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Biomedical subjects

J Lindstrom

Publications and source records attributed to J Lindstrom.

At least 19 recordsLinked to original sources
PubMed2000

N-Methyl-D-aspartate receptor mediated toxicity in nonneuronal cell lines: characterization using fluorescent measures of cell viability and reactive oxygen species production.

Cells transfected with specific N-methyl-D-aspartate (NMDA) receptor subtypes undergo cell death that mimics glutamate-induced excitotoxicity pharmacologically. We have further characterized the mechanisms of cell death resulting from NMDA receptor activation in such cells through development of cell counting methods based on co-transfection with green fluorescent protein. When co-transfected with NMDA receptors, GFP expression was limited to live cells as indicated by the observation that GFP was only detected in cells which were positive for markers of live cells, and was found in no cells which were trypan blue or propidium iodide positive. Using co-transfection with green fluorescent protein and cell counting of viable cells with a fluorescence activated cells sorter, we confirmed the subunit-specific profile of NMDA receptor-mediated cell death in cells transfected with NMDA receptors. Toxicity was greatest in the NR1A/2A receptor, less in the NR1A/2B receptor, and least in NR1A/2C receptors. Cell death also differed pharmacologically between subunit combinations. Cell death in cells transfected with NR 1A/2A was blocked by amino-phosphonovaleric acid at lower concentrations than in cells transfected with NR 1A/2B. In cells transfected with the NR1A/2A or NR1A/2B combinations but not NR1A/2C, cell death was also associated with production of reactive oxygen species. In addition, removal of the final 400 amino acids of the C-terminal region of NR2A decreased cell death. The use of GFP based cell counting provides a sensitive mechanism for assessing the mechanism of excitotoxicity in transfected cell models.

2-Amino-5-phosphonovalerate↗
PubMed2000

Acetylcholine receptor extracellular domain determines sensitivity to nicotine-induced inactivation.

We have shown previously that chronic exposure to submicromolar concentrations of nicotine permanently inactivates alpha4beta2 and alpha7 neuronal nicotinic acetylcholine receptors while alpha3beta2 acetylcholine receptors are resistant to inactivation. Phosphorylation of the large cytoplasmic domain has been proposed to mediate functional inactivation. Chimeric subunits consisting of human alpha4 sequence from their N-terminus to either the beginning of the first transmembrane domain or the large cytoplasmic domain and alpha3 sequences thereafter formed acetylcholine receptors with beta2 subunits which were as susceptible to nicotine-induced inactivation as wild-type alpha4 acetylcholine receptors. The converse chimeras, containing the N-terminal parts of the alpha3 subunit and the C-terminal parts of the alpha4 subunit, formed acetylcholine receptors with beta2 subunits which were as resistant to nicotine-induced inactivation as wild-type alpha3beta2 acetylcholine receptors. Thus, inactivation of acetylcholine receptors produced by chronic exposure to nicotine results primarily from effects of the agonist on the extracellular and transmembrane domains of the alpha subunit.

Animals↗
PubMed2000

Quantitative assessment of nicotinic acetylcholine receptor proteins in the cerebral cortex of Alzheimer patients.

Cholinergic transmission has for long been known to be one of the most severely affected systems in Alzheimer's disease (AD), resulting clinically in massive cognitive deficits. The molecular basis of this dysfunction--on both the pre- and the postsynaptic sites--is still a matter of ongoing investigations. Here, we report on the quantitative assessment of nicotinic acetylcholine receptor isoform expression in AD vs. control cortices. For both subunit proteins assessed, the alpha4 and the alpha7 isoform, highly significant decreases in diseased vs. normal cortices were observed. Both alpha4 and alpha7 subunits are known to be important constituents in hetero- (alpha4beta2) and homooligomeric (alpha7) receptor subtypes. Their decreased expression may contribute to the decreased nicotinic binding known to be accompanied by AD and severe cognitive deficits. The quantitative assessment of nicotinic acetylcholine receptor expression will help to determine those subunits suited as targets for pharmacological stimulation.

Aged↗
PubMed2000

A comparative non-radioactive in situ hybridization and immunohistochemical study of the distribution of alpha7 and alpha8 subunits of the nicotinic acetylcholine receptors in visual areas of the chick brain.

