Biomedical subjects

H Bigalke

Publications and source records attributed to H Bigalke.

At least 37 records · Page 2Linked to original sources

PubMed1995

Molecular aspects of tetanus and botulinum neurotoxin poisoning.

Clostridial neurotoxins, tetanus and the botulinum toxins A-G, are high molecular weight proteins consisting of a heavy chain which is responsible for the internalisation and a light chain possessing a zinc-dependent proteolytic activity. They exclusively proteolyse either the vesicle membrane protein, synaptobrevin or two integral plasma membrane proteins, SNAP 25 and syntaxin. Together with cytosolic proteins these proteins form the SNARE complex involved in vesicle exocytosis, and their cleavage blocks the latter process. Clostridial neurotoxins have now become powerful tools to investigate the final events occurring during secretion in neuronal, endocrine, and non-neuronal cells. They are applied to dissect the specific interactions of the SNARE protein complex with cytosolic fusogens and other modulators of exocytosis. Whereas exocytosis is not essential for the survival of cells, the organism as a whole will fall victim to a few nanograms since interneuronal and neuromuscular transmission is vital to muscular control, especially in respiration. Although all clostridial neurotoxins by their light chains attack proteins of the SNARE complex, tetanus toxin and the various botulinum toxins differ dramatically in their clinical symptoms. The biological information for this difference resides on the respective heavy chains which select different transport routes carrying the light chain from the place of entrance to the final compartment of action. So far the different transport vesicles used either by the various botulinum neurotoxins or by tetanus toxin are not yet defined. Nevertheless at least one of the botulinum toxins serves as a beneficial drug in the treatment of severe neuromuscular spasms.

Botulinum Toxins↗

PubMed1994

Specific antibodies against the Zn(2+)-binding domain of clostridial neurotoxins restore exocytosis in chromaffin cells treated with tetanus or botulinum A neurotoxin.

Although tetanus and botulinum A neurotoxins are ineffective in cultured chromaffin cells, they will inhibit carbachol-induced release of noradrenaline provided they gain access to the cytosol either through artificial pores generated in the plasma membrane or by binding to incorporated exogenous gangliosides. The block of exocytosis persists for weeks followed by a slow recovery of cell function. When specific anti-botulinum A toxin antibodies are introduced into cells through pores after manifestation of the block by botulinum A neurotoxin, restoration of exocytotic function is accelerated and fully reestablished within 4 days. The same time course of restoration is seen with anti-tetanus toxin antibodies in cells poisoned by tetanus toxin. Since the light chains of the toxins are enzymatically active, we have introduced polyclonal and monoclonal anti-light chain antibodies into the cytosol. Of all light chain antibodies tested, only those directed against the peptide homologous to the zinc-binding sequence, which is present in both neurotoxins, restored exocytosis regardless of which toxin caused the block. These results indicate that the zinc-binding domain is directly involved in the interaction of the light chains with their substrates and that the toxins have to be present continuously to maintain the block.

Adrenal Medulla↗

PubMed1992

Restoration of exocytosis occurs after inactivation of intracellular tetanus toxin.

Tetanus toxin blocks carbachol-stimulated release of noradrenaline from bovine adrenal chromaffin cells in culture, provided it can gain access to the cells. This can be achieved by electropermeabilization of the plasma membrane or by enriching the membrane with exogenous gangliosides which serve as carriers of the toxin. The inhibition of noradrenaline release persists for at least 6 days, even in the presence of specific anti-tetanus toxin antibodies in the culture medium. However, the block is preventable, for the most part, when antibodies enter chromaffin cells during electropermeabilization, before the uptake of the toxin is facilitated by inserting exogenous gangliosides into the plasma membrane 2 days later. This indicates that the antibodies pass into the cells through the physically induced pores and that these intracellular antibodies neutralize incoming tetanus toxin. If, on the other hand, exocytosis has been inhibited by tetanus toxin, it will recover within 3 days, provided specific anti-tetanus toxin antibodies are introduced into the cells by electropermeabilization. The recovery is not linked to a specific route of entry of the toxin. It is concluded that the restoration of noradrenaline release requires not only the intracellular neutralization of tetanus toxin but also the reconstitution of the as yet unknown target molecule of the toxin.

