α4-1 Subunit mRNA of the nicotinic acetylcholine receptor in the rat olfactory bulb: cellular expression in adult, pre- and postnatal stages

In addition to their role in signal transduction, nicotinic acetylcholine receptors have been shown in vi-tro to be involved in neuronal growth cone regulation during development. This idea is supported by recent histochemical findings showing that iso- and archicortical nicotinic alpha4-1 receptor mRNA expression precedes cholinergic fiber ingrowth. To test whether this also holds true for rhinencephalic parts of the telencephalon, we have studied the olfactory bulb by digoxigenin-mediated in situ hybridization, using an alpha4-1 isoform-specific riboprobe and an alkaline-phosphatase-based detection system. Development is characterized by early intense alpha4-1 mRNA expression (embryonic d…

Retinoic Acid Induces Apoptosis-Associated Neural Differentiation of a Murine Teratocarcinoma Cell Line

Abstract: Incubation with all-trans retinoic acid (RA) induces PCC7-Mz1 embryonic carcinoma cells to cease proliferation and to develop into a tissue-like pattern of neuronal, astroglial, and fibroblast-like derivatives over a period of several days. Concomitant with the induction of differentiation by RA, a sizable fraction of the Mz1 stem cells detaches and dies, with the maximal level of cell death achieved after 10 h of RA treatment. This RA-induced cell death fulfills all criteria of apoptosis, including nuclear condensation, intranucleosomal DNA degradation, expression of cysteine aspases (caspases), and the formation of apoptotic bodies. Apoptosis could be suppressed by the pan-caspa…

Chapter 8 Nicotinic receptors of the vertebrate CNS: introductory remarks

Publisher Summary This chapter focuses on the nicotinic receptors of the vertebrate central nervous system (CNS). In vertebrates, nicotinic cholinergic neurotransmission is found in both the CNS and the periphery (muscle endplate). Although muscle and neuronal nicotinic acetylcholine receptors (nAChR) have evolved from a common ancestor, it is striking that the muscle receptor has remained rather stable in evolution, whereas the neuronal receptor has evolved to a wide diversity of subtypes. As an attractive hypothesis, neurotransmitters and neurohormones may not only interact with their archetypic cognate receptors but also with other neuroreceptor, albeit in a modulatory fashion. By modula…

A second pathway of activation of the Torpedo acetylcholine receptor channel

We have studied the interaction of the reversible acetylcholine esterase inhibitor (-)physostigmine (D-eserine) with the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue by means of ligand-induced ion flux into nAChR-rich membrane vesicles and of equilibrium binding. We find that (-) physostigmine induces cation flux (and also binds to the receptor) even in the presence of saturating concentrations of antagonists of acetylcholine, such as D-tubocurarine, alpha-bungarotoxin or antibody WF6. The direct action on the acetylcholine receptor is not affected by removal of the methylcarbamate function from the drug and thus is not due to carbamylation of the receptor…

The cGMP-gated channel of the rod photoreceptor — a new type of channel structure?

Recents findings from Numa's laboratory reveal that there might exist a wider variety in channel protein structure than originally anticipated. Recently, the cloning has been reported of the first cGMP-gated ion channel, the vertebrate rod photoreceptor which is activated by cGMP acting from the inside of the rod outer segment membrane

Noncompetitive agonism at nicotinic acetylcholine receptors; functional significance for CNS signal transduction.

The alkaloids (-)physostigmine (Phy), galanthamine (Gal) and codeine (Cod), and several derivatives and homologous compounds, can act as noncompetitive agonists (NCA) of nicotinic acetylcholine receptors (nAChR) from Torpedo electrocytes, frog and mammalian muscle cells, clonal rat pheochromocytoma cells, cultured hippocampal neurons and several ectopic expression systems, by interacting with a binding site on the alpha-subunits of these nAChRs that is insensitive to the natural transmitter, acetylcholine (ACh), and ACh-competitive agonists and antagonists. Several endogenous ligands, including opioid-type compounds, can also act via this site, albeit at higher concentrations than is typica…

Choline is a Selective Agonist of α7 Nicotinic Acetylcholine Receptors in the Rat Brain Neurons

In the present study, we demonstrate that choline, a precursor of acetylcholine (ACh) and a product of acetylcholine hydrolysis by acetylcholinesterase (AChE), acts as an efficient and relatively selective agonist of alpha7-containing nicotinic acetylcholine receptors (nAChR) in neurons cultured from the rat hippocampus, olfactory bulb and thalamus as well as in PC12 cells. Choline was able to activate postsynaptic and presynaptic alpha7 nAChRs, with the latter action resulting in the release of other neurotransmitters. Although choline was approximately one order of magnitude less potent than ACh (EC50 of 1.6 mM for choline and 0.13 mM for ACh), it acted as a full agonist at alpha7 nAChRs.…

Sensitivity of neuronal nicotinic acetylcholine receptors to the opiate antagonists naltrexone and naloxone: receptor blockade and up-regulation

In HEK293 cells stably expressing alpha4beta2 nAChRs, naltrexone, but not naloxone, blocked alpha4beta2 nAChRs via an open-channel blocking mechanism. In primary hippocampal cultures, naltrexone inhibited alpha7 nAChRs up-regulated by nicotine, and in organotypic hippocampal cultures naltrexone caused a time-dependent up-regulation of functional alpha7 nAChRs that was detected after removal of the drug. These results indicate that naltrexone could be used as a smoking cessation aid.

Nicotinic acetylcholine receptors have ligand-specific attachment point patterns.

Employing a panel of synthetic peptides as representative structural elements of the nicotinic acetylcholine receptor from Torpedo electric organ, we recently identified three sequence regions of the receptor (alpha 55-74, alpha 134-153 and alpha 181-200) serving as subsites for the binding of high molecular weight antagonists of acetylcholine (Conti-Tronconi et al. 1990). The relative binding affinities to these subsites of alpha-bungarotoxin and three competitive antibodies varied in a ligand-specific fashion. Employing a set of homologous synthetic peptides differing from alpha 181-200 by the exchange of single amino acid residues along the sequence, we now find that ligand binding cruci…

Identification of Purine Binding Sites on Torpedo Acetylcholine Receptor

Electrophysiological studies from this and other laboratories have suggested a direct action of ATP on nicotinic acetylcholine receptors (nAChR). To determine the site of binding of this purine derivative, we have covalently modified the nAChR from Torpedo marmorata electrocytes employing 2-[3H]-8-azido-ATP as a photoactivable affinity label. Covalently attached radioactivity was predominantly found in the beta-polypeptide of the receptor. Based on the results of protection studies with several nAChR ligands whose target sites at the receptor are known, we conclude that the purine site(s) differ from those of acetylcholine and of physostigmine, galanthamine and related ligands, and those of…

Regulatoren der Apoptose: Ziele für die Arzneimittelentwicklung

Der programmierte Zelltod (Apoptose) ist ein fur die Entwicklung und Aufrechterhaltung eines multizellularen Organismus lebenswichtiger Mechanismus. Ausgeschaltet oder uberaktiviert kann Apoptose aber auch zu schweren Erkrankungen fuhren. Mit dem neuen Protein Diablo/Smac ist kurzlich ein weiterer Regulator der Apoptose entdeckt worden.

