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nhibition of membrane-bound acetylcholinesterase by a new AChE inhibitor from the zoanthid Parazoanthus axinellae
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Abstracts
Inhibition of membrane-bound acetylcholinesterase by a new AChE inhibitor from the zoanthid Parazoanthus axinellae. J. Strupi-Suput,’ L. KamariE,’ T. Turk2 and D. Suput’ (‘School of Medicine, Institute of Pathophysiology, EDT Lab., P.O. Box 11, SLO-61105 Ljubljana, Slovenia; and 2Department of Biology, BFT, Ljubljana, Slovenia). It has been shown recently that the extract from the zoanthid crust coral Parazoanthus axinellae is lethal to crabs and mice. The in vivo effects of the extract were typical of an AChE inhibition. The purified substance isolated from the extract inhibits the purified AChE from electric eel in 1 PM concentration, which is comparable to the potency of physostigmine, the first described natural AChE inhibitor. Since the effects and possible therapeutic applications of this new substance would depend on the specificity of actions on different membrane-bound AChEs, it seemed reasonable to study the effects of the purified inhibitor on BuChE in human plasma, on AChE in bovine superior cervical ganglion and iris, and on human red blood cells. Each sample was homogenized and the activity of AChE was measured by continuous pH-metric titration at constant substrate concentrations, pH 7.4, at 25°C. In a control experiment the inhibition by ethanolic extract of the purified electric eel AChE was measured using the same method. The maximal inhibition of the human erythrocyte AChE was about 80%. The same concentration inhibited only 25% of BuChE activity in plasma. Although the difference was significant it is not sufficient to consider the new inhibitor as a selective AChE inhibitor. The new inhibitor has a structure not previously seen among the cholinesterase inhibitors, Therefore it may be an interesting starting point to develop new drugs to be used in human disease. Preliminary experiments on the insect AChE suggest that the inhibitor could also serve as a model for the development of new pesticides.
ShK is a potassium channel blocker from the Caribbean sea anemone Stichodactyla helianthus. 0. Castaiieda,’ E. G. Rowan, L. Young,’ A. L. Harvey2 and E. Karlsson3 (‘Facultad de Biologia, Universidad de La Habana, Cuba; *Department of Physiology and Pharmacology, Strathclyde Institude for Drug Research, University of Strathclyde, Glasgow, U.K.; and )Department of Biochemistry, Biomedical Center, Box 576, 751 23, Uppsala, Sweden). For many years sea anemones have constituted a source of neuroactive compounds mainly modifying kinetic aspects of sodium channels. In addition, potassium channel blocking activity has been discovered in the aqueous extract of Stichodactyla helianthus and Bunodosoma granulifera (Karlsson et al., 1991), and recently two potassium channel blockers have been isolated: BgK (Aneiros et al., 1993) and ShK (Castafieda ef a/., 1995). ShK is a 35 amino acid residue peptide with three disulphide bonds, and a mass of 4054 Da. The toxin is a presynaptic facilitator, as demonstrated by its action upon neuromuscular transmission in indirectly stimulated chick biventer cervicis neuromuscular preparations. It also inhibits the binding of dendrotoxin (a probe for voltage-dependent potassium channels) to rat brain synaptosomal membranes with an apparent K, of around 0.5 nM. The interaction with dendrotoxin is non-competitive. The toxin also diminishes the potassium channel currents in rat dorsal root ganglion neurons and in NG108-15 neurons in culture. Aneiros, A. et al. (1993) Biochim. biophys. Acta 1157, 8692. Castatieda, 0. et al. (1995) Toxicon 33, 603613. Karlsson, E. et al. (1991) Toxicon 29, 1168.
Cardiovascular effects of equinatoxin III. R. Frangei Toxinology, Institute of Pathophysiology, University Slovenia).
