Effects of the sea anemone Anemonia sulcata toxin II on skeletal muscle and on neuromuscular transmission

Effects of anemone toxin II (ATX II) have been analysed on the neuromuscular junction of the frog and different twitch muscles. Amplitudes of evoked endplate potentials and endplate currents are increased by ATX II, without effects on the amplitudes of miniature endplate potentials and endplate currents resulting from ionophoretically applied transmitter. The increase in evoked transmitter release is due to an increase in quantal content caused by an effect of the toxin on the presynaptic action potentials. ATX II is also effective on muscle fibers. The action potentials of frog twitch muscles are reversibly prolonged by ATX II. Their rate of rise and amplitudes are increased, while there is no effect on resting membrane potential. Similarly, action potentials of fast twitch muscle (extensor digitorum longus, EDL) of the mouse are reversibly prolonged by ATX II. In slow twitch muscle (soleus, SOL) of the mouse the toxin induces repetitive action potentials following the generation of a single action potential. Tetrodotoxin resistant action potentials of both denervated EDL and SOL are greatly and irreversibly prolonged by ATX II. The effects on muscle are due to a Na+ channel specific action of ATX II. Na+ current inactivation is slowed with the time constant tau h increasing towards positive membrane potentials. The steady state inactivation curve hoo was shifted to more positive potentials and its slope reduced.     

The action of equinatoxin,a peptide from the venom of the sea anemone, Actinia equina, on the isolated lung

On perfusion through isolated lungs from male Sprague-Dawley rats, equinatoxin caused a dose-dependent increase in the wet to dry weight ratio. Ratios were significantly elevated above control values at equinatoxin concentrations of 80–200 ng/ml. The increased ratios were accompanied by an increase in the permeability of the lung vasculature. When equinatoxin was perfused through isolated lungs at concentrations of 100 ng/ml or greater, significantly more [³H]polyethylene glycol (PEG; approximately 900 mol. wt) was retained in the extravascular space as compared to controls. Perfusion pressures of the lung were significantly elevated above controls at equinatoxin concentrations greater than 100 ng/ml. These effects of equinatoxin were not mediated by degranulation of mast cells, as preperfusion of the lung with 100 or 200 μM Na cromolyn or 1 μM lanthanum chloride did not modify the pulmonary response to equinatoxin. At concentrations of equinatoxin below 150 ng/ml the fluid movement appears to be restricted primarily to intracellular, or possibly interstitial, spaces, as no significant amounts of [³H]polyethylene glycol were recovered by tracheal lavage. At concentrations of equinatoxin equal to or greater than 150 ng/ml, significant amounts of PEG were washed from the trachea. As it is a potent inducer of pulmonary edema, equinatoxin may become an important probe to study fluid regulation in the lung.     

Cardiovascular and respiratory effects induced by a purified scorpion toxin (tityustoxin) in unanesthetized rats

Intravenous injection of a purified scorpion toxin (tityustoxin) into unanesthetized rats induced acute pulmonary edema, which was directly related to the dose and the time of intoxication. To study the cardiovascular and respiratory effects evoked by an edematogenic dose of the toxin (1 mg/kg), the following parameters were recorded in unanesthetized rats: systolic, diastolic and mean arterial pressure; central venous pressure; electrocardiogram; respiratory movements. The toxin induced acute systolic and diastolic hypertension, bradycardia and bradypnea. During a 1 hr period, the systolic, diastolic and mean arterial pressure fell progressively to control values, whereas the central venous pressure did not change significantly. The cardiac and respiratory rates remained lower than the control values during a 1 hr period. Several changes in the respiratory pattern were recorded, such as gasping, prolonged and labored expiration, ataxic rhythm and noisy inspiration with the mouth open. These respiratory changes were explained, in part, by the presence of edema in the lungs and froth in the trachea. From a group of 24 rats, 6 died 18 – 30 min after tityustoxin injection. The cause of death was apnea. The female rats were more susceptible to pulmonary edema and death than the male rats.     