Adrenal Glands↗

PubMed1991

Distinct targets for tetanus and botulinum A neurotoxins within the signal transducing pathway in chromaffin cells.

Tetanus and botulinum A neurotoxins inhibited exocytosis evoked by various secretagogues in intact and permeabilized chromaffin cells. The block of exocytosis in intact chromaffin cells due to botulinum A neurotoxin could partially be overcome by enhancing nicotine- and veratridine-induced stimulation, whereas the block due to tetanus toxin persisted under the same conditions. The receptor-mediated restoration of 3H-noradrenaline release was specific for nicotinic stimulation, because exocytosis did not occur during muscarinic stimulation. Depolarization of intact chromaffin cells with increasing concentration of K+ failed to restore exocytosis that had been blocked by either toxin. When chromaffin cells, treated with tetanus or botulinum A neurotoxins, were exposed to the Ca2(+)-ionophore A 23187 or permeabilized by staphylococcal alpha-toxin, Ca2(+)-stimulated exocytosis was also inhibited. The inhibition was unaffected by increasing concentrations of free Ca2+. Activation of proteinkinase C and of G-proteins by phorbolester and GMPPNHP, respectively, increased Ca2(+)-induced exocytosis in control cells as well as in cells treated with tetanus and botulinum A neurotoxins. The block, however, could not be relieved by these manipulations, and it could not be relieved by activating the cGMP or cAMP pathways with analoga of cyclic nucleotides, phosphodiesterases inhibitors, and forskolin either. It is concluded that nicotine and veratridine trigger a mechanism within the sequence of events leading to exocytosis that is located beyond the increase in intracellular Ca2(+)-concentration. This pathway may not be affected by botulinum A neurotoxin. The target of tetanus toxin is probably located even closer to the fusion process, i.e. beyond the step upon which botulinum A neurotoxin acts.

Adrenal Glands↗

PubMed1991

The translocation of botulinum A neurotoxin by chromaffin cells is promoted in low ionic strength solution and is insensitive to trypsin.

Botulinum A neurotoxin (BoNtx) produced a partial inhibition of carbachol induced 3H-noradrenaline (3H-NA) release from bovine adrenal chromaffin cells in monolayer culture. Each of the polysialogangliosides GD1a, GT1b and GD1b enhanced the block of exocytosis when they were applied prior to the toxin exposure. The monosialoganglioside GM1 was not effective. Chromaffin cells treated with neuraminidase lost their sensitivity to BoNtx. Application of gangliosides to these cells, however, restored their susceptibility to the toxin. Treatment of the cells with trypsin did not affect the BoNtx-blockade of 3H-NA-release. The potency of botulinum A toxin was increased in a solution of low ionic strength in which sodium chloride was replaced by sucrose. In agreement with the potency of botulinum A neurotoxin in blocking exocytosis under the various conditions, binding of 125I-botulinum A neurotoxin to chromaffin cells was enhanced in low ionic strength solution and by pretreatment of the cells with gangliosides. The binding was decreased by digestion of gangliosides with neuraminidase. It is concluded that botulinum A neurotoxin binds exclusively to polysialogangliosides which subsequently serve as carriers for the toxin. The low ionic strength may increase some physico-chemical interaction of the toxin with the polysialogangliosides.

Animals↗

PubMed1991

Tetanus and botulinum A toxins inhibit stimulated F-actin rearrangement in chromaffin cells.

Tetanus and botulinum A neurotoxins block exocytosis of noradrenaline in chromaffin cells. During exocytosis vesicles fuse with the plasma membrane. This requires an intact cytoskeleton. The cytoskeleton can be displayed by rhodamine-labelled phalloidin binding to F-actin. When chromaffin cells are stimulated with carbachol, F-actin forms clusters close to the plasma membrane. On withdrawal of the secretagogue the stimulated cells revert to their original appearance. The conversions, like exocytosis, depend on the presence of Ca2+, indicating the association of exocytosis with F-actin arrangement. When exocytosis is blocked in chromaffin cells by tetanus or botulinum A neurotoxins, F-actin fails to cluster during nicotinic stimulation. Thus, the toxins act somewhere between the rise in intracellular free Ca2+ and the rearrangement of F-actin.

Actins↗

PubMed1991

Modulation of glycine-activated membrane current by adamantane derivatives.