The distribution of mRNA transcripts corresponding to the alpha7 and alpha8 subunits of the nicotinic acetylcholine receptors (nAChRs) was studied in selected structures of the chick visual system with non-radioactive in situ hybridization and immunohistochemical techniques. The results indicated that the alpha7 and alpha8 nAChR transcripts are widely distributed in the brain, exhibiting differential expression in some structures but also some degree of co-localization. The pattern of localization of alpha7 and alpha8 nAChR transcripts was highly correlated with immunohistochemical data, with very few instances of possible mismatches between the distribution of mRNAs and their corresponding proteins.

Animals↗
PubMed1999

Single channel properties of human alpha3 AChRs: impact of beta2, beta4 and alpha5 subunits.

1. We performed single channel analysis on human alpha3 acetylcholine receptors (AChRs) in Xenopus oocytes and native AChRs from the human neuroblastoma cell line IMR-32. alpha3 AChRs exhibit channel properties that reflect subunit composition. 2. alpha3beta2 AChR open times were 0.71 +/- 0.14 and 3.5 +/- 0.4 ms with a predominant conductance of 26 pS. alpha3beta4 AChRs had open times of 1.4 +/- 0.2 and 6.5 +/- 0.8 ms and a predominant conductance of 31 pS. Burst times were 0.82 +/- 0.12 and 5.3 +/- 0.7 ms for alpha3beta2 and 1.7 +/- 0.1 and 16 +/- 1 ms for alpha3beta4. Desensitization was faster for AChRs with the beta2 subunit than for those with the beta4 subunit. 3. One open time for alpha3alpha5beta2 AChRs (5.5 +/- 0.3 ms) was different from those of alpha3beta2 AChRs. For alpha3alpha5beta4 AChRs, an additional conductance, open time and burst time (36 pS, 22 +/- 3 ms and 43 +/- 4 ms, respectively) were different from those for alpha3beta4 AChRs. 4. alpha3 AChRs were inhibited by hexamethonium or mecamylamine. The rate constants for block of alpha3beta4 by hexamethonium and of alpha3beta2 by mecamylamine were 1.2 x 107 and 4.6 x 107 M-1 s-1, respectively. 5. AChRs from IMR-32 cells had a predominant conductance of 32 pS and open times of 1.5 +/- 0.3 and 9.6 +/- 1.2 ms. These properties were most similar to those of alpha3beta4 AChRs expressed in oocytes. Antibodies revealed that 5 +/- 2 % of IMR-32 alpha3 AChRs contained alpha5 subunits and 6 +/- 2 % contained beta2 subunits. IMR-32 alpha3 AChRs are primarily alpha3beta4 AChRs.

Animals↗
PubMed1999

Spatiotemporal patterns of activity in an intact mammalian network with single-cell resolution: optical studies of nicotinic activity in an enteric plexus.

Multiple Site Optical Recording of Transmembrane Voltage (MSORTV) has been used to measure, continuously and simultaneously, the spontaneous electrical activity from all of the neurons in individual ganglia or up to five interconnected ganglia of the submucous plexus of the guinea pig small intestine. These are the first optical recordings of electrical activity with single-cell resolution from a mammalian nervous system. They are used to investigate the effects of acute and chronic application of nicotine on the firing patterns of this neural network containing important cholinergic components. After washout of acutely applied nicotine, the firing rates of selected neurons were dramatically elevated. These results suggest that nAChRs that reversibly desensitize after exposure to nicotine may be responsible for the enhancement of activity that is observed after a brief application of this agonist. In addition, immunostaining with monoclonal antibodies was used to localize alpha3/alpha5, alpha7, and beta2 nAChR subunits, and the results demonstrate the prevalence of alpha3/alpha5. It is this alpha3-containing nAChR subtype that probably accounts for most of the excess activity elicited by nicotine application.

Animals↗
PubMed1999

Expression of nicotinic acetylcholine receptor subunits in the cerebral cortex in Alzheimer's disease: histotopographical correlation with amyloid plaques and hyperphosphorylated-tau protein.

