Super agonist actions of clothianidin and related compounds on the SAD beta 2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes

To compare the actions of clothianidin, a neonicotinoid acting on insect nicotinic acetylcholine receptors, and related compounds with that of imidacloprid, the compounds were tested on the Drosophila SAD-chicken beta2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. The maximum response of the SAD beta 2 nicotinic receptor to clothianidin was larger than that observed for acetylcholine. Ring breakage of the imidazolidine ring of imidacloprid resulting in the generation of a guanidine group was critical for this super agonist action.     

Properties of Glycomacropeptide and Para-K-casein Derived from Human K-Casein and Comparison of Human and Bovine K-Caseins as to Susceptibility to Chymosin and Pepsin

The nutritional efficiency of quinolinic acid as a substitute for nicotinic acid was investigated using weanling rats Of the Sprague Dawley strain (3-weeks old) fed a nicotinic acid-free, tryptophan-limited diet containing various amounts of nicotinic acid or quinolinic acid. Judging from the growth response, food efficiency ratio, levels of NAD activity in the blood, liver, brain and upper small intestine, and urinary excretion of niacin we have concluded that exogenous quinolinic acid is approximately 1/9 as active as nicotinic acid. As many foods contain quinolinic acid, dietary quinolinic acid cannot be ignored from the standpoint of tryptophan and nicotinic acid replacement.     

Molecular studies of the neuronal nicotinic acetylcholine receptor family

Nicotinic acetylcholine receptors on neurons are part of a gene family that includes nicotinic acetylcholine receptors on skeletal muscles and neuronal alpha bungarotoxin-binding proteins that in many species, unlike receptors, do not have an acetylcholine-regulated cation channel. This gene superfamily of ligand-gated receptors also includes receptors for glycine and gamma-aminobutyric acid. Rapid progress on neuronal nicotinic receptors has recently been possible using monoclonal antibodies as probes for receptor proteins and cDNAs as probes for receptor genes. These studies are the primary focus of this review, although other aspects of these receptors are also considered. In birds and mammals, there are subtypes of neuronal nicotinic receptors. All of these receptors differ from nicotinic receptors of muscle pharmacologically (none bind alpha bungarotoxin, and some have very high affinity for nicotine), structurally (having only two types of subunits rather than four), and, in some cases, in functional role (some are located presynaptically). However, there are amino acid sequence homologies between the subunits of these receptors that suggest the location of important functional domains. Sequence homologies also suggest that the subunits of the proteins of this family all evolved from a common ancestral protein subunit. The ligand-gated ion channel characteristic of this superfamily is formed from multiple copies of homologous subunits. Conserved domains responsible for strong stereospecific association of the subunits are probably a fundamental organizing principle of the superfamily. Whereas the structure of muscle-type nicotinic receptors appears to have been established by the time of elasmobranchs and has evolved quite conservatively since then, the evolution of neuronal-type nicotinic receptors appears to be in more rapid flux. Certainly, the studies of these receptors are in rapid flux, with the availability of monoclonal antibody probes for localizing, purifying, and characterizing the proteins, and cDNA probes for determining sequences, localizing mRNAs, expressing functional receptors, and studying genetic regulation. The role of nicotinic receptors in neuromuscular transmission is well understood, but the role of nicotinic receptors in brain function is not. The current deluge of data using antibodies and cDNAs is beginning to come together nicely to describe the structure of these receptors. Soon, these techniques may combine with others to better reveal the functional roles of neuronal nicotinic receptors.     

A model for the assembly of nicotinic receptors based on subunit-subunit interactions

Neuronal ion-channels are complex multimeric proteins. Within a given family, the variability of their pharmacological responses depends on subunit composition and subunit arrangement. We report here that protein assembly in the pentameric nicotinic acetylcholine receptor family, the best characterized of all neuronal receptors, can be predicted using information derived from homology modeled surface to surface subunit interactions based on the atomic structure of a snail acetylcholine-binding protein. An empirical assembly model is able to establish both subunit stoichiometry and subunit arrangement of known neuronal and muscle nicotinic receptors. This contribution to the understanding of nicotinic receptor assembly and variability might be extended to other types of ion-channels.     

