C.Y. Lee

Distribution of Bungarus multicinctus venom following envenomation

The distribution of the venom of Bungarus multicinctus and its purified toxins (α- and β-Bungarotoxin) in animals has been studied either by bioassay method or using I131-labeled preparations. Although radioactivity could be detected in the cerebrospinal fluid (CSF) of rabbits after intravenous injection of 1 mg per kg of the venom or the toxins, the CSF levels were too low to account for the rapid respiratory paralysis. No measurable amounts of the venom were found in the CSF by the bioassay method at this dose level. The highest radioactivity was found in kidneys, followed in decreasing order by lungs, spleen, heart, stomach, intestine, diaphragm and skeletal muscle, with the lowest in the brain. In mice, injected subcutaneously with 2 ld50 of the venom or the Bungarotoxins, similar distribution patterns were found, except for much higher radioactivity in the stomach. α-Bungarotoxin was found to localize on the end-plate zone of the mouse diaphragm, whereas β-Bungarotoxin did not show such localization.

Coronary vasospasm as the primary cause of death due to the venom of the burrowing asp, Atractaspis engaddensis

The venom from the snake Atractaspis engaddensis has been shown to be cardiotoxic in anesthetized mice. The effects of the venom were further tested on both atrial and Langendorff heart preparations of rats, in addition to its cardiovascular effects in anesthetized mice. The venom (0.1 mg/kg, i.v.) produced a transient hypertension followed by fluctuation of arterial blood pressure, leading to cardiac failure within 20 min. Various kinds of ECG changes, including S-T depression and A-V block were observed within 10 sec after injection. A dose as low as 1 microgram of venom injected into the perfusion system produced a marked coronary vasospasm in the Langendorff heart preparation, whereas no deleterious effect was found in the atrial preparation at a concentration as high as 10 micrograms/ml. It is concluded that the cardiotoxic effects of the venom are primarily due to coronary vasospasm.

A study of cardiotoxic principles from the venom of Bungarus fasciatus (Schneider).

Abstract Two cardiotoxin-like fractions (VI A and VI B) were isolated from the venom of Bungarus fasciatus (Schneider) by means of column chromatography on CM cellulose. The homogeneity of these two fractions was verified by microzone and disc electrophoresis. These two fractions shared with cobra cardiotoxin (CTX) many pharmacological actions. Both toxins produced skeletal muscle contracture, depression on cardiac muscle, blockade of neuromuscular transmission, local irritation and had anticholinesterase activity. However, the molecular weights of VI A and VI B were about two times that of CTX and the amino acid composition was also quite different from that of CTX. Moreover, both VI A and VI B were devoid of direct hemolytic action. The contracture inducing activity of VI B and CTX could be abolished by EDTA or high Ca 2+ (12 mM) and accelerated by low Ca 2+ (0.5 mM) medium. However, a high Mg 2+ (10 mM) medium inhibited VI B induced contracture but accelerated CTX contracture. Spermine, spermidine and protamine were without effect on VI B contracture but accelerated profoundly CTX induced contracture. From these results, it is considered that the mode of contracture inducing activity of VI B may be different from that of CTX.

Comparative studies on the biological activities of cardiotoxin, melittin and prymnesin.

SummaryThe actions of cardiotoxin (CTX), melittin and prymnesin were compared on dog erythrocytes, chicken biventer, chicken biventer cervicis muscle, rabbit conjunctiva, acetylcholinesterase, succinate-cytochrome c reductase and turbidity of the rat liver mitochondrial suspension.1.CTX and melittin were approximately equipotent in the various biological activities, while prymnesin was not.2.The rate of direct hemolysis induced by CTX was slow, while that induced by either melittin or prymnesin was fast.3.Phosphate ions, 10mM Ca++, as well as 1 mM reduced glutathione, considerably inhibited the CTX-induced hemolysis, but only slightly inhibited that induced by melittin or prymnesin.4.CTX, melittin and prymnesin caused contracture of the chicken biventer cervicis muscle. Prymnesin was much less active in this preparation as compared with its hemolytic potency. The CTX contracture was completely inhibited by high Ca++ (10 mM) medium, while the melittin contracture was not.5.The rate of CTX contracture to reach the peak tension was increased when the concentration of CTX was increased, while the rate of melittin contracture did not change very much as the concentrations varied.6.All three toxins caused a local irritation of the conjunctival sac of the rabbit eye.7.Both CTX and melittin inhibited acetylcholinesterase and succinate-cytochrome c reductase activities, and also increased the turbidity of the rat liver mitochondrial suspension, while prymnesin was totally inactive in these respects. It is concluded that the mechanism of actions of these toxins may be different at the molecular level. The role of the detergent properties of these toxins in their biological activities is discussed.

Species differences in susceptibility to elapid venoms

Birds (pigeon, chick and Formosan sharp-tailed munia) are extremely susceptible to the lethal action of the venom of Bungarus multicinctus as compared with mammals. Comparison of the neuromuscular blocking activity of α- and β-bungarotoxin in chicks biventer cervicis muscle reveals that avian muscle is particularly susceptible to β- but not to α-bungarotoxin. It is concluded that the transmitter-releasing mechanism of avian motor nerve endings is particularly vulnerable to the action of β-bungarotoxin. On the other hand, cats are highly resistant to cobra venom, if given s.c. It was found that the skeletal muscle of the cat is highly resistant to the neuromuscular blocking action of cobra venom.

