Localization Of Nicotinic Acetylcholine Receptors Research Articles

Intracellular localization of nicotinic acetylcholine receptors in human cell lines

AimsPreviously we demonstrated that mouse liver mitochondria express functional α7 nicotinic acetylcholine receptors (nAChRs). The aim of this study was to investigate whether the nAChRs are found in mitochondria of non-neuronal human cell lines. Main methodsThree cell lines: U373 (astrocytes), U937 (monocytes) and Daudi (B lymphocytes) were examined by flow cytometry, Cell ELISA and fluorescent confocal microscopy using the antibodies against extracellular epitopes of α3, α4, α7, α9, β2 and β4 nAChR subunits. Key findingsIt is shown that the studied cells expressed different sets of nAChR subunits on the plasma membrane suggesting the presence of α7 nAChRs on all cells, of α3β4 nAChRs on U373 cells and of α4β2/α4β4 nAChRs on U937 cells. In addition to nAChRs exposed on the surface, all cells contained a considerable intracellular pool of α3- and α7-containing nAChRs. The binding of α3-, α7- and β4-specific antibodies partially overlapped with that of mitochondrial outer membrane translocase-specific antibody. Binding of α7-specific antibody also overlapped with that of MitoTracker Green, which binds to active mitochondria. SignificanceThe data obtained suggest that a part of intracellular α3β4 and α7 nAChRs in U373, U937 and Daudi cells is located on mitochondria. This finding complements our previous observation of α7 nAChRs in mouse liver mitochondria and reveals new intracellular targets for cholinergic regulation.

Ultrastructural visualization of the transmembranous and cytomatrix-related part of nicotinic acetylcholine receptor of frog motor endplate by means of an immunochemical avidity of IgG for d-tubocurarine.

In the present study, a fine ultrastructural localization of nicotinic acetylcholine receptor (nAChR) was attempted, using d-tubocurarine (d-TC), a quaternary ammonium compound binding to nAChR. The localization was based on the binding avidity of immunoglobulin G (IgG) for acetylcholine (ACh) and other quaternary ammonium compounds, such as d-TC. d-TC was applied to the frog neuromuscular preparation and caused a blockade of neuromuscular transmission. Then, d-TC was rendered insoluble in situ by silicotungstic acid (STA), a precipitating agent of soluble proteins and quaternary ammonium compounds. After tissue fixation, a normal rabbit serum was applied to the fine precipitate of the insoluble salt of d-TC silicotungstate (quaternary ammonium radical of d-TC) to form the immunochemical complex d-TC- rabbit IgG at ACh binding sites. The IgG of the complex was revealed by means of the conventional immunoperoxidase procedure used for ultrastructural localization. Under the electron microscope, fine diaminobenzidine (DAB) precipitates appeared as regular rod-like structures oriented to cytoplasmic side of the horizontal part (crest) of the postsynaptic membrane (between the junctional folds) which is known to be endowed with nAChR. The rod-like precipitates were not observed in the postsynaptic junctional folds which are devoid of nAChR. The distance separating the rods each other was rather constant (12 - 15 nm), while the length of the rods was variable and exceeded the usual length of nAChR. The present work indicates that the rod-like structures, already observed in association with sarcoplasmic side of the postsynaptic membrane, did correspond to the intramembranous and intracytoplasmic part of nAChR and related proteins. These cytochemical results confirm that d-TC binds to ACh binding sites in the pore of nAChR, and raise the question of DAB staining of cytoskeletal proteins related to the nAChR complex.

Distribution and synaptic localization of nicotinic acetylcholine receptors containing a novel α7 subunit isoform in embryonic rat cortical neurons

Neuronal nicotinic acetylcholine receptors (nAChRs) containing the α7 subunit isoform, α7-2 (α7-2-nAChRs), have previously been found to form functional homopentameric channels that desensitize slowly and bind α-bungarotoxin (αBgt) in a rapidly reversible manner. This isoform incorporates a novel cassette exon in the extracellular, ligand binding domain of the native receptor. Although this α7 subunit isoform has been detected in peripheral ganglia as well as in the central nervous system, little is known about the cellular function of α7-2-nAChRs. Co-localization immunocytochemical studies were conducted in an embryonic rat cultured cortical neuron model using a polyclonal antibody (Ab 87) raised against the amino acid sequence of the cassette exon, in combination with (1) an antibody that recognizes all known α7-nAChRs, (2) αBgt, and (3) antibodies directed against multiple cellular markers. The pattern of α7-2-nAChR expression was consistent with α7 staining in general, based on co-distribution of mAb319 and αBgt signals. However, α7-2-nAChRs clearly represent a distinct subset of α7 receptors. The α7-2-nAChR subtype was found throughout the cell-soma surface and was localized to a subpopulation of dendrites. Punctate staining characteristic of synaptic α7-2 targeting was observed at post-synaptic densities and intermittently at pre-synaptic locations. The α7-2 subunit was expressed on both GABAergic and non-GABAergic neurons. These studies reveal that receptors containing the α7-2 subunit constitute a subpopulation of α7-nAChRs and likely participate in cell-to-cell signaling in developing synapses of central neurons.

