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.

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.

Mutational analysis of roles for extracellular cysteine residues in the assembly and function of human alpha 7-nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChR) containing alpha7 subunits self-assemble into simple, homopentameric complexes. However, successful heterologous expression of functional alpha7-nAChR has only been achieved in a few host cell types, such as the SH-EP1 human epithelial cell line. All ionotropic glycine receptor, GABA(A) receptor, 5-HT(3) receptor, and nAChR subunits contain a pair of highly conserved cysteine residues (C150 and C164 for alpha7 subunits) in their N-terminal extracellular domain. These residues are thought to be involved in the formation of a conserved cystine loop that is critical to the proper folding and assembly of subunits. However, nAChR alpha7 (and alpha8) subunits also contain a third cysteine residue, C138, N-terminal to the conserved cysteine pair. Using SH-EP1 cells as a host for heterologous expression, we evaluated the roles of C138, C150, and C164 in subunit folding, assembly, and cell surface expression and function of alpha7-nAChR. Results indicate that mutation of C138, but not of C150 or C164, yields an nAChR that can assemble to form (125)I-labeled alpha-bungarotoxin binding sites expressed on the cell surface. Further, whole-cell patch clamp recordings demonstrate that mutation of C138 to alanine does not alter the function of the fully assembled alpha7-nAChR. These results indicate that C150 and C164 are required for surface expression, but that C138 is neither necessary for nor inhibitory toward the surface expression and function of human alpha7-nAChR. These results suggest that disulfide bond formation between C138 and either C150 or C164, if it occurs, has no significant effect on alpha7-nAChR assembly or function.

Nicotinic acetylcholine receptors as targets for antidepressants.

While the monoamine deficiency hypothesis of depression is still most commonly used to explain the actions of antidepressant drugs, a growing body of evidence has accumulated that is not adequately explained by the hypothesis. This article draws attention to contributions from another apparently common pharmacological property of antidepressant medications--the inhibition of nicotinic acetylcholine receptors (nAChR). Evidence is presented suggesting the hypercholinergic neurotransmission, which is associated with depressed mood states, may be mediated through excessive neuronal nicotinic receptor activation and that the therapeutic actions of many antidepressants may be, in part, mediated through inhibition of these receptors. In support of this hypothesis, preliminary evidence is presented suggesting that the potent, centrally acting nAChR antagonist, mecamylamine, which is devoid of monoamine reuptake inhibition, may reduce symptoms of depression and mood instability in patients with comorbid depression and bipolar disorder. If this hypothesis is supported by further preclinical and clinical research, nicotinic acetylcholine receptor antagonists may represent a novel class of therapeutic agents for treating mood disorders.

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.

Local anesthetics noncompetitively inhibit function of four distinct nicotinic acetylcholine receptor subtypes.

Local anesthetics (LAs) are considered to act primarily by inhibiting voltage-gated Na(+) channels. However, LAs also are pharmacologically active at other ion channels including nicotinic acetylcholine receptors (nAChR). nAChR exist as a family of diverse subtypes, each of which has a unique pharmacological profile. The current studies established effects of LAs on function of four human nAChR subtypes: naturally expressed muscle-type (alpha1*-nAChR) or autonomic (alpha3beta4*-nAChR) nAChR, or heterologously expressed nAChR containing alpha4 with either beta2- or beta4-subunits (alpha4beta2- or alpha4beta4-nAChR). Of the LAs tested, those with structures containing two separated aromatic rings (e.g., proadifen and adiphenine) had the greatest inhibition potency (IC(50) values between 0.34 and 6.3 microM) but lowest selectivity (approximately 4-fold) across the four nAChR subtypes examined. From the fused, two-ring (isoquinoline backbone) class of LAs, dimethisoquin had comparatively moderate inhibition potency (IC(50) values between 2.4 and 61 microM) and approximately 30-fold selectivity across nAChR subtypes. Lidocaine, a commonly used LA from the single ring category of LAs, blocked nAChR function with IC(50) values of between 52 and 250 microM and had only approximately 5-fold selectivity across nAChR subtypes. Its quaternary triethyl ammonium analog, QX-314, had greater inhibition potency, but the trimethyl ammonium derivative, QX-222, was the least potent LA at all but the alpha4beta2-nAChR subtype. With only a few exceptions, LA effects were consistent with noncompetitive inhibition of nAChR function and occurred at therapeutic doses. These studies suggest structural determinants for LA action at diverse nAChR subtypes and that nAChR likely are clinically relevant targets of LAs.

