Stimulation of calcium influx and platelet activation by canatoxin: methoxyverapamil inhibition and downregulation by cGMP.

Canatoxin (CNTX), a toxic protein isolated from seeds of Canavalia ensiformis, induces Ca2+ influx across the platelet plasma membrane, mobilization of arachidonic acid mediated by phospholipase A2, ATP secretion, and platelet aggregation. All these events depend on the presence of extracellular Ca2+ and are blocked by methoxyverapamil, a calcium-channel blocker. CNTX does not activate phospholipase C, and the intracellular calcium mobilization mediated by IP3 does not play a role in platelet activation by this toxin. Preincubation of rabbit platelets with 8-Br-cGMP inhibited the CNTX-evoked calcium influx, arachidonate release, ATP secretion, and cell aggregation. Our data suggest that the calcium influx is a prior step on platelet activation by CNTX, being modulated by cGMP.

Intraspecific variation in the venoms of the South American rattlesnake (Crotalus durissus terrificus).

The venom of eight individual Crotalus durissus terrificus snakes from the State of Minas Gerais, Brazil, in addition to pooled venom from Butantan Institute, were compared. Snakes were captured in distinct locations, some of them 600 km apart: Conselheiro Lafaiete, Entre Rios de Minas, Itauna, Itapecerica, Lavras, Patos de Minas, Paracatu, and Santo Antonio do Amparo. The crude venoms were tested for proteolytic, phospholipase A2, platelet aggregating, and hemagglutinating activities. The venoms were also analyzed by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF). Chromatographic patterns of venom proteins on both gel-filtration and anion-exchange chromatographies were also performed. All venoms presented high phospholipase A2 and platelet-aggregating activities, but only minimal hemagglutinating or proteolytic activities were found. Gel-filtration chromatography showed a characteristic profile for most venoms where four main peaks were separated, including the typical ones where convulxin and crotoxin were identified; however, peaks with high amounts of lower molecular weight proteins were found in the venoms from the Santo Antonio do Amparo location and Butantan Institute, characterizing these venoms as crotamine positive. Anion-exchange chromatographies presented a similar protein distribution pattern, although the number of peaks (up to ten) distinguished some venom samples. Consistent with these results, polyacrylamide gels that were silver stained after venom separation by PAGE or IEF presented a similar qualitative band distribution, although a quantitative heterogeneity was detected among venoms. Our results suggest that the variability found in venom components of C. d. terrificus venoms captured in Minas Gerais State may be genetically inherited and/or environmentally induced.

Proteolytic activation of canatoxin, a plant toxic protein, by insect cathepsin-like enzymes.

Canatoxin is a protein isolated from jackbean (Canavalia ensiformis), seeds. Injected intraperitoneally, the toxin is lethal to mice but it is inactive if given orally. Canatoxin is also lethal when fed to insects with cathepsin-based digestion while insects with trypsin-based digestion are not affected. The hypothesis that canatoxin is proteolytically activated by cathepsins was investigated. Experiments were performed with 4(th) instar and adult Rhodnius prolixus fed meals containing canatoxin (2.5 microg/mg weight body). While 100% of nymphs died, no effect was observed in adults. Hemolymph taken from nymphs and adults showed the presence of canatoxin's proteolytic fragments. Reduced lethality was seen in R. prolixus 4(th) instars fed meals containing canatoxin and inhibitors of cathepsin enzymes, E-64 (2.0 microM) or Pepstatin-A (2. 0 microM). In another approach, canatoxin was digested in vitro with enzymes from the bruchid, Callosobruchus maculatus, and the resulting peptides were tested in R. prolixus. Three groups of toxic peptides (8,000-12,000 kD range) were separated by gel-filtration. When these peptides were fed to the insects simultaneously with the cathepsin inhibitors, no protective effect was seen. These results confirm the proteolytic activation of canatoxin by insect cathepsin-like enzymes to produce entomotoxic peptide(s). Furthermore, our data point towards overlooked differences in the digestive physiology of distinct life stages of R. prolixus. Arch.