Minireview: Nicotinic Acetylcholine Receptors on Hippocampal Neurons: Distribution on the Neuronal Surface and Modulation of Receptor Activity

The recent development of a technique that uses infrared microscopy for the visualization of well-defined areas on the surface of neurons, and a computerized system of micromanipulators led to the discovery that functional nicotinic acetylcholine receptors (nAChRs) are expressed at higher density on the dendrites than on the soma of rat hippocampal neurons. The finding that the expression of alpha-bungarotoxin-sensitive, alpha 7-bearing, nAChRs and dihydro-beta-erythroidine-sensitive, alpha 4 beta 2 nAChRs tends to increase along the dendritic length suggests that these receptors may be highly involved in the integration of synaptic functions in hippocampal neurons. The present report also …

Neuronal nicotinic receptors in synaptic functions in humans and rats: physiological and clinical relevance.

The present report describes the participation of nicotinic receptors (nAChRs) in controlling the excitability of local neuronal circuitries in the rat hippocampus and in the human cerebral cortex. The patch-clamp technique was used to record responses triggered by the non-selective agonist ACh and the alpha7-nAChR-selective agonist choline in interneurons of human cerebral cortical and rat hippocampal slices. Evidence is provided that functional alpha7- and alpha4beta2-like nAChRs are present on somatodendritic and/or preterminal/terminal regions of interneurons in the CA1 field of the rat hippocampus and in the human cerebral cortex and that activation of the different nAChR subtypes pres…

Modulatory control by non-competitive agonists of nicotinic cholinergic neurotransmission in the central nervous system

Abstract Several exogenous and endogenous compounds have been discovered that act on nicotinic acetylcholine receptors as non-competitive agonists (NCA) of low efficacy, and probably also as allosteric regulators of the receptor's sensitivity to acetylcholine. Nicotinic NCAs may be viewed as another facet in the increasingly complex picture of vertebrate brain organization, i.e. they may be part of a higher level ‘chemical’ network that overlays the neuronal network of the CNS. Furthermore, exogenous NCAs may provide a new therapeutic approach to Alzheimer's dementia.

GABAA-Rezeptorsubtyp-Spezifität von Benzodiazepinen

Eine Punktmutation in der α1-Untereinheit des GABAA-Rezeptors bei Mausen verhindert, dass die durch GABA induzierte Cl- -Kanalaktivitat dieses Subtyp weiterhin durch klassische Benzodiazepine modulierbar ist: Mause, die mit dem mutierten Gen ausgestattet sind, zeigen nicht mehr die beruhigende und angstlosende Wirkung der Benzodiazepine. Die Wirkung dieser allosterischen Modulatoren ist daher Rezeptorsubtyp-spezifisch, was die Hoffnung eroffnet, dass selektive Benzodiazepine entwickelt werden konnen.

The murine nuclear orphan receptor GCNF is expressed in the XY body of primary spermatocytes

AbstractWe have studied the expression of the nuclear orphan receptor GCNF (germ cell nuclear factor) on the mRNA and protein level in pubertal and adult mouse testes. We show by Northern and Western blot analyses and by in situ hybridization that GCNF is expressed in spermatocytes and round spermatids of adult mouse testis suggesting that GCNF may be a transcriptional regulator of spermatogenesis. Since the GCNF protein is accumulated in the XY body of late pachytene spermatocytes, it may be involved in transcriptional inactivation of sex chromosomes.

Allosteric modulation of nicotinic receptors as a treatment strategy for Alzheimer's disease.

Impairment of the central cholinergic system has a pivotal role in the cognitive decline observed in patients with Alzheimer’s disease (AD). One of the most prominent cholinergic deficits is the reduced number of nicotinic acetylcholine receptors (nAChR) in the brain. Since these receptors are important for memory and learning, enhancing nicotinic neurotransmission is a promising treatment strategy for AD. The two most common approaches to correcting these cholinergic deficits are to increase the synaptic availability of acetylcholine (ACh) by inhibiting acetylcholinesterase (AChE), or to mimic the effects of ACh (nicotinic agonists) by acting directly on nicotinic receptors. Clinical studi…

Unconventional ligands and modulators of nicotinic receptors

Evidence gathered from epidemiologic and behavioral studies have indicated that neuronal nicotinic receptors (nAChRs) are intimately involved in the pathogenesis of a number of neurologic disorders, including Alzheimer's disease, Parkinson's disease, and schizophrenia. In the mammalian brain, neuronal nAChRs, in addition to mediating fast synaptic transmission, modulate fast synaptic transmission mediated by the major excitatory and inhibitory neurotransmitters glutamate and GABA, respectively. Of major interest, however, is the fact that the activity of the different subtypes of neuronal nAChR is also subject to modulation by substances of endogenous origin such as choline, the tryptophan …

Galantamine is an allosterically potentiating ligand of neuronal nicotinic but not of muscarinic acetylcholine receptors.

Galantamine (Reminyl), an approved treatment for Alzheimer's disease (AD), is a potent allosteric potentiating ligand (APL) of human alpha 3 beta 4, alpha 4 beta 2, and alpha 6 beta 4 nicotinic receptors (nAChRs), and of the chicken/mouse chimeric alpha 7/5-hydroxytryptamine3 receptor, as was shown by whole-cell patch-clamp studies of human embryonic kidney-293 cells stably expressing a single nAChR subtype. Galantamine potentiates agonist responses of the four nAChR subtypes studied in the same window of concentrations (i.e., 0.1-1 microM), which correlates with the cerebrospinal fluid concentration of the drug at the recommended daily dosage of 16 to 24 mg. At concentrations10 microM, gal…

The expression level of the orphan nuclear receptor GCNF (germ cell nuclear factor) is critical for neuronal differentiation.

