Showing papers by "Jon Lindstrom published in 1994"

Nicotine-induced increase in neuronal nicotinic receptors results from a decrease in the rate of receptor turnover.

Abstract: Chronic nicotine exposure in tobacco smokers or experimental animals is known to cause an increase in brain binding sites for nicotine. It has been proposed that this is an adaptive response of neurons to accumulation of chronically desensitized receptors. Acetylcholine receptors of the same (alpha 4)2(beta 2)3 subunit composition as the predominant subtype of brain nicotinic receptors with high affinity for nicotine have been expressed in Xenopus oocytes and in a permanently transfected fibroblast cell line. Chronic exposure of these cells to nicotine or another agonist is shown to result in an increase in receptor amount, indicating that nicotine-induced up-regulation reflects properties of the alpha 4 beta 2 receptor protein, rather than being an adaptive response unique to the neurons in which these receptors are normally expressed. The nicotine concentration dependence, time course, and extent of receptor up-regulation are similar to those reported for receptors in brain. Up-regulation does not appear to require ion flow through the ion channel, because it is also caused by mecamylamine, which blocks the ion channel, and because after prolonged exposure to nicotine most receptors become permanently unable to open their channels in response to nicotine binding. The noncompetitive antagonist mecamylamine blocks open channels more effectively, and so it is more effective at blocking channels in the presence of nicotine. Mecamylamine and nicotine are also synergistic in causing receptor up-regulation. Ligands that cause up-regulation appear to induce a conformation of the receptor that is removed from the surface and degraded more slowly.

Immunocytochemical localization of the alpha 7 subunit of the nicotinic acetylcholine receptor in the rat central nervous system.

Abstract: Previous molecular cloning studies have revealed that alpha-bungarotoxin binding proteins present in the brain are members of the neuronal nicotinic acetylcholine receptor gene family. The alpha 7 subunit is structurally related to the agonist binding subunits present in the central and peripheral nervous systems and, when expressed in Xenopus oocytes, forms functional channels blockable by alpha-bungarotoxin. In the present study, three different monoclonal antibodies raised against the alpha 7 subunit were used to map its distribution throughout the central nervous system of the rat. Immunohistochemical localization revealed that the alpha 7 subunit is expressed in most regions of the brain, being, overall, well correlated with previous "in situ" localization of alpha 7 transcripts and alpha-bungarotoxin autoradiographic binding studies. Particularly strong immunoreactivity was observed in several sensory and motor nuclei of the brainstem as well as the red nucleus. At the cellular level, alpha 7 immunostaining was usually found both in somata and dendrites, whereas axonal and terminal labeling was not observed. The widespread distribution of the alpha 7 subunit polypeptide is consistent with immunoprecipitation data demonstrating that it is a component of the predominant subtype of brain alpha-bungarotoxin-sensitive nicotinic receptors.

Human alpha 7 acetylcholine receptor: cloning of the alpha 7 subunit from the SH-SY5Y cell line and determination of pharmacological properties of native receptors and functional alpha 7 homomers expressed in Xenopus oocytes.

Abstract: The alpha-bungarotoxin-binding acetylcholine receptors from the human neuroblastoma cell line SH-SY5Y were found to cross-react with some monoclonal antibodies to alpha 7 subunits of nicotinic acetylcholine receptors from chicken brain. The human alpha 7 subunit cDNA from SH-SY5Y was cloned, revealing 94% amino acid sequence identity to rat alpha 7 subunits and 92% identity to chicken alpha 7 subunits. Native human alpha 7 receptors showed affinities for some ligands similar to those previously observed with native chicken alpha 7 receptors, but for other ligands there were large species-specific differences in binding affinity. These results paralleled properties of alpha 7 homomers expressed in Xenopus oocytes. Human alpha 7 homomers exhibited rapidly desensitizing, inwardly rectifying, agonist-induced, cation currents that triggered Ca(2+)-sensitive Cl- channels in the oocytes. A change in efficacy from partial agonist for chicken alpha 7 homomers to full agonist for human alpha 7 homomers was exhibited by 1,1-dimethyl-4-phenylpiperazinium. This result reveals a large species-specific pharmacological difference, despite small differences in alpha 7 sequences. This is important for understanding the effects of these drugs in humans and for identifying amino acids that may contribute to the acetylcholine binding site, for analysis by in vitro mutagenesis. These results also characterize properties of native alpha 7 receptors and alpha 7 homomers that will provide criteria for functional properties expected of structural subunits, when these can be identified, cloned, and coexpressed with alpha 7 subunits.

