Roles of nicotinic acetylcholine receptor ß subunit cytoplasmic loops in acute desensitization and single-channel features.

To evaluate physiological roles of the large, second cytoplasmic loops (C2) situated between the M3 and M4 transmembrane domains of nicotinic acetylcholine receptor (nAChR) subunits. We have constructed chimeric ß2 (ß2χ) and ß4 (ß4χ) subunits in which the "nested" C2 domains (but not the "proximal" sequences of ∼14 residues immediately adjacent to the M3 or M4 domains) of these ß subunits were replaced by the corresponding sequence from the serotonin 5-HT3A receptor subunit. We previously reported that heterologously expressed nAChR containing α4 and ß2χ subunits displayed a faster whole-cell current decay in its agonist response compared to responses of all-wild-type α4ß2-nAChR. This suggests an unexpected, functional role for the C2 domain of the ß2 subunit in α4ß2-nAChR acute desensitization. Here we report that there also is faster desensitization of α4ß4χ-nAChR relative to α4ß4-nAChR stably and heterologously expressed in the human SH-EP1 cell-line. In addition, cell-attached, single-channel recording shows that both acetylcholine-activated α4ß2χ- and α4ß4χ-nAChR have a significantly lower mean open probability, shorter mean open-time, and a longer mean closed-time than their fully wild-type counterparts while not having different conductance amplitudes. These findings reveal microscopic bases for the faster desensitization of α4(∗)-nAChR containing chimeric instead of wild-type ß subunits. Our findings also remain consistent with novel and unexpected roles of ß subunit-nested C2 domains in modulation of α4(∗)-nAChR function.

Functional expression of nicotinic acetylcholine receptors containing rat alpha 7 subunits in human SH-SY5Y neuroblastoma cells.

Neuronal nicotinic acetylcholine receptors (nAChR) are made from different combinations of subunits encoded by a diverse family of genes. However, the recently cloned alpha 7 gene codes for subunits that can form homooligomeric nAChR complexes when expressed in Xenopus oocytes. Electrophysiological studies reveal that these alpha 7-nAChR function as alpha-bungarotoxin (Bgt)-sensitive, quickly activating/inactivating ion channels with a unique pharmacological profile and an unusually high permeability to calcium ions. Although similar observations have been made in studies of Bgt-sensitive, functional nAChR subtypes that are naturally expressed in neuronal cells, all attempts until now to reconstitute functional alpha 7-nAChR in cell lines have failed. Here we report the successful use of SH-SY5Y human neuroblastoma cells, which naturally express low levels of endogenous alpha 7 transcripts, to stably overexpress heterologous rat nAChR alpha 7 transgenes. These transgenes are expressed as the appropriately-sized alpha 7 messages and protein, and stably transfected SH-SY5Y cells have over 30-times higher levels of specific Bgt binding sites than do wild-type cells. Whole cell current recordings confirm that transfected cells express functional nAChR that are sensitive to blockade by Bgt and display the typical physiological and pharmacological profiles of alpha 7-nAChR. We conclude that stable, functional expression of alpha 7 transgenes in a mammalian cell line has been achieved for the first time.

Regulation of nicotinic acetylcholine receptor numbers and function by chronic nicotine exposure.

Recent advances concerning effects of chronic nicotine exposure on nicotinic acetylcholine receptor (nAChR) expression are reviewed. Implications are assessed of these findings for roles of nAChR in health and disease and for design of drugs for treatment of neurological and psychiatric disorders. Most studies continue to show that chronic nicotine exposure induces increases in numbers of nAChR-like binding or antigenic sites ("upregulation") across all nAChR subtypes investigated, but with time- and dose-dependencies and magnitudes for these effects that are unique to subsets of nAChR subtypes. These effects appear to be post-transcriptionally based, but mechanisms involved remain obscure. With notable exceptions, most studies also show that chronic nicotine exposure induces several phases of nAChR functional loss ("desensitization" and longer-lasting "persistent inactivation") assessed in response to acute nicotinic agonist challenges. Times for onset and recovery and dose-dependencies for nicotine-induced functional loss also are nAChR subtype-specific. Some findings suggest that upregulation and functional loss are not causally- or mechanistically-related. It is suggested that upregulation is not as physiologically significant in vivo as functional effects of chronic nicotine exposure. By contrast, brain levels of nicotine in tobacco users, and perhaps levels of acetylcholine in the extracellular space, clearly are in the range that would alter the balance between nAChR in functionally ready or inactivated states. Further work is warranted to illuminate how effects of chronic nicotinic ligand exposure are integrated across nAChR subtypes and the neuronal circuits and chemical signaling pathways that they service to produce nicotine dependence and/or therapeutic benefit.