The germ cell nuclear factor (GCNF) is essential for normal embryonic development and gametogenesis. To test the prediction that GCNF is additionally required for neuronal differentiation, we used the mouse embryonal carcinoma cell line PCC7-Mz1, which represents an advantageous model to study neuronal cells from the stage of fate choice until the acquirement of functional competence. We generated stable transfectants that express gcnf sense or antisense RNA under the control of a tetracycline-regulated promoter. After retinoic acid-induced withdrawal from the cell cycle, sense clones developed a neuron network with changed properties, and the time course of neuron maturation was shortened.…

Expression of the α4 isoform of the nicotinic acetylcholine receptor in the fetal human cerebral cortex

Nicotinic acetylcholine receptors are likely to play an important role in neuronal migration during development. Furthermore, the alpha4 receptor subunit gene is related to a hereditary juvenile form of epilepsy. Only little information is available, however, on the expression of cerebrocortical nicotinic acetylcholine receptors during human fetal development. Using non-isotopic in situ hybridization and immunohistochemistry, we have studied the distribution of the alpha4 subunit of the nicotinic acetylcholine receptor mRNA and protein in the human frontal cortex at middle (17-24 weeks of gestation) and late (34-42 weeks of gestation) fetal stages. Both, alpha4 receptor mRNA and alpha4 rece…

Galantamine modulates nicotinic receptor and blocks Aβ-enhanced glutamate toxicity

Galantamine is a plant alkaloid that is used in the treatment of Alzheimer's disease. We have studied the effects of galantamine on beta-amyloid-enhanced glutamate toxicity using primary rat cultured cortical neurons. Nicotine and galantamine alone, and in combination, protected neurons against this neurotoxicity. The protection was not blocked by alpha4beta2 nicotinic acetylcholine receptor (nAChR) antagonists, but was partially blocked by alpha7 nAChR antagonists. Galantamine induced phosphorylation of Akt, an effector of phosphatidylinositol 3-kinase (PI3K), while PI3K inhibitors blocked the protective effect and Akt phosphorylation. The antibody FK1, which selectively blocks the alloste…

Nicotinic receptor function in the mammalian central nervous system.

The diversity of neuronal nicotinic receptors (nAChRs) in addition to their possible involvement in such pathological conditions as Alzheimer's disease have directed our research towards the characterization of these receptors in various mammalian brain areas. Our studies have relied on electrophysiological, biochemical, and immunofluorescent techniques applied to cultured and acutely dissociated hippocampal neurons, and have been aimed at identifying the various subtypes of nAChRs expressed in the mammalian central nervous system (CNS), at defining the mechanisms by which CNS nAChR activity is modulated, and at determining the ion permeability of CNS nAChR channels. Our findings can be sum…

α7 Nicotinic acetylcholine receptors and modulation of gabaergic synaptic transmission in the hippocampus

The present report provides new findings regarding modulation of gamma-aminobutyric acid (GABA) transmission by alpha7 nicotinic receptor activity in CA1 interneurons of rat hippocampal slices. Recordings were obtained from tight-seal cell-attached patches of the CA1 interneurons, and agonists were delivered to the neurons via a modified U-tube. Application for 6 s of the alpha7 nicotinic receptor-selective agonist choline (or =1 mM) to all CA1 interneurons tested triggered action potentials that were detected as fast current transients. The activity triggered by choline terminated well before the end of the agonist pulse, was blocked by the alpha7 nicotinic receptor antagonist methyllycaco…

Galantamin und die nikotinisch-cholinerge Neurotransmission: Neue Theorien!

Eines der klarsten neurochemischen Kennzeichen fur die Alzheimer'sche Demenz ist die verringerte Zahl an nikotinischen Acetylcholinrezeptoren in Cortex und Hippokampus. Der Verlust an nAChRs ist dabei eng korreliert mit dem Verlust an kognitiver Hirnleistung und bietet sich daher als Ziel fur eine Arzneimitteltherapie an. Von den moglichen Therapieansatzen haben sich weder eine Forderung der Synthese von Acetylcholin noch eine Behandlung mit nikotinischen oder muskarinischen Agonisten bewahrt. Die Behandlung mit schwachen Hemmern der ersten Generation hat sich uber kurzere Zeitraume bewahrt, wobei die klinischen Verbesserungen jedoch nur moderater Art sind. Uber langere Zeitraume verliert d…

Biochemistry. Von C. K. Mathews und K. E. van Holde. The Benjamin/Cummings Publishing Co. Inc., Redwood City, CA (USA), 1990. XXIX, 1129 S., geb. $ 48.95. – ISBN 0-8053-5015-2

Synthesis and in vitro evaluation of galanthamine derivatives for examination of nicotinic acetylcholine receptor system

ISunmrary: The syntheses and radioactive labeling of several galanthamine derivatives, 6-O-demethyl-6-O-fluoroethylgalanthamine, 10-N-demethyl-lO-Nfluoroethylgalanthamine and N-methylgalanthaminium are reported. First ill vitro evaluations were carried out to determine their properties as allosterically potentiating ligands of nicotinic receptors. N-methylgalanthaminium was found to be a promising candidate for further investigations. The most commonly applied therapeutic approach to balance nicotinic cholinergic deficits in Alzheimer’s disease (AD) patients is the administration of acetylcholinesterase inhibitors (AChE-I) although they have been proven to be of limited therapeutic value [ …

Gentechnik in der Schule

The Fonds of German Chemical Industry (FCI) has produced an instrumental kit named „Blue Genes” which enables High School teachers to perform in their school environment basic experiments of gene analysis and cloning. In particular, the kit contains all necessary equipment and reagents to perform a restriction analysis, and cloning and expression of a bacterial gene.

Differentiation-associated apoptosis of neural stem cells is effected by Bcl-2 overexpression: impact on cell lineage determination

Apoptosis is an integral part of neural development. To elucidate the importance of programmed cell death on cell lineage determination we utilized murine PCC7-Mzl cells, a model system for neural differentiation. Treatment of pluripotent PCC7-Mzl stem cells with 0.1 microM all-trans retinoic acid (RA) causes a cease of proliferation and an initiation of differentiation into neurons, glial cells and fibroblasts. Simultaneously, a fraction of the cell culture (ca. 25%) dies within 24 h by apoptosis. We transfected PCC7-Mzl cells with the human bcl-2 cDNA and generated PCC7-Mz-Bcl-2 cell lines expressing two- to tenfold higher levels of Bcl-2 than parental cells. Overexpression of Bcl-2 resul…

Cellular Distribution and Expression of Cortical Acetylcholine Receptors in Aging and Alzheimer's Disease

Ligand binding studies show marked reductions of nicotinic, but not of muscarinic binding sites in Alzheimer's disease. Using monoclonal antibodies we studied immunohistochemically the expression of the respective receptor proteins in the frontal cortex of middle-aged (55 +/- 5 yr) controls, age-matched controls (73 +/- 6 yr), and patients with Alzheimer's disease (74 +/- 5 yr). Density of nicotinic cholinoceptive neurons was 8000/mm3 for middle-aged controls and 4000/mm3 for age-matched controls, but only 900/mm3 in Alzheimer's brains (p less than 0.0001). Densities of muscarinic cholinoceptive and of Nissl-stained neurons were not significantly different between the groups, pointing to a …

Expression of protein kinase C gene family members is temporally and spatially regulated during neural development in vitro.

