THESIS: E.L.A. Malpezzi submitted this thesis for the degree of Ph.D. publicly examined at the Department of Physiology of the Institute of Biosciences, University of São Paulo, Brazil, in November 1996.
Advisor: Prof. Dr. José Carlos de Freitas
ABSTRACT. Biologically active compounds from the Brazilian sea anemone Bunodosoma caissarum have been studied chemically and pharmacologically for many years. The purpose of this work was to continue these studies focusing on the toxins contained in the nematocysts, as well as their activities, because many toxic effects were attributed to sea anemones, but without interest in the origin of these substances in animal tissues or cells. The nematocyst venom was extracted by electrical stimulation of the animal maintained under artificial sea water. This method allows the extraction of a practically pure venom and may be considered a physiological technique. Therefore, the animal can be reused or returned to the sea. The experiments performed with the venom showed new activities of this species such as the phospholipase activity and the forming of channels in artificial lipid bilayers. Both activities are related to the hemolytic fraction of the venom. The fractionation by Sephadex G-50 resin allowed a specific study in terms of mechanism of action with the isolated components. After monitoring the activities by chromatograms, four regions were combined: a big peak without any determined activity, an hemolytic fraction and two neurotoxic fractions with action on voltage dependent sodium (N1) and potassium channels (N2). The results obtained with the neurotoxic fractions N1 and N2 in the crustacean nerve by sucrose-gap technique and in the chick neuro-muscular biventer cervicis preparation showed the effects resulting from the main activity of each fraction. In the first method, it was observed that the increase in the duration of the action potential induced by N1 was due to the maintenance of the sodium channel in the open state for a longer period; and it is increased by the addition of the N2 fraction that blocks the potassium channels. But, if the order of addition of the toxins were inverted, the final response would be lower since the blockade of potassium channels depolarizes the membrane that consequently inactivates the voltage-dependent sodium channels. So, as the N1 toxin needs open channels to act, a smaller number of channels will be affected. It was also observed that the response of the preparation to the N2 toxin is similar to the response to TEA (Tetraethylammonium). In the chick preparation, the response was due to the same effect since N1 increases the muscular contraction while N2 is acting, the resulting response with the time is a decrease in the contraction. The binding assays were important in the verification of the presence of a toxin with blocking action on potassium channels, in the same manner as the 
-dendrotoxin isolated from the green mamba, Dendroaspis angusticeps. Therefore, it was very important the purification and the sequencing of the Bc-III, the toxin with action on sodium channel, because the high homology with the other long type 1 toxins isolated from sea anemones. Focusing on the hemolytic fraction, the ability in forming pores in artificial lipid bilayers was verified and the loss of activity with the purification was also verified. These pores behave like ionic channels in some instances, showing conductances proportional to the tested toxin concentration; also showing voltage dependence. They are channels that, in general, when formed stay open and it seems that they are the responsible for the hemolytic activity, already verified during the monitoring phase and also observed under microscope analysis. Phospholipase A2 activity was verified in the hemolytic fraction of the venom that may participate in the hemolysis with the pore-forming toxin. The fraction containing these pore-forming polypeptides was purified and one of them was submitted to sequencing. This hemolysin was called bunodolysin or Bc-H. In relation to the cardiotonic effects, it seems that the hemolytic fraction, also the neurotoxic N1 fraction are responsible for the effects. These effects would be due to the cationic entrance in the cells, both by maintenance of the sodium channels in the open state and/or by pore opening in the membrane. This effect would result in an increase of the activity of the exchanger sodium-calcium, resulting in an increase of cytosolic calcium with consequent cardiotonic effect.
REFERENCES
01 MALPEZZI ELA., FREITAS JC., MURAMOTO K., KAMIYA H. Characterization of peptides in sea anemone venom isolated by a novel procedure. Toxicon, 1993, 31, 853-64.
CORRESPONDENCE TO:
E.L.A. MALPEZZI - Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, Travessa 14, nº. 10, CEP 05.508-900, São Paulo, SP, Brasil.
Publication Dates
-
Publication in this collection
08 Jan 1999 -
Date of issue
1997