Effect of calcium on proteolytic activity and conformation of hemorrhagic toxin I from five pace snake (Agkistrodon acutus) venom

J. J. Zhang, Z. X. Chen, Y. Z. He and X. Xu. Effect of calcium on proteolytic activity and conformation of hemorrhagic toxin I from five pace snake (Agkistrodon acutus) venom. Toxicon22, 931–935, 1984. — AaHI, a proteolytic hemorrhagic toxin from A. acutus venom, contains one g-atom Zn per mole protein and 2 g-atoms Ca per mole protein. AaHI is activated by calcium and slightly inhibited by zinc. When treated with EDTA, AaHI is completely inactivated. When dialyzed against 1,10-O-phenanthroline, 50–80% of the metal content and activities are lost, while 90% of the hemorrhagic and proteolytic activities are retained when dialyzed against 1,10-O-phenanthroline containing 5mM Ca²⁺. The results suggest that calcium is essential for the hemorrhagic and proteolytic activities. The circular dichroism spectra show that calcium may play an important role in maintaining a proper structure for AaHI. The function of zinc is not clear.     

Purification and properties of a toxin from the South African sea anemone, Pseudactinia varia

A comparison was made of the hemolytic potency of aqueous extracts prepared from five species of intertidal sea anemones from the coast of South Africa. The active agent in an extract of Pseudactinia varia was purified by ammonium sulfate precipitation, gel permeation chromatography and isoelectric focusing. The hemolytic toxin, termed variolysin, is a protein having a molecular weight of 19,500 and an isoelectric pH of 9.8. It retained appreciable activity after heating to 70 degrees for 40 min. Amino acid analysis revealed that it lacked methionine and cysteine. Its hemolytic activity was inhibited by sphingomyelin. The properties of variolysin show that it is broadly similar to cytolytic toxins isolated from a number of other anthozoans.     

Pharmacological activity of alkaloids from poison-dart frogs (Dendrobatidae)

Batrachotoxin, pumiliotoxin B, isodihydrohistrionicotoxin, pumiliotoxin C and gephyrotoxin represent five different classes of skin alkaloids from the dendrobatid poison dart frogs. In quinea pig ileum segments batrachotoxin and pumiliotoxin B caused rhythmic contractures which were prevented by tetrodotoxin. In quinea pig atria, batrachotoxin and pumiliotoxin B had positive inotropic and chronotropic effects, followed in the case of batrachotoxin by irregular arrhythmias and atrial arrest. Isodihydrohistrionicotoxin has no apparent effect in ileum or atrium, while gephyrotoxin was a potent muscarinic antagonist. In rat phrenic nerve-diaphragm, batrachotoxin blocked both direct and indirect-elicited twitch; pumiliotoxin B potentiated both direct and indirect-elicited twitch, isodihydrohistrionicotoxin blocked indirect-elicited twitch and gephyrotoxin had no apparent effect. Pumiliotoxin C had no apparent effect in any of the preparations.     

The presynaptic neuromuscular blocking effect and phospholipase A2 activity of textilotoxin, a potent toxin isolated from the venom of the Australian brown snake, Pseudonaja textilis

In the presence of Ca²⁺ and deoxycholate, textilotoxin has, on a molar basis, about the same phospholipid-splitting activity as β-bungarotoxin and other presynaptically acting toxins. Sr²⁺ inhibited the enzyme activity in the presence of Ca²⁺ by about 90%. Textilotoxin blocked neuromuscular transmission without significant change of muscle contractility. As with taipoxin, the mouse diaphragm was more susceptible than the chick biventer cervicis and the rat diaphragm preparations. After complete neuromuscular blockade, the amplitude of spontaneous miniature endplate potentials (m.e.p.p.) and the membrane potential of the mouse diaphragm remained unchanged, as did the contractile response of the chick biventer cervicis muscle to bath-applied acetylcholine. Textilotoxin caused an immediate facilitation of transmitter release in the mouse diaphragm lasting for about 4 min, followed by depression lasting for about 15 min and then a second phase of facilitation lasting for about 25 min, before causing final inhibition. The frequency of m.e.p.p.s was first depressed, then increased twice before the final depression. The time-course, however, was slower than the change of evoked transmitter release. Like all other presynaptic phospholipase A neurotoxins, textilotoxin bound rapidly to the nerve terminal and its blocking effect was antagonized by high Mg²⁺ and Ca²⁺ media, but was markedly enhanced on increasing the stimulus frequency from 0.1 Hz to 1.0 Hz. Sr²⁺ strongly antagonized the neuromuscular blocking effect of textilotoxin only at 1.0 Hz stimulation, but not at 0.1 Hz. It is concluded that textilotoxin acts mainly on the presynaptic nerve terminal, with multiphasic changes of transmitter release. The final blockade is due to its phospholipase A activity acting inside the terminal axolemma.     