We used primary cultures of rat hippocampal neurons and PCC7-Mz1 cells to correlate the expression of the protein kinase C (PKC) gene family with specific events during neural differentiation. Multipotent PCC7-Mz1 embryonic carcinoma stem cells develop into a tissue-like pattern of neuronal, fibroblast-like and astroglial cells by all-trans retinoic acid (RA) treatment. Western blot analyses demonstrate that PKCalpha, betaI, gamma, theta, mu, lambda, and zeta were constitutively expressed but the expression of PKCbetaII, delta, epsilon, and eta was up-regulated three days after addition of RA when cells mature morphologically. While the protein levels of the PKC isoforms betaII, delta and e…

Expression of α4-1 and α5 Nicotinic Cholinoceptor mRNA in the Aging Rat Cerebral Cortex

Although important in neurodegeneration, systematic studies of nicotinic acetylcholine receptor expression in normal aging human brains are difficult to perform. We have studied the expression of nicotinic receptor alpha 4-1 and alpha 5 mRNA in the frontal and parietal isocortex of 3- (young adult), 24- (late middle aged), and 33-month-old (old) rats by nonisotopic in situ hybridization. In all groups transcripts were mainly present in layer II/III and V pyramidal neurons. The numerical densities of alpha 4-1 mRNA-containing neurons with respect to those of cresyl violet-stained neurons decreased with aging in the rat frontal and parietal cortex, while those of alpha 5 mRNA-containing neuro…

Species- and Subtype-Specific Recognition by Antibody WF6 of a Sequence Segment Forming an α-Bungarotoxin Binding Site on the Nicotinic Acetylcholine Receptor α Subunit

The monoclonal antibody WF6 competes with acetylcholine and alpha-bungarotoxin (alpha-BGT) for binding to the Torpedo nicotinic acetylcholine receptor (nAChR) alpha 1 subunit. Using synthetic peptides corresponding to the complete Torpedo nAChR alpha 1 subunit, we previously mapped a continuous epitope recognized by WF6, and the prototope for alpha-BGT, to the sequence segment alpha 1(181-200). Single amino acid substitution analogs have been used as an initial approach to determine the critical amino acids for WF6 and alpha-BGT binding. In the present study, we continue our analysis of the structural features of the WF6 epitope by comparing its cross-reactivity with synthetic peptides corr…

Nicotinic cholinoceptors in the rat pineal gland as analyzed by Western blot, light- and electron microscopy

Abstract The monoclonal antibody WF6, raised against purified Torpedo nicotinic acetylcholine receptor (nAChR) was used to study the distribution of cholinoceptors in the rat pineal gland by means of Western blot analysis, light- and electron microscopy. The immunoblot analysis using homogenized pineal gland revealed a labeled protein band of apparent molecular weight 40 kDa which was identified as α-subunits of a nAChR. In the light microscope, approximately one-fourth of the pinealocytes exhibited cytoplasmic immunoreactivity (IR) of varying density. In the electron microscope, IR was seen as patchy staining of cell membranes of pinealocyte somata and processes. Presynaptic IR material wa…

Cellular distribution of nicotinic acetylcholine receptor subunit mRNAs in the human cerebral cortex as revealed by non-isotopic in situ hybridization.

The pharmacology of telencephalic nicotinic acetylcholine receptors (nAChRs) has become an important issue in recent years. While in the human brain a direct pharmacological assessment is difficult to achieve the visualization of nAChRs has been enabled by histochemical techniques providing an ever increasing and improving resolution. Receptor autoradiography was used to visualize binding sites on the level of cortical layers whereas immunohistochemistry has allowed for the cell type-specific and ultrastructural localization of receptor protein. Further investigations have to elucidate the cellular sites of NAChR biosynthesis by visualizing subunit-specific transcripts. Using autopsy sample…

Biochemical Characterization of a Novel Channel-Activating Site on Nicotinic Acetylcholine Receptors

We have studied the interaction of the reversible acetylcholine esterase inhibitor (-)physostigmine and several structurally related compounds with the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue by means of ligand-induced ion flux into nAChR-rich membrane vesicles, direct binding studies and photoaffinity labeling. (-)Physostigmine acts as a channel-activating ligand at low concentrations and as a direct channel blocker at elevated concentrations. Channel activation is not inhibited by desensitizing concentrations of ACh or ACh-competitive ligands (including alpha-bungarotoxin and D-tubocurarine) but is inhibited by antibody FK1 and several other compoun…

Transkriptionskontrolle und Altersdiabetes

Bei Diabetikern mit schwerer Insulinresistenz wurden Mutationen in der Ligandenbindedomane des Kernrezeptors und Transkriptionsregulators PPARγ gefunden. Die Mutationen weisen darauf hin, das PPARγ eine wichtige Rolle bei der Kontrolle der Insulinempfindlichkeit, des Blutzuckerspiegels und des Blutdrucks spielt.

Chili und Hitze: Molekularer Schmerzrezeptor identifiziert

Knock-out-Mause, in denen das Gen fur den Vanilloidrezeptor VR1 inaktiviert wurde, reagieren sehr viel unempfindlicher als normale Mause auf Capsaicin und Hitze. Damit scheint erstmals ein molekularer Schmerzrezeptor eindeutig identifiziert zu sein.

Chapter 27: Peptidergic and cholinergic receptors on cultured astrocytes of different regions of the rat CNS

Publisher Summary This chapter discusses whether astrocytes possess peptidergic and cholinergic receptors. There is strong evidence that in addition to neurons, astrocytes also express receptors for neurotransmitters and neuromodulators. The investigations were carried out in explant cultures of the spinal cord, brain-stem, cerebellum, and neocortex of fetal and newborn rats. By the means of autoradiography, it was demonstrated that astrocytes possess binding sites for the vasoactive peptides 3 H-Ang II, 125 I- VIP, 125 I-ET-l, ET-3, and 125 I-AVP. These findings are consistent with electrophysiological studies demonstrating that Ang II, ET, and AVP cause a depolarization of the glial membr…

Neuronal Cell Nuclear Factor. A Nuclear Receptor Possibly Involved in the Control of Neurogenesis and Neuronal Differentiation

We have cloned from a cDNA library of neuronal derivatives of retinoic-acid-induced embryonic carcinoma cells a nuclear receptor that may be involved in the control of late neurogenesis and early neuronal differentiation. The receptor which is practically identical in sequence with germ cell nuclear factor, has been designated neuronal cell nuclear factor (NCNF). NCNF is exclusively expressed in the neuronal derivatives of PCC7-Mz1 cells, with the expression beginning within hours of exposure to retinoic acid. In the developing mouse brain, NCNF is expressed in the marginal zones of the neuroepithelium which are known to contain young postmitotic neurons. NCNF binds to the DRO sequence ther…

Photoaffinity labeling of Torpedo acetylcholine receptor by physostigmine.

The plant alkaloid physostigmine, an established anti-cholinesterase agent of the carbamate type, has recently been shown to bind to the nicotinic acetylcholine receptor from Torpedo marmorata electrocytes [Okonjo, K. O., Kuhlmann, J.Maelicke, A. (1991) Eur. J. Biochem. 200, 671-677]. Pharmacological studies of physostigmine-induced ion flux into nicotinic-acetylcholine-receptor-rich membrane vesicles, indicated distinct binding sites for physostigmine and acetylcholine. As shown in this study by photoaffinity labeling with [phenyl-(n)-3H](-)physostigmine, the physostigmine-binding site is located within the same subunit (alpha polypeptide) of the receptor as the acetylcholine-binding site.…

Acetylcholine esterase: the structure.