Monoclonal rat anti-Torpedo electroplax nicotinic acetylcholine receptor antibodies: immunochemical characterization.

The interaction of Torpedo californica nicotinic acetylcholine receptor (nAcChoR) with three rat monoclonal antibodies (mcab) directed against nAcChoR (Gomez et al., 1979) was studied by use of four different radioimmunoassay protocols. Each mcab reacts poorly with formalin-fixed Staphylococcus aureus or S. aureus Protein A, which requires that an additional incubation with second antibody (goat anti-rat immunoglobulin G) is included in each radioimmunoassay paradigm. One mcab inhibits 125I-labeled alpha-bungarotoxin binding to nAcChoR, recognizes a subset of solubilized nAcChoR-toxin complexes, and shows higher titer against nAcChoR in the absence of toxin. Thus, it appears to be directed against nAcChoR antigenic determinants that at least partially overlap with toxin binding sites. Two other mcab react with nAcChoR in a toxin-independent manner. Receptor-mcab dissociation constants are less than 10 nM, according to each assay paradigm. Estimates of antibody titer or concentrations of antibody in stock hybridoma supernatants vary according to the assay used. This predictable result is attributed to differences in design and sensitivity of assay protocols. The data provide a basis for further utilization of monospecific antibodies as probes in characterization of nAcChoR structure and function.

Effects of diltiazem on human nicotinic acetylcholine and GABA(A) receptors.

Effects of the L-type calcium channel antagonist diltiazem on recombinant human GABA(A) receptor (alpha1beta2gamma2s) or on muscle (alpha1beta1deltagamma and alpha1beta1delta(epsilon)) or neuronal (alpha7 and alpha4beta2) nicotinic acetylcholine receptors expressed in Xenopus oocytes were examined using two-electrode voltage-clamp. Diltiazem inhibited the function of both muscle and neuronal nicotinic receptors, but it had no effect on GABA(A) receptors. The extent of functional inhibition of nicotinic receptors depended on the receptor subtype, and the order of inhibition potency by diltiazem was alpha7>alpha4beta2 approximately alpha1beta1deltagamma approximately alpha1beta1delta(epsilon). Inhibition of alpha7 receptor function was non-competitive and voltage-independent, and it occurred at concentrations far lower than those needed to inhibit (never completely) binding of (125)I-alpha-bungarotoxin to heterologously expressed alpha7 receptors in mammalian cells. Pre-incubation in diltiazem before concomitant application with acetylcholine increased inhibition of function and slowed recovery from inhibition. Verapamil, a phenylalkylamine antagonist of L-type Ca(2+) channels also fully inhibited alpha7 receptor function and partially inhibited (125)I-alpha-bungarotoxin binding to alpha7 receptors, but was less potent than diltiazem. Effects on both alpha7 receptor function and (125)I-alpha-bungarotoxin binding by verapamil plus diltiazem suggest separate sites for verapamil and diltiazem on alpha7 receptors. These results provide further evidence that L-type Ca(2+) channel drugs inhibit ligand-gated cationic channels and suggest that caution should be applied when using these compounds to study systems in which L-type Ca(2+) channels and ligand-gated cationic channels co-exist.

Halogenated cytisine derivatives as agonists at human neuronal nicotinic acetylcholine receptor subtypes.