Activity of cytisine and its brominated isosteres on recombinant human alpha7, alpha4beta2 and alpha4beta4 nicotinic acetylcholine receptors.

Effects of cytisine (cy), 3-bromocytisine (3-Br-cy), 5-bromocytisine (5-Br-cy) and 3,5-dibromocytisine (3,5-diBr-cy) on human (h) alpha7-, alpha4beta2- and alpha4beta4 nicotinic acetylcholine (nACh) receptors, expressed in Xenopus oocytes and cell lines, have been investigated. Cy and its bromo-isosteres fully inhibited binding of both [alpha-(125)I]bungarotoxin ([alpha-(125)I]BgTx) to halpha7- and [(3)H]cy to halpha4beta2- or halpha4beta4-nACh receptors. 3-Br-cy was the most potent inhibitor of both [alpha-(125)I]BgTx and [(3)H]cy binding. Cy was less potent than 3-Br-cy, but 5-Br-cy and 3,5-diBr-cy were the least potent inhibitors. Cy and 3-Br-cy were potent full agonists at halpha7-nACh receptors but behaved as partial agonists at halpha4beta2- and halpha4beta4-nACh receptors. 5-Br-cy and 3,5-diBr-cy had low potency and were partial agonists at halpha7- and halpha4beta4-nACh receptors, but they elicited no responses on halpha4beta2-nACh receptors. Cy and 3-Br-cy produced dual dose-response curves (DRC) at both halpha4beta2- and halpha4beta4-nACh receptors, but ACh produced dual DRC only at halpha4beta2-nACh receptors. Low concentrations of cy, 3-Br-cy and 5-Br-cy enhanced ACh responses of oocytes expressing halpha4beta2-nACh receptors, but at high concentrations they inhibited the responses. In contrast, 3,5-diBr-cy only inhibited, in a competitive manner, ACh responses of halpha4beta2-nACh receptors. It is concluded that bromination of the pyridone ring of cy produces marked changes in effects of cy that are manifest as nACh receptor subtype-specific differences in binding affinities and in functional potencies and efficacies.

Characterization of human alpha4beta2 neuronal nicotinic receptors stably expressed in SH-EP1 cells.

These studies characterized human alpha4beta2 neuronal nicotinic receptors stably expressed in a human epithelial cell line (SH-EP1). Receptors in transfected SH-EPI-halpha4beta2 cells were functional, as determined by increases in intracellular Ca2+ in response to a nicotine stimulus. Nicotine increased Fura-2 fluorescence in a concentration-dependent manner with an apparent EC50 of 2.4 microM, a response that was blocked by the specific antagonist mecamylamine. When cells were incubated in 50 nM nicotine for 24 hours, the Ca2+ response inactivated by 44%, an effect that recovered within 24 hours. SH-EP1-halpha4beta2 cells expressed a single class of high affinity binding sites for [3H]cytisine with a Kd of 0.63 +/- 0.08 nM and a Bmax of 6,797 +/- 732 femtomoles/mg protein. Incubation of cells with 50 nM nicotine for 24 hours increased the Bmax by 45% without changing affinity, a concentration-dependent effect with an EC50, of 58.6 nM. The nicotine-induced up regulation was reversible, and control values were achieved within 24 hours. Results indicate that SH-EPI-halpha4beta2 cells may be a good model system to study regulation of human alpha4beta2 receptors, the most abundant nicotinic receptor subtype in brain.

