scholarly journals Acute Nicotine Reduces Brain Arachidonic Acid Signaling in Unanesthetized Rats

2009 ◽  
Vol 29 (3) ◽  
pp. 648-658 ◽  
Author(s):  
Lisa Chang ◽  
Stanley I Rapoport ◽  
Henry N Nguyen ◽  
Dede Greenstein ◽  
Mei Chen ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Brain Regions ◽  
Membrane Phospholipids ◽  
Dose Equivalent ◽  
Central Effects ◽  
Postsynaptic Receptors ◽  
Cytosolic Phospholipase ◽  
Presynaptic Release

Nicotine exerts its central effects by activating pre- and postsynaptic nicotinic acetylcholine receptors (nAChRs). Presynaptic nAChRs modulate the release of many neurotransmitters that bind to postsynaptic receptors. These may be coupled to the activation of cytosolic phospholipase A2 (cPLA2), which hydrolyzes arachidonic acid (AA) from membrane phospholipids. We hypothesized that nicotine would modify brain signaling involving AA by binding to nAChRs. Nicotine (0.1 mg/kg, subcutaneously) or saline was injected 2 or 10 mins before infusing [1-14C]AA in unanesthetized rats. The AA incorporation coefficient k∗ (a marker of the AA signal) was measured in 80 brain regions by quantitative autoradiography. Nicotine, compared to saline, when administrated 2 mins before [1-14C]AA infusion, significantly decreased k∗ for AA in 26 regions, including cerebral cortex, thalamus, and habenula—interpeduncular regions, by 13% to 45%. These decreases could be entirely prevented by pretreatment with mecamylamine (1.0 mg/kg, subcutaneously). When administered 10 mins before [1-14C]AA infusion, nicotine did not alter any value of k∗. In summary, nicotine given to unanesthetized rats rapidly reduces signaling involving AA in brain regions containing nAChRs, likely by modulating the presynaptic release of neurotransmitters. The effect shows rapid desensitization and is produced at a nicotine dose equivalent to smoking one cigarette in humans.

scholarly journals High-Throughput Patch Clamp Screening in Human α6-Containing Nicotinic Acetylcholine Receptors

2017 ◽  
Vol 22 (6) ◽  
pp. 686-695 ◽  
Author(s):  
Lucas C. Armstrong ◽  
Glenn E. Kirsch ◽  
Nikolai B. Fedorov ◽  
Caiyun Wu ◽  
Yuri A. Kuryshev ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Cell Line ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Brain Regions ◽  
Tobacco Product ◽  
Tobacco Products ◽  
Nicotinic Acetylcholine ◽  
Automated Patch Clamp ◽  
Subtype Selective

Nicotine, the addictive component of tobacco products, is an agonist at nicotinic acetylcholine receptors (nAChRs) in the brain. The subtypes of nAChR are defined by their α- and β-subunit composition. The α6β2β3 nAChR subtype is expressed in terminals of dopaminergic neurons that project to the nucleus accumbens and striatum and modulate dopamine release in brain regions involved in nicotine addiction. Although subtype-dependent selectivity of nicotine is well documented, subtype-selective profiles of other tobacco product constituents are largely unknown and could be essential for understanding the addiction-related neurological effects of tobacco products. We describe the development and validation of a recombinant cell line expressing human α6/3β2β3V273S nAChR for screening and profiling assays in an automated patch clamp platform (IonWorks Barracuda). The cell line was pharmacologically characterized by subtype-selective and nonselective reference agonists, pore blockers, and competitive antagonists. Agonist and antagonist effects detected by the automated patch clamp approach were comparable to those obtained by conventional electrophysiological assays. A pilot screen of a library of Food and Drug Administration–approved drugs identified compounds, previously not known to modulate nAChRs, which selectively inhibited the α6/3β2β3V273S subtype. These assays provide new tools for screening and subtype-selective profiling of compounds that act at α6β2β3 nicotinic receptors.