Impairment of cholinergic transmission and decreased numbers of nicotinic binding sites are well-known features accompanying the cognitive dysfunction seen in Alzheimer's disease (AD). In order to elucidate the underlying cause of this cholinoceptive dysfunction, the expression of two pharmacologically different nicotinic acetylcholine receptor (nAChR) subunits (alpha4, alpha7) was studied in the cerebral cortex of Alzheimer patients as compared to controls. Patch-clamp recordings of 14 dissociated neurons of control cortices showed responses suggesting the existence of alpha4- and alpha7-containing functional nAChRs in the human cortex. In cortices of Alzheimer patients and controls, the pattern of distribution and the number of alpha4 and alpha7 mRNA-expressing neurons were similar, whereas at the protein level a decrease in the density of alpha4- and alpha7-expressing neurons of approximately 30% was observed in Alzheimer patients. The histotopographical correlation of nAChR expression with accompanying pathological changes, e.g. accumulation of hyperphosphorylated-tau (HP-tau) protein and beta-amyloid showed that neurons in the vicinity of beta-amyloid plaques bore both nAChR transcripts. Neurons heavily labelled for HP-tau, however, expressed little or no alpha4 and alpha7 mRNA. These results point to an impaired synthesis of nAChRs on the protein level as a possible cause of the cholinoceptive deficit in AD. Further investigations need to elucidate whether interactions of HP-tau with nAChR mRNA, or alterations in the quality of alpha4 and alpha7 transcripts give rise to decreased protein expression at the level of individual neurons.

Aged↗
PubMed1999

Prenatal nicotine increases pulmonary alpha7 nicotinic receptor expression and alters fetal lung development in monkeys.

It is well established that maternal smoking during pregnancy is a leading preventable cause of low birth weight and prematurity. Less appreciated is that maternal smoking during pregnancy is also associated with alterations in pulmonary function at birth and greater incidence of respiratory illnesses after birth. To determine if this is the direct result of nicotine interacting with nicotinic cholinergic receptors (nAChRs) during lung development, rhesus monkeys were treated with 1 mg/kg/day of nicotine from days 26 to 134 of pregnancy. Nicotine administration caused lung hypoplasia and reduced surface complexity of developing alveoli. Immunohistochemistry and in situ alpha-bungarotoxin (alphaBGT) binding showed that alpha7 nAChRs are present in the developing lung in airway epithelial cells, cells surrounding large airways and blood vessels, alveolar type II cells, free alveolar macrophages, and pulmonary neuroendocrine cells (PNEC). As detected both by immunohistochemistry and by alphaBGT binding, nicotine administration markedly increased alpha7 receptor subunit expression and binding in the fetal lung. Correlating with areas of increased alpha7 expression, collagen expression surrounding large airways and vessels was significantly increased. Nicotine also significantly increased numbers of type II cells and neuroendocrine cells in neuroepithelial bodies. These findings demonstrate that nicotine can alter fetal monkey lung development by crossing the placenta to interact directly with nicotinic receptors on non-neuronal cells in the developing lung, and that similar effects likely occur in human infants whose mothers smoke during pregnancy.

Animals↗
PubMed1998

Chronic nicotine treatment up-regulates human alpha3 beta2 but not alpha3 beta4 acetylcholine receptors stably transfected in human embryonic kidney cells.

Human nicotinic acetylcholine receptor (AChR) subtypes alpha3 beta2, alpha3 beta2 alpha5, alpha3 beta4, and alpha3 beta4 alpha5 were stably expressed in cells derived from the human embryonic kidney cell line 293. alpha3 beta4 AChRs were found in prominent 2-micrometer patches on the cell surface, whereas most alpha3 beta2 AChRs were more diffusely distributed. The functional properties of the alpha3 AChRs in tsA201 cells were characterized by whole cell patch clamp using both acetylcholine and nicotine as agonists. Nicotine was a partial agonist on alpha3 beta4 AChRs and nearly a full agonist on alpha3 beta2 alpha5 AChRs. Chronic exposure of cells expressing alpha3 beta2 AChRs or alpha3 beta2 alpha5 AChRs to nicotine or carbamylcholine increased their amount up to 24-fold but had no effect on the amount of alpha3beta4 or alpha3 beta4 alpha5 AChRs, i.e. the up-regulation of alpha3 AChRs depended on the presence of beta2 but not beta4 subunits in the AChRs. This was also found to be true of alpha3 AChRs in the human neuroblastoma SH-SY5Y. In the absence of nicotine, alpha3 beta2 AChRs were expressed at much lower levels than alpha3 beta4 AChRs, but in the presence of nicotine, the amount of alpha3 beta2 AChRs exceeded that of alpha3 beta4 AChRs. Up-regulation was seen for both total AChRs and surface AChRs. Up-regulated alpha3beta2 AChRs were functional. The nicotinic antagonists curare and dihydro-beta-erythroidine also up-regulated alpha3 beta2 AChRs, but only by 3-5-fold. The channel blocker mecamylamine did not cause up-regulation of alpha3 beta2 AChRs and inhibited up-regulation by nicotine. Our data suggest that up-regulation of alpha3 beta2 AChRs in these lines by nicotine results from both increased subunit assembly and decreased AChR turnover.