The Bushman arrow toxin, diamphidia toxin: Isolation from pupae of Diamphidia nigro-ornata

J. McK. R. Woollard, F. A. Fuhrman and H. S. Mosher. The Bushman arrow toxin, diamphidia toxin: isolation from pupae of Diamphidia nigro-ornata. Toxicon22, 937–946, 1984. — The Bushmen of the Kalahari Desert in Botswana use the pupae of the beetle Diamphidia nigro-ornata Ståhl to poison their arrows. Sequential aqueous extraction, ammonium sulfate precipitation, ultrafiltration and chromatofocusing have given an apparently homogeneous active protein from these pupae with an approximate mol. wt of 54,000, an isolectric point of about 8.0 pH and a lethal potency (minimum lethal dose, mld) between 5 and 20 μg/kg (i.p. mouse). Preliminary pharmacological studies on less purified material show that, after a delay, this diamphidia toxin causes sustained contraction of isolated intestinal smooth muscle. This contraction is not blocked by atropine or mepyramine and, therefore, is not due to release of acetylcholine or histamine. Results on the phrenic nerve - hemidiaphragm preparation demonstrate that in the presence of the toxin, contraction in response to indirect stimulation gradually fails and is accompanied by contracture. Since direct stimulation of the muscle still elicits a contraction, the toxin apparently does not affect the contractile mechanism itself. We conclude that diamphidia pupae contain a protein toxin that is responsible for its lethality. Although this toxin appears to differ in some properties from the toxins reported by Mebs et al., de la Harpe et al. and Kündig, these protein preparations undoubtedly correspond to each other. We did not find any evidence of the low molecular weight toxic component reported by Mebs et al.     

Capillary permeability-increasing enzyme from the venom of Agkistrodon caliginosus (kankoku-mamushi): activity due to the release of peptide material from a protein in bovine plasma

When a mixture of the purified capillary permeability-increasing enzyme from A. caliginosus venom and bovine plasma or heated bovine plasma was injected into the depilated skin of the back of a rabbit, the capillary permeability-increasing activity was much greater than that induced by injection of the enzyme alone. The substance which increases capillary permeability was extracted from the incubated mixture of bovine plasma and enzyme with 50 – 70% ethanol. Its activity was lost when treated with carboxypeptidase A. Thus, it is supposed that the increase in capillary permeability induced by the enzyme is due to a low molecular weight peptide released from a protein in bovine plasma by the action of the enzyme. No liberation by the enzyme of histamine or anaphylatoxins of the complement system was found.     

Purification of a proteinase (Ac5-proteinase) and characterization of hemorrhagic toxins from the venom of the hundred-pace snake (Agkistrodon acutus)

Ac5-Proteinase (15.2 mg) was isolated from Agkistrodon acutus venom (1 g) by column chromatography on Sephadex G-75, CM-Sephadex C-50 and CM-Cellulose. Ac5-Proteinase was homogeneous by disc electrophoresis on polyacrylamide gel at pH 4.3 and also by SDS-disc polyacrylamide gel electrophoresis. Ac1-, Ac2-, Ac3- and Ac5-proteinases possessed lethal and hemorrhagic activities, but Ac4-proteinase had no lethal activity. These activities were inhibited completely by ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline or cysteine. The molecular weights of Ac1-, Ac2-, Ac3-, Ac4- and Ac5-proteinases were approximately 24,500, 25,000, 57,000, 33,000 and 24,000, respectively. Ac1-, Ac2-, Ac4- and Ac5-proteinases did not contain any carbohydrates, but Ac3-proteinase contained 0.1% carbohydrate by weight.     

