Chronic Ethanol Treatment Decreases [3H]Epibatidine and [3H]Nicotine Binding and Differentially Regulates mRNA Levels of Nicotinic Acetylcholine Receptor Subunits Expressed in M10 and SH-SY5Y Neuroblastoma Cells

Abstract: For a study of the underlying mechanisms of a possible interaction between ethanol and nicotinic receptors during ethanol dependence, the aim of this work was to investigate the effect of chronic ethanol exposure on nicotinic receptor subtypes in a transfected fibroblast cell line (M10 cells) stably expressing α4β2 nicotinic receptor subtype and an SH-SY5Y neuroblastoma cell line expressing α3, α5, α7, β2, and β4 nicotinic acetylcholine receptor (nAChR) subunits. A significant dose-related decrease (−30–80%) in number of [³H]nicotine binding sites was observed in ethanol-treated (25–240 m M ) compared with untreated M10 cells. Similarly, 4-day treatment with ethanol in concentrations relevant to chronic alcoholism (100 m M ) decreased the number of nicotinic receptor binding sites in the SH-SY5Y cells when measured using [³H]epibatidine. When M10 cells were chronically treated with nicotine, ethanol partly inhibited the up-regulation of nicotinic receptors when present in the cells together with nicotine. Chronic treatment for 4 days with 100 m M ethanol significantly decreased the mRNA level for the α3 nAChR subunit (−39%), while the mRNA levels for the α7 (+30%) and α4 (+22%) subunits were significantly increased. Chronic ethanol treatment did not affect the mRNA levels for the β2 nAChR subunit. Changes in the levels of nAChR protein and mRNA may have adaptive significance and be involved in the development of dependence, tolerance, and addiction to chronic ethanol and nicotine exposure. They also may be targets for therapeutic strategies in the treatment of ethanol and nicotine dependence.     

Increased levels of tau protein in SH-SY5Y cells after treatment with cholinesterase inhibitors and nicotinic agonists

Several cholinesterase inhibitors used in the treatment of Alzheimer's disease (AD) have been shown to interact with an allosteric site on the nicotinic acetylcholine receptor (nAChR). A possible linkage between the phosphorylation state of tau, the major component of paired helical filaments found in AD brain, and stimulation of nAChRs by cholinesterase inhibitors and nicotinic agonists was investigated. Western blot analysis showed that treatment of SH-SY5Y cells for 72 h with the cholinesterase inhibitors tacrine (10(-5) M), donepezil (10(-5) M), and galanthamine (10(-5) M), nicotine (10(-5) M), and epibatidine (10(-7) M) increased tau levels as detected with Tau-1, AT 8, and AT 270 monoclonal antibodies and binding of [3H]epibatidine. The increase in tau immunoreactivity induced by nicotine, epibatidine, and tacrine, but not the up-regulation of nAChRs, was prevented by the antagonists d-tubocurarine and mecamylamine. Both antagonists were synergistic with the nicotinic agonists in causing up-regulation, but only d-tubocurarine showed a synergistic effect with tacrine. The increased tau immunoreactivity induced by tacrine was not prevented by atropine, indicating that in terms of cholinergic receptors, tacrine modulates tau levels mainly through interactions with nAChRs and not with muscarinic receptors. Additional work is needed to determine the exact mechanism by which cholinesterase inhibitors and nicotinic agonists modulate phosphorylation and levels of tau protein.     

Methadone increases intracellular calcium in SH-SY5Y and SH-EP1-halpha7 cells by activating neuronal nicotinic acetylcholine receptors