大鼠颈上神经节烟碱电流的整流及失敏

在培养的新生大鼠颈上神经节交感神经元标本上,用全细胞膜片钳技术研究了烟碱型乙酰胆碱受体（ｎＡＣｈＲ）通道的整流及失敏现象。烟碱激动剂引起的全细胞电流在膜电位为负值时随膜电位呈线性改变,而在膜电位达＋６０ｍＶ时仍不出现外向电流,表现出强烈的内向整流。ｎＡＣｈＲ通道电流存在失敏现象,即持续恒压喷射激动剂所引起的全细胞电流随时间呈指数衰减,不能保持在峰值水平,失敏随激剂浓度呈量效关系,膜电位的超极化也加     

恶臭假单胞菌NA-1菌体培养转化和静息细胞转化联合工艺生产6-羟基烟酸研究

现有微生物羟基化烟酸采用的是静息细胞转化工艺。但研究揭示,恶臭假单胞菌NA-1(Pseudomonas putidaNA-1)在培养过程中不降解发酵液中由诱导剂烟酸转化形成的6-羟基烟酸,这是由于烟酸的存在抑制了羟基烟酸降解酶的作用,而不是因为细胞停止生长不利用羟基烟酸的缘故。因而尝试利用菌体诱导培养过程进行烟酸转化生产,建立了一种新的生产工艺,即菌体培养转化和静息细胞转化联合工艺。该工艺在恶臭假单胞菌NA-1培养过程中持续补充烟酸以维持1%(W/V)浓度,使烟酸被生长细胞转化为羟基化烟酸并在发酵液中线性积累,而不被进一步降解;培养转化结束后,发酵液中的静息细胞依然拥有很高的羟基化酶活力,能够再次用于转化反应。该联合转化工艺与传统的静息细胞转化工艺相比,不仅节约了诱导剂烟酸,而且6-羟基烟酸的产量提高了65%。     

Kinetic analysis of the transglycosidation reaction catalyzed by rabbit spleen pyridine nucleotide glycohydrolase

Properties of the transglycosidation reaction catalyzed by rabbit spleen pyridine nucleotide glycohydrolase were characterized using a modified cyanide addition method by which initial velocities of the transglycosidation (vT) and hydrolysis (vH) of pyridine nucleotides could be monitored simultaneously. (1) The vT was routinely determined with NMN and nicotinic acid used as substrates and was observed to be maximal at pH 6. Arrhenius plots of vT and vH indicated that the activation energies for transglycosidation and hydrolysis were 8.7 and 10.7 kcal/mol, respectively. (2) The enzyme showed a broad spectrum of substrate specificity with respect to both pyridine nucleotides and bases. Of the compounds tested, NMN and nicotinic acid were shown to be the best substrates when compared on the basis of Vmax/Km values. Kinetic constants for the enzyme-catalyzed transglycosidation reaction were as follows; Km(NMN) = 0.53 mM, Km(nicotinic acid), as acid form = 15 mM, apparent Vmax = 7.8 mumol/min/mg protein, in the presence of 0.2 M nicotinic acid. (3) The ratio of vT/vH was shown to be dependent on both pH and nicotinic acid concentration. However, transglycosidation versus hydrolysis partition at a fixed pH was constant regardless of the nicotinic acid concentration employed and approximated to be 1.2 x 10(4) at the maximal pH. (4) Nicotinamide, one of the most potent inhibitors for the enzyme-catalyzed hydrolysis, was shown to function as an antagonist for the transglycosidation reaction with NMN and nicotinic acid used as substrates. The inhibition mechanism with nicotinamide was purely noncompetitive with respect to nicotinic acid; on the other hand, the double reciprocal plot of the transglycosidation velocity against NMN concentration at a fixed concentration of nicotinamide was concave downwards. (5) The equilibrium constant of the reaction, NMN + 3-acetylpyridine----3-acetylpyridine mononucleotide + nicotinamide, was 0.61, whereas the conversion of NMN with nicotinic acid to nicotinic acid mononucleotide was essentially irreversible. These enzymatic properties of rabbit spleen pyridine nucleotide glycohydrolase suggested that the enzyme should not function as a glycohydrolase but as a transglycosidase and could serve in an important mechanism for an alternative biosynthetic pathway of nicotinic acid mononucleotide, one of the precursors for NAD synthesis, when nicotinic acid is supplied.     