Gentechnik stellt erneut Tabus in Frage

Ist mit der Geburt des Adam Nash der Weg frei fur Designer-Babies jeglicher Art, und was ist von Stammzellkulturen der gleichen oder gar verschiedener Arten als Gewebeersatz zu halten? Wieder einmal ist die Ethik vor Tatsachen gestellt, auf die sie nicht vorbereitet war. Die deutsche Offentlichkeit, auch die wissenschaftliche, reagiert verunsichert und zieht sich auf dogmatisches Verhalten zuruck.

Chapter 9 Nicotinic acetylcholine receptors on hippocampal neurons: cell compartment-specific expression and modulatory control of channel activity

Publisher Summary The neuronal nicotinic acetylcholine receptors (nAChRs) are differentially expressed on the somato-dendritic surface of hippocampal neurons. This chapter demonstrates that various ions and drugs play a crucial role in modulating the activity of neuronal nAChRs. Considering the diversity of the neurotransmitter receptors and their binding sites and the diversity of substances, which can act simultaneously as a primary agonist of one receptor and an allosteric modulator of a different receptor, an enormous variety of combinatorial possibilities can be achieved in the brain giving rise to very complex neuronal networks. The characterization of the diversity of many receptors …

Desensitization is a property of the cholinergic binding region of the nicotinic acetylcholine receptor, not of the receptor-integral ion channel

AbstractThe reversible acetylcholine esterase inhibitor (−)-physostigmine (eserine) is the prototype of a new class of nicotinic acetylcholine receptor (nAChR) activating ligands: it induces cation fluxes into nAChR-rich membrane vesicles from Torpedo marmorala electric tissue even under conditions of antagonist blocked acetylcholine binding sites (Okonjo, Kuhlmann, Maclicke, Neuron, in press). This suggests that eserine exerts its channel-activating property via binding sites at the nAChR separate from those of the natural transmitter. We now report that eserine can activate the channel even when the receptor has been preincubated (desensitized) with elevated concentrations of acetylcholin…

Cloning of Several Genes Coding for Retinoic Acid Nuclear Receptors in the Mouse Embryonal Carcinoma Cell Line PCC7–MZ1

Mouse embryonal carcinoma cell line PCC7-Mz1 can be induced by retinoic acid (RA) to differentiate into several well defined phenotypes of neuroectodermal origin (Lang, E. et al. (1989) J. Cell. Biol. 109, 2481-2493). Several subclones of the cell line (clonal variants) differ from each other in their developmental potential. To test whether these differences in cellular fate are due to somatic mutations in specific genes of these cells, we have cloned full length cDNAs coding for the alpha 1 and beta 2 isoforms, and partial length cDNAs coding for the alpha 2, beta 1 and beta 3 isoforms of the retinoic acid nuclear receptor (RAR). The cloned cDNAs did not differ in sequence from those of n…

Cellular expression of α4 subunit mRNA of the nicotinic acetylcholine receptor in the developing rat telencephalon

By contrast to adult brain, little is known on the development of nicotinic acetylcholine receptor (nAChR) expression. Using a digoxigenin-labeled riboprobe for in situ hybridization, alpha 4 nAChR subunit mRNA expression was studied in embryonic and postnatal rat neocortex and hippocampus where it was transiently increased in neuronal subpopulations and preceded cholinergic fiber ingrowth. alpha 4 expression was increased in neocortical layer VIb between E20 and P2 and, about birth, in dentate gyrus granule cells subsequently decreasing to adult levels. nAChR mRNA expression is increased at the developing neuromuscular endplate preceding cholinergic innervation which triggers changes in no…

An ion channel-gated adenylyl cyclase

Expression of retinoic acid nuclear receptors in the mouse embryonal carcinoma cell line PCC7-Mz1

Mouse embryonal carcinoma cell line PCC7-Mz1 can serve as a model of mammalian neural development [1989, J. Cell. Biol. 109, 2481-2493]. Upon exposure to all-trans retinoic acid (RA), Mz1 cells differentiate into a stable pattern of neurons, astroglia and fibroblasts whereas variants of the parental cell line either are restricted in their patterns of derivatives or do not respond at all to RA. Using gene probes specific for the alpha 1, alpha 2 and beta 2 isoforms of the retinoic acid nuclear receptor, we have studied by Northern blot analysis the expression of these transcription factors in uninduced and induced cells of clone Mz1 and in variants with different developmental potential. al…

Expression of functional α7 nicotinic acetylcholine receptor during mammalian muscle development and denervation

We have studied, on the transcriptional, protein and functional level, the expression of alpha7 nicotinic acetylcholine receptors (nAChR) in the course of rat muscle development, denervation and renervation. At foetal day 13, alpha7 nAChR expression was observed in somites and developing muscles of the back, but not yet in migrating myoblasts. Two days later, concomitant with myoblast aggregation, the alpha7 isoform began to be expressed in isolated myoblasts, with the highest level of expression in the frontal zone of the migrating wave. On foetal day 18, a time when the myoblasts in the upper hindleg have fused, alpha7 nAChR expression was most prominent in the outer layer of muscle tissu…

Synthesis and characterization of biotinylated and photoactivatable neuroleptics. Novel bifunctional probes for dopamine receptors

Abstract We have synthesized and characterized a series of novel derivatives of established antagonists of the neurotransmitter dopamine, i.e. butyrophenones, hexahydrocarbolines and phenothiazenes. All derivatives were biotinylated, some of them carried an additional (photoactivatable) azido group. In the case of butyrophenones, the structural modifications were introduced at the aliphatic keto group and/or the heterocyclic ring system, both modifications resulting in significant decreases in binding affinity to dopamine D 2 and dopamine D 1 receptor subtypes. Biotinylation of hexahydrocarbolines significantly increased their binding affinity to D 1 receptors, with the affinity for D 2 rec…

Inhibitor der Neuroregeneration kloniert

Wahrend Nervenfasern des peripheren Nervensystems recht gut regenerieren konnen, gelten diejenigen des Zentralen Nervensystems gemeinhin als nicht regenerierfahig, solange sie sich in ihrer naturlichen Umgebung befinden. Grund dafur sind insbesondere bestimmte Markscheide-assoziierte Proteine, Nogo genannt. Nun ist das wichtigste dieser inhibitorischen Proteine identifiziert und auf Genebene kloniert worden. Damit eroffnen sich auch neue Wege fur die Behandlung von Hirnschlag und Ruckenmarkschadigungen und von neurodegenerativen Erkrankungen.