Endothelin-induced activation of phosphoinositide turnover, calcium mobilization, and transmitter release in cultured neurons and neurally related cell types

Endothelin (ET)-related peptides robustly stimulated [3H]-inositol phosphate (IP) formation in cultured cerebellar granule cells, astrocytes, and C6 glioma cells. Their agonist selectivities were ET-1 = ET-2 greater than or equal to sarafotoxin S6b greater than ET-3 greater than big ET-1 for granule cells and ET-1 greater than or equal to ET-2 greater than or equal to S6b greater than big ET-1 greater than ET-3 for cerebellar astrocytes and C6 glioma cells. These effects were Ca(2+)-dependent but insensitive to antagonists of L-type Ca2+ channels and the Na+/Ca2+ antiporter. Pretreatment of cells with ET-1 or S6b induced homologous desensitization of phosphoinositide (PI) response mediated by ET receptors. Long-term pertussis toxin (PTX) treatment attenuated the phosphoinositide (PI) response in astrocytes and glioma but not in granule cells. ET-1 and its related peptides increased [Ca2+]i in C6 glioma by two distinct pathways: IP3-induced Ca2+ mobilization or receptor-operated Ca2+ influx. La3+, Mn2+, and Cd2+ inhibited the Ca2+ influx and sustained PI turnover, while Ca2+ mobilization was attenuated by phorbol ester and TMB-8. ET-induced Ca2+ influx was essential for the sustained [Ca2+]i increase and PI turnover. Homologous desensitization of [Ca2+]i increase was also noted. In cerebellar granule cells, ET evoked the release of [3H]D-aspartate from these neurons. This action appears to be dependent on PI hydrolysis and [Ca2+]i increase and modulated by protein kinase C.

Electrophysiological effects of cobra cardiotoxin on rabbit heart cells

Abstract Effects on transmembrane potentials of cardiotoxin isolated from Formosan cobra venom were studied on rabbit atrial cells by means of electrophysiological techniques. It was found that cardiotoxin at a concentration of 10 −5 g per ml caused a progressive and irreversible decrease of the resting potential in myocardial cells starting 5 min after addition of the toxin. The decrease in maximum diastolic repolarization of the pacemaker type cells occurred much later and to a much less extent. Associated with the decrease of resting potential, the magnitude and rate of rise of the action potential, the overshoot, the time to 80% repolarization and the spike ionic conductance were also decreased. The effect of the toxin on the membrane potential was not altered by either tetrodotoxin or sodium removal but was inhibited by high calcium. It is suggested that disintegration of the membrane structure is responsible for the membrane depolarization.

A nucleoside isolated from the venom of Bungarus multicinctus

During isolation of neurotoxins from the venom of Bungarus multicinctus by means of zone electrophoresis on starch, a non-protein fraction was found in addition to the 4 protein fractions obtained previously. It shows maximum absorbance at 252 mμ, positive Molischs test for sugar and orcinol test for pentose, but negative Lorings test for phosphate. It shows the same Rf value as guanosine on the paper-chromatogram, and its spectral data at various pH values are also identical with those of guanosine. Its content in the venom was estimated to be 1.1 ± 0.14 per cent. So far no biological effects could be attributed to the presence of guanosine in the venom.

A study on the cause of death due to waglerin-I, a toxin from Trimeresurus wagleri.

Waglerin-I, a lethal toxin isolated from the venom of Trimeresurus wagleri, consists of 22 amino acid residues with a proline-rich sequence. In the present study, we investigated the cause of death and the possible site of action of this toxin. In anesthetized mice, i.v. administration of waglerin-I at 0.5 microgram/g produced respiratory failure within 5 min. The blood pressure could be maintained by the application of artificial respiration immediately after respiratory arrest. Waglerin-I at 4 microM reversibly blocked the indirect twitch of the mouse phrenic nerve-hemidiaphragm preparation, but had no effect on the direct twitch. It is concluded that respiratory failure, resulting from neuromuscular block, is the primary cause of death due to waglerin-I in mice. In contrast to the results obtained in mice, in anesthetized rats no toxic effects on respiration and blood pressure were observed except for a transient hypotension up to 10 micrograms/g (i.v.). In accordance with the in vivo experiments, waglerin-I at concentrations up to 40 microM did not block the indirect twitch of the rat diaphragm. It thus appears that rats are resistant to the toxic effects of waglerin-I.

Effect of sea nettle (Chrysaora quinquecirrha) venom on isolated rat aorta

The venom from sea nettle (Chrysaora quinquecirrha) (1-10 micrograms/ml) produced an irreversible contraction of the isolated rat aortic ring that was slow in onset, increased with time, and reached maximum in about 10-20 min. The contraction was not inhibited by pretreatment with phenoxybenzamine, atropine, indomethacin, tetrodotoxin, ouabain, low Na+ or Na+-free medium, however, it was markedly decreased by the Ca2+ channel blockers, nifedipine and verapamil. In Ca2+-free medium, no increase in tension was produced by the venom. It is concluded that sea nettle venom causes a contraction of the rat aortic ring by increasing Ca2+ influx through the voltage-dependent Ca2+ channels.