and D. Suput (Laboratory for Cell Physiology and of Ljubljana, School of Medicine, 61105 Ljubljana,
Equinatoxin III is one of the three isotoxins isolated from the Adriatic sea anemone Actinia equina. Its effects in viuo and on isolated organs have not yet been studied. Cardiorespiratory arrest due to the coronary vasoconstriction is the main reason for the lethality of a similar equinatoxin (II). Equinatoxin III is more haemolytic but less toxic than equinatoxin II. The aim of our study was to compare mechanisms responsible for the lethal activity of the two toxins. The toxin was tested in vivo on rats and in vitro on isolated pig coronary arteries. Equinatoxin III was injected intravenously and the arterial blood pressure, the electrocardiogram, and respiratory activity were measured. The tension of the vascular smooth muscle in vitro was measured isometrically by a mechanoelectrical transducer. In viva the effects of equinatoxin III were similar to the effects of equinatoxin II. Immediately after application of the toxin the animal stopped breathing, and soon a marked arrhythmia developed. Analysis of the blood samples revealed haemolysis, elevated plasma potassium levels and degranulation of neutrophils and basophils. Artificial respiration and vagotomy had no effect on the survival of the experimental animal. Intravenous application of potassium chloride solution had no significant effects. Neither equinatoxin III in 1.5 PM concentration, nor potassium (15 mM final concentration) in the bath had any effects on the contraction of the pig coronary artery. We conclude that hyperkaliemia can contribute to the lethality of the toxin but is not its primary cause. It also seems that the drop in coronary perfusion observed on
Abstracts
Inhibition of membrane-bound acetylcholinesterase by a new AChE inhibitor from the zoanthid Parazoanthus axinellae. J. Strupi-Suput,’ L. KamariE,’ T. Turk2 and D. Suput’ (‘School of Medicine, Institute of Pathophysiology, EDT Lab., P.O. Box 11, SLO-61105 Ljubljana, Slovenia; and 2Department of Biology, BFT, Ljubljana, Slovenia). It has been shown recently that the extract from the zoanthid crust coral Parazoanthus axinellae is lethal to crabs and mice. The in vivo effects of the extract were typical of an AChE inhibition. The purified substance isolated from the extract inhibits the purified AChE from electric eel in 1 PM concentration, which is comparable to the potency of physostigmine, the first described natural AChE inhibitor. Since the effects and possible therapeutic applications of this new substance would depend on the specificity of actions on different membrane-bound AChEs, it seemed reasonable to study the effects of the purified inhibitor on BuChE in human plasma, on AChE in bovine superior cervical ganglion and iris, and on human red blood cells. Each sample was homogenized and the activity of AChE was measured by continuous pH-metric titration at constant substrate concentrations, pH 7.4, at 25°C. In a control experiment the inhibition by ethanolic extract of the purified electric eel AChE was measured using the same method. The maximal inhibition of the human erythrocyte AChE was about 80%. The same concentration inhibited only 25% of BuChE activity in plasma. Although the difference was significant it is not sufficient to consider the new inhibitor as a selective AChE inhibitor. The new inhibitor has a structure not previously seen among the cholinesterase inhibitors, Therefore it may be an interesting starting point to develop new drugs to be used in human disease. Preliminary experiments on the insect AChE suggest that the inhibitor could also serve as a model for the development of new pesticides.
ShK is a potassium channel blocker from the Caribbean sea anemone Stichodactyla helianthus. 0. Castaiieda,’ E. G. Rowan, L. Young,’ A. L. Harvey2 and E. Karlsson3 (‘Facultad de Biologia, Universidad de La Habana, Cuba; *Department of Physiology and Pharmacology, Strathclyde Institude for Drug Research, University of Strathclyde, Glasgow, U.K.; and )Department of Biochemistry, Biomedical Center, Box 576, 751 23, Uppsala, Sweden). For many years sea anemones have constituted a source of neuroactive compounds mainly modifying kinetic aspects of sodium channels. In addition, potassium channel blocking activity has been discovered in the aqueous extract of Stichodactyla helianthus and Bunodosoma granulifera (Karlsson et al., 1991), and recently two potassium channel blockers have been isolated: BgK (Aneiros et al., 1993) and ShK (Castafieda ef a/., 1995). ShK is a 35 amino acid residue peptide with three disulphide bonds, and a mass of 4054 Da. The toxin is a presynaptic facilitator, as demonstrated by its action upon neuromuscular transmission in indirectly stimulated chick biventer cervicis neuromuscular preparations. It also inhibits the binding of dendrotoxin (a probe for voltage-dependent potassium channels) to rat brain synaptosomal membranes with an apparent K, of around 0.5 nM. The interaction with dendrotoxin is non-competitive. The toxin also diminishes the potassium channel currents in rat dorsal root ganglion neurons and in NG108-15 neurons in culture. Aneiros, A. et al. (1993) Biochim. biophys. Acta 1157, 8692. Castatieda, 0. et al. (1995) Toxicon 33, 603613. Karlsson, E. et al. (1991) Toxicon 29, 1168.
Cardiovascular effects of equinatoxin III. R. Frangei Toxinology, Institute of Pathophysiology, University Slovenia).
and D. Suput (Laboratory for Cell Physiology and of Ljubljana, School of Medicine, 61105 Ljubljana,
Equinatoxin III is one of the three isotoxins isolated from the Adriatic sea anemone Actinia equina. Its effects in viuo and on isolated organs have not yet been studied. Cardiorespiratory arrest due to the coronary vasoconstriction is the main reason for the lethality of a similar equinatoxin (II). Equinatoxin III is more haemolytic but less toxic than equinatoxin II. The aim of our study was to compare mechanisms responsible for the lethal activity of the two toxins. The toxin was tested in vivo on rats and in vitro on isolated pig coronary arteries. Equinatoxin III was injected intravenously and the arterial blood pressure, the electrocardiogram, and respiratory activity were measured. The tension of the vascular smooth muscle in vitro was measured isometrically by a mechanoelectrical transducer. In viva the effects of equinatoxin III were similar to the effects of equinatoxin II. Immediately after application of the toxin the animal stopped breathing, and soon a marked arrhythmia developed. Analysis of the blood samples revealed haemolysis, elevated plasma potassium levels and degranulation of neutrophils and basophils. Artificial respiration and vagotomy had no effect on the survival of the experimental animal. Intravenous application of potassium chloride solution had no significant effects. Neither equinatoxin III in 1.5 PM concentration, nor potassium (15 mM final concentration) in the bath had any effects on the contraction of the pig coronary artery. We conclude that hyperkaliemia can contribute to the lethality of the toxin but is not its primary cause. It also seems that the drop in coronary perfusion observed on