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H Breer

Publications and source records attributed to H Breer.

At least 145 records · Page 8Linked to original sources
PubMed1989

Reconstitution of acetylcholine receptors into planar lipid bilayers.

Obviously, bilayer reconstitution experiments have largely contributed to the understanding of the AChR-channel function. Nevertheless, at present there are many unanswered questions concerning the minimum structural requirements for AChR-channel function, agonist cooperativity, and different types of AChR. Another complex of parameters important for receptor function which must be explored in much more detail, is the dependence of AChR-channel function on membrane composition and its physical state. This important but rather neglected field is predestined to be explored by reconstitution techniques. All the results on AChRs reconstituted in planar lipid bilayers cannot adequately be discussed without the data obtained by other techniques, thus coming back to the statements already mentioned in the introductory section about strategies for investigating ion channels in general. Only such a strategy can lead to a molecular understanding of channel function.

Lipid Bilayers↗
PubMed1989

Immunocytochemical localization of glutamate decarboxylase in the mesothoracic ganglion of Locusta migratoria.

Glutamate decarboxylase immunoreactivity has been located in the thoracic ganglia of the locust, Locusta migratoria, using an antiserum raised from rat brain. At the light microscopic level clusters of nerve cell somata as well as nerve fibres were positively labelled by the antiserum. Electron microscopy showed that glutamic acid decarboxylase was localized in numerous synaptic terminals.

Animals↗
PubMed1989

Comparison of acetylcholine and alpha-bungarotoxin binding sites in insects and vertebrates.

1. The nervous tissue of locusts contains high affinity as well as low affinity binding sites for acetylcholine which display a similar nicotinic pharmacology. 2. Hill plot analysis indicated a non-cooperative binding of acetylcholine. 3. In membrane preparations from locust ganglia and mouse brain the number of binding sites for ACh was about ten fold lower than for BGTX, whereas in membranes from electric tissue both sites occurred in similar concentrations. 4. Drug binding studies suggest that the high affinity binding sites for ACh and BGTX in preparations from insect and mouse are different; whereas in electric tissue both sites are very similar. 5. Precipitation experiments using immobilized BGTX and specific antibodies indicated that in insect nervous tissue as in electric tissue the ACh and BGTX binding sites are located on the same receptor molecule and occupy distinct partially overlapping binding sites, whereas in the vertebrate brain both sites are located on distinct binding proteins.

Animals↗
PubMed1989

Muscarinic receptors modulating acetylcholine release from insect synaptosomes.

1. Cholinergic synapses in the central nervous system of insects contain inhibitory muscarinic receptors whose stimulation by agonists leads to a diminished output of acetylcholine; antagonists, like atropine, facilitate acetylcholine release. 2. The receptors involved appear to be of the M2-subtype. Upon activation of presynaptic receptors a significant reduction of the intrasynaptosomal cyclic AMP level as well as a significantly increased membrane potential was observed. 3. The observed membrane hyperpolarization is apparently not a consequence of a lower cyclic AMP level, thus both effects may offer alternative or synergistical mechanisms for modulating transmitter release.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Expression of neuronal acetylcholine receptor polypeptides in vitro.

1. Translation of poly(A) RNA extracted from the nervous tissue of locusts in a reticulocyte lysate system led to polypeptides with a broad spectrum of molecular weights. 2. Using anti-locust acetylcholine receptor (AChR) antisera, polypeptides with a molecular weight of about 50,000 were immunoprecipitated. These peptides comprised about 0.3% of the total translation products. 3. Cotranslational incubation with pancreatic rough microsomes resulted in a glycosylated 60,000-dalton immunoprecipitate. 4. Density-gradient analysis of in vitro synthesized and glycosylated receptor polypeptides indicated that no assembly of subunits had taken the place under the in vitro conditions.

Animals↗
PubMed1987

Characterization of the channel properties of a neuronal acetylcholine receptor reconstituted into planar lipid bilayers.