Presynaptic effects of snake venom toxins which have phospholipase A2 activity (beta-bungarotoxin, taipoxin, crotoxin)

The presynaptic effects of beta-bungarotoxin, crotoxin and taipoxin were studied in the mouse phrenic nerve-diaphragm preparation (27 degrees C). The phospholipase A2 activity, assayed by pH-stat titration, was reduced to 4-10% at 27 degrees C compared with that at 37 degrees C. The late neuromuscular blocking activity was also reduced by more than three fold for all toxins. In contrast, the early biphasic response to the toxins, i.e. immediate depression followed by facilitation, was not delayed. The evoked quantal release of acetylcholine was enhanced by all toxins at low Ca2+-concentrations during the phase of facilitation, without an increase of the maximal release. At the late phase of treatment with beta-bungarotoxin and taipoxin, the curve relating the quantal contents of endplate potentials with Ca2+-concentration was shifted parallel to the right at low Ca2+, but marked depression of the maximal release occurred at high Ca2+. When diaminopyridine was added at the time of the late phase block by beta-bungarotoxin, the quantal release could still be enhanced at low Ca2+-concentrations, even beyond control; however, the maximal release was not simultaneously restored. It is concluded that the late phase block, but not the early biphasic response, is due to an enzymatic action and the release mechanism is abolished when the hydrolysis of membrane phospholipids proceeds to a certain critical level.     

Effect of the catecholamine-depleting agent 1-phenyl-3-(2-thiazolyl)-2-thiourea (U-14,624) on drug metabolism in the rat

Male rats injected with 1-phenyl-3-(2-thiazolyl)-2-thiourea (U-14,624) (25 mg/kg/day i.p.) for 3 days prior to induction of anesthesia with pentobarbital (40 mg/kg, i.p.) slept significantly (P < 0.05) longer than control animals. Plasma and brain half-lives of pentobarbital were also prolonged in the treated animals, but both control and treated groups awakened with similar brain levels of pentobarbital. In addition, the plasma half-life of antipyrine in treated animals was also prolonged significantly. Subacute administration of U-14,624 (50 mg/kg/day i.p.) to male rats for 5–7 days suppressed the activities of aminopyrine N-demethylase, aniline hydroxylase and p-nitroanisole O-demethylase enzymes in vitro; this effect could not be demonstrated at lower doses. Single doses of U-14,624 (100–200 mg/kg, i.p.) also suppressed the activities of the three oxidative enzymes. The suppression was positively correlated with reduced levels of hepatic microsomal cytochrome P-450. Levels of cytochrome b₅ and NADPH-cytochrome c reductase activity were not affected consistently by acute dosage with U-14,624. The inhibitory effects of single doses (100–400 mg/kg, i.p.) on all enzymatic systems were reversible, and recovery was complete within 48 hr. Whereas all three oxidative drug-metabolizing enzymes were inhibited in a mixed manner by in vitro exposure to U-14,624 (10^?5–10^?2 M), neotetrazolium diaphorase was not inhibited by U-14,624 at concentrations as high as 5 mM. Inhibition of oxidative drug metabolism by U-14,624 is mechanistically related to depletion of cytochrome P-450, but inhibition of these enzymes in vitro indicates that a second inhibitory mechanism may also be operative.     

Basic phospholipase A2 from Naja nigricollis snake venom: phospholipid hydrolysis and effects on electrical and contractile activity of the rat heart

To clarify the mechanism of the cardiotoxic action of the basic phospholipase A₂ from Naja nigricollis snake venom, its effects were tested on the rat heart, including hydrolysis of cardiac phospholipids, electrocardiogram, (Na + K)-ATPase activity, resting and action potentials, and cardiac contractility. Its effects contrasted with those of a noncardiotoxic acidic phospholipase from Naja naja atra snake venom. Only the N. nigricollis enzyme exhibited cardiotoxic effects in isolated perfused rat hearts. The cardiotoxic effects occurred with low levels of phospholipid hydrolysis, phosphatidylserine being hydrolyzed to the greatest extent. Although high concentrations of either phospholipase decreased (Na + K)-ATPase activity in vitro, this reaction did not account for the cardiotoxic effects observed in isolated perfused hearts. In rat atrial preparations the cardiotoxic enzyme depolarizes the cells, decreases action potential amplitude and overshoot, decreases time to 50 and 90% repolarization of action potential, and prolongs the latency to initiation of the action potential. Simultaneously recorded contractile activity showed prolonged latency to initiation of contraction, increased time to peak force of contraction, and decreased amplitude of contraction. These effects were mimicked by caffeine and 7.5 mm Ca²⁺ in the bathing medium. The cardiotoxic effects were blocked by Mn²⁺ (Mn²⁺ also reversed cardiotoxic actions) and 4.0 mm Ca²⁺, but not by lanthanum, tetrodotoxin, lidocaine, acetylcholine, or cyanide. Manganese added to rat atrial preparations before N. nigricollis phospholipase decreased hydrolysis of phosphatidylserine. These results suggest that the N. nigricollis enzyme exerts its cardiotoxic action by increasing intracellular Ca²⁺ levels. The cardiotoxic effects correlate with, but may not be related to, phosphatidylserine hydrolysis.     

