The Potentiometric Biosensor Acetylcholine Esterase as a Model System

 In a methodical investigation of pH-detection systems (glass and polymer membrane electrodes and ion selective field effect transistor (ISFET)), the possible use of the acetylcholine esterase biosensor as a model for all pH-based biosensors is shown. It revealed that the biomembrane properties are more important for the result than the technology itself. All techniques resulted in a ΔH⁺ detection limit of 6*10⁻⁷ mol/L. Received January 20, 2001; accepted December 18, 2001; published online July 15, 2002     

Activation of nicotinic acetylcholine receptor prevents the production of reactive oxygen species in fibrillar beta amyloid peptide (1-42)-stimulated microglia

Recent studies have reported that the cholinergic anti-inflammatory pathway regulates peripheral inflammatory responses via alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) and that acetylcholine and nicotine regulate the expression of proinflammatory mediators such as TNF-alpha and prostaglandin E2 in microglial cultures. In a previous study we showed that ATP released by beta-amyloid-stimulated microglia induced reactive oxygen species (ROS) production, in a process involving the P2X(7) receptor (P2X(7)R), in an autocrine fashion. These observations led us to investigate whether stimulation by nicotine could regulate fibrillar beta amyloid peptide (1-42) (fAbeta1-42)-induced ROS production by modulating ATP efflux-mediated Ca(2+) influx through P2X(7)R. Nicotine inhibited ROS generation in fAbeta(1-42)-stimulated microglial cells, and this inhibition was blocked by mecamylamine, a non-selective nAChR antagonist, and a-bungarotoxin, a selective alpha7 nAChR antagonist. Nicotine inhibited NADPH oxidase activation and completely blocked Ca(2+) influx in fAbeta(1-42)-stimulated microglia. Moreover, ATP release from fAbeta(1-42)-stimulated microglia was significantly suppressed by nicotine treatment. In contrast, nicotine did not inhibit 2',3'-O-(4-benzoyl)-benzoyl ATP (BzATP)-induced Ca(2+) influx, but inhibited ROS generation in BzATP-stimulated microglia, indicating an inhibitory effect of nicotine on a signaling process downstream of P2X(7)R. Taken together, these results suggest that the inhibitory effect of nicotine on ROS production in fAbeta1-42-stimulated microglia is mediated by indirect blockage of ATP release and by directly altering the signaling process downstream from P2X(7)R.     

Alpha4/3 conotoxins: phylogenetic distribution, functional properties, and structure-function insights

This review examines the alpha4/3 conotoxins as an example of molecular diversity in a class of compounds that have evolved in a group of closely related species in a single phylogenetic lineage. The species examined belong to Stephanoconus, a clade of Conus, a genus that contains 500-700 different species of carnivorous marine snails. We examine earlier work that describes the identification and characterization of alpha-ImI, the founding alpha4/3 toxin, and two other alpha4/3 toxins, alpha-ImII and alpha-RgIA. These three toxins all inhibit nicotinic acetylcholine receptors (nAChRs) belonging to a subset of nAChRs that are composed of only alpha subunits; they are, however, diverse in terms of the all-alpha subtype they preferentially antagonize and the receptor site that they bind to. We thus speculate that the alpha4/3 toxins may be a rich source of functionally diverse all-alpha subunit nAChR inhibitors. We review extensive work that has established a detailed model for alpha-ImI binding to one of its preferred nAChR subtypes (the alpha7 nAChR) and, by comparing the alpha-ImI, alpha-ImII and alpha-RgIA sequences demonstrate how structural features of alpha4/3 peptides that account for their diverse functional properties can be identified. This approach is extended to derive models of receptor-toxin binding that may account for the different subtype specificities of alpha4/3 peptides. We also speculate on how rational modification of alpha4/3 toxins may allow engineering of ligands with desired subtype specificities. The chemical diversity produced by the closely related animals in Stephanoconus is thus functionally differentiated, although structurally homologous.     

Studies on sea snake venom

Erabutoxins a and b are neurotoxins isolated from venom of a sea snake Laticauda semifasciata (erabu-umihebi). Amino acid sequences of the toxins indicated that the toxins are members of a superfamily consisting of short and long neurotoxins and cytotoxins found in sea snakes and terrestrial snakes. The short neurotoxins to which erabutoxins belong act by blocking the nicotinic acetylcholine receptor on the post synaptic membrane in a manner similar to that of curare. X-ray crystallography and NMR analyses showed that the toxins have a three-finger structure, in which three fingers made of three loops emerging from a dense core make a gently concave surface of the protein. The sequence comparison and the location of essential residues on the protein suggested the mechanism of binding of the toxin to the acetylcholine receptor. Classification of snakes by means of sequence comparison and that based on different morphological features were inconsistent, which led the authors to propose a hypothesis "Evolution without divergence."     