Cytisine (cy) is a potent and competitive partial agonist at alpha4 subunit-containing nicotinic acetylcholine (nACh) receptors while at homomeric alpha7-nACh receptors it behaves as a full agonist with a relatively lower potency. In the present study, we assessed the effects of bromination or iodination of the pyridone ring of cy and N-methylcytisine (N-Me-cy) on the effects of these compounds on recombinant human (h) alpha7, halpha4beta2 and halpha4beta4 nACh receptors expressed in clonal cell lines and Xenopus oocytes. Halogenation at C(3) of cy or N-Me-cy usually brings about a marked increase in both affinity and efficacy at halpha7, halpha4beta2 and halpha4beta4 nACh, the extent of which depends on whether the halogen is bromine or iodine, and upon receptor subtype. The effects of halogenation at C(5) are strongly influenced by the specific halogen substituent so that bromination causes a decrease in both affinity and efficacy while iodination decreases affinity but its effects on efficacy range from a decrease (halpha7, halpha4beta4 nACh receptors) to a marked increase (halpha4beta2 nACh receptors). Based on these findings, which differ from those showing that neither the affinity nor efficacy of nicotine, 3-(2-azetidinylmethoxy)-pyridine or epibatidine are greatly affected by halogenation, dehalogenation or halogen exchange at equivalent positions, we suggest that cy, N-Me-cy and their halo-isosteres bind to neuronal nACh receptors in a different orientation allowing the halogen atom to interact with a hydrophobic halogen-accepting region within the predominantly hydrophobic agonist-binding pocket of the receptors.

Photoaffinity labeling of muscle-type nicotinic acetylcholine receptors and neuronal/nicotinic alpha-bungarotoxin binding sites with a derivative of alpha-bungarotoxin.

Neuronal/nicotinic alpha-bungarotoxin binding sites (nBgtS) found in the nervous system are not well characterized. In this study, photolabile toxin derivatives have been used in affinity labeling protocols to investigate the subunit composition of nBgtS expressed by different neuron-like cell lines. Data obtained was compared to the known subunit composition of toxin-binding muscle-type nicotinic acetylcholine receptors (nAChR). Muscle-type nAChR-rich membranes prepared from Torpedo electroplax contain components with corrected apparent molecular sizes of 41, 46, 50, 62 and 66 kDa that are reactive with toxin. The photoaffinity labeling patterns for preparations derived from cells of the TE671 clone, which express muscle-type nAChR, are very similar to that of cells of the IMR-32 or SH-SY5Y clonal lines, which express nBgtS. There is consistent labeling of four polypeptides with corrected apparent molecular weights of 40, 43, 47 and 56 kDa. These results suggest that both mammalian muscle-type nAChR and mammalian nBgtS are similarly composed of at least four kinds of subunits.

Diversity and patterns of regulation of nicotinic receptor subtypes.

In our studies we explored the functional relevance of nAChR diversity, in part from the perspective of nAChR as ideal targets for regulatory influences, including those mediated via actions of ligands at other "interacting" receptors. We explored possible mechanisms for nAChR regulation and roles played by nAChR subtype and subunit diversity in those processes. We showed that regulatory factors can influence nAChR numbers at transcriptional and posttranscriptional levels and can affect nAChR function and subcellular distribution. We also demonstrated that nAChR expression can be influenced (1) by nicotinic ligands, (2) by second messengers, (3) by growth factors, (4) by agents targeting the nucleus, and (5) by agents targeting the cytoskeleton. We found common effects of some regulatory influences on more than one nAChR subtype, and we found instances where regulatory influences differ for different cell and nAChR types. Even from the very limited number of these initial studies, it is evident that nAChR subunit and subtype diversity, which alone can provide diversity in nAChR functions, localization, and ligand sensitivity, dovetails with diversity in cellular signaling mechanisms that can affect nAChR expression to amplify the potential functional plasticity of cholinoceptive cells. As examples, we discussed potential roles for nAChR diversity and regulatory plasticity in synapse remodeling and in changes in neuronal circuit conditions. These examples illustrate how nAChR diversity could play important roles in the regulation of nervous system function.