(-)-Methadone acts as an agonist at opioid receptors. Both (+)- and (-)-enantiomers of methadone have been suggested to be potent non-competitive antagonists of alpha3beta4 neuronal nicotinic acetylcholine receptors (nAChRs). In the present study, we have examined interactions of methadone with nAChRs by using receptor binding assays, patch-clamp recording and calcium fluorometry imaging with SH-SY5Y cells naturally expressing alpha7 and alpha3* nAChR subtypes and SH-EP1-halpha7 cells heterologously expressing human alpha7 nAChRs. Methadone potently inhibited binding of [3H]methyllycaconitine to alpha7 nAChRs and that of [3H]epibatidine to alpha3* nAChRs. Methadone pretreatment induced up-regulation of epibatidine binding sites in SH-SY5Y cells. Using whole-cell patch-clamp recording, both isomers of methadone activated cation currents via mecamylamine-sensitive nAChRs in SH-SY5Y cells. Nicotine and both (+)- and (-)-methadone evoked increases in [Ca2+]i in both fluo-3AM loaded cell lines, and these effects were blocked by mecamylamine and by the alpha7 selective antagonist methyllycaconitine, suggesting effects of methadone as alpha7-nAChR agonist. Sensitivity of sustained nicotine and methadone effects to blockade by CdCl2, ryanodine and xestospongin-c implicates voltage-operated Ca2+ channels and intracellular Ca2+ stores as downstream modulators of elevated [Ca2+]i. Collectively, our results suggest that methadone engages in complex and potentially pharmacologically significant interactions with nAChRs.     

Effects of Steroid Exposure on Ligand Binding and Functional Activities of Diverse Nicotinic Acetylcholine Receptor Subtypes

Abstract: Nicotinic acetylcholine receptors (nAChR) are diverse members of the ligand-gated ion channel superfamily of neurotransmitter receptors and play critical roles in chemical signaling throughout the nervous system. The present study tests whether nAChR are potential targets for steroids. Acute or short-term (5 min) preexposure to steroids such as progesterone (which acts most potently), estradiol, corticosterone, or dexamethasone inhibits function of human muscle-type (α1β1γδ) or ganglionic (α3β4) nAChR measured using ⁸⁶Rb⁺ efflux assays in TE671/RD clonal or SH-SY5Y neuroblastoma cells. Absolute (high nanomolar to intermediate micromolar range) and rank-order potencies for steroid-mediated functional inhibition are similar across nAChR subtypes but differ for some steroid derivatives. At concentrations that produce blockade of nAChR function, steroids do not affect binding of radioligands such as ¹²⁵I-labeled α-bungarotoxin or [³H]acetylcholine to muscle-type or ganglionic nAChR or to neuronal toxin-binding nAChR that contain α7 subunits (α7-nAChR). Steroid-mediated blockade of nAChR function is insurmountable by increasing agonist concentrations, and cell-impermeant progesterone:bovine serum albumin conjugates have full potency as inhibitors of ganglionic or muscle-type nAChR function. Chronic (48 h) exposure to progesterone or estradiol, but not the other steroids, also produces blockade of nAChR function, without significant effects on numbers of nAChR radioligand-binding sites. Collectively, these results suggest that steroids act noncompetitively at extracellular sites to inhibit nAChR function with unique potencies for different steroid-nAChR subtype combinations. Thus, nAChR could be among the targets mediating physiologically relevant effects of steroid action in the nervous system.     

Nicotinic receptor agonists and antagonists increase sAPPα secretion and decrease Aβ levels in vitro

We have earlier reported that Aβ were significantly reduced in brains of smoking Alzheimer patients and control subjects compared with non-smokers, as well as in nicotine treated APPsw transgenic mice. To examine the mechanisms by which nicotine modulates APP processing we here measured levels of secreted amyloid precursor protein (sAPPα), total sAPP, Aβ40 and Aβ42 in different cell lines expressing different nicotinic receptor (nAChR) subtypes or no nAChRs. Treatment with nicotine increased release of sAPPα and at the same time lowered Aβ levels in both SH-SY5Y and SH-SY5Y/APPsw cells expressing α3 and α7 nAChR subtypes. These effects could also be evoked by co-treatment with the competitive α7 nAChR antagonists α-bungarotoxin and methyllycaconitine (MLA), and by these antagonists alone, suggesting that binding to the agonist binding site, rather than activation of the receptor, may be sufficient to trigger changes in APP processing. The nicotine-induced increase in sAPPα could only be blocked by co-treatment with the open channel blocker mecamylamine. In addition to nicotine, the agonists epibatidine and cytisine both significantly increased the release of sAPP in M10 cells expressing the α4/β2 nAChR subtype, and this effect was blocked by co-treatment with mecamylamine but not by the α4/β2 competitive antagonist dihydro-β-erythroidine. The lack of effect of nicotine on sAPPα and Aβ levels in HEK 293/APPsw cells, which do not express any nAChRs, demonstrates that the nicotine-induced attenuation of β-amyloidosis is mediated by nAChRs and not by a direct effect of nicotine. Our data show that nicotinic compounds stimulate the non-amyloidogenic pathway and that α4 and α7 nAChRs play a major role in modulating this process. Nicotinic drugs directed towards specific nAChR subtypes might therefore be beneficial for the treatment of AD not only by lowering Aβ production but also by enhance release of neuroprotective sAPPα.     