An alpha-toxin-binding membrane protein, isolated from the head and thoracic ganglia of the locus (Locusta migratoria), was reconstituted into planar lipid bilayers. Cholinergic agonists such as acetylcholine, carbamylcholine, and suberyldicholine induced fluctuations of single channels, which suggests that the protein represents a functional cholinergic receptor channel. The antagonist d-tubocurarine blocked the activation of the channels, whereas hexamethonium had only a weak effect; similar properties have been described for nicotinic insect receptors in situ. The channel was selectively permeable to monovalent cations but was impermeable to anions. The conductance of the channel (75 pS in 100 mM NaCl) was independent of the type of agonist used to activate the receptor. Kinetic analysis of the channel gating revealed that, at high agonist concentrations (50 microM carbamylcholine), more than one closed state exists and that multiple gating events, bursting as well as fast flickering, appeared. At very high agonist concentrations (500 microM carbamylcholine), desensitization was observed. Channel kinetics were dependent on the transmembrane potential. Comparing the conductance, the kinetics, and the pharmacology of nicotinic acetylcholine receptor from insect ganglia and fish electroplax reconstituted into bilayers revealed obvious similarities but also significant differences.

Animals↗
PubMed1986

Regulation of the high affinity choline transport in locust synaptosomes by adenosine triphosphate.

The effect of various nucleotides on the high affinity choline accumulation by synaptosomes from locusts has been studied. Extracellular adenosine triphosphate (ATP) was found to inhibit the transport of choline, whereas the accumulation of gamma-aminobutyric acid was not affected. The ATP-effect could not be mimicked by other purine derivatives, and seems not be mediated by purinergic receptors, but rather hydrolysis of the phosphate appears to be essential.

Adenosine Triphosphate↗
PubMed1986

Messenger RNA from insect nervous tissue induces expression of neuronal acetylcholine receptors in Xenopus oocytes.

mRNA isolated from the nervous tissue of young insects microinjected in Xenopus oocytes induced the expression of alpha-toxin binding sites which were inserted into the surface membrane. Immunoprecipitation experiments revealed that the coded receptor polypeptides were processed to the authentic size, and ion flux studies demonstrated that functional nicotinic acetylcholine receptors were produced and inserted into the oocyte membrane.

Animals↗
PubMed1986

Black widow spider venom-induced release of neurotransmitters: mammalian synaptosomes are stimulated by a unique venom component (alpha-latrotoxin), insect synaptosomes by multiple components.

Synaptosomes isolated from the rat brain corpus striatum and locust head and thoracic ganglia were loaded with radioactive neurotransmitter ([3H]dopamine and [3H]acetylcholine, respectively) and then treated with alpha-latrotoxin and other fractions (fractions C, D and E of Frontali et al.8) obtained by Sephadex G200 column chromatography from black widow spider venom gland homogenates. As shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, alpha-latrotoxin is a high Mr protein, whereas fractions C-E are mixtures of several proteins, that include small amounts of contaminating alpha-latrotoxin (especially in fraction C). In rat synaptosomes alpha-latrotoxin induced massive neurotransmitter release, and some release was induced also by high concentrations of fractions C and D. These responses were blocked almost completely by a monospecific anti-alpha-latrotoxin serum, indicating that they were all due to alpha-latrotoxin. Release of [3H]acetylcholine from locust synaptosomes was induced by the various preparations investigated. alpha-Latrotoxin was about 10-fold less potent in locust than in rat synaptosomes. The effects of fractions C-E tended to disappear with storage. The most active batches of fractions C and E were even more potent than alpha-latrotoxin, while the D fraction was approximately 5-fold less potent. The anti-alpha-latrotoxin antiserum inhibited part of the responses elicited by fractions C and E, but left fraction D almost unaffected. Release by D and E fractions was maintained even when Ca2+ was removed from the incubation medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1985

Choline fluxes in synaptosomal membrane vesicles.