scholarly journals Nicotine exploits a COPI-mediated process for chaperone-mediated up-regulation of its receptors

2013 ◽  
Vol 143 (1) ◽  
pp. 51-66 ◽  
Author(s):  
Brandon J. Henderson ◽  
Rahul Srinivasan ◽  
Weston A. Nichols ◽  
Crystal N. Dilworth ◽  
Diana F. Gutierrez ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Brain Regions ◽  
Substantia Nigra Pars Compacta ◽  
Common Mechanism ◽  
Binding Motif ◽  
Chronic Nicotine ◽  
Dependent Process

Chronic exposure to nicotine up-regulates high sensitivity nicotinic acetylcholine receptors (nAChRs) in the brain. This up-regulation partially underlies addiction and may also contribute to protection against Parkinson’s disease. nAChRs containing the α6 subunit (α6* nAChRs) are expressed in neurons in several brain regions, but comparatively little is known about the effect of chronic nicotine on these nAChRs. We report here that nicotine up-regulates α6* nAChRs in several mouse brain regions (substantia nigra pars compacta, ventral tegmental area, medial habenula, and superior colliculus) and in neuroblastoma 2a cells. We present evidence that a coat protein complex I (COPI)-mediated process mediates this up-regulation of α6* or α4* nAChRs but does not participate in basal trafficking. We show that α6β2β3 nAChR up-regulation is prevented by mutating a putative COPI-binding motif in the β3 subunit or by inhibiting COPI. Similarly, a COPI-dependent process is required for up-regulation of α4β2 nAChRs by chronic nicotine but not for basal trafficking. Mutation of the putative COPI-binding motif or inhibition of COPI also results in reduced normalized Förster resonance energy transfer between α6β2β3 nAChRs and εCOP subunits. The discovery that nicotine exploits a COPI-dependent process to chaperone high sensitivity nAChRs is novel and suggests that this may be a common mechanism in the up-regulation of nAChRs in response to chronic nicotine.

Nonsynaptic Chemical Transmission Through Nicotinic Acetylcholine Receptors

2008 ◽  
Vol 88 (2) ◽  
pp. 333-349 ◽  
Author(s):  
Balázs Lendvai ◽  
E. Sylvester Vizi
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Neural Plasticity ◽  
Acetylcholine Receptors ◽  
Human Subjects ◽  
Brain Regions ◽  
Nicotinic Acetylcholine ◽  
Basic Activity ◽  
The Central Nervous System ◽  
Steady State Levels ◽  
Fast Activation

This review attempts to organize the different aspects of nicotinic transmission in the context of nonsynaptic interactions. Nicotinic acetylcholine receptors (nAChRs) dominantly operate in the nonsynaptic mode in the central nervous system despite their ligand-gated ion-channel nature, which would otherwise be better suited for fast synaptic transmission. This fast form of nonsynaptic transmission, most likely unique to nAChRs, represents a new avenue in the communication platforms of the brain. Cholinergic messages received by nAChRs, arriving at a later phase following synaptic activation, can interfere with dendritic signal integration. Nicotinic transmission plays a role in both neural plasticity and cellular learning processes, as well as in long-term changes in basic activity through fast activation, desensitization of receptors, and fluctuations of the steady-state levels of ACh. ACh release can contribute to plastic changes via activation of nAChRs in neurons and therefore plays a role in learning and memory in different brain regions. Assuming that nAChRs in human subjects are ready to receive long-lasting messages from the extracellular space because of their predominantly nonsynaptic distribution, they offer an ideal target for drug therapy at low, nontoxic drug levels.

scholarly journals Cellular Plasmalogen Content Does Not Influence Arachidonic Acid Levels or Distribution in Macrophages: A Role for Cytosolic Phospholipase A2γ in Phospholipid Remodeling