Alterations of Alzheimer's disease in the cholesterol-fed rabbit, including vascular inflammation. Preliminary observations.

We determined the levels of endothelial inflammation using MECA-32 antibody and alpha 4 nicotinic receptor subunit densities employing [3H]epibatidine binding in the brains of Alzheimer's disease (AD) patients, cholesterol-fed rabbits, and appropriate controls. We also assessed rabbit brain for beta-amyloid levels and immunohistochemical localization, and for evidence of blood-brain barrier breach using normally-excluded Evans Blue dye. Dietary cholesterol induced a twofold increase in beta-amyloid concentration in rabbit hippocampal cortex, which may be related to the appearance of beta-amyloid immunoreactivity in the neuropil. Epibatidine binding was significantly decreased in AD superior frontal cortex, but unchanged in the superior frontal cortex of cholesterol-fed rabbits. Increased vascular MECA-32 immunoreactivity occurred in AD and cholesterol-fed rabbit brain. Evans Blue dye could be found in the parenchyma of cholesterol-fed rabbits only, and appeared as pockets of dye surrounding small blood vessels. The data suggest that vascular inflammation can lead to breach of the blood-brain barrier, which may produce biochemical derangements in surrounding brain tissue that are conducive to production of beta-amyloid.

Interactions of anti-nicotinic acetylcholine receptor antibodies at alpha-bungarotoxin binding sites across species and tissues.

Two antisera prepared against the nicotinic acetylcholine receptor (nAcChoR) from Electrophorus exhibit comparable ability to inhibit high-affinity alpha-bungarotoxin binding to membrane fractions from rat brain or muscle, PC12 or TE671 cells, or Torpedo electric tissue. Only one of several monoclonal antibodies raised against nAcChoR from Torpedo inhibits toxin binding to membranes from rat brain or muscle or TE671 cells, but is considerably more potent as an inhibitor of toxin binding to Torpedo nAcChoR. These results indicate that some antibodies prepared against nAcChoR from electric tissue recognize epitopes near the high-affinity toxin binding sites. Some of these toxin binding site epitopes are preserved across species and tissues. The positive outcome of this study supports the continued use of toxin as a probe for at least a subset of mammalian neuronal nAcChoR.

Allosteric modification of alpha-bungarotoxin binding by the 'calcium channel antagonist' verapamil.

The putative calcium channel antagonist verapamil has been shown to affect a variety of other ion channels as well as neurotransmitter receptors. We report that verapamil reversibly inhibits binding of the nicotinic receptor probe 125I-alpha-bungarotoxin to membranes from rat brain, Torpedo californica, the rat pheochromocytoma cell line PC12 and the human medulloblastoma cell line TE671, with Ki values of 24.0, 90.5, 165.4 and 869.6 microM, respectively. These effects are calcium independent and insensitive to a variety of organic and inorganic calcium channel antagonists. While Hill coefficients suggest mechanistic differences for verapamil action in the tissues examined, derivative graphical analysis demonstrates a common mechanism of heterotypic, allosteric inhibition. Verapamil may be useful as a probe to elucidate aspects of receptor-effector coupling.

Basic fibroblast growth factor (bFGF) regulates tyrosine hydroxylase and proenkephalin mRNA levels in adrenal chromaffin cells.

