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The Bothrops jararaca venom and the cardiovascular function
494
Abstracts/Toxicon 38 (2000) 487-595
been described as "neurotoxin like" PLA2 inhibitors. The mechanism of action of these toxin neutralizing factors has not been yet completely elucidated, but the antibothropic factor isolated by us from opossum serum acts as a metalloproteinase inhibitor. It forms non-covalent complexes with venom hemorrhagins, inhibits its hemorrhagic activity, but seems to have no action on either venom serine-proteinases or non-venom enzymes. The opossum antihemorrhagic factor inhibits Bothrops jararaca venom hydrolyses of fibrin, fibrinogen, gelatines I and V, laminin, fibronectin and collagen IV. This effective inhibition of the B. jararaca venom digestion of extracellular matrix proteins by the opossum antihemorrhagic factor could indicate that this hydrolytic process may play an important role in the hemorrhagic phenomenon. Besides antihemorrhagic capacity, the opossum antibothropic factor showed antilethal, antimyonecrotic, antioedematogenic and antihyperalgesic activities. On the other hand, several phospholipase A~ (PLA2) inhibitors have been isolated from the plasma of snakes, some of them participate in the mechanism of neutralization of the toxic effect of snake venoms. We isolated a crotoxin inhibitor from Crotahts durissus terrificus serum (CICS) which neutralizes the toxicity and inhibits the PLA2 activity of crotoxin, as well as of its CB subunit. We demonstrated that the inhibitor induces the dissociation of the two crotoxin subunits, forming a stable inactive complex with the CB subunit and releasing the acidic nonenzymatic subunit CA. Once the interaction between crotoxin and CICS is very similar to that of crotoxin with its acceptor, we proposed that CICS acts physiologically as a soluble crotoxin acceptor to retain the toxin in the snake vascular system, preventing its action on the neuromuscular system.
Round Table The Bothrops jararaca venom and the cardiovascular function. Antonio C.M. Camargo, Luiza A.F. Ferreira, Solange M.T. Serrano, Mirian A.F. Hayashi (Laborat6rio de Bioquimica e Biofisica, Instituto Butantan, S~o Paulo, Brazil). The discovery of bradykinin, in 1949, by Rocha e Silva et al., and, as a consequence, the discovery of the bradykinin-potentiating peptides (BPPs) present in Bothrops jararaca (Bj) venom, by Ferreira and Rocha e Silva in 1965, are probably the most important Brazilian scientific contributions to the biomedical sciences. These latter peptides were shown to be strong competitive inhibitors of the angiotensin-converting enzyme (ACE) which additionally degrades bradykinin. One of the Bj-BPPs led to the synthesis of Captopril by Squibb Laboratories used by millions of people to treat hypertension. Both discoveries derived from the studies of the hypotensive effect caused by envenoming with Bj venom. The ACE has been the critical metabolic target used by the pharmaceutical industry to generate antihypertensive drugs through the development of specific inhibitors. The structure-activity studies of the BPPs and analogs were essential for the development of the non-peptidic ACE inhibitors. Thirteen Bj-BPPs, containing
Abstracts / Toxicon 38 (2000) 487-595
495
from 5 to 13 amino acid residues, have been purified and sequenced so far. Recently, we described a 1.8 kb cDNA clone isolated from a Bothrops jararaca venom gland cDNA library which encodes a 256 amino acids precursor for bradykinin potentiating peptides and a C-type natriuretic peptide (PNAS, 94: 1189-1193,1997). Direct evidence for the presence of the natriuretic peptide in the crude low molecular weight fraction of the B. jararaca venom was shown both by the detection of its biological activity and by RIA. Northern blot analysis indicated the predominant expression of a 1.8 kb mRNA in the venom glands as well as in the spleen, the brain, and the pancreas. Our results indicate that the hypotention provoked by the Bj-evenoming could be, in part, explained by the rapid diffusion of toxic substances due to the local vasodilatation and increased capillary permeability leading eventually to a cardiovascular shock. Once in the circulatory system, a Bj-kininogenase releases bradykinin from plasma kininogen causing vasodilatation which is highly potentiated by the ACE inhibitory effect of the BPPs. Besides protecting bradykinin from degradation, the BPPs may also increase the sensitivity of the vascular smooth muscle bradykinin receptor to bradykinin intensifying the local and systemic vasodilatation. Moreover, the presence of the Bj-CNP may represent an additional factor aggravating the cardiovascular damages. Acknowledgements: Funded by FAPESP, CNPq and FINEP