Pressure application of the inhibitory neurotransmitters glycine and GABA, respectively, evoked a Cl- current in spinal cord neurons of fetal mice in culture. Memantine, in low concentrations (0.3-5 microM) enhanced the glycine-mediated current at both negative and positive holding potentials. At concentrations of more than 5 microM, memantine increased the glycine-evoked current only when the membrane potential was positive. At negative potentials the current was inhibited. 3-isopropylamantadine and 3,5-diethylamantadine augmented the glycine-activated membrane current irrespective of the concentrations applied, whereas amantadine was ineffective. The GABA-activated Cl- current was not altered by the adamantanes. These data are evidence of a specific glycinergic interaction with putative antispastic agents and can explain some conflicting effects of memantine in experimentally induced seizures.

Adamantane↗

PubMed1990

Memantine reduces repetitive action potential firing in spinal cord nerve cell cultures.

(1) The anticonvulsant effects of memantine were examined and compared with those of baclofen in monolayer primary cultures of murine nerve cells using conventional intracellular recordings. (2) Memantine and baclofen (each 10-100 microM) decreased spontaneous synaptic activity when action potential frequencies exceeded 6 Hz. Neurons firing action potentials at frequencies below 6 Hz (about 90% of all impaled cells), however, were not affected by the drugs. (3) Memantine reduced the duration of strychnine-elicited bursts and the firing rate of action potentials within a burst. In contrast, baclofen lowered the frequency of the bursts without reducing intra-burst firing. The duration of the bursts was increased. (4) Memantine, but not baclofen, reduced the extent of sustained repetitive firing evoked by pulses of depolarizing current. (5) In the presence of memantine, the second of two electrically evoked action potentials increasingly failed to appear as the intervals between successive stimulating pulses were shortened. Such an effect was not seen when baclofen was applied. Thus, both antispastic agents, memantine and baclofen, reduce hyperactivity of spinal cord neurons in culture, although their mechanisms of action are different.

Action Potentials↗

PubMed1990

Tetanus antitoxin binds to intracellular tetanus toxin in permeabilized chromaffin cells without restoring Ca2(+)-induced exocytosis.

Tetanus toxin blocks Ca2(+)-evoked catecholamine release from permeabilized bovine adrenal chromaffin cells preloaded with gangliosides. Tetanus toxin preincubated with its specific antibodies F(ab')2 is without any effect on exocytosis. Specific antitetanus F(ab')2 presented to chromaffin cells which are pretreated with tetanus toxin and permeabilized by digitonin cannot restore exocytosis. Under the same conditions, however, 125I-labeled F(ab')2 accumulates in chromaffin cells. The accumulation depends on the presence and concentration of tetanus toxin and can be prevented by an excess of unlabeled F(ab')2. Once tetanus toxin has initiated block of exocytosis, it cannot be neutralized by binding to its specific antibody.

Animals↗

PubMed1990

Carbamazepine blocks NMDA-activated currents in cultured spinal cord neurons.

The antiepileptic agents, carbamazepine and phenytoin, suppress seizures in man and convulsant-induced hyperactivity in spinal cord nerve cell cultures. In the present study, we have shown by whole cell recording that carbamazepine, in contrast to phenytoin, blocks N-methyl-D-aspartate (NMDA)-activated membrane currents in cultured neurons in a dose-dependent fashion. The NMDA receptor-activated channel, which is blocked at physiological concentrations of Mg2+ at resting membrane potential, can be activated by glutamate in depolarized neurons and thus be involved in epileptogenesis. Therefore, the block of NMDA-evoked membrane currents in cultured neurons may contribute to the clinical effectiveness of carbamazepine.

2-Amino-5-phosphonovalerate↗

PubMed1990

Uptake of antitetanus F(ab')2 fragments into eukaryotic cells.

1. In order to introduce antitetanus immunoglobulin fragments into eukaryotic cells, either antitetanus F(ab')2 or Fab' fragments have been linked to carrier molecules. Aciclovir, horseradish peroxidase, wheat germ agglutinin, and transferrin were tried as carriers. 2. F(ab')2-aciclovir and Fab'-horseradish peroxidase were not internalized by NG108-15 neurohybridoma cells. 3. [Fab']2-wheat germ agglutinin and F(ab')2-transferrin conjugates were internalized into various cells. 4. F(ab')2-transferrin conjugates were made with three different linkers: N-succinimidyl 3-(2-pyridyldithio) propionate, bis-maleimido hexane, and bis-maleimidoethoxy propane. All three conjugates were internalized but had a different fate inside the cells.