bFGF is a neurotrophic protein expressed in various regions of the adult peripheral and central nervous system. The present study was undertaken to examine the role of bFGF in multihormonal, catecholaminergic and enkephalinergic cells of the adrenal medulla (AM). Western blot analysis revealed the presence of at least three bFGF isoforms (18, 22/23, and 24 kDa) in cultured bovine AM cells. Incubation of AM cells with the exogenous 18 kDa bFGF produced time-dependent increases in tyrosine hydroxylase (TH) and proenkephalin (PEK) mRNA, with maximal changes occurring at 12 h (TH) or 24 h (PEK) of bFGF exposure. Effects of bFGF on TH and PEK mRNA were non-additive with increases induced by exposure of AM cells to nicotine, the depolarizing agent veratridine, or the adenylate cyclase activator forskolin. These data indicate that bFGF effects may occur through intracellular pathways accessed during transsynaptic induction of TH and PEK genes. The increases in PEK mRNA induced by nicotine or bFGF were inhibited by the calcium antagonist TMB-8. TMB-8 also inhibited bFGF-induced increases in TH mRNA as well. However, treatment with TMB-8 increased basal levels of TH mRNA. The addition of bFGF increased endogenous levels of c-fos mRNA, c-Fos and c-Fos-related proteins, suggesting that bFGF may activate TH and PEK gene expression through a calcium-AP1 transcriptional regulatory pathway. Immunohistochemical analysis revealed the presence of bFGF-immunoreactivity (bFGF-IR) in the cytoplasm and in the nucleus of AM cells. Incubation of cells with exogenous bFGF produced time-dependent increases of nuclear bFGF-IR.(ABSTRACT TRUNCATED AT 250 WORDS)

Inducible, heterologous expression of human alpha7-nicotinic acetylcholine receptors in a native nicotinic receptor-null human clonal line.

Tetracycline-regulated expression of recombinant nicotinic acetylcholine receptors (nAChR) composed of human alpha7 subunits is achieved in native nAChR-null SH-EP1 human epithelial cells. alpha7 subunits are heterologously expressed as messenger RNA and as components of 125I-labeled alpha-bungarotoxin (I-Bgt)-binding nAChR ( approximately 10 pmol per milligram of membrane protein) at levels sensitive to the amount of tetracycline in cell growth medium. I-Bgt-binding alpha7-nAChR appear on the cell surface pool and in intracellular pools. The pharmacological profile for drug competition toward I-Bgt binding to these recombinant alpha7-nAChR matches that of human native alpha7-nAChR naturally expressed in SH-SY5Y human neuroblastoma cells (rank order potency methyllycaconitine>1, 1-dimethyl-4-phenylpiperazinium>(-)nicotine>cytisine>carbamylch oli ne> /=d-tubocurarine). Chronic exposure to nicotine induces up-regulation of human recombinant alpha7-nAChR (80% up-regulation at 10 microM nicotine) just as it does native alpha7-nAChR in other human cell lines. These studies confirm expression of nAChR as homooligomers of human alpha7 subunits from transgenes, establish a native nAChR-null background for such expression, and demonstrate that this expression can be regulated to facilitate studies of human alpha7-nAChR.

5-hydroxytryptamine interaction with the nicotinic acetylcholine receptor.

The present study examines the interaction of the neurotransmitter 5-hydroxytryptamine (5-HT) with muscle-type nicotinic acetylcholine receptors. 5-HT inhibits the initial rate of [125I]alpha-bungarotoxin binding to Torpedo acetylcholine receptor membranes (IC(50)=8.5+/-0.32 mM) and [3H]5-HT can be photoincorporated into acetylcholine receptor subunits, with labeling of the alpha-subunit inhibitable by both agonists and competitive antagonists. Within the agonist-binding domain, [3H]5-HT photoincorporates into alphaTyr(190), alphaCys(192) and alphaCys(193). Functional studies using the human clonal cell line TE671/RD, show that 5-HT is a weak inhibitor (IC(50)=1.55+/-0.25 mM) of acetylcholine receptor activity. In this regard, agonist-response profiles in the absence and presence of 5-HT indicate a noncompetitive mode of inhibition. In addition, 5-HT displaces high affinity [3H]thienylcyclohexylpiperidine binding to the desensitized Torpedo acetylcholine receptor channel (IC(50)=1.61+/-0.07 mM). Collectively, these results indicate that 5-HT interacts weakly with the agonist recognition site and inhibits receptor function noncompetitively by binding to the acetylcholine receptor channel.