Animals↗
PubMed1998

Inhibition of glucose trimming with castanospermine reduces calnexin association and promotes proteasome degradation of the alpha-subunit of the nicotinic acetylcholine receptor.

To identify factors involved in the expression of ligand-gated ion channels, we expressed nicotinic acetylcholine receptors in HEK cells to characterize roles for oligosaccharide trimming, calnexin association, and targeting to the proteasome. The homologous subunits of the acetylcholine receptor traverse the membrane four times, contain at least one oligosaccharide, and are retained in the endoplasmic reticulum until completely assembled into the circular arrangement of subunits of delta-alpha-gamma-alpha-beta to enclose the ion channel. We previously demonstrated that calnexin is associated with unassembled subunits of the receptor, but appears to dissociate when subunits are assembled in various combinations. We used the glucosidase inhibitor castanospermine to block oligosaccharide processing, and thereby inhibit calnexin's interaction with the oligosaccharides in the receptor subunits. Castanospermine treatment reduces the association of calnexin with the alpha-subunit of the receptor, and diminishes the intracellular accumulation of unassembled receptor subunit protein. However, treatment with castanospermine does not appear to alter subunit folding or assembly. In contrast, co-treatment with proteasome inhibitors and castanospermine enhances the accumulation of polyubiquitin-conjugated alpha-subunits, and generally reverses the castanospermine induced loss of alpha-subunit protein. Co-transfection of cDNAs encoding the alpha- and delta-subunits, which leads to the expression of assembled alpha- and delta- subunits, also inhibits the loss of alpha-subunits expressed in the presence of castanospermine. Taken together, these observations indicate that calnexin association reduces the degradation of unassembled receptor subunits in the ubiquitin-proteasome pathway.

Acetylcysteine↗
PubMed1998

Water-soluble nicotinic acetylcholine receptor formed by alpha7 subunit extracellular domains.

Water-soluble models of ligand-gated ion channels would be advantageous for structural studies. We investigated the suitability of three versions of the N-terminal extracellular domain (ECD) of the alpha7 subunit of the nicotinic acetylcholine receptor (AChR) family for this purpose by examining their ligand-binding and assembly properties. Two versions included the first transmembrane domain and were solubilized with detergent after expression in Xenopus oocytes. The third was truncated before the first transmembrane domain and was soluble without detergent. For all three, their equilibrium binding affinities for alpha-bungarotoxin, nicotine, and acetylcholine, combined with their velocity sedimentation profiles, were consistent with the formation of native-like AChRs. These characteristics imply that the alpha7 ECD can form a water-soluble AChR that is a model of the ECD of the full-length alpha7 AChR.

Acetylcholine↗
PubMed1998

Analysis of local structure in the D2/S1-S2 region of the rat skeletal muscle type 1 sodium channel using insertional mutagenesis.

A reporter epitope was inserted at 11 positions in a region encompassing proposed transmembrane segments S1 and S2 in the second repeat domain (D2) of the rat skeletal muscle type 1 sodium channel. All mutations produced full-length membrane-associated protein following transfection into cultured cells, although the level of expression varied with insertion position. Characterization of cognate cRNAs for each mutation in Xenopus oocytes by two-electrode voltage clamp defined a permissive region between the proposed transmembrane regions in which these large insertions did not interfere with channel function. Two of the mutations, in which the point of insertion was within the proposed S1-S2 loop, demonstrated extracellular membrane labeling when studied either by antibody binding in oocytes or by confocal analysis following transfection into primary muscle cells. Our results define the likely boundaries of an extramembrane region linking the S1 and S2 transmembrane segments in D2 and confirm the extracellular location of this S1-S2 loop predicted by current models of channel tertiary structure.

Amino Acid Sequence↗
PubMed1998

alpha 5 Subunit alters desensitization, pharmacology, Ca++ permeability and Ca++ modulation of human neuronal alpha 3 nicotinic receptors.