Acyclovir↗

PubMed1989

Tetanus toxin: inhibitory action in chromaffin cells is initiated by specified types of gangliosides and promoted in low ionic strength solution.

Adrenal medullary chromaffin cells preloaded with [3H]noradrenaline release the hormone by exocytosis upon stimulation with carbachol. This exocytosis is not influenced by tetanus toxin (Tetx) which can block the release of transmitters from nerve endings. Preincubation of chromaffin cells with gangliosides followed by toxin exposure in low ionic strength solution results in a decrease of stimulated catecholamine release. The inhibition of exocytosis is most evident when cells are pretreated with the ganglioside GD lb. Cells treated with GTlb are 10 times less sensitive, those treated with GDla and GMl even 30 times less sensitive to Tetx. Thus GDlb appears to be the principal carrier for Tetx in chromaffin cells whereas the other gangliosides only play a minor role.

Adrenal Medulla↗

PubMed1989

Gangliosides mediate inhibitory effects of tetanus and botulinum A neurotoxins on exocytosis in chromaffin cells.

Bovine chromaffin cells in monolayer culture were preloaded with 3H-NA (noradrenaline) and subsequently stimulated with carbachol. Botulinum A neurotoxin partially inhibited the evoked release of 3H-NA and the basal efflux of the hormone. The inhibition of evoked release did not exceed 40%, although the cells were exposed to 10 micrograms/ml of toxin for 6 days. The inhibitory effect of botulinum A neurotoxin was neutralized by its antibodies. In contrast to botulinum A neurotoxin, tetanus toxin at even higher concentrations did not influence evoked release. This difference in sensitivity could be explained by the ganglioside pattern of chromaffin cells. Ganglioside GD1a, a putative receptor for botulinum A neurotoxin, could be identified in lipophilic extracts, whereas the tetanus toxin binding gangliosides GT1b and GD1b could not be detected by means of thin-layer chromatography. Treatment of the cells with neuraminidase abolished both, GD1a and the inhibitory effect of botulinum A neurotoxin. Incubation of chromaffin cells with a mixture of gangliosides (21% GM1, 44% GD1a, 15% GD1b, 20% GT1b) not only increased the efficacy of botulinum A neurotoxin but also made the cells sensitive towards tetanus toxin. The concentration-response curve of botulinum A neurotoxin was shifted to the left about five-fold and the maximum inhibition of evoked release was increased up to 60%, even though the cells were exposed to the toxin for 3 days only. In contrast, the maximum inhibition that could be achieved by tetanus toxin was 40%. The results indicate that polysialogangliosides are important for the intracellular accumulation of these clostridial neurotoxins.

Adrenal Medulla↗

PubMed1987

Differential effects of various secretagogues on quantal transmitter release from mouse motor nerve terminals treated with botulinum A and tetanus toxin.

Electrophysiological and electron microscopic techniques were used to investigate the actions of potassium depolarization, black widow venom (BWSV), Ca2+-ionophore A 23187 and hyperosmotic solution on mouse hemidiaphragms poisoned in vitro with botulinum A toxin (BoTx) and tetanus toxin (TeTx). These neurotoxins reduced the frequency of miniature endplate potentials (m.e.p.ps) from 5/s of the control to 2/min and 21/min, respectively. High potassium (25 mmol/l) increased the m.e.p.p.-frequency at BoTx- and TeTx-poisoned endplates to 30/min and 50/s, respectively. The ultrastructure of endplates showed no obvious changes. BWSV (0.04 glands/ml) was just as effective in promoting transmitter release from BoTx-treated endplates as in control preparations. Electron micrographs revealed depletion of vesicles as well as swollen and disrupted mitochondria. When preparations were pretreated with TeTx, BWSV only moderately increased transmitter release and no alterations of the ultrastructure could be observed. At TeTx- or BoTx-poisoned endplates Ca2+-ionophore A 23187 usually produced an extreme reduction of m.e.p.p.-frequency (0.005/s), sometimes preceded by a short burst-like release. The ultrastructure of these endplates was not obviously affected. Application of hyperosmotic solution to BoTx- or TeTx-poisoned preparations further reduced the already low m.e.p.p.-frequency. These results further demonstrate that TeTx and BoTx act at different sites in the transmitter releasing process.

