Hyperalgesic and edematogenic effects of Secapin-2, a peptide isolated from Africanized honeybee (Apis mellifera) venom.

Honeybee stings are a severe public health problem. Bee venom contains a series of active components, including enzymes, peptides, and biogenic amines. The local reactions observed after envenoming include a typical inflammatory response and pain. Honeybee venom contains some well-known polycationic peptides, such as Melittin, Apamin, MCD peptide, Cardiopep, and Tertiapin. Secapin in honeybee venom was described 38 years ago, yet almost nothing is known about its action. A novel, variant form of this peptide was isolated from the venom of Africanized honeybees (Apis mellifera). This novel peptide, named Secapin-2, is 25 amino acid residues long. Conformational analyses using circular dichroism and molecular dynamics simulations revealed a secondary structure rich in strands and turns, stabilized by an intramolecular disulfide bridge. Biological assays indicated that Secapin-2 did not induce hemolysis, mast cell degranulation or chemotactic activities. However, Secapin-2 caused potent dose-related hyperalgesic and edematogenic responses in experimental animals. To evaluate the roles of prostanoids and lipid mediators in the hyperalgesia and edema induced by this peptide, Indomethacin and Zileuton were used to inhibit the cyclooxygenase and lipoxygenase pathways, respectively. The results showed that Zileuton partially blocked the hyperalgesia induced by Secapin-2 and decreased the edematogenic response. In contrast, Indomethacin did not interfere with these phenomena. Zafirlukast, a leukotriene receptor antagonist, blocked the Secapin-2 induced hyperalgesia and edematogenic response. These results indicate that Secapin-2 induces inflammation and pain through the lipoxygenase pathway in both phenomena.

Involvement of formyl peptide receptors in the stimulatory effect of crotoxin on macrophages co-cultivated with tumour cells.

Crotoxin (CTX) is the main neurotoxic component of Crotalus durissus terrificus snake venom. It inhibits tumour growth and modulates the function of macrophages, which are essential cells in the tumour microenvironment. The present study investigated the effect of CTX on the secretory activity of monocultured macrophages and macrophages co-cultivated with LLC-WRC 256 cells. The effect of the macrophage secretory activities on tumour cell proliferation was also evaluated. Macrophages pre-treated with CTX (0.3 µg/mL) for 2 h were co-cultivated with LLC-WRC 256 cells, and the secretory activity of the macrophages was determined after 12, 24 and 48 h. The co-cultivation of CTX-treated macrophages with the tumour cells caused a 20% reduction in tumour cell proliferation. The production of both H2O2 and NO was increased by 41% and 29% after 24 or 48 h of co-cultivation, respectively, compared to the values for the co-cultures of macrophages of control. The level of secreted IL-1ß increased by 3.7- and 3.2-fold after 12 h and 24 h of co-cultivation, respectively. Moreover, an increased level of LXA4 (25%) was observed after 24 h of co-cultivation, and a 2.3- and 2.1-fold increased level of 15-epi-LXA4 was observed after 24 h and 48 h, respectively. Boc-2, a selective antagonist of formyl peptide receptors, blocked both the stimulatory effect of CTX on the macrophage secretory activity and the inhibitory effect of these cells on tumour cell proliferation. Taken together, these results indicate that CTX enhanced the secretory activity of macrophages, which may contribute to the antitumour activity of these cells, and that activation of formyl peptide receptors appears to play a major role in this effect.

Hyperalgesic and edematogenic effects of peptides isolated from the venoms of honeybee (Apis mellifera) and neotropical social wasps (Polybia paulista and Protonectarina sylveirae).

Stings by bees and wasps, including Brazilian species, are a severe public health problem. The local reactions observed after the envenoming includes typical inflammatory response and pain. Several studies have been performed to identify the substances, including peptides that are responsible for such phenomena. The aim of the present study is to characterize the possible nociceptive (hyperalgesic) and edematogenic effects of some peptides isolated from the venoms of the honeybee (Apis mellifera) and the social wasps Polybia paulista and Protonectarina sylveirae, in addition to characterize some of the mechanisms involved in these phenomena. For this purpose, different doses of the peptides mellitin (Apis mellifera), Polybia-MP-I, N-2-Polybia-MP-I (Polybia paulista), Protonectarina-MP-NH2 and Protonectarina-MP-OH (Protonectarina sylveirae) were injected into the hind paw of mice. Hyperalgesia and edema were determined after peptide application, by using an electronic von Frey apparatus and a paquimeter. Carrageenin and saline were used as controls. Results showed that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH(2) and Protonectarina-MP-OH peptides produced a dose- and time-related hyperalgesic and edematogenic responses. Both phenomena are detected 2 h after melittin, Polybia-MP-I, N-2-Polybia-MP-I injection; their effects lasted until 8 h. In order to evaluate the role of prostanoids and the involvement of lipidic mediators in hyperalgesia induced by the peptides, indomethacin and zileuton were used. Results showed that zileuton blocked peptide-induced hyperalgesia and induced a decrease of the edematogenic response. On the other hand, indomethacin did not interfere with these phenomena. These results indicate that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH(2), and Protonectarina-MP-OH peptides could contribute to inflammation and pain induced by insect venoms.