Functional effects of human alpha 5 nicotinic ACh receptor (AChR) subunits coassembled with alpha 3 and beta 2 or with alpha 3 and beta 4 subunits, were investigated in Xenopus oocytes. The presence of alpha 5 subunits altered some properties of both alpha 3 AChRs and differentially altered other properties of alpha 3 beta 2 AChRs vs. alpha 3 beta 4 AChRs. alpha 5 subunits increased desensitization and Ca++ permeability of all alpha 3 AChRs. The Ca++ permeabilities of both alpha 3 beta 2 alpha 5 and alpha 3 beta 4 alpha 5 AChRs were comparable to that of alpha 7 AChRs. As we have shown previously, alpha 5 subunits increased the ACh sensitivity of alpha 3 beta 2 AChRs 50-fold but had little effect on alpha 3 beta 4 AChRs. alpha 5 caused only subtle changes in the activation potencies of alpha 3 AChRs for nicotine, cytisine and 1,1-dimethyl-4-plenylpiperazinium (DMPP). However, alpha 5 increased the efficacies of nicotine and DMPP on alpha 3 beta 2 AChRs but decreased them on alpha 3 beta 4 AChRs. Immunoisolation of cloned human AChRs expressed in oocytes showed that alpha 5 efficiently coassembled with alpha 3 plus beta 2 and/or beta 4 subunits. As expected, human AChRs immunoisolated from SH-SY5Y neuroblastoma cells showed that AChRs containing alpha 3 and probably alpha 5 subunits were present, but alpha 4 AChRs were not. In brain, by contrast, alpha 4 beta 2 AChRs were shown to predominate over alpha 3 AChRs. Some of the brain alpha 4 beta 2 AChRs were found to contain alpha 5 subunits.

Animals↗
PubMed1998

Determinants of channel gating located in the N-terminal extracellular domain of nicotinic alpha7 receptor.

We identified regions within the N-terminal extracellular domain of alpha7 nicotinic acetylcholine receptors that affect channel gating. By single-channel analysis of alpha7 nicotinic acetylcholine receptors currents, we show that the difference in efficacy between the two agonists acetylcholine and 1,1-dimethyl-4-phenylpiperazinium (DMPP) is due to a slower channel activation rate by DMPP. The partial efficacy of DMPP was not caused by channel block or faster desensitization of alpha7 AChRs by DMPP. In addition, the efficacy and, by inference, the activation rate were found to be voltage dependent. Using chimeras of the two closely related subunits alpha7 and alpha8, we map residues that affect channel activation rate and agonist affinity to two different regions of the extracellular domain. Residues that affect channel activation rate are within the sequence 1-179, whereas residues that affect agonist affinity are within the sequence 180-208.

Animals↗
PubMed1998

Analysis of 3-(4-hydroxy, 2-Methoxybenzylidene)anabaseine selectivity and activity at human and rat alpha-7 nicotinic receptors.

3-(2,4-dimethoxybenzylidene)anabaseine (GTS-21) is a selective partial agonist for rat alpha-7 nicotine receptors with reportedly much lower efficacy for human alpha-7 receptors. Because this drug improves memory-related performance in nonhuman primates, and is presently in a clinical trial for Alzheimer's disease, we investigated the potential effects of its primary human metabolite, 3-(4-hydroxy, 2-methoxy-benzylidene)anabaseine) on human as well as rat nicotinic acetylcholine receptor. 4OH-GTS-21 exhibited a similar level of efficacy for both rat and human alpha-7 receptors expressed in Xenopus oocytes. It displaced high affinity [125I]alpha-bungarotoxin binding to human SK-N-SH cell-membranes (Ki 0.17 microM) and rat PC12 cell-membranes (Ki 0.45 microM). GTS-21 also displaced [125I]alpha-bungarotoxin binding to PC12 cell membranes with high potency (Ki 0.31 microM), but was much less potent in this regard in SK-N-SH cells (23 microM). 4OH-GTS-21 produced less residual inhibition of either the human or rat AChR subtypes than GTS-21 did. To compare the neuroprotective efficacies of GTS-21 and 4OH-GTS-21 in both species, an amyloid-toxicity model (Abeta 25-35) was used. 4OH-GTS-21 was protective in both human and rat cell lines, although GTS-21 was effective only in the latter. These studies suggest that the efficacy of GTS-21 in primates may depend on a pro-drug function.

Anabasine↗