Introduction: Mechanisms of neurotransmission

Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures

Nicotinic acetylcholine receptors play important roles in numerous cognitive processes as well as in several debilitating central nervous system (CNS) disorders. In order to fully elucidate the diverse roles of nicotinic acetylcholine receptors in CNS function and dysfunction, a detailed knowledge of their cellular and subcellular localizations is essential. To date, methods to precisely localize nicotinic acetylcholine receptors in the CNS have predominantly relied on the use of anti-receptor subunit antibodies. Although data obtained by immunohistology and immunoblotting are generally in accordance with ligand binding studies, some discrepancies remain, in particular with electrophysiolog…

Genomics und Postgenomics

Mit der weitgehend vollstandigen Sequenzierung des Erbmaterials von derzeit etwa 30 Organismen, einschlieslich des Menschen, ist ein Gipfelpunkt der Genomforschung erreicht. Nun kommt es darauf an, die genetische Information in biologisches und medizinisches Wissen umzusetzen.

Stable expression in HEK-293 cells of the rat alpha3/beta4 subtype of neuronal nicotinic acetylcholine receptor.

The alpha3/beta4 subtype of neuronal nicotinic acetylcholine receptor (nAChR) was stably expressed in human embryonic kidney (HEK) 293 cells that co-expressed a voltage-gated Ca2+ channel. alpha3/beta4-nAChR-expressing clones were identified using the fura-2 Ca2+ imaging technique, and were further characterised by single-cell and whole-cell patch-clamp studies. Acetylcholine (ACh) induced fast activating currents which showed desensitisation and inward rectification. The conductance of the ACh-activated channel was 29 pS. The order of potency of the nicotinic agonists tested was cytisine approximately = nicotine > acetylcholine. The EC50 value for ACh was 145 microM; the Hill coefficient w…

Mapping of a binding site for ATP within the extracellular region of the Torpedo nicotinic acetylcholine receptor beta-subunit.

Using 2,8,5'-[H-3]ATP as a direct photoaffinity label for membrane-bound nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata, we have identified a binding site for ATP in the extracellular region of the beta-subunit of the receptor. Photolabeling was completely inhibited in the presence of saturating concentrations of nonradioactive ATP, whereas neither the purinoreceptor antagonists suramin, theophyllin, and caffeine nor the nAChR antagonists alpha-bungarotoxin and d-tubocurarine affected the labeling reaction. Competitive and noncompetitive nicotinic agonists and Ca2+ increased the yield of the photoreaction by up to 50%, suggesting that the respective binding sites are allost…

Analysis of NO synthase expression in neuronal, astroglial and fibroblast-like derivatives differen-tiating from PCC7-Mzl embryonic carcinoma cells

We studied the expression of the NO synthase isoforms in an in vitro model of neural development using RT-PCR, Western blot and immu- nohistochemistry. Murine PCC7-Mzl cells (Jostock et al., Eur. J. Cell Biol. 76, 63–76,1998) differentiate in the presence of all-trans retinoic acid and dibutyryl cAMP along the neural pathway into neuron-like, fibroblast-like and astroglia-like cells. Undifferentiated cells showed immunofluorescent staining for neuronal-type NOSI and endothelial- type NOS III. This expression pattern was retained in those cells differ entiating into neurofilament- and tau protein-positive neuronal cells. Thymocyte alloantigen (Thyl.2/CD 90.2)-positive Fibroblasts, appearing …

<title>Spectroscopic investigation of the nicotinic acetylcholine receptor for application in medical diagnosis</title>

Native vesicles containing the nicotinic acetylcholine receptor (nAChR) prepared from the electric organ of the ray Torpedo marmorata were used to obtain fluorescence signal sin dependence of different concentrations of the local anesthetics procaine. Nonlinear concentration dependent spectral changes are found using ethidium bromide as a fluorescence marker. Structural changes are found for the proteins including the nAChR in the vesicles during immobilization onto surfaces such as IR transparent germanium (GE) crystal, Ge crystal coated with silver (Ag) cluster to use the SEIRA effect and/or crystals covered with a lipid subphase. A comparison between Ge and Ge coated with Ag (Ge/Ag) clus…

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 p…

Allosteric sensitization of nicotinic receptors by galantamine, a new treatment strategy for Alzheimer’s disease

Cholinesterase inhibitors are the only approved drug treatment for patients with mild to moderately severe Alzheimer's disease. Interestingly, the clinical potency of these drugs does not correlate well with their activity as cholinesterase inhibitors, nor is their action as short lived as would be expected from purely symptomatic treatment. A few cholinesterase inhibitors, including galantamine, produce beneficial effects even after drug treatment has been terminated. These effects assume modes of action other than mere esterase inhibition and are capable of inducing systemic changes. We have recently discovered a mechanism that could account, at least in part, for the above-mentioned unex…

Neuronal Nicotinic Receptors in the Locust Locusta migratoria

We have identified five cDNA clones that encode nicotinic acetylcholine receptor (nAChR) subunits expressed in the nervous system of the locust Locusta migratoria. Four of the subunits are ligand-binding α subunits, and the other is a structural β subunit. The existence of at least one more nAChR gene, probably encoding a β subunit, is indicated. Based on Northern analysis and in situ hybridization, the five subunit genes are expressed. locα1, locα3, andlocβ1 are the most abundant subunits and are expressed in similar areas of the head ganglia and retina of the adult locust. Because Locα3 binds α-bungarotoxin with high affinity, it may form a homomeric nAChR subtype such as the mammalian α7…

Cell fate specification in an in vitro model of neural development

We have studied in an in vitro model of neural development the effect of neighboring cells on the fate of single fluorescently labeled precursor cells. In one line of experiments, PCC7-Mz1 embryonal carcinoma cells were transiently transfected with "green fluorescent protein" (GFP) and, following incubation with 0.1 microM all-trans retinoic acid (RA), the number and morphology of derivatives (neuronal or non-neuronal) was determined that form groups of GFP-expressing cells in a surrounding of unlabeled cells. Because single PCC7-Mz1 cells can produce single-lineage and mixed-lineage derivatives, they are individually pluripotent. In another line of experiments, we have analyzed the fate of…

Introductory Lecture: Allosteric Modulation of Torpedo Nicotinic Acetylcholine Receptor Ion Channel Activity by Noncompetitive Agonists

AbstractSimilar to other neuroreceptors of the vertebrate central nervous system, the nicotinic acetylcholine receptor (nAChR) is subject to modulatory control by allosterically acting ligands. Of particular interest in this regard are allosteric ligands that enhance the sensitivity of the receptor to its natural agonist acetylcholine (ACh), as such ligands could be useful as drugs in diseases associated with impaired nicotinic neurotransmission. Here we discuss the action of a novel class of nAChR ligands which act as allosterically potentiating ligands (APL) on the nicotinic responses induced by ACh and competitive agonists. In addition, APLs also act as noncompetitive agonists of very lo…

Allosterically potentiating ligands of nicotinic receptors as a treatment strategy for Alzheimer's disease.