Interaction of α-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor

α-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α-Conotoxins blocking muscle-type or α7 nAChRs compete with α-bungarotoxin. However, α-conotoxin ImII, a close homolog of the α7 nAChR-targeting α-conotoxin ImI, blocked α7 and muscle nAChRs without displacing α-bungarotoxin ( Ellison et al. 2003, 2004 ), suggesting binding at a different site. We synthesized α-conotoxin ImII, its ribbon isomer (ImII iso ), 'mutant' ImII(W10Y) and found similar potencies in blocking human α7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [¹²⁵I]-α-bungarotoxin from human α7 nAChRs in the cell line GH₄C₁ (IC₅₀ 17 and 23 μM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC₅₀ 2.0–9.0 μM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized α-bungarotoxin ( K _d and IC₅₀ 2.5–8.2 μM). On Torpedo nAChR, α-conotoxin [¹²⁵I]-ImII(W10Y) revealed specific binding ( K _d 1.5–6.1 μM) and could be displaced by α-conotoxin ImII, ImII iso and ImII(W10Y) with IC₅₀ 2.7, 2.2 and 3.1 μM, respectively. As α-cobratoxin and α-conotoxin ImI displaced [¹²⁵I]-ImII(W10Y) only at higher concentrations (IC₅₀≥ 90 μM), our results indicate that α-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.     

Interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with nicotinic acetylcholine receptor subtypes expressed in cell lines and rat cortex

The interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with different nicotinic acetylcholine receptor (nAChR) subtypes was studied in cell lines and rat cortex. MPA showed an affinity (Ki = 1.21 nM) which was higher than anatoxin-a > (−)-nicotine > (+)-[R]nornicotine > (−)-[S]nornicotine > and (+)-nicotine, but lower than cytisine (Ki = 0.46 nM) in competing for (−)-[³H]nicotine binding in M10 cells, which stably express the recombinant ₄β₂ nAChR subtype. A one-binding site model was observed in all competing experiments between (−)-[³H]nicotine binding and each of the agonists studied in M10 cells. MPA showed a 13-fold higher affinity for (−)-[³H]nicotine binding sites compared to the [³H]epibatidine binding sites in rat cortical membranes. In human neuroblastoma SH-SY5Y cells, which predominantly express the ₃ nAChR subunit mRNA, MPA displaced [³H]epibatidine binding from a single population of the binding sites with an affinity in the same nM range as that observed MPA in displacing [³H]epibatidine binding in rat cortical membranes. Chronic treatment of M10 cells with MPA significantly up-regulated the number of (−)-[³H]nicotine binding sites in a concentration dependent manner. Thus MPA appears to have higher affinity to 4-subunit containing receptor subtype than ₃-subunit containing receptor subtype of nAChRs. Furthermore MPA binds to ₄β₂ receptor subtype with higher affinity than (−)-nicotine and behaves, opposite to cytisine, as a full agonist in up-regulating the number of nAChRs. © 1998 Elsevier Science Ltd. All rights reserved.     

Characterization and Localization of Adrenal Nicotinic Acetylcholine Receptors: Evidence that mAb35-Nicotinic Receptors Are the Principal Receptors Mediating Adrenal Catecholamine Secretion