Hyperalgesic and edematogenic effects of peptides isolated from the venoms of honeybee (Apis mellifera) and neotropical social wasps (Polybia paulista and Protonectarina sylveirae)

Stings by bees and wasps, including Brazilian species, are a severe public health problem. The local reactions observed after the envenoming includes typical inflammatory response and pain. Several studies have been performed to identify the substances, including peptides that are responsible for such phenomena. The aim of the present study is to characterize the possible nociceptive (hyperalgesic) and edematogenic effects of some peptides isolated from the venoms of the honeybee (Apis mellifera) and the social wasps Polybia paulista and Protonectarina sylveirae, in addition to characterize some of the mechanisms involved in these phenomena. For this purpose, different doses of the peptides mellitin (Apis mellifera), Polybia-MP-I, N-2-Polybia-MP-I (Polybia paulista), Protonectarina-MP-NH2 and Protonectarina-MP-OH (Protonectarina sylveirae) were injected into the hind paw of mice. Hyperalgesia and edema were determined after peptide application, by using an electronic von Frey apparatus and a paquimeter. Carrageenin and saline were used as controls. Results showed that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH2 and Protonectarina-MP-OH peptides produced a dose- and time-related hyperalgesic and edematogenic responses. Both phenomena are detected 2 h after melittin, Polybia-MP-I, N-2-Polybia-MP-I injection; their effects lasted until 8 h. In order to evaluate the role of prostanoids and the involvement of lipidic mediators in hyperalgesia induced by the peptides, indomethacin and zileuton were used. Results showed that zileuton blocked peptide-induced hyperalgesia and induced a decrease of the edematogenic response. On the other hand, indomethacin did not interfere with these phenomena. These results indicate that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH2, and Protonectarina-MP-OH peptides could contribute to inflammation and pain induced by insect venoms.

Lipoxygenase-derived eicosanoids are involved in the inhibitory effect of Crotalus durissus terrificus venom or crotoxin on rat macrophage phagocytosis.

Crotalus durissus terrificus snake venom and its major toxin, crotoxin or type II PLA2 subunit of this toxin, induce an inhibitory effect on spreading and phagocytosis in 2h incubated macrophages. The involvement of arachidonate-derived mediators on the inhibitory action of the venom or toxins on rat peritoneal macrophage phagocytosis was presently investigated. Peritoneal cells harvested from naive rats and incubated with the venom or toxins or harvested from the peritoneal cavity of rats pre-treated with the toxins were used. Zileuton, a 5-lipoxygenase inhibitor but not indomethacin, a cyclooxygenase inhibitor, given in vivo and in vitro abolished the inhibitory effect of venom or toxins on phagocytosis. Resident peritoneal macrophages incubated with the venom or toxins showed increased levels of prostaglandin E2 and lipoxin A4, with no change in leukotriene B4. These results suggest that lipoxygenase-derived eicosanoids are involved in the inhibitory effect of C.d. terrificus venom, crotoxin or PLA2 on macrophage phagocytosis.

Contribution of crotoxin for the inhibitory effect of Crotalus durissus terrificus snake venom on macrophage function.

Previous work of our group demonstrated that Crotalus durissus terrificus venom has a dual effect on macrophage function: it inhibits spreading and phagocytosis and stimulates hydrogen peroxide and nitric oxide production, antimicrobial activity and glucose and glutamine metabolism of these cells. Crotalid venom also induces analgesia and this effect is mediated by opioid receptors. The involvement of opioidergic mechanism and the determination of the active component responsible for the inhibitory effect of crotalid venom on macrophage function were investigated. The venom reduced the spreading and phagocytic activities of peritoneal macrophages. This effect was observed in vitro, 2 h after incubation of resident peritoneal macrophages with the venom, and in vivo, 2 h after subcutaneous injection of the venom. The inhibition of phagocytosis was not modified by naloxone, an antagonist of opioid receptors. Venom neutralization with crotalid antivenom abolished the inhibitory effect of the venom, indicating that venom toxins are involved in this effect. Crotoxin, the main toxin of crotalid venom, s.c. injected to rats or added to the medium of peritoneal cell incubation, inhibited macrophage function in a similar manner to that observed for crude venom. The present results suggest that crotoxin causes a direct inhibition of macrophage spreading and phagocytic activities and may contribute to the inhibitory effect of crotalid venom on macrophage function.

Tolerance to the antinociceptive effect of Crotalus durissus terrificus snake venom in mice is mediated by pharmacodynamic mechanisms.

Crotalus durissus terrificus venom exerts central and peripheral antinociceptive effect mediated by opioid receptors. The present work investigated the tolerance to the antinociceptive effect of the venom and characterised the mechanisms involved in this phenomenon. The hot plate test, applied in mice, was used for pain threshold determination. The venom (200 microg/kg) was administered by oral route, daily, for 14 days, and the nociceptive test was applied before and on days 1, 7 and 14 of the treatment. Prolonged treatment with venom lead to the development of tolerance to the antinociceptive effect. Tolerant animals exhibited increased sodium pentobarbital-induced sleeping time, although total hepatic microsomal cytochrome P450 was not altered. The antinociceptive effect of a single dose of venom (200 microg/kg) is mediated by kappa opioid receptors. Mice long-term-treated with venom showed cross-tolerance to U-TRANS, an agonist of kappa-opioid receptor, but not to morphine or DAMGO, two mu-opioid receptor agonists. Prolonged administration of venom did not cause symptoms of abstinence syndrome. These data indicate that prolonged treatment with C. durissus terrificus venom induces tolerance to the antinociceptive effect and that pharmacodynamic mechanisms are involved in the genesis of this phenomenon.