Morphological and biochemical differentiation of the human medulloblastoma cell line TE671.

Cells of the human medulloblastoma clonal line TE671 exhibit polymorphism when grown in vitro in serum-supplemented medium. Under these conditions, cell numbers double every 18 h during log phase growth. These tumorigenic precursors of cerebellar interneurons are not contact-inhibited and approach densities of one million cells per cm2. TE671 cells in proliferative growth express a class of nicotinic acetylcholine receptors that are fully sensitive to functional blockade by the neurotoxin alpha-bungarotoxin (Bgt). TE671 cells grown in medium containing dibutyryl cyclic adenosine monophosphate (dbcAMP) rapidly undergo a distinctive morphological transformation characterized by neurite extension and formation of cell-cell contacts. The rate of cell division and cell saturation densities are diminished coordinately with these treatments. Sodium fluoride and forskolin induce similar changes in cell division and morphology as does dbcAMP, and these effects are potentiated by aluminum and the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, respectively. The high-affinity binding of Bgt to TE671 cells also is reduced on exposure to dbcAMP in a time and dose-dependent manner. The results suggest that activation of adenylate cyclase and the concomitant elevation of intracellular cAMP levels may be involved in the morphological transformation of TE671 cells to a mature, neuronal phenotype and in changes in the level of expression of a subtype of human neuronal nicotinic receptors. These studies establish a unique, neural tube-derived model system for investigation of the mechanisms involved in these processes.

Vanadate amplifies receptor-mediated accumulation of inositol trisphosphates and inhibits inositol tris- and tetrakis-phosphatase activities.

Lithium ion, which inhibits hydrolytic degradation of inositol monophosphates, is the most common therapeutic agent used in the control of bipolar disorder. There exists evidence that elevated elemental vanadium levels may play an etiological role in at least some forms of manic-depression. Here we demonstrate that vanadate treatment of intact cells from several different clonal lines synergistically induces substantial augmentation in neurotransmitter receptor-mediated or growth factor receptor-triggered inositol trisphosphate accumulation in situ. Furthermore, studies done using cellular extracts indicate that effects of vanadate treatment in situ may be due to its ability to inhibit hydrolysis of inositol 1,4,5-trisphosphate inositol 1,3,4-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate in vitro. These results suggest that vanadate treatment may facilitate characterization of inositol phosphate metabolism and intracellular signaling.

Characterization of nicotinic acetylcholine receptor channels of the TE671 human medulloblastoma clonal line.

Acetylcholine (ACh)-gated single channel events were studied on the TE671 human medulloblastoma clonal cell line by the use of the cell-attached patch clamp technique. Channel activity was detected (86% probability) in the presence of 0.1-2 microM ACh but not (0% probability) in the absence of agonist or in the presence of 1 microM alpha-bungarotoxin (Bgt). This effect of Bgt was reversible within 1 h. The most prominent channel type had a conductance of 50 pS. The kinetics of opening and closing of the channel were similar to that for skeletal muscle nicotinic acetylcholine receptors.

Immunohistochemical localization of nicotinic beta2 and alpha4 receptor subunits in normal human brain and individuals with Lewy body and Alzheimer's disease: preliminary observations.

Optimum immunohistochemical methods were established to immuno-localize nicotinic acetylcholine receptor alpha4 and beta2 subunits in temporal cortex and substantia nigra of normal aged and diseased human brain. In normal aged brain, fibers were immunoreactive for both the alpha4 and beta2 subunits of the nicotinic receptor in the temporal cortex and the substantia nigra. In the cortex of normal aged brain, rare neurofibrillary tangles occurring could be identified with either anti-alpha4 or anti-beta2 antibodies, but existing senile plaques were demonstrable with neither. In Alzheimer's disease temporal cortex, there were diminished numbers of nicotinic receptor subunit immunoreactive fibers, and there were appreciable numbers of neuropil threads, neurofibrillary tangles and senile plaques immunoreactive with both the alpha4 and beta2 antibodies.