Studies of natural toxins--a forward look. Franc Gubensek (Department of
Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana and Department of Biochemistry and Molecular Biology, J. Stefan Institute, Ljubljana, Slovenia). Our experience with snake toxins is based on 30 years of studies of venom proteins, mainly from the European long nosed viper Vipera ammodytes. Our investigations have centered on the presynaptically toxic and myotoxic phospholipase A2 (PLA2) isotoxins, named ammodytoxins and ammodytin, respectively. We have used a variety of approaches including protein isolation and sequencing, pharmacological and immunochemical studies, protein engineering, acceptor identification, analysis of cDNA and gene structure and molecular evolution of the PLA2 multigene family. Our view of future trends in snake venom research, although based on studies of a relatively non-problematic European species, is unlikely to differ significantly from studies of the majority of the more poisonous and lethal species from other continents. The molecular mechanism of action has been clarified for a number of postsynaptically acting alpha-neurotoxins which bind to acetylcholine receptors, for K + ion channel binding toxins and for some others, where binding of the toxin directly blocks the physiological function of the target protein on the presynaptic or postsynaptic side of the synaptic cleft. The neurotransmission blocking presynaptic action of PLA~ is, however, considerably more complicated. Although these toxins appear to be targeted to different membrane proteins their mode of action is essentially the same and probably a consequence of a series of events that follow the still not
Abstracts/Toxicon 38 (2000) 487-595
been described as "neurotoxin like" PLA2 inhibitors. The mechanism of action of these toxin neutralizing factors has not been yet completely elucidated, but the antibothropic factor isolated by us from opossum serum acts as a metalloproteinase inhibitor. It forms non-covalent complexes with venom hemorrhagins, inhibits its hemorrhagic activity, but seems to have no action on either venom serine-proteinases or non-venom enzymes. The opossum antihemorrhagic factor inhibits Bothrops jararaca venom hydrolyses of fibrin, fibrinogen, gelatines I and V, laminin, fibronectin and collagen IV. This effective inhibition of the B. jararaca venom digestion of extracellular matrix proteins by the opossum antihemorrhagic factor could indicate that this hydrolytic process may play an important role in the hemorrhagic phenomenon. Besides antihemorrhagic capacity, the opossum antibothropic factor showed antilethal, antimyonecrotic, antioedematogenic and antihyperalgesic activities. On the other hand, several phospholipase A~ (PLA2) inhibitors have been isolated from the plasma of snakes, some of them participate in the mechanism of neutralization of the toxic effect of snake venoms. We isolated a crotoxin inhibitor from Crotahts durissus terrificus serum (CICS) which neutralizes the toxicity and inhibits the PLA2 activity of crotoxin, as well as of its CB subunit. We demonstrated that the inhibitor induces the dissociation of the two crotoxin subunits, forming a stable inactive complex with the CB subunit and releasing the acidic nonenzymatic subunit CA. Once the interaction between crotoxin and CICS is very similar to that of crotoxin with its acceptor, we proposed that CICS acts physiologically as a soluble crotoxin acceptor to retain the toxin in the snake vascular system, preventing its action on the neuromuscular system.