Effect of honeybee (Apis mellifera) venom on the course of adjuvant-induced arthritis and depression of drug metabolism in the rat

The ability of honeybee venom to suppress Mycobacterium butyricum-induced arthritis was studied in Lewis rats. Bee venom, 2 mg.kg-1.day-1 for 24 days, suppressed but did not abolish the primary and secondary inflammatory responses to the adjuvant as monitored by decreases in the swelling of the left and right hind paws and adjuvant-induced arthritis on heme metabolism were also examined. Bee venom or adjuvant had no effect on hepatic delta-aminolevulinic acid synthase, porphyrin content, or ferrochelatase activity. However, with both treatments cytochrome P-450 and the associated enzymic activities of ethylmorphine N-demethylase and benzo[a]pyrene hydroxylase were depressed markedly. In contrast, both treatments caused several-fold enhancement of hepatic microsomal heme oxygenase activity. Adjuvant-treated rats receiving bee venom showed changes in heme metabolism which were of a magnitude similar to those observed when either agent was administered to the experimental animals. Although the bee venom appears to suppress adjuvant-induced arthritis to a greater extent in female than in male rats, the alterations in heme metabolism were similar in bee venom-treated male and female rats. The observed changes in heme metabolism elicited by the venom or by the adjuvant are strongly suggestive of perturbations of the immune system causing alterations in hepatic microsomal enzymes.     

The activity of membrane enzymes in homogenate fractions of rat kidney after administration of lead

Rats received one or two consecutive daily ip injections, each 0.5 mg Pb2+/100 g body weight, and the kidneys were studied 48 or 24 hr, respectively, after the injection. Renal brush border preparations from Pb2+-treated rats exhibited significant decreases in the activity of alanine aminopeptidase and gamma-glutamyl transpeptidase, greater after two injections, yet the amount of brush border protein remained unchanged. Moreover, the activity of alkaline phosphatase in the brush border was significantly increased after Pb2+. No significant changes in urine volume, urinary protein, or enzymes could be detected in these experiments. The enzymatic changes observed in the brush border after acute exposure to Pb2+ contrasted with those after exposure to Hg2+ where both the structure and enzymatic functions were severely damaged and after exposure to Cd2+ where enzymatic alterations were not accompanied by cytological changes.     

The effects of nitrogen mustard (HN2) on activities of the plasma membrane of PC6A mouse plasmacytoma cells

Nitrogen mustard, HN2 (10^?5 M), inhibited the transport of the potassium congener ⁸⁶rubidium into PC6A mouse plasmacytoma cells by 45% after a 4 hr incubation at 37° in vitro. HN2 (10^?3 M) had a rapid effect on the profile of ⁸⁶rubidium transport into PC6A cells when added simultaneously with the ⁸⁶rubidium whereas a monofunctional analogue of HN2((2-chloroethyl)dimethylamine) had no effect at 10^?3 M. The transport of the amino acid analogues α-aminoisobutyric acid and cycloleucine into PC6A cells was inhibited by 19% and 5% respectively after a 4 hr incubation with 10^?5 M HN2. The results suggest that the activity of plasma membrane Na⁺K⁺-ATPase may be affected by HN2. This enzyme may play a pivotal role in controlling cell growth and division. Crude cell membrane preparations from PC6A cells had variable Na⁺K⁺-ATPase activity which was possibly due to contamination with mitochondrial Mg²⁺-ATPase. Incubation of a crude cell membrane preparation in the presence of 40 nM dicyclohexylcarbodiimide gave constant Na⁺K⁺-ATPase activity which was inhibited by 44% on incubation with HN2 (10^?3 M) for 0.5 hr. The monofunctional analogue of HN2 inhibited this preparation by only 7% under the same conditions. It is suggested that inhibition of Na⁺K⁺-ATPase by HN2 may be an important facet of its cytotoxic activity.     

