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The molecular pathways that promote the proliferation and maintenance of pituitary somatotrophs and other cell types of the anterior pituitary gland are not well understood at present. However, such knowledge is likely to lead to the development of novel drugs useful for the treatment of various human growth disorders. Although muscarinic cholinergic pathways have been implicated in regulating somatotroph function, the physiological relevance of this effect and the localization and nature of the receptor subtypes involved in this activity remain unclear. We report the surprising observation that mutant mice that selectively lack the [M.sub.3] muscarinic acetylcholine receptor subtype in the brain (neurons and glial cells; Br-M3-KO mice) showed a dwarf phenotype associated with a pronounced hypoplasia of the anterior pituitary gland and a marked decrease in pituitary and serum growth hormone (GH) and prolactin. Remarkably, treatment of Br-M3-KO mice with CJC-1295, a synthetic GH-releasing hormone (GHRH) analog, rescued the growth deficit displayed by Br-M3-KO mice by restoring normal pituitary size and normal serum GH and IGF-1 levels. These findings, together with results from [M.sub.3] receptor/ GHRH colocalization studies and hypothalamic hormone measurements, support a model in which central (hypothalamic) [M.sub.3] receptors are required for the proper function of hypothalamic GHRH neurons. Our data reveal an unexpected and critical role for central [M.sub.3] receptors in regulating longitudinal growth by promoting the proliferation of pituitary somatotroph cells. anterior pituitary gland | dwarfism | knockout mice

The structural information of acetylcholine and its two agonists, nicotine and muscarine is obtained from the interpretation of infrared spectra recorded in the gas-phase or in low pH aqueous solutions. The shift of protonation site in nicotine ions is provided by the change of their infrared spectrum induced by hydration, whereas the structures of muscarine ions are only slightly influenced by the presence of water.

The key pathological features of Alzheimer's disease include synaptic dysfunction, profound changes in the cholinergic system, and deposition of [beta]-amyloid peptides generated by proteolytic processing of the amyloid-[beta] precursor protein (APP). However, the pathways linking APP with synaptic activity and cholinergic neuronal function are poorly understood. We report here that APP is essential in regulating the presynaptic expression and activity of the high-affinity choline transporter (CHT), a molecule that mediates the rate-limiting step of cholinergic synaptic transmission in both the neuromuscular junction and central cholinergic neurons. Loss of APP leads to aberrant localization of CHT at the neuromuscular synapses and reduced CHT activity at cholinergic projections. At the cellular level, we show that APP and CHT can be found in Rab5-positive endosomal compartments and that APP affects CHT endocytosis. Furthermore, we demonstrate that APP interacts with CHT through the C-terminal domain, providing support for a specific and direct regulation of CHT by APP through protein-protein interactions. These results identify a physiological activity of APP in cholinergic neurons, and our data indicate that deregulation of APP function may contribute to cholinergic impairment and AD pathogenesis. neuromuscular synapse | Alzheimer's disease | cholinergic neurons | knockout mice | endocytosis

A photoaffinity probe and radioligand prepared from the homopentameric mollusk nicotine acetylcholine (ACh) binding protein (AChBP) which was derivatized by the highly potent agonist azidoepibatidine (AzEPI) is described.

Alcohol dependence is a disease that impacts millions of individuals worldwide. There has been some progress with pharmacotherapy for alcohol-dependent individuals; however, there remains a critical need for the development of novel and additional therapeutic approaches. Alcohol and nicotine are commonly abused together, and there is evidence that neuronal nicotinic acetylcholine receptors (nAChRs) play a role in both alcohol and nicotine dependence. Varenicline, a partial agonist at the [alpha]4[beta]2 nAChRs, reduces nicotine intake and was recently approved as a smoking cessation aid. We have investigated the role of varenicline in the modulation of ethanol consumption and seeking using three different animal models of drinking. We show that acute administration of varenicline, in doses reported to reduce nicotine reward, selectively reduced ethanol but not sucrose seeking using an operant self-administration drinking paradigm and also decreased voluntary ethanol but not water consumption in animals chronically exposed to ethanol for 2 months before varenicline treatment. Furthermore, chronic varenicline administration decreased ethanol consumption, which did not result in a rebound increase in ethanol intake when the varenicline was no longer administered. The data suggest that the [alpha]4[beta]2 nAChRs may play a role in ethanol-seeking behaviors in animals chronically exposed to ethanol. The selectivity of varenicline in decreasing ethanol consumption combined with its reported safety profile and mild side effects in humans suggest that varenicline may prove to be a treatment for alcohol dependence. addiction | treatment | alcohol | nicotine | dependence

