developmental neurotoxicity of postnatal chlorpyrifos directed toward cholinergic systems: better, worse, or just "different?".

This study examines whether prenatal nicotine exposure sensitizes the developing brain to subsequent developmental neurotoxicity evoked by chlorpyrifos, a commonly-used insecticide. We gave nicotine to pregnant rats throughout gestation at a dose (3mg/kg/day) producing plasma levels typical of smokers; offspring were then given chlorpyrifos on postnatal days 1-4, at a dose (1mg/kg) that produces minimally-detectable inhibition of brain cholinesterase activity. We evaluated indices for acetylcholine (ACh) synaptic function throughout adolescence, young adulthood and later adulthood, in brain regions possessing the majority of ACh projections and cell bodies; we measured nicotinic ACh receptor binding, hemicholinium-3 binding to the presynaptic choline transporter and choline acetyltransferase activity, all known targets for the adverse developmental effects of nicotine and chlorpyrifos given individually. By itself nicotine elicited overall upregulation of the ACh markers, albeit with selective differences by sex, region and age. Likewise, chlorpyrifos alone had highly sex-selective effects. Importantly, all the effects showed temporal progression between adolescence and adulthood, pointing to ongoing synaptic changes rather than just persistence after an initial injury. Prenatal nicotine administration altered the responses to chlorpyrifos in a consistent pattern for all three markers, lowering values relative to those of the individual treatments or to those expected from simple additive effects of nicotine and chlorpyrifos. The combination produced global interference with emergence of the ACh phenotype, an effect not seen with nicotine or chlorpyrifos alone. Given that human exposures to nicotine and chlorpyrifos are widespread, our results point to the creation of a subpopulation with heightened vulnerability.

Choline kinase active site provides features for designing versatile inhibitors.

Choline kinase (CK) is a homodimeric enzyme that catalyses the transfer of the ATP γ-phosphate to choline, generating phosphocholine and ADP in the presence of magnesium. Several isoforms of CK are present in humans but only the HsCKα has been associated with cancer and validated as a drug target to treat this disease. As a consequence a large number of compounds based on Hemicholinium (HC-3) have been described. Two compounds, previously reported to inhibit the human enzyme, have recently been shown to inhibit P. falciparum CK (PfCK) and therefore their potential applications might be anticipated to other pathogens. Herein, using molecular dynamic simulations, we have firstly observed that the ATP and the choline binding site of different CK in pathogens and human are conserved, suggesting that previous compounds inhibiting the human enzyme may also interact with CKs from different pathogens. We have substantiated such observation with experimental assays showing that HsCKα1, PfCK and CpCK bind to two compounds with distinct structural features in the low µM range. Collectively, these results uncover similarities among the choline kinase binding site from different pathogenic species and the human enzyme, highlighting the feasibility of designing novel inhibitors based on the choline binding pocket.

Cigarette smoke alters non-neuronal cholinergic system components inducing MUC5AC production in the H292 cell line.

Cigarette smoke extract (CSE) affects the expression of Choline Acetyl-Transferase (ChAT), muscarinic acetylcholine receptors, and mucin production in bronchial epithelial cells. Mucin 5AC (MUC5AC), muscarinic acetylcholine receptor M3, ChAT expression, acetylcholine levels and acetylcholine binding were measured in a human pulmonary mucoepidermoid carcinoma cell line (H292) stimulated with CSE. We performed ChAT/RNA interference experiments in H292 cells stimulated with CSE to study the role of ChAT/acetylcholine in MUC5AC production. The effects of Hemicholinium-3 (HCh-3) (50 µM) (a potent and selective choline uptake blocker) and Tiotropium bromide (Spiriva(®)) (100 nM), alone or in combination with Salmeterol (SL) and Fluticasone propionate (FP), were tested in this model. MUC5AC, muscarinic acetylcholine receptor M3, ChAT, acetylcholine expression and acetylcholine binding significantly increased in H292 cells stimulated with CSE (5%) compared to untreated cells. HCh-3 reduced acetylcholine binding and MUC5AC production in H292 cells stimulated with CSE. ChAT/RNA interference eliminated the effect of CSE on MUC5AC production. FP reduced ChAT and acetylcholine binding in unstimulated cells, while showing a partial effect in CSE stimulated cells. SL increased the ChAT expression and acetylcholine binding in H292 cells stimulated with or without CSE. Tiotropium, alone or together with FP and SL, reduced acetylcholine binding and MUC5AC production in H292 cells stimulated with CSE. CSE affects the ChAT/acetylcholine expression, increasing MUC5AC production in H292 cells. Pharmacological treatment with anticholinergic drugs reduces the secretion of MUC5AC generated by autocrine acetylcholine activity in airway epithelial cells.

Phosphatidylcholine metabolism and choline kinase in human osteoblasts.

There is a paucity of information about phosphatidylcholine (PC) biosynthesis in bone formation. Thus, we characterized PC metabolism in both primary human osteoblasts (HOB) and human osteosarcoma MG-63 cells. Our results show that the CDP-choline pathway is the only de novo route for PC biosynthesis in both HOB and MG-63 cells. Both CK activity and CKα expression in MG-63 cells were significantly higher than those in HOB cells. Silencing of CKα in MG-63 cells had no significant effect on PC concentration but decreased the amount of phosphocholine by approximately 80%. The silencing of CKα also reduced cell proliferation. Moreover, pharmacological inhibition of CK activity impaired the mineralization capacity of MG-63 cells. Our data suggest that CK and its product phosphocholine are required for the normal growth and mineralization of MG-63 cells.