Bispyridinium Compounds Inhibit Both Muscle and Neuronal Nicotinic Acetylcholine Receptors in Human Cell Lines

Standard treatment of poisoning by organophosphorus anticholinesterases uses atropine to reduce the muscarinic effects of acetylcholine accumulation and oximes to reactivate acetylcholinesterase (the effectiveness of which depends on the specific anticholinesterase), but does not directly address the nicotinic effects of poisoning. Bispyridinium molecules which act as noncompetitive antagonists at nicotinic acetylcholine receptors have been identified as promising compounds and one has been shown to improve survival following organophosphorus poisoning in guinea-pigs. Here, we have investigated the structural requirements for antagonism and compared inhibitory potency of these compounds at muscle and neuronal nicotinic receptors and acetylcholinesterase. A series of compounds was synthesised, in which the length of the polymethylene linker between the two pyridinium moieties was increased sequentially from one to ten carbon atoms. Their effects on nicotinic receptor-mediated calcium responses were tested in muscle-derived (CN21) and neuronal (SH-SY5Y) cells. Their ability to inhibit acetylcholinesterase activity was tested using human erythrocyte ghosts. In both cell lines, the nicotinic response was inhibited in a dose-dependent manner and the inhibitory potency of the compounds increased with greater linker length between the two pyridinium moieties, as did their inhibitory potency for human acetylcholinesterase activity in vitro. These results demonstrate that bispyridinium compounds inhibit both neuronal and muscle nicotinic receptors and that their potency depends on the length of the hydrocarbon chain linking the two pyridinium moieties. Knowledge of structure-activity relationships will aid the optimisation of molecular structures for therapeutic use against the nicotinic effects of organophosphorus poisoning.     

Nicotinic alpha5 subunit deletion locally reduces high-affinity agonist activation without altering nicotinic receptor numbers

Neuronal nicotinic acetylcholine receptor subunit alpha5 mRNA is widely expressed in the CNS. An alpha5 gene polymorphism has been implicated in behavioral differences between mouse strains, and alpha5-null mutation induces profound changes in mouse acute responses to nicotine. In this study, we have examined the distribution and prevalence of alpha5* nicotinic acetylcholine receptor in mouse brain, and quantified the effects of alpha5-null mutation on pre-synaptic nicotinic acetylcholine receptor function (measured using synaptosomal (86)Rb(+) efflux) and overall [(125)I]epibatidine binding site expression. alpha5* nicotinic acetylcholine receptor expression was found in nine of fifteen regions examined, although < 20% of the total nicotinic acetylcholine receptor population in any region contained alpha5. Deletion of the alpha5 subunit gene resulted in localized loss of function (thalamus, striatum), which was itself confined to the DHbetaE-sensitive receptor population. No changes in receptor expression were seen. Consequently, functional changes must occur as a result of altered function per unit of receptor. The selective depletion of high agonist activation affinity sites results in overall nicotinic function being reduced, and increases the overall agonist activation affinity. Together, these results describe the receptor-level changes underlying altered behavioral responses to nicotine in nicotinic acetylcholine receptor alpha5 subunit-null mutants.     