Abstract: Adrenal chromaffin cells contain at least two subtypes of nicotinic acetylcholine receptors (nAChRs). These studies were designed to identify and characterize the subtype of nAChR mediating adrenal catecholamine release using the monoclonal antibody mAb35, which recognizes the α-subunit of muscle nAChRs and cross-reacts with some neuronal nAChRs. Immunocytochemical studies demonstrated that mAb35 interacts with specific sites on cultured chromaffin cells. Pretreatment with mAb35 reduced nAChR-stimulated catecholamine release (IC₅₀ of ∼10 n M ). mAb35 had no effects on release stimulated through non-nAChR mechanisms. Unlike agonist-induced nAChR desensitization, the mAb35-induced reduction in nAChR-mediated secretion developed slowly. Although not immediately reversible, nAChR-stimulated release recovered after mAb35 removal. However, unlike recovery from agonist pretreatment, recovery from mAb35 pretreatment was relatively slow and was partially blocked by vinblastine. Hybridization of adrenal chromaffin RNA with a rat α3 cDNA revealed two strong bands and two fainter bands: two higher-molecular-weight bands, 6.9 and 8.5 kb; a strong band of 3.2 kb; and a lower amount of a 2.3-kb RNA. With recovery of nAChR function after agonist or mAb35 treatment, no significant effects on α3 subunit mRNA levels were seen. In summary, these studies demonstrate the presence of mAb35-nAChRs on adrenal chromaffin cells and provide evidence that these receptors represent the major population that regulates secretory events in adrenal chromaffin cells.     

Novel Neonicotinoid-Agarose Affinity Column for Drosophila and Musca Nicotinic Acetylcholine Receptors

Abstract: Neonicotinoids such as the insecticide imidacloprid (IMI) act as agonists at the insect nicotinic acetylcholine receptor (nAChR). Head membranes of Drosophila melanogaster and Musca domestica have a single high-affinity binding site for [³H]IMI with K _D values of 1–2 n M and B _max values of 560–850 fmol/mg of protein. Locusta and Periplaneta nAChRs isolated with an α-bungarotoxin (α-BGT)-agarose affinity column are known to be α-subunit homooligomers. This study uses 1 - [ N - (6 - chloro - 3 - pyridylmethyl) - N - ethyl]amino - 1 - amino-2-nitroethene (which inhibits [³H]IMI binding to Drosophila and Musca head membranes at 2–3 n M ) to develop a neonicotinoid-agarose affinity column. The procedure—introduction of Triton-solubilized Drosophila or Musca head membranes into this neonicotinoid-based column, elution with IMI, and analysis by lithium dodecyl sulfate-polyacrylamide gel electrophoresis—gives only three proteins (69, 66, and 61 kDa) tentatively assigned as putative subunits of the nAChR; the same three proteins are obtained with Musca using the α-BGT-agarose affinity column. Photoaffinity labeling of the Drosophila and Musca putative subunits from the neonicotinoid column with ¹²⁵I-α-BGT-4-azidosalicylic acid gives a labeled derivative of 66–69 kDa. The yield is 2–5 µg of receptor protein from 1 g of Drosophila or Musca heads. Neonicotinoid affinity chromatography to isolate native Drosophila and Musca receptors will facilitate studies on the structure and function of insect nAChRs.     

Effects of Chronic Nicotinic Ligand Exposure on Functional Activity of Nicotinic Acetylcholine Receptors Expressed by Cells of the PC12 Rat Pheochromocytoma or the TE671/RD Human Clonal Line

Studies were conducted to ascertain the temporal and dose-dependent effects of nicotinic ligand exposure on functional activity of different nicotinic acetylcholine receptor (nAChR) subtypes, as expressed by cells of the PC12 rat pheochromocytoma (ganglia-type nAChR) or the TE671/RD human (muscle-type nAChR) clonal line. Chronic (3-72-h) agonist (nicotine or carbamylcholine) treatment of cells led to a complete (TE671) or nearly complete (PC12) loss of functional nAChR responses, which is referred to as "functional inactivation." Some inactivation of nAChR function was also observed for the nicotinic ligands d-tubocurarine (d-TC), mecamylamine, and decamethonium. Half-maximal inactivation of nAChR function was observed within 3 min for TE671 cells and within 10 min for PC12 cells treated with inactivating ligands. Functional inactivation occurred with dose dependencies that could not always be reconciled with those obtained for acute agonist activation of nAChR function or for acute inhibition of those responses by d-TC, decamethonium, or mecamylamine. Treatment of TE671 or PC12 cells with the nicotinic antagonist pancuronium or alcuronium alone had no effect on levels of expression of functional nAChRs. However, evidence was obtained that either of these antagonists protected TE671 cell muscle-type nAChRs or PC12 cell ganglia-type nAChRs from functional inactivation on long-term treatment with agonists. Recovery of TE671 cell nAChR function following treatment with carbamylcholine, nicotine, or d-TC occurred with half-times of 1-3 days whether cells were maintained in situ or harvested and replated after removal of ligand. By contrast, 50% recovery of functional nAChRs on PC12 cells occurred within 2-6 h after drug removal. In either case the time course for recovery from nAChR functional inactivation is much slower than recovery from nAChR "functional desensitization," which is a reversible process that occurs on shorter-term (0-5-min) agonist exposure of cells. These results indicate that ganglia-type and muscle-type nAChRs are similar in their sensitivities to functional inactivation by nicotinic ligands but differ in their rates of recovery from and onset of those effects. The ability of drugs such as the agonists d-TC, decamethonium, and mecamylamine to induce functional inactivation may relate to their activities as partial/full agonists, channel blockers, and/or allosteric regulators. Effects of drugs such as pancuronium and alcuronium are likely to reflect simple competitive inhibition of primary ligand binding at functional activation sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phorbol Ester-Enhanced Noradrenaline Secretion Correlates with the Presence and Activity of Protein Kinase C-α in Human SH-SY5Y Neuroblastoma Cells