Cells ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 799 ◽  
Author(s):  
Patricia Lebrero ◽  
Alma M. Astudillo ◽  
Julio M. Rubio ◽  
Lidia Fernández-Caballero ◽  
George Kokotos ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Phospholipid Composition ◽  
Limiting Factor ◽  
Membrane Phospholipids ◽  
Cytosolic Phospholipase ◽  
Rich Media ◽  
Plasmalogen Content ◽  
Free Arachidonic Acid ◽  
Ethanolamine Plasmalogens

Availability of free arachidonic acid (AA) constitutes a rate limiting factor for cellular eicosanoid synthesis. AA distributes differentially across membrane phospholipids, which is largely due to the action of coenzyme A-independent transacylase (CoA-IT), an enzyme that moves the fatty acid primarily from diacyl phospholipid species to ether-containing species, particularly the ethanolamine plasmalogens. In this work, we examined the dependence of AA remodeling on plasmalogen content using the murine macrophage cell line RAW264.7 and its plasmalogen-deficient variants RAW.12 and RAW.108. All three strains remodeled AA between phospholipids with similar magnitude and kinetics, thus demonstrating that cellular plasmalogen content does not influence the process. Cell stimulation with yeast-derived zymosan also had no effect on AA remodeling, but incubating the cells in AA-rich media markedly slowed down the process. Further, knockdown of cytosolic-group IVC phospholipase A2γ (cPLA2γ) by RNA silencing significantly reduced AA remodeling, while inhibition of other major phospholipase A2 forms such as cytosolic phospholipase A2α, calcium-independent phospholipase A2β, or secreted phospholipase A2 had no effect. These results uncover new regulatory features of CoA-IT-mediated transacylation reactions in cellular AA homeostasis and suggest a hitherto unrecognized role for cPLA2γ in maintaining membrane phospholipid composition via regulation of AA remodeling.

scholarly journals Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 470 ◽  
Author(s):  
Anish Deshpande ◽  
Remitha M. Vinayakamoorthy ◽  
Brijesh K. Garg ◽  
Jaya Prakash Thummapudi ◽  
Gauri Oza ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Splice Variants ◽  
Brain Regions ◽  
Nucleotide Polymorphisms ◽  
Western Blots ◽  
Alpha Bungarotoxin

Alpha7 nicotinic acetylcholine receptors (α7nAChRs) are interesting not only because of their physiological effects, but because this receptor requires chaperones to traffic to cell surfaces (measured by alpha-bungarotoxin [αBGT] binding). While knockout (KO) animals and antibodies that react across species exist for tmem35a encoding the protein chaperone NACHO, commercially available antibodies against the chaperone RIC3 that allow Western blots across species have not been generally available. Further, no effects of deleting RIC3 function (ric3 KO) on α7nAChR expression are reported. Finally, antibodies against α7nAChRs have shown various deficiencies. We find mouse macrophages bind αBGT but lack NACHO. We also report on a new α7nAChR antibody and testing commercially available anti-RIC3 antibodies that react across species allowing Western blot analysis of in vitro cultures. These antibodies also react to specific RIC3 splice variants and single-nucleotide polymorphisms. Preliminary autoradiographic analysis reveals that ric3 KOs show subtle αBGT binding changes across different mouse brain regions, while tmem35a KOs show a complete loss of αBGT binding. These findings are inconsistent with effects observed in vitro, as RIC3 promotes αBGT binding to α7nAChRs expressed in HEK cells, even in the absence of NACHO. Collectively, additional regulatory factors are likely involved in the in vivo expression of α7nAChRs.

scholarly journals Phospholipid Arachidonic Acid Remodeling During Phagocytosis in Mouse Peritoneal Macrophages

Biomedicines ◽  
2020 ◽  
Vol 8 (8) ◽  
pp. 274
Author(s):  
Luis Gil-de-Gómez ◽  
Patricia Monge ◽  
Juan P. Rodríguez ◽  
Alma M. Astudillo ◽  
María A. Balboa ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Peritoneal Macrophages ◽  
Lipid Mediator ◽  
Membrane Phospholipids ◽  
Lipid Profiling ◽  
Surface Receptors ◽  
Cytosolic Phospholipase ◽  
Mouse Peritoneal Macrophages ◽  
Phospholipid Species ◽  
Potential Use