Effect of nicotinic acid on zinc and iron metabolism

Nicotinic acid has functional groups capable of forming complexes with trace metals. The present study examines the effect of nicotinic acid supplementation on absorption and utilization of zinc and iron. In vitro zinc uptake by human erythrocytes was studied using blood samples of 10 healthy subjects. It was found that 8 moles nicotinic acid or NADP increased ⁶⁵Zn uptake by 38.9% and 43.1% in fasting, and by 70.9% and 28.1% in postprandial conditions. In animal experiments, nicotinic acid supplementation to finger millet based diet resulted in significant enhancement of percent zinc absorption, liver zinc and growth of weanling mice (P < 0.05). When mice were fed with nicotinic acid-deficient, -adequate and -excess synthetic diets for four weeks it was observed that, in comparison with the nicotinic acid-deficient diet, percent zinc absorption, intestinal zinc, percent haeomoglobin and liver iron increased significantly under nicotinic acid-adequate and -excess conditions. The results obtained suggested that nicotinic acid, in addition to its known effect on growth and metabolism, may be playing an important role in enhancing zinc and iron utilization.     

Intracellular recording in avian brain of a nicotinic response that is insensitive to K-bungarotoxin

We examined nicotinic acetylcholine receptors in the avian brain using a combination of autoradiographic and intracellular electrophysiological techniques. We found that the lateral spiriform nucleus (SPL) in the mesencephalon has a very high density of 3H-nicotine binding sites but no detectable 125I-K-bungarotoxin (125I-K-BuTx) or 125I-alpha-bungarotoxin (125I-alpha-BuTx) bindings sites. Intracellular recordings in brain slices revealed that SPL neurons depolarize in response to nicotine and carbachol (in the presence of atropine). These depolarizations were blocked by the classic nicotinic antagonists d-tubocurarine and dihydro-beta-erythroidine. As predicted for nicotinic receptors with a high affinity for nicotine, neither K-BuTx nor alpha-BuTx blocked these nicotinic responses. Thus, although the existence of high-affinity 3H-nicotine binding sites has been known for some time, we now report the in situ detection of a functional nicotinic receptor that has a high affinity for nicotine and is K-BuTx-insensitive.     

Nitrilase-Catalyzed Production of Nicotinic Acid from 3-Cyanopyridine in Rhodococcus rhodochrous J1

The nitrilase which occurs abundantly in cells of Rhodococcus rhodochrous J1 catalyzes the direct hydrolysis of 3-cyanopyridine to nicotinic acid without forming nicotinamide. By using resting cells, the reaction conditions for nicotinic acid production were optimized. Under the optimum conditions, 100% of the added 3-cyanopyridine could be converted to nicotinic acid, the highest yield achieved being 172 mg of nicotinic acid per 1.0 ml of reaction mixture containing 2.89 mg (dry weight) of cells in 26 h.     

Timing and location of nicotinic activity enhances or depresses hippocampal synaptic plasticity

This study reveals mechanisms in the mouse hippocampus that may underlie nicotinic influences on attention, memory, and cognition. Induction of synaptic plasticity, arising via generally accepted mechanisms, is modulated by nicotinic acetylcholine receptors. Properly timed nicotinic activity at pyramidal neurons boosted the induction of long-term potentiation via presynaptic and postsynaptic pathways. On the other hand, nicotinic activity on interneurons inhibited nearby pyramidal neurons and thereby prevented or diminished the induction of synaptic potentiation. The synaptic modulation was dependent on the location and timing of the nicotinic activity. Loss of these synaptic mechanisms may contribute to the cognitive deficits experienced during Alzheimer's diseases, which is associated with a loss of cholinergic projections and with a decrease in the number of nicotinic receptors.     

Myometrial maxi-K channel beta1 subunit modulation during pregnancy and after 17beta-estradiol stimulation

In Saccharomyces cerevisiae the nicotinic acid moiety of NAD+ can be synthesized from tryptophan using the kynurenine pathway or incorporated directly using nicotinate phosphoribosyl transferase (NPT1). We have identified the genes that encode the enzymes of the kynurenine pathway and for BNA5 (YLR231c) and BNA6 (YFR047c) confirmed that they encode kynureninase and quinolinate phosphoribosyl transferase respectively. We show that deletion of genes encoding kynurenine pathway enzymes are co-lethal with the Deltanpt1, demonstrating that no other pathway for the synthesis of nicotinic acid exists in S. cerevisiae. Also, we show that under anaerobic conditions S. cerevisiae is a nicotinic acid auxotroph.     