Homomers of alpha 8 and alpha 7 subunits of nicotinic receptors exhibit similar channel but contrasting binding site properties.

Abstract: alpha 8 subunits of alpha-bungarotoxin-sensitive chick neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes from cRNA are shown to form homomeric, acetylcholine-gated, rapidly desensitizing, inwardly rectifying, Ca(2+)-permeable cation channels similar to those of alpha 7 homomers. alpha 8 forms oligomers of several sizes, of which < 14% are expressed on the oocyte surface, which is less efficient than for alpha 7 homomers. alpha 8 homomers are more sensitive to agonists but less sensitive to antagonists than are alpha 7 homomers, and some agonists for alpha 8 homomers are partial agonists or antagonists for alpha 7 homomers. The pharmacological properties of homomers of alpha 8 and alpha 7 subunits generally reflect those of native alpha 8 and alpha 7 receptors.

Physostigmine and galanthamine: probes for a novel binding site on the alpha 4 beta 2 subtype of neuronal nicotinic acetylcholine receptors stably expressed in fibroblast cells.

Abstract: In the present study, we demonstrated that the chicken alpha 4 beta 2 neuronal nicotinic receptor stably expressed in transfected mouse fibroblasts (M10 cells) can be activated via the acetylcholine-binding site or via a site that is distinct from that for acetylcholine and recognizes physostigmine and galanthamine as agonists. In outside-out patches excised from dexamethasone-induced M10 cells, (+)-anatoxin-a, physostigmine and galanthamine (each at 1 microM) activated single channels with conductances of 18 and 30 pS. Dihydro-beta-erythroidine (1-30 nM), but not the nicotinic receptor-specific monoclonal antibody FK1, reduced the frequency of channels activated by anatoxin (1 microM). On the other hand, the frequency of channel activity induced by physostigmine (1 microM) was unaffected by dihydro-beta-erythroidine and was markedly decreased by FK1. In uninduced M10 cells and in dexamethasone-treated untransfected fibroblasts, we observed that physostigmine, galanthamine and nicotinic agonists did not evoke whole-cell or single-channel currents. Also, neither [3H]L-nicotine nor FK1 was able to bind to uninduced M10 cells. In dexamethasone-induced M10 cells, the nicotinic agonists acetylcholine, anatoxin, 1,1-dimethyl-4-phenylpiperazinium, (-)-nicotine, and cytisine (each at 100 microM) activated whole-cell currents that showed a marked inward rectification and were sensitive to blockade by dihydro-beta-erythroidine (100 nM). However, neither galanthamine nor physostigmine could evoke whole-cell currents in cells that were responsive to nicotinic agonists. Other effects of physostigmine and galanthamine on the nicotinic receptor that outweight the agonist properties of these compounds could account for their inability to evoke whole-cell currents.

Differential development of alpha-bungarotoxin-sensitive and alpha-bungarotoxin-insensitive nicotinic acetylcholine receptors in the chick retina.

Abstract: The development of cells containing neuronal nicotinic receptors (nAChRs) in the chick retina was investigated by means of immunohistochemical techniques with antibodies directed against the alpha 3 and alpha 8 nAChR subunits. The alpha 3 subunit is one of the major alpha-bungarotoxin-insensitive nicotinic receptor subunits in the chick retina, whereas alpha 8 appears to be the most common alpha-bungarotoxin-sensitive subunit in the same structure, alpha 3-like immunoreactivity (alpha 3-LI) was first detected in cells of the vitreal margin, on the embryonic day 4.5 (E4.5). alpha 8-LI was first detected in the same type of cell almost a day later. However, the processes of alpha 8-LI cells developed much faster than those of alpha 3-LI cells, generating visible stained laminae in the prospective inner plexiform layer as early as E7. alpha 3-LI was only clearly seen in laminae of the inner plexiform layer by E12. By this date, both alpha 3 and alpha 8-LI were seen in the same types of cells as in the adult retina, i.e., amacrines, displaced ganglion cells, and cells of the ganglion cell layer for alpha 3-LI; and amacrines, bipolar cells, and cells of the ganglion cell layer for alpha 8-LI. These results reveal different patterns of development of cells containing the alpha 3 and alpha 8 nAChR subunits in the chick retina and indicate that those nAChR subunits are expressed in the chick retina before choline acetyltransferase-positive cells can be detected and well before synaptogenesis. These data also suggest that nAChRs may have a developmental function in the retina.

