Comparison of the presynaptic actions of botulinum toxin and β-bungarotoxin on neuromuscular transmission

Summary Comparison was made between the presynaptic actions of type A botulinum toxin (BoTX) and -bungarotoxin (-BuTX) on isolated nerve-muscle preparations. On the mouse and rat diaphragms, BoTX is about 100 and 10 times more potent than -BuTX, respectively, whereas on the chick biventer cervicis muscle, -BuTX is 3–10 times more potent. The paralytic actions of both toxins are preceded by latency, antagonized by high concentrations of calcium or magnesium and by deficiency of calcium, accelerated by high frequencies of nerve stimulation and retarded by decrease of temperature. The paralytic actions of BoTX as well as -BuTX appear to take place in two processes: first, binding with their respective target sites and second, the inhibitory changes of the target macromolecule of the nerve terminals leading to failure of transmitter release. The latter process is not reversed by washing but is retarded greatly by low calcium, high magnesium or low temperature. Binding of -BuTX is faster than that of BoTX.Miniature end-plate potentials of unreduced amplitude could be recorded in junctions blocked by either toxin. End-plate potentials were depressed and the successive decline of their amplitude during train of pulses was abolished by both toxins.In contrast to the initial facilitatory actions after -BuTX, BoTX has no sign of facilitation such as increase of the frequency of miniature end-plate potential, restoration of neuromuscular transmission, increase of quantal content of end-plate potential and occurrence of spontaneous fasciculations in low calcium media. Another difference between the two toxins is the typical Wedensky inhibition on repetitive stimulation and post-tetanic potentiation in -BuTX paralysed muscles. By contrast, after BoTX, sustained contraction without post-tetanic potentiation was observed.The two toxins show a mutual antagonism especially when -BuTX is added before or simultaneously with BoTX. The action of the latter was completely antagonized in the presence of -BuTX. Once it is bound to the target site, however, BoTX seems not to be antagonized by -BuTX. On the other hand, BoTX appears to be able to retard the effect of bound -BuTX.     

α-Bungarotoxin aggregates on radioiodination with chloramine-T but not with iodogen

The presence of polymer(s) of radioiodinated bungarotoxin (Bgt) in preparations iodinated with chloramine-T or with iodogen, was investigated by chromatography on Sephadex-G50. In addition to a monomeric peak (P2), chloramine-T preparations showed a high content of polymeric forms (16–43%) eluting in the void volume (P1), present only to the extent of 1–3% in iodogen preparations. In purified [¹²⁵I]Bgt prepared by the iodogen method, the content of P1 increased on treatment with chloramine-T, in the absence of radioiodine. The nonspecific binding of [¹²⁵I]Bgt to DE-81 filter discs was high in the case of chloramine-T preparations and proportional to the content of P1 in each batch. Purified P1 (polymer) but not P2 (monomer) showed a concentration-dependent high degree of binding to DE-81 discs.     

Studies on the trophic influence of nerve on skeletal muscle

Colchicine is a drug known to block axoplasmic transport in myelinated nerves. When injected under the perineurium of the sciatic nerve of the rat, the drug induced denervation-like changes in the extensor digitorum longus muscle. These changes, consisting of the appearance of extra-junctional cholinergic sensitivity and of action potentials resistant to the blocking effect of tetrodotoxin, developed despite the fact that nerve impulse conduction, transmitter release and neuromuscular transmission were intact. The results indicate that trophic substances, necessary for the prevention of denervation changes, are transported by axoplasmic flow.

β-Bungarotoxin, a purified neurotoxin from Bungarus multicinctus acts selectively on motor nerve terminals blocking the release of acetylcholine from the nerve. S.c. injection of the toxin into the hind leg of rats produced paralysis and complete blockade of spontaneous transmitter release in the extensor digitorum longus muscle. The toxin also induced the aforementioned denervation-like changes in the muscle membrane. It is suggested that β-bungarotoxin in addition to blocking transmitter release blocks the release of trophic substances from the nerve or alternatively that neuromuscular transmission and resulting muscle activity constitute trophic influences necessary for the prevention of the appearance of denervation changes.     