Localization of Nicotinic Acetylcholine Receptors on Somatic Spines of Chick Ciliary Ganglion Neurons

Nicotinic acetylcholine receptors (nAChRs) are pentameric membrane proteins that function as cation selective, ligand-gated ion channels and are widely distributed throughout the vertebrate nervous system. One of the most abundant nAChRs is a species that contains the α7 gene product (α7-AChRs), binds the snake venom oc-bungarotoxin, and rapidly desensitizes. These receptors have been shown to function at presynaptic sites to modulate transmitter release, as well as on postsynaptic cells where they mediate transmission. Interestingly, these receptors have an exceptionally high relative permeability to calcium.In the chick ciliary ganglion, the α 7-AChRs play a prominent role, by generating large synaptic currents, but the receptors appear to be excluded from postysnaptic densities on the cell. Immunohistochemical studies have shown that the receptors form large clusters on the surface of the ciliary ganglion neurons. We have recently shown that the α 7-containing receptors are concentrated on mats of somatic spines in close proximity to putative sites of presynaptic transmitter release.

Immunohistochemical localization of nicotinic acetylcholine receptors in the guinea pig bowel and pancreas

Immunohistochemical localization of nicotinic acetylcholine receptors in the guinea pig bowel and pancreas

Immunohistochemical localization of nicotinic acetylcholine receptors in the guinea pig bowel and pancreas

Although nicotinic acetylcholine receptors (nAChRs) are known to be present on neural elements in both the bowel and the pancreas, the precise location of these receptors has not previously been determined. Immunocytochemistry, by using a rat monoclonal antibody (mAb35), which recognizes alpha-bungarotoxin (alpha-Bgt)-insensitive nAChRs, and a polyclonal antibody raised against the alpha-Bgt-sensitive receptor subunit, alpha7, was used to locate receptor protein in guinea pig gut and pancreas. mAb35-receptor (mAb35-R) immunoreactivity was abundant in both enteric plexuses, enterochromaffin cells, and pancreatic ganglia. Immunostaining was associated with the cell membrane, and clusters of mAb35-R were observed on cell somas and dendrites. Receptor immunoreactivity was also observed on terminals and axons, suggesting that a subset of nAChRs is presynaptic. Internal sites of mAb35-R were observed in permeabilized ganglia. Cells expressing the receptors were closely associated with ChAT-immunoreactive nerve fibers. In addition, the majority of ChAT-positive neurons expressed both cell surface and internal stores of mAb35-R. In the bowel, clusters of mAb35-R were present on the soma and dendrites of Dogiel type I motorneurons and secretomotor neurons. Receptors were detected at the plasma membrane of calbindin-immunoreactive myenteric neurons. In contrast, calbindin-immunoreactive submucosal neurons did not express cell surface mAb35-R, supporting the idea that they are sensory neurons. A subset of enteric neurons expressed both mAb35-R and glutamate receptor (GluR1) immunoreactivity. In the pancreas, mAb35-R immunoreactivity was only observed in ganglia. Alpha7-immunoreactivity was found on both enteric cell bodies and nerve fibers. Based on these results, it appears that visceral nAChRs are composed of at least four subunits and that both pre- and postsynaptic nAChRs are present in the gut and pancreas.