Abstract One of the most prominent cholinergic deficit in Alzheimer’s disease (AD) is the reduced number of nicotinic acetylcholine receptors (nAChR) in the hippocampus and cortex of AD patients, as compared to age-matched controls. This deficit results in reduced nicotinic cholinergic excitation which may not only impair postsynaptic depolarization but also presynaptic neurotransmitter release and Ca 2+ -dependent intracellular signaling, including transcriptional activity. Presently, the most common approach to correct the nicotinic cholinergic deficit in AD is the application of cholinesterase inhibitors. Due to the resulting increase in synaptic acetylcholine levels, both in concentrati…

Physostigmine and Neuromuscular Transmission

Single channel studies carried out in cultured rat myoballs and cultured hippocampal neurons, and ion flux studies performed on Torpedo electrocyte membrane vesicles, showed that physostigmine (Phy), a well-established acetylcholinesterase inhibitor, interacts directly with nicotinic acetylcholine receptors (nAChR). Low concentrations (0.1 microM) of Phy activate the receptor integral channel, whereas higher concentrations blocked the channel in its opened state. In contrast to channel activation by acetylcholine (ACh) and classical cholinergic agonists, however, Phy was capable of activating the nAChR channel even when the ACh binding sites were blocked by competitive antagonists, such as …

Cellular distribution in the rat telencephalon of mRNAs encoding for the alpha 3 and alpha 4 subunits of the nicotinic acetylcholine receptor.

Pharmacological and electrophysiological studies provide evidence for the involvement of different nicotinic acetylcholine receptor isoforms in rat neocortical and hippocampal signal transduction. Yet, rather little is known on the cellular localization of these isoforms. With the availability of isoform specific nucleic acid probes and sensitive non-isotopic detection systems, nicotinic receptors can be studied on the mRNA level in individual neurons. In this way, we have paradigmatically studied the distribution of the alpha 3 and alpha 4 isoform mRNAs of the nicotinic receptor in the rat telencephalon. In the cerebral cortex, alpha 3 transcripts were mainly located in pyramidal neurons o…

α-Conotoxins EpI and AuIB switch subtype selectivity and activity in native versus recombinant nicotinic acetylcholine receptors

The Xenopus laevis oocyte expression system was used to determine the activities of alpha-conotoxins EpI and the ribbon isomer of AuIB, on defined nicotinic acetylcholine receptors (nAChRs). In contrast to previous findings on intracardiac ganglion neurones, alpha-EpI showed no significant activity on oocyte-expressed alpha3beta4 and alpha3beta2 nAChRs but blocked the alpha7 nAChR with an IC50 value of 30 nM. A similar IC50 value (103 nM) was obtained on the alpha7/5HT3 chimeric receptor stably expressed in mammalian cells. Ribbon AuIB maintained its selectivity on oocyte-expressed alpha3beta4 receptors but unlike in native cells, where it was 10-fold more potent than native alpha-AuIB, had…

Neurodegenerative Erkrankungen: Auslösung durch zufälligen Zelltod?

Auch genetisch bedingte neurodegenerative Erkrankungen brechen gewohnlich erst in spateren Lebensphasen aus. Erfordert der Krankheitsausbruch - wie bisher angenommen -vorausgehende kumulative Zellschadigungen oder ist er eher ein singulares Zufallsereignis?

„Gene prospecting”︁ - die Jagd nach Geninformation

Mikroarrayverfahren haben die Analyse von genetischem Material revolutioniert. Schon kann man im „Netz” mit dem MGuide eine Selbstbauanleitung fur einen Microarrayroboter finden. Das „gene prospecting”, die Suche nach Bioinformation im Land der Gene und im Stil der fruheren Gold- und Olsucher ist auf seinem Hohepunkt angelangt.

Monoclonal antibodies FK1 and WF6 define two neighboring ligand binding sites on Torpedo acetylcholine receptor alpha-polypeptide.

Previous studies have identified the sequence region flanking the invariant vicinal cysteinyl residues at positions 192 and 193 of the nicotinic acetylcholine receptor alpha-subunit as containing major elements of the binding site for acetylcholine and its agonists and antagonists, including antibody WF6 (Conti-Tronconi, B. M., Diethelm, B. M., Wu, X., Tang, F., Bertazzon, T., Schroder, B., Reinhardt-Maelicke, A., and Maelicke, A. (1991) Biochemistry 30, 2575-2584). Recently we have shown that the sequence region flanking lysine alpha 125 contains elements of the binding site for physostigmine and related ligands, including antibody FK1 (Schrattenholz, A., Godovac-Zimmerman, J., Schafer, H.…

Production of ceramides causes apoptosis during early neural differentiation in vitro.

To investigate signal transduction pathways leading to apoptosis during the early phase of neurogenesis, we employed PCC7-Mz1 cells, which cease to proliferate and begin to differentiate into a stable pattern of neurons, astroglial cells, and fibroblasts upon incubation with retinoic acid (RA). As part of lineage determination, a sizable fraction of RA-treated cultures die by apoptosis. Applying natural long-chain C(16)-ceramides as well as membrane-permeable C(2)/C(6)-ceramide analogs caused apoptosis, whereas the biologically nonactive C(2)-dihydroceramide did not. Treating PCC7-Mz1 stem cells with a neutral sphingomyelinase or with the ceramidase inhibitor N-oleoylethanolamine elevated t…

Cellular Acetylcholine Receptor Expression in the Brain of Patients with Alzheimer’s and Parkinson’s Dementia

Binding studies and receptor autoradiography reveal the overall changes of acetylcholine receptors (AChR) in Alzheimer’s and Parkinson’s dementia cortices.2,5 A detailed account of these changes requires a study of neurochemical phenotype of individual neurons as basic elements of networks constituting the substrate of cortical functions.4 Examples will be given for cell-type specific AChR localization in normal and diseased human cerebral cortex.

Synthesis and evaluation of tritium labelled 10-methylgalanthamine iodide: a novel compound to examine the mechanism of interaction of galanthamine derivatives with the nicotinic acetylcholine receptors

Summary A new promising galanthamine derivative, 10-[ 3 H]methylgalanthamine iodide, was synthesized for binding studies to nicotinic acetylcholine receptors expressed in Torpedo electric ray electroplaques. Galanthamine was reacted with [ 3 H]methyl iodide to yield 10-[ 3 H]methylgalanthamine iodide with a radiochemical yield of >70% and a specific activity of 32 Ci/mmol after purification via solid phase extraction. To test the ligand properties of the radioligand, calcium imaging and electrophysiology of the non-radioactive analogue were performed to obtain an EC50 of 270 nM, a Hill coefficient of 1.9 and the induced cell current. Copyright # 2003 John Wiley & Sons, Ltd.

Binding Sites for Neurotoxins and Cholinergic Ligands in Peripheral and Neuronal Nicotinic Receptors Studies with Synthetic Receptor Sequencesa

Expression of nicotinic acetylcholine receptors in the rat superior cervical ganglion on mRNA and protein level.