Macrophages contain large amounts of arachidonic acid (AA), which distributes differentially across membrane phospholipids. This is largely due to the action of coenzyme A-independent transacylase (CoA-IT), which transfers the AA primarily from diacyl choline-containing phospholipids to ethanolamine-containing phospholipids. In this work we have comparatively analyzed glycerophospholipid changes leading to AA mobilization in mouse peritoneal macrophages responding to either zymosan or serum-opsonized zymosan (OpZ). These two phagocytic stimuli promote the cytosolic phospholipase A2-dependent mobilization of AA by activating distinct surface receptors. Application of mass spectrometry-based lipid profiling to identify changes in AA-containing phospholipids during macrophage exposure to both stimuli revealed significant decreases in the levels of all major choline phospholipid molecular species and a major phosphatidylinositol species. Importantly, while no changes in ethanolamine phospholipid species were detected on stimulation with zymosan, significant decreases in these species were observed when OpZ was used. Analyses of CoA-IT-mediated AA remodeling revealed that the process occurred faster in the zymosan-stimulated cells compared with OpZ-stimulated cells. Pharmacological inhibition of CoA-IT strongly blunted AA release in response to zymosan but had only a moderate effect on the OpZ-mediated response. These results suggest a hitherto undescribed receptor-dependent role for CoA-independent AA remodeling reactions in modulating the eicosanoid biosynthetic response of macrophages. Our data help define novel targets within the AA remodeling pathway with potential use to control lipid mediator formation

scholarly journals A Lipidomic Perspective of the Action of Group IIA Secreted Phospholipase A2 on Human Monocytes: Lipid Droplet Biogenesis and Activation of Cytosolic Phospholipase A2α

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 891 ◽  
Author(s):  
Juan P. Rodríguez ◽  
Elbio Leiguez ◽  
Carlos Guijas ◽  
Bruno Lomonte ◽  
José M. Gutiérrez ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Lipid Droplet ◽  
Human Peripheral Blood ◽  
Fatty Acid Content ◽  
Blood Monocytes ◽  
Membrane Phospholipids ◽  
Cytosolic Phospholipase ◽  
Secreted Phospholipase A2 ◽  
Cytosolic Phospholipase A2α

Phospholipase A2s constitute a wide group of lipid-modifying enzymes which display a variety of functions in innate immune responses. In this work, we utilized mass spectrometry-based lipidomic approaches to investigate the action of Asp-49 Ca2+-dependent secreted phospholipase A2 (sPLA2) (MT-III) and Lys-49 sPLA2 (MT-II), two group IIA phospholipase A2s isolated from the venom of the snake Bothrops asper, on human peripheral blood monocytes. MT-III is catalytically active, whereas MT-II lacks enzyme activity. A large decrease in the fatty acid content of membrane phospholipids was detected in MT III-treated monocytes. The significant diminution of the cellular content of phospholipid-bound arachidonic acid seemed to be mediated, in part, by the activation of the endogenous group IVA cytosolic phospholipase A2α. MT-III triggered the formation of triacylglycerol and cholesterol enriched in palmitic, stearic, and oleic acids, but not arachidonic acid, along with an increase in lipid droplet synthesis. Additionally, it was shown that the increased availability of arachidonic acid arising from phospholipid hydrolysis promoted abundant eicosanoid synthesis. The inactive form, MT-II, failed to produce any of the effects described above. These studies provide a complete lipidomic characterization of the monocyte response to snake venom group IIA phospholipase A2, and reveal significant connections among lipid droplet biogenesis, cell signaling and biochemical pathways that contribute to initiating the inflammatory response.

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