Synaptic plasma membrane vesicles isolated from the highly cholinergic nervous tissue of insects were used to study the translocation of choline across the membrane via a high-affinity carrier-mediated mechanism energized by ion gradients as the sole driving force. The uphill movement of choline, energized mainly by the Na+ gradient, attained levels of choline severalfold the final equilibrium value at the peak of the overshoot. Efflux of choline required the presence of internal sodium ions and was promoted by external choline if Na+ was present. External choline inhibited choline efflux in the absence of sodium. It is concluded that the efflux of choline is in many aspects symmetrical with its uptake.

Animals↗
PubMed1985

Molecular forms and subunit structure of the acetylcholine receptor in the central nervous system of insects.

The nicotinic acetylcholine receptor as probed by alpha-bungarotoxin binding has been isolated from detergent-solubilized ganglionic membrane preparations from the insect, Locusta migratoria. The isolation and characterization of the receptor protein was achieved by preparation of membrane fragments, extraction by sodium deoxycholate, centrifugation on sucrose density gradient, affinity chromatography, gel electrophoresis, and immunoblotting. The purified receptor protein migrated as a single band on polyacrylamide when native (Mr = 250,000 to 300,000) but also under denaturing conditions (Mr = 65,000) and cross-reacted with some monoclonal antibodies against the Torpedo receptor. In immunohistochemical approaches using polyclonal antibodies the acetylcholine receptor antigenic sites could topochemically be identified at very distinct zones in the neuropil of locust ganglia. The results suggest that the acetylcholine receptor in the central nervous system of insects represents an oligomeric complex composed of four identical or very similar subunits and thus may represent a prototype of the recently proposed homo-oligomeric ancestral acetylcholine receptor.

Animals↗
PubMed1984

Isolation of a putative nicotinic acetylcholine receptor from the central nervous system of Locusta migratoria.

The alpha-bungarotoxin binding component from locust central nervous tissue was solubilized and purified by affinity chromatography on alpha-bungarotoxin Sepharose 4B. On sucrose density gradients containing Triton X-100, the toxin binding site sedimented with an apparent sedimentation coefficient of about 10 S. As revealed by sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified receptor protein was composed predominantly of 65,000 molecular weight polypeptides.

Animals↗
PubMed1982

Uptake of [N-Me-3H]-choline by synaptosomes from the central nervous system of Locusta migratoria.

The accumulation of 3H-choline by isolated synaptosomes from the central nervous system of locust was studied at concentrations varying from 0.05 to 40 microM. Kinetic analysis of the saturable process revealed a high-affinity and a low-affinity system. The high-affinity uptake was competitively inhibited by hemicholinium-3 and was absolutely dependent on external sodium. Elevated potassium concentrations inhibited choline uptake. The choline uptake by insect synaptosomes was found to be remarkably resistant to a variety of metabolic inhibitors. The reduced choline uptake under depolarizing conditions (high potassium concentration or veratridine) in the absence of calcium implies that electrochemical gradients are important for high-affinity choline uptake. Depolarization of preloaded synaptosomes under appropriate conditions resulted in a significant release of newly accumulated choline radioactivity.

Animals↗
PubMed1981

Effect of exogenous gangliosides on synaptosomal membrane ATPase activity.

Changes in the activity of (Na+, K+)-ATPase of synaptosomal membranes induced by exogenous gangliosides were studied. Depending on the ganglioside-protein ratio, the enzyme activity was finally reduced to 40% when the ratio was about 1. By analysis of the reaction kinetics the effect was characterized as a noncompetitive inhibition. Moreover the ganglioside effect was clearly dependent on the incubation temperature. Since exogenous gangliosides thereby caused a shifting in the optimum temperature of (Na+, K+)-ATPase, the effect is discussed in terms of changes of the membrane properties. In preincubation experiments it was revealed that the interaction of the glycolipids with synaptosomal membranes itself was temperature dependent and enhanced by ATP. It is suggested that ganglioside micelles might have been incorporated by the membranes in a way comparable to a fusion process.

Adenosine Triphosphate↗