Immunocytochemical localization of nicotinic acetylcholine receptor in the rat cerebellar cortex

Although nicotinic acetylcholine receptors (nAChRs) have been reported to function in the cerebellar cortex, nicotinic acetylcholine receptor subtypes contributing to nicotinic currents in the cortex have not been well characterized at the subcellular level. This study deals with immunocytochemical localization of the nicotinic acetylcholine receptor α4 subunit using a monoclonal antibody against the α4 subunit. α4-LI (α4-like immunoreactivity) was detected in the cell bodies of molecular, Purkinje cell and granular layers. In particular, the cell bodies of Purkinje cells were extensively immunostained. In Purkinje cells, α4-LI was found in perikarya mainly associated with rough endoplasmic reticulum, plasma membrane, and cytoplasmic matrix. At higher magnification, the immunoreaction product was densely localized along with somatic plasma membranes at the axo-somatic synapse and the plasma membranes at extrasynaptic regions of cell bodies. α4-LI was also found in the axon terminals which form synapses with Purkinje cells. In the granular layer, somatic cell membranes of granular cells were immunostained. These morphological observations suggest that α4-containing nAChRs contribute nicotinic currents reported in Purkinje cells, and that presynaptic α4-containing nAChRs regulate the release of neurotransmitters on the axon terminals found near Purkinje cells.

Localization of nicotinic acetylcholine receptors in cat carotid body and petrosal ganglion.

How the carotid body converts hypoxic stimuli into neural activity remains largely unknown. Several neurotransmitters, which have been demonstrated in type I cells in the carotid body, are still candidates for primary neurotransmitter(s) of chemotransduction.KeywordsTyrosine HydroxylaseNicotinic Acetylcholine ReceptorCarotid BodyGlomus CellSuperior Cervical Ganglion NeuronThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Immunocytochemical localization of nicotinic acetylcholine receptor in rat cerebral cortex.

Localization of nicotinic acetylcholine receptor (nAChR) alpha 4 subunits was investigated in rat cerebral cortex using a monoclonal antibody against alpha 4 subunits. The antibody depleted more than 70% of the [3H]methylcarbamylcholine choline binding activity of the solubilized membrane fraction. By light microscopy alpha 4-like immunoreactivity (alpha 4-LI) was found through layers II to VI. The immunostaining was the most prominent in cell bodies and apical dendrites of pyramidal cells in layer V. By electron microscopy most immunoreaction products were observed in the rough endoplasmic reticulum, cytoplasmic matrix and synaptic membranes. Alpha 4-LI was detected in the postsynaptic membranes of neuronal cell bodies and apical dendrites. These findings suggest that alpha 4-containing subtypes serve as one possibly the postsynaptic nAChR in rat cerebral cortex.

Immunocytochemical localization of nicotinic acetylcholine receptor in rat hypothalamus

Immunocytochemical localization of neuronal nicotinic acetylcholine receptor (nAChR) was examined in rat hypothalamus. Monoclonal antibody against α4 ACh-binding subunits of nAChR was used in the avidin-biotin-peroxidase complex (ABC) immunocytochemical method at both the light and electron microscopic levels. By light microscopy nAChR-like immunoreactivity was found in many neuronal cell bodies and their fibers in the paraventricular nucleus (PVN) and in many axons and axon terminals in the median eminence (ME). The immunoreactivity of nAChR was the most intense in the ME. By electron microscopy immunoreaction products occurred on the rough endoplasmic reticulum, nuclear envelope, cytoplasmic matrices and postsynaptic densities of synaptic junctions in some neurons in the parvocellular part of the PVN. In the external layer of the ME, nAChR-like immunoreactivity was found over the entire plasma membranes of many axon terminals. Involvement of nAChRs in the release of neurotransmitters and neuropeptides both in the PVN and the ME is discussed.

O-283 - Localization of nicotinic acetylcholine receptor and corticotropin releasing factor in rat hypothalamus

O-283 - Localization of nicotinic acetylcholine receptor and corticotropin releasing factor in rat hypothalamus

Expression of RAPsyn (43K protein) and nicotinic acetylcholine receptor genes is not coordinatelv regulated in mouse muscle

RAPsyn (also known as 43K protein), a mouse muscle protein localized to the synaptic membrane, is thought to be involved in the localization of nicotinic acetylcholine receptors at the neuromuscular junction. We have characterized the transcriptional regulation of the RAPsyn gene and the synthesis of the RAPsyn protein during muscle cell differentiation. Nuclear run-on experiments and RNAase protection analyses showed that mRNA encoding RAPsyn, but not the acetylcholine receptor subunits, is present in undifferentiated muscle cells. The RAPsyn protein present in undifferentiated and differentiated muscle cells cannot be distinguished by peptide maps, turnover rates, cellular subfractionation, or ability to incorporate myristate. Whereas the amount of acetylcholine receptor subunit mRNA is increased approximately 100-fold after denervation, the amount of RAPsyn mRNA is increased just 2- to 3-fold. We conclude that the expression of RAPsyn and the acetylcholine receptor is not coordinately regulated in mouse muscle.