Determination of Toxins Using Enzyme-Amplified Receptor Assay

Abstract

A new receptor based assay is described for the determination of toxins which have high affinities for the acetylcholine receptor. The method is based upon the hindrance of the normal binding of a synthetic enzyme-drug conjugate with a high affinity for the acetylcholine receptor protein by the presence of toxins acting as antagonists. The activity of the enzyme marker system, glucose-6-phosphate dehydrogenase covalently conjugated to desipramine, is monitored by colorimetric detection of the rate for NADH formation at 340 nm. The procedure proposed is designed to provide a simple toxin screen which can be done in a minimally equipped laboratory while achieving the required sensitivity. The technique is illustrated for snake venoms from Bungarus multicintus, Naja naja, and the alkaloid tubocurarine. Aspecific binding responses are shown to have minimal effect on the assay.     

Participation of the Cholinergic System in the Development of Polycystic Ovary Syndrome

In rats with polycystic ovary syndrome (PCOS) induced by injection of estradiol valerate (EV), unilateral or bilateral section of the vagus nerve restores ovulatory function in 75% of animals, suggesting that the vagus nerve participates in the development of PCOS. Since the vagus nerve is a mixed nerve through which mainly cholinergic-type information passes, the objective of the present study was to analyze whether acetylcholine (ACh) is involved in the development of PCOS. Ten-day-old rats were injected with 2.0 mg EV, and at 60 days of age, they were microinjected on the day of diestrus in the bursa of the left or right ovary with 100 or 700 mg/kg of ovarian weight atropine, a blocker of muscarinic receptors, and sacrificed for histopathological examination after the surgery. Animals with PCOS microinjected with 100 mg of atropine showed a lack of ovulation, lower serum concentrations of progesterone and testosterone, and cysts. Histology of the ovaries of animals microinjected with 700 mg of atropine showed corpus luteum and follicles at different stages of development, which was accompanied by a lower concentration of progesterone and testosterone. These results allow us to suggest that in animals with PCOS, ACh, which passes through parasympathetic innervation, is an important component in the persistence and development of the pathophysiology.     

Sensing Acetylcholine and Anticholinesterase Compounds

Acetylcholine is a key neurotransmitter, and anticholinesterase agents are essential compounds used as medical drugs, pesticides, and chemical warfare agents. A semisynthetic fluorescence‐based probe for the direct, real‐time detection of acetylcholine and anticholinesterase compounds is introduced. The probe possesses good sensitivity, tunable detection range, and can be selectively targeted to cell surfaces, thereby making it an attractive tool for applications in analytical chemistry and quantitative biology.     

Effects of Muscle Electrical Activity on the Transmission of Developing Neuromuscular Junction

Miniature endplate potentials (MEPPS) caused by the spontaneous release of ACh from the growth cone of cholinergic neurons, are recorded by the whole-cell patch-clamp technique on a large number of 1-day cultured myoballs which have contact neurites of co-cultured neurons. Both muscle cell and neuron are dissociated from the 1-day-old (about stage 20) Xenopus embryo. Frequency and/or amplitude of MEPPs can obviously increase after the repetitive high-level depolarization caused by the stimuli on muscle cells. No detectable changes of single ACh receptor channel property are observed by using the single-channel recording technique. These results suggest that the mechanism of the increase of MEPPs after electrical activity of postsynaptic muscle cells probably involve some alteration of presynaptic membrane.     

高效液相色谱-柱后固定化酶反应器-电化学检测法分析大鼠脑微透析液中的乙酰胆碱和胆碱

建立了用高效液相色谱分离－柱后固定化酶反应器酶解－电化学检测器检测酶解最终产物Ｈ２Ｏ２的方法，分析了麻醉和自由活动大鼠脑微透析液中乙酰胆碱（ＡＣｈ）和胆碱（Ｃｈ）的含量。至少在０．２～１００μｍｏｌ／Ｌ范围内ＡＣｈ和Ｃｈ的浓度与其响应的线性关系良好，它们的检测极限都可达５０ｆｍｏｌ。对高效液相色谱结合固定化酶反应器的分析方法作了简要的讨论。