Abstract: The effect of inhibition and down-regulation of protein kinase C (PKC) subtypes α, ε, and ζ on noradrenaline (NA) secretion from human SH-SY5Y neuroblastoma cells was investigated. The PKC inhibitor Ro 31-7549 inhibited carbachol-evoked NA release (IC₅₀ 0.6 µ M ) but not 100 m M K⁺-evoked release. In addition, Ro 31-7549 inhibited the enhancement of carbachol- and K⁺-evoked release after pretreatment with 12- O -tetradecanoylphorbol 13-acetate (TPA; 100 n M ) for 8 min, with IC₅₀ values of 0.7 and 2.4 µ M , respectively. Immunoblotting studies showed that prolonged exposure (48 h) of SH-SY5Y cells to phorbol 12,13-dibutyrate (PDBu) or bryostatin-1 caused down-regulation of PKC-α and PKC-ε but not PKC-ζ. Under these conditions, the acute TPA enhancement of NA release was inhibited. Moreover, the inhibition of TPA-enhanced secretion was also apparent after only 2-h exposure to either PDBu or bryostatin-1, conditions that caused down-regulation of PKC-α, but not PKC-ε or ζ. The PKC inhibitor Gö-6976 (2 µ M ), which has been shown to inhibit selectively PKC-α and β in vitro, also inhibited the TPA enhancement of carbachol- and K⁺-evoked NA release by >50%. These data suggest that in SH-SY5Y cells, the ability of TPA to enhance carbachol- and K⁺-evoked NA secretion is due to activation of PKC-\ga.     

Chronic nicotine administration does not increase nicotinic receptors labeled by [125I]epibatidine in adrenal gland,superior cervical ganglia,pineal or retina

Neuronal nicotinic acetylcholine receptors (nAChRs) were measured in CNS and peripheral tissues following continuous exposure to saline or nicotine hydrogen tartrate (3.3 or 10 mg/kg/day) for 14 days via osmotic pumps. Initially, binding of [3H](-)nicotine, [3H]cytisine and [3H]epibatidine to nAChRs was compared to determine the suitability of each for these kinds of studies. The predominant nAChR labeled by agonists in the cerebral cortex is an alpha 4 beta 2 subtype, whereas the predominant nicotinic receptors in the adrenal gland, superior cervical ganglia and pineal gland contain an alpha 3 subunit, and they do not bind either [3H](-)nicotine or [3H]cytisine with high affinity. In retina some nAChRs bind all three ligands with high affinity, and others appear to bind only [3H]epibatidine. Thus, only [3H]epibatidine had high enough affinity to be useful for measuring the nAChRs in all of the tissues. The receptors from nicotine-treated rats were then measured using [125I]epibatidine, which has binding characteristics very similar to [3H]epibatidine. Treatment with the two doses of nicotine hydrogen tartrate increased binding sites in the cerebral cortex by 40% and 70%, respectively. In contrast, no significant changes in the density of receptor binding sites were found in the adrenal gland, superior cervical ganglia, pineal gland or retina. These data indicate that chronic administration of nicotine even at high doses does not increase all nicotinic receptor subtypes, and that receptors containing alpha 3 subunits may be particularly resistant to this nicotine-induced change.     