Autoradiographic localization of nicotinic acetylcholine receptors in the brain of the zebra finch (Poephila guttata).

We have localized nicotinic acetylcholine receptors in the zebra finch brain by using three 125I-labelled ligands: alpha bungarotoxin and two monoclonal antibodies to neuronal nicotinic receptors (MAb 35 of Tzardos et al., J. Biol. Chem., 250: 8635-8645, '81; and MAb 270 of Whiting and Lindstrom: J. Neurosci. 6: 3061-3069, '86). Unfixed brains from intact adult male and female zebra finches were prepared for in vitro autoradiography. Low-resolution film autoradiograms and high-resolution emulsion autoradiograms were prepared for each of the three ligands. The major brain structures that bind all three of the ligands are hippocampus; hyperstriatum dorsalis; hyperstriatum ventralis; nucleus lentiformis mesencephali; nucleus pretectalis, some layers of the optic tectum; nucleus mesencephalicus lateralis; pars dorsalis; locus ceruleus; and all cranial motor nuclei except nucleus nervi hypoglossi. The major structures labelled only by [125I]-alpha bungarotoxin binding included hyperstriatum accessorium and the nuclei: preopticus medialis, medialis hypothalami posterioris, semilunaris, olivarius inferior, and the periventricular organ. Of the song control nuclei, nucleus magnocellularis of the anterior neostriatum; hyperstriatum ventralis, pars caudalis; nucleus intercollicularis; and nucleus hypoglossus were labelled. The binding patterns of the two antibodies were similar to one another but not identical. Both labelled nucleus spiriformis lateralis and nucleus geniculatus lateralis, pars ventralis especially heavily and also labelled the nucleus habenula medialis; nucleus subpretectalis; nucleus isthmi, pars magnocellularis; nucleus reticularis gigantocellularis; nucleus reticularis lateralis; nucleus tractus solitarii; nucleus vestibularis dorsolateralis; nucleus vestibularis lateralis; nucleus descendens nervi trigemini; and the deep cerebellar nuclei. Lobus parolfactorius and nucleus vestibularis medialis were labelled by only MAb 270, whereas only MAb 35 labelled nucleus laminaris and the medial and lateral pontine nuclei. These data extend previous reports of cholinergic participation in the song system (Ryan and Arnold: J. Comp. Neurol. 202: 211-219, '81) to suggest that the zebra finch song system may contain several closely related nicotinic receptors. In several brain nuclei it appeared that certain anatomical portions of a nucleus or a certain class of neurons were specifically labelled. Furthermore, in certain cases, the labelling appeared to be clustered around Nissl-stained cell nuclei, thus suggesting that the receptors are concentrated on or in somata.

Immunocytochemical localization of α-bungarotoxin receptors in the chick ciliary ganglion: Synaptic and extrasynaptic sites?

The immunocytochemical localization of nicotinic acetylcholine receptors in the chick ciliary ganglion was investigated at the ultrastructural level with a procedure utilizing binding of α-bungarotoxin visualized by immunoperoxidase histology. Both ganglionic cell populations, i.e. the ciliary and choroid neurons, showed specific immunoreactivity for receptors. In both types of neurons evident stain was found on the postsynaptic membrane. Often the reaction product filled discrete regions of the synaptic cleft. Furthermore, specific staining was also observed on large areas of the neuronal surface devoid of presynaptic nerve endings. These data probably indicate the occurrence of both synaptic and extrasynaptic nicotinic receptors on the neuronal plasma membrane. Immunoreactivity was also observed on the membrane of the presynaptic nerve endings, and on the part of the satellite plasma membrane which is adjacent to the neuron. These last results are discussed in relation to the occurrence of possible artifacts or, alternatively, to the presence of presynaptic and glial receptors. Immunoreactivity at all sites was prevented almost completely by ganglion incubation in 1 mM d-tubocurarine prior to and during treatment with toxin.

Localization of cholinergic synapses in mammalian retina with peroxidase-conjugated α-bungarotoxin

A conjugate of α-bungarotoxin with horseradish peroxidase was used for cytochemical localization of nicotinic acetylcholine receptors in the mouse retina. Reactive synapses were seen at contacts of amacrine cells with bipolar cells and other amacrine cells. The data indicates that a relatively small population of amacrine cells are involved in cholinergic transmission.