Tolerance, cross-tolerance, and receptors after chronic nicotine or oxotremorine

Saline, 8.0 mg/kg/hr nicotine, or 1.0 mg/kg/hr oxotremorine was continuously infused into the jugular veins of DBA female mice. After 10 days of treatment, respiratory rate, Rotarod performance, Y-maze crossings, Y-maze rears, heart rate, and body temperature were measured after challenge with 2.0 mg/kg nicotine or saline or 0.2 mg/kg oxotremorine. Nicotine-infused mice were tolerant to the effects of nicotine for all six tests and oxtremorine-infused mice were tolerant to the effects of oxotremorine for all six tests and to the effects of nicotine on heart rate and body temperature. Oxotremorine infusion reduced the Bmax for [3H]-L-QNB binding, but had no effect on Bmax for either [3H]-DL-nicotine or [125I]-alpha-BTX binding. Conversely nicotine infusion did not alter the Bmax for [3H]-L-QNB binding, but increased the Bmax for both [3H]-DL-nicotine and [125I]-alpha-BTX binding. These results indicate that tolerance developed to the effects of two cholinergic agents, nicotine and oxotremorine, and that some cross-tolerance to the effects of nicotine occurred in oxotremorine-treated mice. Treatment with oxotremorine caused down-regulation of muscarinic receptors, while treatment with nicotine caused up-regulation of nicotinic receptors. Although some cross-tolerance to the effects of nicotine occurred in oxotremorine-treated mice, this did not appear to result from changes in nicotinic receptors.     

Inactivity induces muscle hypertrophy and redistribution of myosin isozymes in chicken anterior latissimus dorsi muscle

Denervation of the anterior latissimus dorsi (ALD) muscle causes transient muscle fibre hypertrophy and leads to an increase of the SM1 myosin isoform. We tested whether the changes that take place after denervation can be attributed to loss of muscle activity which follows denervation. Neuromuscular activity was prevented by blocking the acetylcholine receptors with -bungarotoxin and thereby paralysing the muscle. Following this treatment, we found increased muscle weight and pronounced hypertrophy of muscle fibres. Also, the proportion of SM1 isomyosin was decreased. Due to the multiple innervation of ALD muscle fibres it is possible to paralyse only part of the muscle. When only a region of the muscle was paralysed a local hypertrophy of fibres was detected, and the change from SM1 to SM2 was most pronounced in the area where activity was blocked. Removal of muscle activity resulted in changes similar to those that occurred after denervation.     

Beta-bungarotoxin-induced depletion of synaptic vesicles at the mammalian neuromuscular junction

The neurotoxic phospholipase A(2), beta-bungarotoxin, caused the failure of the mechanical response of the indirectly stimulated rat diaphragm. Exposure to beta-bungarotoxin had no effect on the response of the muscle to direct stimulation. Resting membrane potentials of muscle fibres exposed to the toxin were similar to control values, and the binding of FITC-labelled alpha-bungarotoxin to nAChR at the neuromuscular junction was unchanged. Motor nerve terminal boutons at a third of cell junctions were destroyed by exposure to beta-bungarotoxin leaving only a synaptic gutter filled with Schwann cell processes and debris. At other junctions, some or all boutons survived exposure to the toxin. Synaptic vesicle density in surviving terminal boutons was reduced by 80% and synaptophysin immunoreactivity by >60% in preparations exposed to beta-bungarotoxin, but syntaxin and SNAP-25 immunoreactivity was largely unchanged. Terminal bouton area was also unchanged. The depletion of synaptic vesicles was completely prevented by prior exposure to botulinum toxin C and significantly reduced by prior exposure to conotoxin omega-MVIIC. The data suggest that synaptic vesicle depletion is caused primarily by a toxin-induced entry of Ca(2+) into motor nerve terminals via voltage gated Ca(2+) channels and an enhanced exocytosis via the formation of t- and v-SNARE complexes.     

KChIP3: a binding protein for Taiwan banded krait beta-bungarotoxin.