Interaction of the catecholamine release-inhibitory peptide catestatin (human chromogranin A(352-372)) with the chromaffin cell surface and Torpedo electroplax: implications for nicotinic cholinergic antagonism

The catecholamine release-inhibitory chromogranin A fragment catestatin (chromogranin A(344-364)) exhibits non-competitive antagonism of nicotinic cholinergic signaling in chromaffin cells. A previous homology model of catestatin's likely structure suggested a mode of interaction of the peptide with the nicotinic receptor, but direct evidence has been lacking. Here we found that [125I]-catestatin binds to the surface of intact PC12 and bovine chromaffin cells with high affinity (K(D)=15.2+/-1.53 nM) and specificity (lack of displacement by another [N-terminal] fragment of chromogranin A). Nicotinic agonist (carbamylcholine) did not displace [125I]-catestatin from chromaffin cells, nor did catestatin displace the nicotinic agonist [3H]-epibatidine; these observations indicate a catestatin binding site separate from the agonist binding pocket on the nicotinic receptor, a finding consistent with catestatin's non-competitive nicotinic mechanism. [125I]-catestatin could be displaced from chromaffin cells by substance P (IC(50) approximately 5 microM), though at far lower potency than displacement by catestatin itself (IC(50) approximately 350-380 nM), suggesting that catestatin and substance P occupy an identical or overlapping non-competitive site on the nicotinic receptor, at different affinities (catestatin > substance P). Small, non-peptide non-competitive nicotinic antagonists (hexamethonium or clonidine) did not diminish [125I]-catestatin binding, suggesting distinct non-competitive binding sites on the nicotinic receptor for peptide and non-peptide antagonists. Similar binding and inhibitory profiles for [125I]-catestatin were observed on chromaffin cells as well as nicotinic receptor-enriched Torpedo membranes. Covalent cross-linking of [125I]-catestatin to Torpedo membranes suggested specific contacts of [125I]-catestatin with the delta, gamma, and beta subunits of the nicotinic receptor, a finding consistent with prior homology modeling of the interaction of catestatin with the extracellular face of the nicotinic heteropentamer. We conclude that catestatin occludes the nicotinic cation pore by interacting with multiple nicotinic subunits at the pore vestibule. Such binding provides a physical explanation for non-competitive antagonism of the peptide at the nicotinic receptor.     

Effect of chromium nicotinic acid supplementation on selected cardiovascular disease risk factors

The effects of daily supplemental chromium (200 μg) complexed with 1.8 mg nicotinic acid on plasma glucose and lipids, including total cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides, were assessed in 14 healthy adults and 5 adults with noninsulin-dependent diabetes mellitus (NIDDM) using a double-blind crossover study with 8-wk experimental periods. Eight of the 14 healthy subjects and all 5 subjects with NIDDM also underwent an oral glucose tolerance test with assessment of 90 min postprandial plasma glucose and insulin concentrations. No statistically significant effects of chromium nicotinic acid supplementation were found on plasma insulin, glucose, or lipid concentrations, although chromium nicotinic acid supplementation slightly lowered fasting plasma total and LDL cholesterol, triglycerides, and glucose concentrations, and 90-min postprandial glucose concentrations in individuals with NIDDM.     