Neurotoxicity of channel mutations in heterologously expressed alpha7-nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChR) composed of chick alpha7 subunits mutated to threonine at amino acid valine-251 in the putative channel-lining M2 domain were expressed heterologously in several neuron-like and non-neuronal mammalian cell lines. Expression of mutant alpha7-nAChR is toxic to neuron-like cells of the human neuroblastoma cell lines SH-SY5Y and IMR-32, but not to several other cell types. Growth in the presence of the alpha7-nAChR antagonist methyllycaconitine (MLA) protects against neurotoxicity, as does gradual downregulation of functional, mutant alpha7-nAChR in surviving transfected SH-SY5Y cells. Relative to wild-type alpha7-nAChR, functional alpha7-nAChR mutants show a higher affinity for agonists, slower rates of desensitization, and sensitivity to dihydro-beta-erythroidine (DHbetaE) as an agonist, but they retain sensitivity to MLA as a competitive antagonist. These findings demonstrate that expression of hyperfunctional, mutant forms of Ca2+-permeable alpha7-nAChR is toxic to neuron-like cells.

Tobacco cembranoids block behavioral sensitization to nicotine and inhibit neuronal acetylcholine receptor function.

Cembranoids are cyclic diterpenoids found in tobacco and in marine invertebrates. The present study established that tobacco cembranoids inhibit behavioral sensitization to nicotine in rats and block several types of nicotine acetylcholine receptors (AChRs). 1) At the behavioral level, rat locomotor activity induced by nicotine was significantly increased after seven daily nicotine injections. This sensitization to nicotine was blocked by mecamylamine (1 mg/kg) and by the cembranoids eunicin, eupalmerin acetate (EUAC), and (4R)-2,7,11-cembratriene-4-6-diol (4R), each at 6 mg/kg. None of these compounds modified locomotor activity of nonsensitized rats. 2) In cells expressing human AChRs, cembranoids blocked carbamoylcholine-induced (86)Rb(+) flux with IC(50) in the low micromolar range. The cell lines used were the SH-EP1-halpha4beta2 cell line heterologously expressing human alpha4beta2-AChR, the SH-SY5Y neuroblastoma line naturally expressing human ganglionic alpha3beta4-AChR, and the TE671/RD cell line naturally expressing embryonic muscle alpha1beta1gammadelta-AChR. The tobacco cembranoids tested were 4R and its diastereoisomer 4S, and marine cembranoids tested were EUAC and 12,13-bisepieupalmerin. 3) At the molecular level, tobacco (4R and 4S) and marine (EUAC) cembranoids blocked binding of the noncompetitive inhibitor [(3)H]tenocyclidine to AChR from Torpedo californica electric organ. IC(50) values were in the submicromolar to low-micromolar range, with 4R displaying an order of magnitude higher potency than its diastereoisomer, 4S.

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.

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.

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.

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.

Dependence of nicotinic acetylcholine receptor recovery from desensitization on the duration of agonist exposure.

When subjected to prolonged exposure to nicotinic agonists, nicotinic acetylcholine receptors undergo desensitization, resulting in an inactive receptor that does not allow for the passage of ions. The induction of desensitization of diverse nicotinic acetylcholine receptor subtypes in muscle, ganglia, or brain is likely to play important modulatory roles in synaptic transmission. Furthermore, nicotinic receptor desensitization may contribute to behavioral changes in humans or animals subjected to prolonged nicotine exposure pharmacologically or through the use of tobacco products. We investigated the recovery from desensitization of muscle-type nicotinic acetylcholine receptors in TE671/RD cells induced by exposure to acetylcholine or nicotine. Rates of recovery from desensitization are dependent on the length of agonist exposure and on the agonist used to induce desensitization. Increasing the time of exposure results in an increase in the time constant of recovery for both agonists. The recovery from nicotine-induced desensitization is consistently faster than the recovery from acetylcholine-induced desensitization regardless of whether nicotine or acetylcholine is used to assess levels of desensitization. These findings suggest the existence of more than one state of receptor desensitization and that nicotinic agonists vary in their efficiency of inducing receptors to states of differing depths of desensitization.

Antidepressants noncompetitively inhibit nicotinic acetylcholine receptor function.