Animals↗

PubMed1987

Differential effects of tetanus toxin on inhibitory and excitatory synaptic transmission in mammalian spinal cord neurons in culture: a presynaptic locus of action for tetanus toxin.

Tetanus toxin reduces monosynaptic inhibitory and excitatory synaptic transmission in mouse spinal cord neurons in culture. Inhibitory transmission is preferentially reduced by the toxin; however, excitatory transmission is also ultimately reduced and blocked by the concentrations of toxin used in these studies. Recordings from monosynaptically connected cell pairs revealed a marked diminution in amplitude of evoked monosynaptic inhibitory postsynaptic potentials coincident with the onset of convulsant action at a time when evoked monosynaptic EPSPs were relatively unaffected. Increased polysynaptic excitation occurred as a result of diminished inhibition. This supports the reduction of inhibition as an important mechanism in the convulsant action of tetanus toxin. Quantal analysis of the late effects of tetanus toxin on the monosynaptic excitatory postsynaptic potential revealed a reduction in quantal number with no reduction in quantal size, thus demonstrating a presynaptic locus of action for the toxin on spinal neurons.

Animals↗

PubMed1986

Microelectrode measurement of cell membrane potential in isolated hepatocytes attached to collagen.

Isolated spherical rat hepatocytes attached to collagen-coated cover slips generate a mean membrane potential (Em) of -78 +/- 9 mV as measured with high-resistance microelectrodes. The recordings were biphasic and were stable for upto 20 minutes. The correlation between external potassium concentration and Em was not linear. Several potassium-channel blockers did not effect the membrane potential. Addition of ouabain added to the incubation solution slowly depolarized the cells. The results indicate a high potassium permeability of the isolated spherical hepatocytes attached to collagen.

Animals↗

PubMed1986

Botulinum A neurotoxin unlike tetanus toxin acts via a neuraminidase sensitive structure.

The binding and effects of tetanus and botulinum A neurotoxins were studied on mouse spinal cord cultures treated with neuraminidase. In untreated cultures both neurotoxins blocked synaptic transmission. Treatment of the cell cultures with neuraminidase, 25 mU/ml for 24 hr, decreased the potency of botulinum A neurotoxin. At 7 X 10(-11) M no toxin effect on inhibitory or excitatory synapses was observed, whereas at higher concentrations of the toxin the concentration-response curve was shifted to the right by a factor of about 30. Surprisingly, the action of tetanus toxin over a large concentration range was unaffected by pretreatment of the neurones with the enzyme. Accordingly, neurones treated with neuraminidase failed to bind 125I-botulinum A neurotoxin, whereas labelled tetanus toxin was still fixed by cell bodies, as well as by neurites, as shown by histoautoradiography. Chromatographic extraction of gangliosides from cultures prelabelled with 14C-glucosamine showed a dramatic loss in the contents of polysialogangliosides following treatment with neuraminidase. Our results indicate that neuraminidase-sensitive structures might be important for the action of botulinum A neurotoxin. The effect of tetanus toxin appears to be mediated by a different site which is insensitive to neuraminidase.

Action Potentials↗

PubMed1986

Tetanus toxin in dissociated spinal cord cultures: long-term characterization of form and action.

The clinical course of tetanus is notable, in addition to its often dramatic clinical presentation, by the long duration of the neuromuscular symptoms. Survivors may have tetanic manifestations for several weeks after the onset of the disease. In this article we correlate the duration of specific electrophysiologic effects produced by tetanus toxin with the degradation of cell-associated toxin in primary cultures of mouse spinal cord neurons. From these studies we can conclude that the toxin has a half-life of 5-6 days. Both the heavy and the light chains of tetanus toxin degrade at similar rates. Labeled toxin, visualized by radioautography, is associated with neuronal cell bodies and neurites, and its distribution is not altered during a 1-week period following toxin exposure. Blockade of synaptic activity persists for weeks at the concentration of radiolabeled toxin used in these studies. This blockade of transmission is reversed as the toxin is degraded, suggesting that degradation of toxin may be a sufficient mechanism for recovery from tetanus.

Action Potentials↗