Autoradiographic distribution of high affinity muscarinic and nicotinic cholinergic receptors labeled with [3H]acetylcholine in rat brain

The relative distribution of muscarinic and nicotinic cholinergic receptors labeled with [3H]acetylcholine was determined using autoradiography. [3H]Acetylcholine binding to high affinity muscarinic receptors was similar to what has been described for an M-2 distribution: highest levels of binding occurred in the pontine and brainstem nuclei, anterior pretectal area and anteroventral thalamic nucleus, while lower levels occurred in the caudate-putamen, accumbens nucleus and primary olfactory cortex. Nicotinic receptors were labeled with [3H]acetylcholine to the greatest extent in the interpeduncular nucleus, several thalamic nuclei, medial habenula, presubiculum and superior colliculus, and to the least extent in the hippocampus and inferior colliculus. By using autoradiography to localize cholinergic binding sites throughout the brain it was observed that the distributions of high affinity muscarinic and nicotinic sites labeled with the endogenous ligand, [3H]acetylcholine are different from each other and are different from distributions of muscarinic and nicotinic sites labeled with muscarinic and nicotinic antagonists.     

Ca(2+) changes induced by different presynaptic nicotinic receptors in separate populations of individual striatal nerve terminals

Presynaptic nicotinic acetylcholine receptors likely play a modulatory role in the nerve terminal. Using laser-scanning confocal microscopy, we have characterized physiological responses obtained on activation of presynaptic nicotinic receptors by measuring calcium changes in individual nerve terminals (synaptosomes) isolated from the rat corpus striatum. Nicotine (500 nM) induced Ca(2+) changes in a subset (10-25%) of synaptosomes. The Ca(2+) responses were dependent on extracellular Ca(2+) and desensitized very slowly (several minutes) on prolonged exposure to agonist. The nicotine-induced Ca(2+) responses were dose-dependent and were completely blocked by dihydro-beta-erythroidine (5 microM), differentially affected by mecamylamine (10 microM) and alpha-conotoxin MII (100 nM), and not affected by alpha-bungarotoxin (500 nM). Immunocytochemical studies using well-characterized monoclonal antibodies revealed the presence of the alpha4 and alpha3/alpha5 nicotinic subunits. The nicotine-induced responses were unaffected by prior depolarization or by a mixture of Ca(2+) channel toxins including omega-conotoxin MVIIC (500 nM), omega-conotoxin GVIA (500 nM) and agatoxin TK (200 nM). Our results indicate that nicotinic receptors present on striatal nerve terminals induce Ca(2+) entry largely without involving voltage-gated Ca(2+) channels, most likely by direct permeation via the receptor channel itself. In addition, at least two subpopulations of presynaptic nicotinic receptors reside on separate terminals in the striatum, suggesting distinct modulatory roles.     

Limitations of RNAi of α6 nicotinic acetylcholine receptor subunits for assessing the in vivo sensitivity to spinosad

Abstract Spinosad is a widely used insecticide that exerts its toxic effect primarily through interactions with the nicotinic acetylcholine receptor. The α6 nicotinic acetylcholine receptor subunit is involved in spinosad toxicity as demonstrated by the high levels of resistance observed in strains lacking α6. RNAi was performed against the Dα6 nicotinic acetylcholine receptor subunit in Drosophila melanogaster using the Gal4‐UAS system to examine if RNAi would yield results similar to those of Dα6 null mutants. These Dα6‐deficient flies were subject to spinosad contact bioassays to evaluate the role of the Dα6 nicotinic acetylcholine receptor subunit on spinosad sensitivity. The expression of Dα6 was reduced 60%–75% as verified by quantitative polymerase chain reaction. However, there was no change in spinosad sensitivity in D. melanogaster. We repeated RNAi experiments in Tribolium castaneum using injection of dsRNA for Tcasα6. RNAi of Tcasα6 did not result in changes in spinosad sensitivity, similar to results obtained with D. melanogaster. The lack of change in spinosad sensitivity in both D. melanogaster and T. castaneum using two routes of dsRNA administration shows that RNAi may not provide adequate conditions to study the role of nicotinic acetylcholine receptor subunits on insecticide sensitivity due to the inability to completely eliminate expression of the α6 subunit in both species. Potential causes for the lack of change in spinosad sensitivity are discussed.