Round Table The Bothrops jararaca venom and the cardiovascular function. Antonio C.M. Camargo, Luiza A.F. Ferreira, Solange M.T. Serrano, Mirian A.F. Hayashi (Laborat6rio de Bioquimica e Biofisica, Instituto Butantan, S~o Paulo, Brazil). The discovery of bradykinin, in 1949, by Rocha e Silva et al., and, as a consequence, the discovery of the bradykinin-potentiating peptides (BPPs) present in Bothrops jararaca (Bj) venom, by Ferreira and Rocha e Silva in 1965, are probably the most important Brazilian scientific contributions to the biomedical sciences. These latter peptides were shown to be strong competitive inhibitors of the angiotensin-converting enzyme (ACE) which additionally degrades bradykinin. One of the Bj-BPPs led to the synthesis of Captopril by Squibb Laboratories used by millions of people to treat hypertension. Both discoveries derived from the studies of the hypotensive effect caused by envenoming with Bj venom. The ACE has been the critical metabolic target used by the pharmaceutical industry to generate antihypertensive drugs through the development of specific inhibitors. The structure-activity studies of the BPPs and analogs were essential for the development of the non-peptidic ACE inhibitors. Thirteen Bj-BPPs, containing
Abstracts / Toxicon 38 (2000) 487-595
495
from 5 to 13 amino acid residues, have been purified and sequenced so far. Recently, we described a 1.8 kb cDNA clone isolated from a Bothrops jararaca venom gland cDNA library which encodes a 256 amino acids precursor for bradykinin potentiating peptides and a C-type natriuretic peptide (PNAS, 94: 1189-1193,1997). Direct evidence for the presence of the natriuretic peptide in the crude low molecular weight fraction of the B. jararaca venom was shown both by the detection of its biological activity and by RIA. Northern blot analysis indicated the predominant expression of a 1.8 kb mRNA in the venom glands as well as in the spleen, the brain, and the pancreas. Our results indicate that the hypotention provoked by the Bj-evenoming could be, in part, explained by the rapid diffusion of toxic substances due to the local vasodilatation and increased capillary permeability leading eventually to a cardiovascular shock. Once in the circulatory system, a Bj-kininogenase releases bradykinin from plasma kininogen causing vasodilatation which is highly potentiated by the ACE inhibitory effect of the BPPs. Besides protecting bradykinin from degradation, the BPPs may also increase the sensitivity of the vascular smooth muscle bradykinin receptor to bradykinin intensifying the local and systemic vasodilatation. Moreover, the presence of the Bj-CNP may represent an additional factor aggravating the cardiovascular damages. Acknowledgements: Funded by FAPESP, CNPq and FINEP
Studies of natural toxins--a forward look. Franc Gubensek (Department of
Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana and Department of Biochemistry and Molecular Biology, J. Stefan Institute, Ljubljana, Slovenia). Our experience with snake toxins is based on 30 years of studies of venom proteins, mainly from the European long nosed viper Vipera ammodytes. Our investigations have centered on the presynaptically toxic and myotoxic phospholipase A2 (PLA2) isotoxins, named ammodytoxins and ammodytin, respectively. We have used a variety of approaches including protein isolation and sequencing, pharmacological and immunochemical studies, protein engineering, acceptor identification, analysis of cDNA and gene structure and molecular evolution of the PLA2 multigene family. Our view of future trends in snake venom research, although based on studies of a relatively non-problematic European species, is unlikely to differ significantly from studies of the majority of the more poisonous and lethal species from other continents. The molecular mechanism of action has been clarified for a number of postsynaptically acting alpha-neurotoxins which bind to acetylcholine receptors, for K + ion channel binding toxins and for some others, where binding of the toxin directly blocks the physiological function of the target protein on the presynaptic or postsynaptic side of the synaptic cleft. The neurotransmission blocking presynaptic action of PLA~ is, however, considerably more complicated. Although these toxins appear to be targeted to different membrane proteins their mode of action is essentially the same and probably a consequence of a series of events that follow the still not