Sulphinpyrazone prevents in vivo the inhibitory effect of aspirin on rat platelet cyclo-oxygenase activity

Sulphinpyrazone reportedly inhibits in vitro platelet cyclo-oxygenase activity. This study shows that Sulphinpyrazone administration (200 mg/kg) to rats was followed by long lasting inhibition of platelet cyclo-oxygenase, as measured by malondialdehyde generation by sodium arachidonate. The inhibition was apparently competitive and could in fact be ascribed to supposed metabolites of the drug. When given 30min–6 hours before aspirin (5–25mg/kg), Sulphinpyrazone and to a considerable extent its metabolites significantly prevented permanent inhibition of platelet cyclo-oxygenase activity normally produced in vivo by aspirin. Sulphinpyrazone at 100 mg/kg was unable by itself to modify platelet malondialdehyde or thromboxane B₂ generation, yet if effectively interfered with aspirin activity. This suggests that Sulphinpyrazone and its metabolites may interact with a binding site on cyclo-oxygenase not directly involved with the enzyme activity. Interaction of aspirin with this binding site would be a prerequisite for its inhibitory effect on the enzyme active site.The clinical implications of this study include a reappraisal of the pharmacological basis for the association of Sulphinpyrazone and aspirin in thrombosis prevention trials.     

Species and organ specificity of the trans-stilbene oxide induced effects on epoxide hydratase and benzo(a)pyrene monooxygenase activity in rodents.

It was investigated, whether the selective induction of epoxide hydratase by trans-stilbene oxide (TSO) represents a general phenomenon or is confined to the liver of male rats where it was discovered. Therefore the effect of treatment with TSO on epoxide hydratase and benzo(a)pyrene mono-oxygenase activities were investigated in other organs (kidney, lung, skin, testis), other species (mice, hamsters) and also in female rats. In female rat livers the effect of TSO on the measured enzyme activities was very similar to that found in the male rat liver, i.e. a large induction of epoxide hydratase activity to 300–400 per cent of controls without affecting the benzo(a)pyrene monooxygenase activity. The potency of TSO to induce liver epoxide hydratase activity expressed as per cent of controls was 350:180:140 in rat, mouse and hamster, respectively. Selective induction of epoxide hydratase was found in rat and hamster liver, but not in the mouse liver, where benzo(a)pyrene monooxygenase activity was induced to about the same extent as the epoxide hydratase activity. The only extrahepatie organ in which an increased epoxide hydratase activity was found after TSO treatment was the rat kidney. Subcutaneous and topical treatment with TSO for 12 and 10 days respectively did not induce rat skin epoxide hydratase activity, instead a decrease of the enzyme activity to about 70 per cent ofthat found in control animals was found. Thus, TSO which was demonstrated to be a selective inducer of epoxide hydratase in rat liver can be utilized so far only in a limited number of carcinogenicity test systems, since it failed to induce the skin epoxide hydratase activity, which would have been an excellent tool to study directly the role of epoxide hydratase in the mechanism of skin tumor formation caused by polycyclic hydrocarbons. Interestingly, the epoxide hydratase of the hamster, investigated for the first time in this study, proved quite different from that of rat and mouse in that it hydrated styrene oxide remarkably faster than benzo(a)pyrene 4,5-oxide. This was true for all organs investigated. Also, the organ distribution of epoxide hydratase proved to be very different from that in rat and mouse. In the mouse the activity (with benzo(a)pyrene 4,5-oxide as substrate) was amongst all organs investigated highest in the testis (2.5 fold as compared to liver) but in the hamster the activity was more than 100 fold lower in testis as compared to liver. On the other hand, the activity in kidney was about 50 fold higher in hamster as compared to mouse.