Suppression of the Nicotinic Acetylcholine Response in Rat Superior Cervical Ganglionic Neurons by Steroids

Abstract : The effects of various types of steroids on the nicotinic acetylcholine (ACh) receptor (nAChR)-mediated responses were investigated in superior cervical ganglionic neurons acutely dissociated from rats using nystatin perforated patch recording. ACh induced a peak followed by a gradual decrease in the inward current at a holding potential of -40 mV. Nicotine, but not muscarine, mimicked ACh. Hydrocortisone at a concentration of > 10^-6 M reversibly suppressed both the peak and steady-state nicotine-induced currents ( I _nic) in a noncompetitive manner. The inhibition of I _nic by hydrocortisone did not show any voltage dependency and persisted in the presence of either cyclic AMP modulators, forskolin and 3-isobutyl-1-methylxanthine, or a protein kinase A inhibitor, N -[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89). β-Estradiol, androsterone, aldosterone, and 17α-estradiol mimicked hydrocortisone in its inhibitory action on ACh-induced currents ( I _ACh). The potency for the inhibitory actions on I _Ach was as follows : androsterne > β-estradiol > hydrocortisone ≥ aldosterone =17α-estradiol. Cholesterol had no effect on the I _ACh. In conclusion, the structural characteristics of steroid are thus considered to be necessary to block nicotinic I _ACh in rat superior cervical ganglionic cells, whereas the cholesterol side chain might disturb the inhibitory action of the steroid skeleton on nAChRs.     

Molecular characterization and localization of the RIC-3 protein, an effector of nicotinic acetylcholine receptor expression

The RIC-3 protein acts as a regulator of acetylcholine nicotinic receptor (nAChR) expression. In Xenopus laevis oocytes the human RIC-3 (hRIC-3) protein enhances expression of α7 receptors and abolishes expression of α4β2 receptors. In vitro translation of hRIC-3 evidenced its membrane insertion but not the role as signal peptide of its first transmembrane domain (TMD). When the TMDs of hRIC-3 were substituted, its effects on nAChR expression were attenuated. A certain linker length between the TMDs was also needed for α7 expression enhancement but not for α4β2 inhibition. A combination of increased α7 receptor steady state levels, facilitated transport and reduced receptor internalization appears to be responsible for the increase in α7 membrane expression induced by hRIC-3. Antibodies against hRIC-3 showed its expression in SH-SY5Y and PC12 cells and its induction upon differentiation. Immunohistochemistry demonstrated the presence of RIC-3 in rat brain localized, in general, in places where α7 nAChRs were found.     

Host Cell-Specific Folding and Assembly of the Neuronal Nicotinic Acetylcholine Receptor α7 Subunit

Abstract: Expression of the cloned neuronal nicotinic acetylcholine receptor (nAChR) α7 subunit in several cultured mammalian cell lines has revealed that the folding, assembly, and subcellular localization of this protein are critically dependent upon the nature of the host cell. In all cell lines that were examined, high levels of α7 protein were detected by metabolic labelling and immunoprecipitation after transfection with the cloned α7 cDNA. In contrast, elevated levels of α-bungarotoxin binding could be detected in only two of the nine cell lines. Both of these "α7-permissive" cell lines [rat phaeochromocytoma (PC12) and human neuroblastoma (SH-SY5Y)] express an endogenous α7 subunit. However, by expression of an epitope-tagged α7 subunit, it has been possible to show that the elevation in surface α-bungarotoxin binding in these two cell lines is due to expression of cDNA-encoded α7. The cell-specific misfolding of the neuronal nAChR α7 subunit is a phenomenon that is not shared by either the hetero-oligomeric muscle nAChR or the homo-oligomeric serotonin receptor 5-HT₃ subunit. Our data also indicate that the cell-specific misfolding cannot be explained by a requirement for the coassembly with other known nAChR subunits and cannot be alleviated by treatments that have been reported to affect the assembly efficiency of other neurotransmitter-gated ion channels.     