The expression of nicotinic acetylcholine receptors (nAChR) in the rat superior cervical ganglion was investigated by Western blotting, immunohistochemistry and non-radioactive in situ hybridization applying probes for the alpha 4-1 and beta 2 subunit mRNA. Immunoblot analysis of homogenized ganglia using the anti-nAChRs antibody WF6 revealed a labeled protein band of apparent molecular weight of 40 kDa which is typical for the alpha subunit of nAChRs. Applying double-labeling immunofluorescence with antibodies against tyrosine hydroxylase, nAChR-like molecules were identified in most postganglionic neurons and in a subpopulation of small intensely fluorescent (SIF) cells. alpha 4-1 and bet…

Corrigendum to: α-Conotoxins EpI and AuIB switch subtype selectivity and activity in native versus recombinant nicotinic acetylcholine receptors (FEBS 27779)

Corrigendum to: K-Conotoxins EpI and AuIB switch subtype selectivity and activity in native versus recombinant nicotinic acetylcholine receptors (FEBS 27779) [FEBS Letters 554 (2003) 219^223]C Annette Nickea;1, Marek Samochockib, Marion L. Loughnana, Paramjit S. Bansala, Alfred Maelickeb, Richard J. Lewisa; aInstitute for Molecular Bioscience, University of Queensland, Brisbane, Qld. 4072, Australia bInstitute for Physiological Chemistry and Pathobiochemistry, University of Mainz, D-55099 Mainz, Germany First published online 24 December 2003

Nicotinic cholinoceptive neurons of the frontal cortex are reduced in Alzheimer's disease.

The cellular distribution of nicotinic acetylcholine receptors was studied in the frontal cortex (area 10) of 1) Alzheimer patients and compared to 2) age-matched and 3) middle-aged controls using the monoclonal antibody WF 6 and an immunoperoxidase protocol. Statistical analysis revealed significant differences between the number of labeled neurons among all three groups tested (middle-aged controls greater than aged controls greater than Alzheimer cases). No differences were seen for cresyl violet-stained samples. These findings underline that the nicotinic receptor decrease found with radioligand binding may reflect a postsynaptic in addition to a presynaptic component.

Galantamine is an allosterically potentiating ligand of the human α4/β2 nAChR

Galantamine (ReminyI ) is a novel drug treatment for mild to moderate Alzheimer's disease (AD). Originally established as a reversible inhibitor of the acetylcholine-degrading enzyme acetylcholinesterase (AChE), galantamine also acts as an allosterically potentiating ligand (APL) on nicotinic acetylcholine receptors (nAChR). Having previously established this second mode of action on nAChRs from murine brain, we demonstrate here the same action of galantamine on the most abundant nAChR in the human brain, the α4/β2 subtype. This nAChR-sensitizing action is not a common property of all, or most, AChE inhibitors, as is shown by the absence of this effect for other therapeutically applied AChE…

Cloning of the human NCNF gene.

We have cloned from a cDNA library of human testis tissue the human homologue to the mouse nuclear orphan receptor NCNF (neuronal cell nuclear factor). The open reading frame encodes a protein of 480 amino acids, the sequence of which (EMBL accession no. X99975) is 98.3% identical to the mouse homologue. Northern blot analysis of adult human tissues revealed a broad pattern of tissue expression. Similar to NCNF expression in mouse testis, two transcript forms of the single copy gene are expressed in human tissues. The two transcript forms which differ only in their 3'UTR, result in human from differential polyadenylation, in mouse from alternative splicing. Based on the high level of sequen…

Adsorption of functionalized biomembrane vesicles on crystalline surfaces monitored by FTIR- and Surface Enhanced FTIR Spectroscopy

A special class of integral membrane proteins are the ion channels. To use them for technical purposes they have to be transferred with their lipid surrounding onto artificial materials, i.e. crystals. In this study we used vesicles containing the nicotinic acetylcholine receptor (nAChR) and porin. The first allows the ligand gated flow and the second allows the voltage gated flow of cations across the membrane. We used ATR-FTIR spectroscopy and surface enhanced FTIR techniques (SEIRA) to monitor the direct contact of the vesicles with the crystal.

Nicotinic Receptors in Human Brain

A vast knowledge is currently available on the molecular biology and the pharmacology of nicotinic acetylcholine receptors (nAChR) in the mammalian central nervous system (CNS) (Sargent, 1993). Only few attempts have been made to approach the expression of nAChRs at the level of functional systems, considering the different cell types involved and their connectivity. This aspect is of particular importance in order to evaluate nAChR expression under pathological conditions. Histochemical techniques have proven to be useful since immunohistochemistry and in situ hybridization can be performed on human autopsy tissue and allow for a cell type-specific localization of nAChR proteins and nAChR …

Molecular Histochemistry of Nicotinic Receptors in Human Brain

Only a decade ago the existence and functional significance of central nervous nicotinic acetylcholine receptors (nAChR) was still a subject of controversy. Today, the importance of this receptor class for signal transduction in human brain in normal and pathological conditions has become quite evident. nAChRs have turned out to be important pharmacological targets in disorders like Alzheimer’s disease (AD) (Arneric et al., 1994). One prerequisite to understand nAChR function is a detailed study of the cellular distribution of nAChR subtypes. In recent years several human-specific data have been made available. This paper attempts to show actual developments in this field, summarizing the e…

Expression and renaturation of the N-terminal extracellular domain of torpedo nicotinic acetylcholine receptor alpha-subunit.

The N-terminal extracellular region (amino acids 1-209) of the alpha-subunit of the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue was expressed as inclusion bodies in Escherichia coli using the pET 3a vector. Employing a novel protocol of unfolding and refolding, in the absence of detergent, a water-soluble globular protein of 25 kDa was obtained displaying approximately 15% alpha-helical and 45% beta-structure. The fragment bound alpha-[3H]bungarotoxin in 1:1 stoichiometry with a KD value of 0.5 nM as determined from kinetic measurements (4 nM from equilibrium binding). The kinetics of association of toxin and fragment were of second order, with a similar …

Allosteric modulation of nicotinic acetylcholine receptors as a treatment strategy for Alzheimer's disease

The basic symptoms of Alzheimer's dementia, i.e., a loss in cognitive function, are due to impaired nicotinic cholinergic neurotransmission. To compensate for this impairment by drug treatment, blockers of the acetylcholine-degrading enzyme acetylcholinesterase are applied, even though this approach obviously is prone to many side-effects, including those of muscarinic nature. We have recently described a novel class of nicotinic acetylcholine receptor ligands which, similar to the action of benzodiazepines on GABA(A) receptors, allosterically potentiate submaximal nicotinic responses. The sensitizing effect is a consequence of facilitated channel opening in the presence of allosterically p…

Pharmacokinetic rationale for switching from donepezil to galantamine.

Galantamine, the most recently approved acetylcholinesterase inhibitor (AChEI) for use in the United States, has allosteric modulating activity at nicotinic receptors and inhibits acetylcholinesterase. This dual mechanism of action may make galantamine an attractive option for patients with Alzheimer's disease who have not benefited from their current therapy; thus, methods for switching patients from donepezil or rivastigmine to galantamine are needed. Protocols for switching patients from one AChEI to another must consider both the time required for washout of the first drug and the rate of dose escalation of the second drug. Both issues depend on the pharmacodynamics, pharmacokinetics, a…