A novel alpha-conotoxin, alpha-conotoxin RgIA (alpha-RgIA), is a specific blocker of the alpha9alpha10 nAChR subtype, which is formed by the assembly of the alpha9 and alpha10 nicotinic acetylcholine receptor (nAChR) subunits. Sequence and subtype specificity studies indicate that the amino acids in the C-terminal half of alpha-RgIA are responsible for its preferential inhibition of the alpha9alpha10 nAChR.

The flexibility of alpha-neurotoxin bound to the acetylcholine-binding protein (AChBP) is examined to obtain a better understanding of alpha-neurotoxin-nAChR interaction. Results of the examination indicate that bound alpha-neurotoxin is not rigidly oriented on the surface of AChBO but rather exhibits segmental motion by virtue of flexibility in its fingerlike structure.

The roles of 7 conserved ionic residues in the 12 putative transmembrane domains (TMDs) of vesicular acetylcholine transporter (VAChT) were investigated, and transport and ligand binding were characterized at different pH values using filter assays. The pH effects indicate that sites that must be unprotonated for transport and vesamicol binding remain unidentified.

The first direct experimental evaluation of the hydrogen-bond affinity of the nicotinium pyridine nitrogen is described. The equilibrium constant of its association with a phenol is 1 order of magnitude greater than the association of the acetylcholine carbonyl oxygen.

The binding of three distinct agonists- acetylcholine (ACh), nicotine, and epibatidine- to the nicotinic acetylcholine receptor is probed using unnatural amino acid mutagenesis. ACh makes a cation-Pi interaction with Trp alpha149, while nicotine employs a hydrogen bond to a backbone carbonyl in the same region of the agonist binding site.

The conus peptides alpha-conotoxin ImI (alpha-ImI) and ImII (alpha-ImII) differ by only three of 11 residues in their primary sequences and yet are shown to inhibit the human alpha7 nicotinic acetylcholine receptor (nAChR) by targeting different sites. Mutations at both faces of the classical ligand binding site of the alpha7 nAChR strongly affect antagonism by alpha-ImI but not alpha-ImII.

The redesigning of the structure of a cyclophanic tetraester that was used to study the cation - Pi interaction in order to improve its binding ability is presented. Calculations suggested that the cyclophanic tetraether formed by the removal of the four carbonyl groups accommodated tetramethylammonium better.

The neurotransmitter acetylcholine along with nicotine the drug that mimics it interacts with a wide range of biological binding sites like acetylcholine esterases and nicotine acetylcholine receptors. Gas-phase bascities of nomicitine, nicotine as well as model pyrrolidines are measured and compared with other studies conducted using different methods.

A complete conformational analysis of the model peptide Ac-ox-[Cys-Cys]-NH2 in water and limited studies of tetra peptides and undecapeptide which are fragments of the alpha7 nAChR has been presented by researchers. The study was carried out by NMR in water computationally by Monte Carlo simulations using OPLS-AA force field and GB/SA water model.

The acetylcholine-binding protein (AChBP) from Lymnaea stagnalis as a model protein and acetylcholine, nicotine, epibatidine and a series of substituted quinuclidines as ligands were used to study the mechanism of agonist interactions with Cys-loop ligand-gated ion channels. The developed procedures would facilitate further exploration of the mechanism of Cys-loop receptor function and the identification of specific ligands suitable for pharmacological application.

A study was conducted to identify the dynamic intramolecular interaction between the G[[beta].sub.5] and DEP domains in the G protein signaling (RGS) required for stabilizing and binding of DEP-G[[beta].sub.5] and their effects of on the interaction of G[[beta].sub.5]-RGS7 with the M3R. The data was found to be consistent with the model interaction of the M3R with G[[beta].sub.5]- RGS7 which caused the DEP domain and G[[beta].sub.5] to dissociate from each other and bind to the C-tail, and the binding of DEP domain to the third loop, which inhibited M3R-mediated signaling.

A study was conducted to identify a homologue of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella teleta, from the annelid phylum. The finding for a functional AChBP in a marine annelid demonstrated that AChBPs might exhibit variations in hallmark motifs such as ligand-binding residues and Cys loop length suggested conclusively that AChBP neurotransmitter binding protein is not limited to the phylum Mollusca.

