Verapamil potentiation of Chironex (box-jellyfish) antivenom.

'Rescue' experiments were performed on mice to compare the efficacy of i.v. verapamil, box-jellyfish antivenom, and a combination of both agents to counter the effects of toxic material extracted from the tentacles of Chironex fleckeri. In each of three series of experiments, four sets of mice were injected with doses of Chironex tentacle extracts containing approximately 4 mg/ml protein which were equivalent to twice the LD50. Three of these groups also received verapamil alone, antivenom alone, or a combination of both treatments. The combination therapy of drug and antivenom was superior to either agent alone. Treatment of verapamil alone appeared to be equivalent to that with only antivenom.

Involvement of neuronal acceptors for dendrotoxin in its convulsive action in rat brain.

Dendrotoxin, a snake-venom polypeptide, is a potent convulsant that facilitates transmitter release apparently by inhibition of voltage-sensitive K+ channels responsible for A-currents. A biologically active 125I-iodinated derivative of this toxin was prepared and used to characterize kinetically homogeneous non-interacting high-affinity acceptors in synaptic membranes from rat cerebral cortex and hippocampus. Binding of radiolabelled toxin from Dendroaspis angusticeps to its membrane acceptor protein was inhibitable by homologous polypeptides from other mamba snakes; most importantly, their rank order of potency was identical with that for their central neurotoxicities in rats, furnishing evidence for involvement of this binding component in the convulsive symptoms observed. Beta-Bungarotoxin, a presynaptically acting neurotoxin whose action on neurotransmitter release at the neuromuscular junction and effects on brain synaptosomes are antagonized by dendrotoxin, was only able to inhibit the binding of the 125I-labelled toxin with low efficacy, although dendrotoxin apparently interacts avidly with the acceptor sites for beta-bungarotoxin. This weak interaction of beta-bungarotoxin with the acceptor was not attributable to its phospholipolytic action. Other neurotoxins and ion-channel antagonists failed to affect the binding of dendrotoxin. The findings presented here, together with recent electrophysiological data, favour the interpretation that dendrotoxin binds to a membrane protein comprising, or closely associated with, this one group of voltage-dependent K+ channels.

Central action of dendrotoxin: selective reduction of a transient K conductance in hippocampus and binding to localized acceptors.

Dendrotoxin, a small single-chain protein from the venom of Dendroaspis angusticeps, is highly toxic following intracerebroventricular injection into rats. Voltage-clamp analysis of CA1 neurons in hippocampal slices, treated with tetrodotoxin, revealed that nanomolar concentrations of dendrotoxin reduce selectively a transient, voltage-dependent K conductance. Epileptiform activity known to be induced by dendrotoxin can be attributed to such an action. Membrane currents not affected directly by the toxin include (i) Ca-activated K conductance; (ii) noninactivating voltage-dependent K conductance; (iii) inactivating and noninactivating Ca conductances; (iv) persistent inward (anomalous) rectifier current. Persistence of the effects of the toxin when Cd was included to suppress spontaneous transmitter release indicates a direct action on the neuronal membrane. Using biologically active, 125I-labeled dendrotoxin, protein acceptor sites of high affinity were detected on cerebrocortical synaptosomal membranes and sections of rat brain. In hippocampus, toxin binding was shown autoradiographically to reside in synapse-rich and white matter regions, with lower levels in cell body layers. This acceptor is implicated in the action of toxin because its affinities for dendrotoxin congeners are proportional to their central neurotoxicities and potencies in reducing the transient, voltage-dependent K conductance.

Botulinum neurotoxin and dendrotoxin as probes for studies on transmitter release.

Acceptors for BoNT have been detected autoradiographically on the terminal membrane of motor nerves at a density of approximately 150/micron2 and shown to mediate toxin internalization, a process deemed essential for its inhibition of transmitter release. DTX, a protein with pronounced central neurotoxicity, was shown to induce convulsive states in hippocampal slices from guinea-pig. Synaptic transmission was facilitated and spontaneous epileptiform activity produced in intact cell populations. Voltage clamp analysis of hippocampal neurones revealed that DTX specifically attenuated a transient voltage-dependent K+ conductance (A-current) and this could account for the excitatory effects observed. Proteinaceous acceptors with high affinity for DTX were identified on brain synaptosomal membranes and found to contain a 65 000 Mr polypeptide. Their location in rat brain regions was established and contrasted with that of the binding sites for beta-bungarotoxin. These findings indicate the usefulness of DTX as a probe for a protein associated with one variety of K+ channel while the larger subunit of BoNT was found to interact with a membraneous component that resides at cholinergic nerve terminals and, hence, is likely to have a unique role.

Synaptic binding sites in brain for [(3)H]?-bungarotoxin - A specific probe that perturbs transmitter release.

A neurotoxic, [(3)H]labelled derivative of ?-bungarotoxin, known to inhibit neurotransmitter release and to be free of phospholipase activity, was used to demonstrate autoradiographically the distribution and ultrastructural location of its saturable binding component in brain. Light-microscope autoradiography of rat cerebellum and hippocampus showed that it resides primarily in synaptic-rich areas, with much lower densities of sites in other regions containing cell bodies; also, little binding was associated with myelinated tracts. Ultrastructural localisation and sub-fractionation studies on purified cerebrocortical synaptosomes showed that [(3)H]toxin binding sites are located predominantly on brain synaptosomal membranes, consistent with their possible association with transmitter release.

Preparation of neurotoxic 3H-beta-bungarotoxin: demonstration of saturable binding to brain synapses and its inhibition by toxin I.

1. Homogeneous beta-bungarotoxin, isolated from the venom of Bungarus multicinctus was radiolabelled with N-succinimidyl-[2.3-(3) H]propionate. Stable, di-propionylated material was obtained which was tritiated on both subunits and had a specific radioactivity of 102 Ci/mmol. 2. After separation from unlabelled toxin by isoelectric focussing, it was shown to exhibit significant biological activity in both the peripheral and central nervous systems but had negligible phospholipase A2 activity towards lecithin or cerebrocortical synaptosomes. 3. The labeled neurotoxin binds specifically to a single class of non-interacting sites of high affinity (Kd = 0.6 nM) on rat cerebral cortex synaptosomes; the content of sites is about 150 fmol/mg protein. This binding was inhibited by unlabelled beta-bungarotoxin with a potency which indicates that tritiation does not alter the affinity significantly. 4. The association of toxin with its binding component and its dissociation were monophasic; rate constants observed were 7.8 x 10(5) M-1 s-1 and 5.6 x 10(-4) s-1 at 37 C, respectively. 5. beta-Bungarotoxin whose phospholipase activity had been inactivated with p-bromophenacyl bromide inhibited to some extent the binding of tritiated toxin but with low efficacy. Taipoxin and phospholipase A2 from bee venom, but not Naja melanoleuca, inhibited the synaptosomal binding of toxin with low potencies in the presence, but not the absence, of Ca2+. 6. Toxin I, a single-chain protein from Dendroaspis polylepis known to potentiate transmitter release at chick neuromuscular junction, completely inhibited the binding of 3H-beta-bungarotoxin with a Ki of 0.07 nM; this explains its ability to antagonise the neuroparalytic action of beta-bungarotoxin. Other pure presynaptic neurotoxins, alpha-latrotoxin and botulinum neurotoxin failed to antagonise the observed binding; likewise tityustoxin, which is known to affect sodium channels, had no effect on 3H-beta-bungarotoxin binding. 7. Trypsinization of synaptosomes completely destroyed the binding activity, suggesting that the binding component is a protein; the functional role of the latter is discussed in relation to the specificity of toxin binding.