Effects of retinal lesions upon the distribution of nicotinic acetylcholine receptor subunits in the chick visual system.

Abstract: Immunohistochemistry was used in this study to evaluate the effects of retinal lesions upon the distribution of neuronal nicotinic acetylcholine receptor subunits in the chick visual system. Following unilateral retinal lesions, the neuropil staining with an antibody against the beta 2 receptor subunit, a major component of alpha-bungarotoxin-insensitive nicotinic receptors, was dramatically reduced or completely eliminated in all of the contralateral retinorecipient structures. On the other hand, neuropil staining with antibodies against two alpha-bungarotoxin-sensitive receptor subunits, alpha 7 and alpha 8, was only slightly affected after retinal lesions. Decreased neuropil staining for alpha 7-like immunoreactivity was only observed in the nucleus of the basal optic root and layers 2-4 and 7 of the optic tectum. For alpha 8-like immunoreactivity, slight reduction of neuropil staining could be observed in the ventral geniculate complex, griseum tecti, nucleus lateralis anterior, nucleus lentiformis mesencephali, layers 4 and 7 of the tectum, and nucleus suprachiasmaticus. Taken together with previous data on the localization of nicotinic receptors in the retina, the present results indicate that the beta 2 subunit is transported from retinal ganglion cells to their central targets, whereas the alpha 7 and alpha 8 subunit immunoreactivity appears to have a central origin. The source of these immunoreactivities could be, at least in part, the stained perikarya that were observed to contain alpha 7 and alpha 8 subunits in all retinorecipient areas. In agreement with this hypothesis, the beta 2 subunit of the nicotinic acetylcholine receptors was not frequently found in perikarya of the same areas.

Bipolar cells of the chick retina containing α-bungarotoxin-sensitive nicotinic acetylcholine receptors

Abstract: Two cDNA clones for nicotinic acetylcholine receptor (nAChR) subunits sensitive to alpha-bungarotoxin (alpha-Bgt) have been isolated, the so-called alpha-Bgt binding proteins alpha 1 (or alpha 7 nAChR subunit) and alpha 2 (or alpha 8 nAChR subunit). Immunohistochemical experiments have shown that both alpha 7 and alpha 8 subunits, as well as subunits insensitive to alpha-Bgt (beta 2 and alpha 3), are present in amacrine and ganglion cells of the chick retina. However, only the alpha 8 subunit was observed in presumptive bipolar cells. The present study investigated in detail the pattern of distribution of the bipolar cells containing the alpha 8 nAChR subunit and its relation to the pattern of distribution of bipolar cells immunoreactive to protein kinase C (PKC). Presumptive alpha 8- and PKC-like immunoreactive (alpha 8-LI and PKC-LI) bipolar cells were observed sending their dendrites to the outer plexiform layers and their axons to the inner plexiform layer. Whereas alpha 8-LI bipolar cells corresponded to 40-53% of the whole population of bipolar cells, PKC-LI bipolar cells represented only 6-8% of the same population. The soma sizes of the alpha 8-LI bipolar cells were slightly smaller (mean +/- S.D.; 4.9 +/- 0.8 microns) than the soma sizes of the PKC-LI bipolar cells (5.4 +/- 0.9 microns). Double-labeling experiments indicated that probably all PKC-LI bipolar cells also contain alpha 8-LI. This indicates that two distinct groups of cholinoceptive bipolar cells exist in the chick retina, one that contains PKC-LI, and another one that does not.

T cell receptor V beta 15 dominates the antiacetylcholine receptor response in Lewis rat T cell lines.

Abstract: Ten Lewis rat T cell lines responsive to Torpedo californica acetylcholine receptor (AChR) were assayed for TCR V beta usage. All lines were CD4+, OX-22-, and exhibited reactivity to one or more AChR chains. Several different V beta s were expressed by these lines, but V beta 15 was dominant in 5 of 10 lines. Unique CDR3 sequences were observed among the 10 lines, although three of the V beta 15 rearrangements used J beta 1.4. These data suggest that V beta 15+ T cells are selected in the in vitro response to the antigenically complex AChR in the Lewis rat.