The binding of (-)-vesamicol from pH 5 to pH 10 is characterized by using filter assays, (-)-[[super 3]H]vesamicol, and human vesicular acetylcholine transporter (VAChT) expressed in [PC12.sup.123.7] cells. The contrasting phasicity has shown that the pathway to and from the [[super 3]H]vesamicol binding site is present in open and at least partially closed states.

Synthesis and binding properties of a new hydrosoluble triphenylene are described. Selective recognition of acetylcholine (ACh) against other aliphatic ammoniums is achieved by this flat receptor and ionic pairing and hydrophobic effects between host and ACh are analyzed by infrared spectroscopy.

The crystal structures of the receptor binding domains (HCR) of BoNT/A and BoNT/F and the characterization of the dual receptors for BoNT/F is reported. The neuronal discrimination of BoNT/F which involves the recognition of ganglioside and protein (glycosylated SV2) carbohydrate moieties provide a structural basis for the high affinity and specificity of BoNT/F for neurons.

The ability of the G[[beta].sub.5]-RGS7 complex (RGS = Regulators of G protein signaling) to selectively inhibit [Ca.sup.2+] mobilization by muscarinic acetylcholine receptor type 3 (M3R), but not by other Gq-coupled receptors such as M1, M5, histamine H1, and GNRH receptors is demonstrated. The study identified a novel molecular mechanism that could impart receptor subtype selectivity on signal transduction via Gq-coupled muscarinic receptors.

The application of unnatural amino acid mutagenesis for physical organic chemistry studies related to complex proteins of neuroscience is reported. The results from the studies provide insight into the significant role of nAChR, [alpha]Va146 in receptor gating by pin-into-socket mechanism.

The muscle-type acetylcholine receptor (AChR) was used as a test system to determine if the blockade kinetics could be modulated by specifying the length of a poly(ethylene glycol) (PEG) spacer incorporated into the blocker. The results obtained offer useful insights into the future design of kinetically tunable open-channel blockers for the AChR.

Computational advances together with the availability of user-friendly software have promoted the development of simple computer-assisted molecular-modeling experiments for bioactive molecules. The students draw biologically-active molecules, analyze their conformations, compare the conformations with different molecules, match the preferred conformations of the Ach with the proposed structure of the actylcholine receptor and compare the results with literature data. .

Molecular dynamics are used to determine the unbinding of acetylcholine from the ligand-binding domain of human alpha7 nicotinic acetylcholine receptor along four different predetermined pathways. The binding energy obtained are in good agreement with that derived from the experimental binding constant for acetylcholine binding protein, but significantly higher than that for the complete human alpha7 nicotinic acetylcholine receptor.

A report on the measurements of secondary beta-deuterium kinetic isotope effects with istopomers of acetylthiocholine is presented. Secondary deuterium isotope effects are inverse when an sp(super 2) hybridized reactant is converted to a quasi-tetrahedral transition state and normal when an sp(super 3) reactant state is converted to a transition state that has greater trigonal planar character.

The purification and characterization of a novel peptide from the venom of Conus radiatus with the same Cys pattern as sigma-conotoxin GVIIIA (sigma-GVIIIA) which diverges in its primary amino acid sequence, post-translational modification, and molecular targets are described. This peptide defines a new family of conotoxins within the S superfamily namely the alphaS family of conotoxins.

The dynamic structural behavior of the whole transmembrane (TM) domain of the nicotine acetylcholine receptor (AChR) and a possible gating mechanism of the channel are studied on the basis of a 35-ns MD simulation. The availability of isolated AChRTM peptides and larger proteolytic fragments containing the AChRTM region together with site-directed fluorescence labeling techniques might provide the time and spatial resolution required to explore the conformational transitions predicted in the MD simulations.

The acetylcholine (ACh) binding site in the vesicular acetycholine transporter (VAChT) is analyzed. The results show that analysis of microscopic kinetics is required for the correct interpretation of mutational effects in VAChT, which are discussed in terms of the three-dimensional structures for other transporters in the major facilitator superfamily.

A practical preparation of the acetylcholine release-enhancing agent 1 is described. The mesylate used for the bisalkylation of anthrone under carefully defined conditions to prepare the cognition enhancer drug candidate 1 has resulted in much-improved yields and permits progression to commercial manufacture.