Sustained Nicotine Exposure Differentially Affects α3β2 and α4β2 Neuronal Nicotinic Receptors Expressed in Xenopus Oocytes

Abstract: To determine whether prolonged exposure to nicotine differentially affects α3β2 versus α4β2 nicotinic receptors expressed in Xenopus oocytes, oocytes were coinjected with subunit cRNAs, and peak responses to agonist, evoked by 0.7 or 7 µ M nicotine for α4β2 and α3β2 receptors, respectively, were determined before and following incubation for up to 48 h with nanomolar concentrations of nicotine. Agonist responses of α4β2 receptors decreased in a concentration-dependent manner with IC₅₀ values in the 10 n M range following incubation for 24 h and in the 1 n M range following incubation for 48 h. In contrast, responses of α3β2 receptors following incubation for 24–48 h with 1,000 n M nicotine decreased by only 50–60%, and total ablation of responses could not be achieved. Attenuation of responses occurred within the first 5 min of nicotine exposure and was a first-order process for both subtypes; half-lives for inactivation were 4.09 and 2.36 min for α4β2 and α3β2 receptors, respectively. Recovery was also first-order for both subtypes; half-lives for recovery were 21 and 7.5 h for α4β2 and α3β2 receptors, respectively. Thus, the responsiveness of both receptors decreased following sustained exposure to nicotine, but α4β2 receptors recovered much slower. Results may explain the differential effect of sustained nicotine exposure on nicotinic receptor-mediated neurotransmitter release.     

Influence of Y151F mutation in loop B on the agonist potency in insect nicotinic acetylcholine receptor

Abstract  Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels, which mediate fast cholinergic synaptic transmission in insect and vertebrate nervous systems. The nAChR agonist-binding site is present at the interface of adjacent subunits and is formed by loops A–C present in α subunits together with loops D–F present in either non-α subunits or homomer-forming α subunits. Although Y151 in loop B has been identified as important in agonist binding, various residues at the 151-site are found among vertebrate and invertebrate nAChR α subunits, such as F151. In Xenopus oocytes expressing Nlα1 or Nlα1^Y151F plus rat β2, Y151F mutation was found to significantly change the rate of receptor desensitization and altered the pharmacological properties of acetylcholine, but not imidacloprid, including the decrease of I _max, the increase of EC₅₀ (the concentration causing 50% of the maximum response) and the fast time-constant of decay (τ_f). By comparisons of residue structure, the hydroxyl group in the side chain of Y151 was thought to be important in the interaction between Nlα1/β2 nAChRs and acetylcholine, and the phenyl group to be important between Nlα1/β2 nAChRs and imidacloprid.     

Differential Expression and Subcellular Localization of Protein Kinase C α, β, γ, δ, and ɛ Isoforms in SH-SY5Y Neuroblastoma Cells: Modifications During Differentiation

Abstract: A decrease in protein kinase C activity caused either by treatment with inhibitors, such as staurosporine or H-7, or by prolonged exposure to phorbol diesters has been proposed to be involved in the early events of SH-SY5Y neuroblastoma cell differentiation. Because eight distinct isoforms of protein kinase C with discrete subcellular and tissue distributions have been described, we determined which isoforms are present in SH-SY5Y cells and studied their modifications during differentiation. The α, β, δ, and ɛ isoforms were present in SH-SY5Y cells, as well as in rat brain. Protein kinase C-α and -β₁ were the most abundant isoforms in SH-SY5Y cells, and immunoreactive protein kinase C-δ and -ɛ were present in much smaller amounts than in rat brain. Subcellular fractionation and immunocytochemistry demonstrated that all four isoforms are distributed bimodally in the cytoplasm and the membranes. Immunocytochemical analysis showed that the α isoform is associated predominantly with the plasma membrane and the processes extended during treatment with 12-tetradecanoyl-13-acetyl-β-phorbol or staurosporine, and that protein kinase C-ɛ is predominantly membrane-bound. Its localization did not change during differentiation. Western blots of total SH-SY5Y cell extracts and of subcellular fractions probed with isoform-specific polyclonal antibodies showed that when SH-SY5Y cells acquired a morphologically differentiated phenotype, protein kinase C-α and -ɛ decreased, and protein kinase C-β₁, did not change. These data suggest distinct roles for the different protein kinase C isoforms during neuronal differentiation, as well as possible involvement of protein kinase α and ɛ in neuritogenesis.