Nicotinic acetylcholine receptors (nAChRs) are diverse members of the neurotransmitter-gated ion channel superfamily and play critical roles in chemical signaling throughout the nervous system. The present study establishes for the first time the acute functional effects of sertraline (Zoloft), paroxetine (Paxil), nefazodone (Serzone), and venlafaxine (Effexor) on two human and one chick nAChR subtype. This study also confirms previous findings of nAChR functional block by fluoxetine (Prozac). Function of human muscle-type nAChR (alpha1/beta gammadelta) in TE671/RD cells, human autonomic nAChR (alpha3/beta4alpha5 +/- beta2) in SH-SY5Y neuroblastoma cells, or chick V274T mutant alpha7-nAChR heterologously expressed in native nAChR-null SH-EP1 epithelial cells was measured using 86Rb+ efflux assays. Functional blockade of human muscle-type and autonomic nAChRs is produced by each of the drugs in the low to intermediate micromolar range, and functional blockade of chick V274T-alpha7-nAChR is produced in the intermediate to high micromolar range. Functional blockade is insurmountable by increasing agonist concentrations at each nAChR subtype tested for each of these drugs, suggesting noncompetitive inhibition of nAChR function. These studies open the possibilities that nAChR subtypes in the brain could be targets for therapeutic antidepressants and could play roles in clinical depression.

Noncompetitive functional inhibition at diverse, human nicotinic acetylcholine receptor subtypes by bupropion, phencyclidine, and ibogaine.

Nicotinic acetylcholine receptors (nAChR) are diverse members of the neurotransmitter-gated ion channel superfamily and play critical roles in chemical signaling throughout the nervous system. The present study establishes the acute functional effects of bupropion, phencyclidine, and ibogaine on two human nAChR subtypes. Function of muscle-type nAChR (alpha1 beta gamma delta) in TE671/RD cells or of ganglionic nAChR (alpha3 beta4 alpha5+/-beta2) in SH-SY5Y neuroblastoma cells was measured with 86Rb+ efflux assays. Functional blockade of human muscle-type and ganglionic nAChR is produced by each of the drugs in the low to intermediate micromolar range. Functional blockade is insurmountable by increasing agonist concentrations in TE671/RD and SH-SY5Y cells for each of these drugs, suggesting noncompetitive inhibition of nAChR function. Based on these findings, we hypothesize that nAChR are targets of diverse substances of abuse and agents used in antiaddiction/smoking cessation strategies. We also hypothesize that nAChR play heretofore underappreciated roles in depression and as targets for clinically useful antidepressants.

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.

Effects of chronic nicotine treatment on expression of diverse nicotinic acetylcholine receptor subtypes. I. Dose- and time-dependent effects of nicotine treatment.

Nicotinic acetylcholine receptors (nAChRs) exist as a diverse family of physiologically important ligand-gated ion channels active in classic, excitatory neurotransmission and perhaps in more novel forms of neurochemical signaling. Because of their critical functional roles centrally and peripherally, nAChRs are ideal targets for the regulation of nervous system function. nAChRs also are targets of nicotine, which acts acutely like acetylcholine to stimulate nAChR function. Here, we report studies using model cell culture systems testing the general hypothesis that more chronic nicotine exposure has unique effects on nAChRs. Chronic nicotine treatment induces increases in numbers of human muscle-type nAChRs containing alpha-1, beta-1, gamma and delta subunits, a human ganglionic nAChR subtype containing alpha-3 and beta-4 subunits and a human ganglionic nAChR containing alpha-7 subunits in intracellular and (except for alpha-7 nAChRs) in cell surface pools. However, the half-maximal potency with which nicotine has these effects differs across these nAChR subtypes, as do rates and magnitudes of the "nicotine-induced nAChR up-regulation." These changes in nAChR numbers are not attributable to either transient or sustained changes in nAChR subunit mRNA levels. Nicotine exposure more potently, more rapidly, and with nAChR-subtype specificity, induces two phases of losses in functional responsiveness of muscle-type nAChRs and alpha-3 beta-4 nAChRs, including a "persistent inactivation" that is distinct from classicly defined "desensitization." Based on these results, we hypothesize that chronic nicotine treatment induces persistent functional inactivation and numerical up-regulation of all nAChR subtypes via distinct post-transcriptional mechanisms and with potencies, at rates and with magnitudes that are nAChR-subtype specific. We also hypothesize that chronic nicotine exposure produces long-lasting changes in nervous system function, at least in part, by disabling rather than activating nicotinic cholinergic signaling.