Using B1 chain of beta-bungarotoxin (beta-Bgt) as bait in yeast two-hybrid screen, we found that KChIP3 was a binding protein of B1 chain. Thus, protein-protein interaction between beta-Bgt and KChIP3 is investigated in the present study. Pull-down assay showed that recombinant KChIP3 proteins were associated with beta-Bgt as well as B1 chain, whereas the inability of KChIPs 1, 2 and 4 to bind with beta-Bgt was observed. Although Ca2+ was not a crucial factor essential for the binding of KChIP3 with beta-Bgt and B1 chain, their interaction could be enhanced by the addition of Ca2+. Alternatively, the association of A1 chain of beta-Bgt with KChIP3 was marginally detected. The dissociation constant of beta-Bgt with KChIP3 were 12.2 and 6.08 microM in the absence and presence of 2mM Ca2+, respectively. Moreover, native KChIP3 from rat brain was to be isolated by beta-Bgt-Sepharose. These observations indicate that KChIP3 is a binding protein of beta-Bgt. In view of the multiple functions of KChIP3 in neuronal cells, the interaction of KChIP3 with beta-Bgt may represent an event for the manifestation of the biological activities of beta-Bgt.     

Neurons of the chick brain and retina expressing both α-bungarotoxin-sensitive and α-bungarotoxin-insensitive nicotinic acetylcholine receptors: an immunohistochemical analysis

Immunohistochemical methods were used to study the possible co-localization of two α-bungarotoxin-sensitive (α7 and α8) and two α-bungarotoxin-insensitive (β2 and α3) subunits of the nicotinic acetylcholine receptors in neurons of the chick brain and retina. Several structures contained neurons that were doubly-labeled with antibodies against the α7 subunit and the β2 subunit. These structures included, for example, the interpeduncular nucleus, nucleus spiriformis lateralis, optic tectum, pretectal visual nuclei, and the lateral hypothalamus. Double-labeling with antibodies against the α7 and α8 subunits was also seen in several regions, which included the interpeduncular nucleus, visual pretectum, lateral hypothalamus, dorsal thalamus, and the habenular complex. In the retina, many cells in the inner nuclear layer were observed to contain α8 and α3 subunits, whereas neurons in the ganglion cell layer were seen to contain α7 and α8 or, less frequently, α7 and α3 subunits. These results indicate that α-bungarotoxin-sensitive and α-bungarotoxin-insensitive subunits of the nicotinic receptors are co-expressed by neurons of the chick brain and retina.     

Rapid synthesis of acetylcholine receptors at neuromuscular junctions

The rate of acetylcholine receptor (AChR) degradation in mature, innervated mammalian neuromuscular junctions has recently been shown to be biphasic; up to 20% are rapidly turned over (RTOs; half life less than 1 day) whereas the remainder are lost more slowly ('stable' AChRs; half life 10-12 days). In order to maintain normal junctional receptor density, synthesis and insertion of AChRs should presumably be sufficiently rapid to replace both the RTOs and the stable receptors. We have tested this prediction by blocking pre-existing AChRs in the mouse sternomastoid muscle with alpha-bungarotoxin (alpha-BuTx), and monitoring the subsequent appearance of 'new' junctional AChRs at intervals of 3 h to 20 days by labeling them with 125I-alpha-BuTx. The results show that new receptors were initially inserted rapidly (16% at 24 h and 28% at 48 h). The rate of increase of 'new' 125I-alpha-BuTx binding sites gradually slowed down during the remainder of the time period studied. Control observations excluded possible artifacts of the experimental procedure including incomplete blockade of AChRs, dissociation of toxin-receptor complexes, or experimentally induced alteration of receptor synthesis. The present demonstration of rapid synthesis and incorporation of AChRs at innervated neuromuscular junctions provides support for the concept of a subpopulation of rapidly turned over AChRs. The RTOs may serve as precursors for the larger population of stable receptors and have an important role in the metabolism of the neuromuscular synapse.     

The evolution and ancestors of toxic proteins.

A large number of sequences of digestive enzymes, hormones and a large series of toxins from organisms representing different levels of evolutionary development have been compared and the results juxtaposed with data about their functions and active sites. This allows random coincidences to be excluded; they serve as a background, against which the real albeit weak analogies are outlined. The results show proinsulin as a protein closest to the common ancestor of the proteins compared. Next in this respect are ribonuclease and phospholipase. It is established that most of the examined proteins are related and constitute a superfamily of a higher level—hypersuperfamily. Evidence in favour of the evolutionary order: cardiotoxins→short and long snake neurotoxins is also presented.