Jack bean urease alters serotonin-induced effects on Rhodnius prolixus anterior midgut F. Stanisc ¸uaski a,b, *, V. Te Brugge c , C.R. Carlini a,b , I. Orchard c a Department of Biophysics, Institute of Biosciences, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil 1 b Center of Biotechnology, Institute of Biosciences, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil c Department of Biology, University of Toronto Mississauga, Mississauga, ON, L5L 1C6, Canada 1. Introduction Ureases (EC 3.5.1.5, urea amidohydrolase) are nickel-dependent metalloenzymes that catalyze the conversion of urea to two molecules of ammonia and one of carbon dioxide (Dixon et al., 1975). Ureases are widespread in plants, fungi and bacteria but are not synthesized by animals (Mobley and Hausinger, 1989). An isoform of jack bean urease (JBU), named canatoxin (Carlini and Guimara ˜es, 1981; Follmer et al., 2001), has been characterized as part of a family of urease-related genes in jack bean (Pires-Alves et al., 2003). Beyond roles related to ureolytic activity, canatoxin (CNTX) exhibits toxic effects in mammals, in addition to possessing fungitoxic and insecticidal activities (Carlini and Polacco, 2008). The major isoform of urease JBU, found in the same seeds, is also toxic to insects, such as the cotton stainer bug Dysdercus peruvianus, and this toxicity is independent of the ureolytic activity (Follmer et al., 2004). The entomotoxic activity is mainly due to the hydrolysis of CNTX/JBU and release of a peptide by the insect’s digestive enzymes (Carlini et al., 1997; Ferreira-DaSilva et al., 2000; Piovesan et al., 2008). This peptide, called pepcanatox, was isolated and characterized (Ferreira-DaSilva et al., 2000). A recombinant peptide, Jaburetox-2Ec, equivalent to that produced by hydrolysis of CNTX with insect cathepsins, has been obtained by heterologous expression in Escherichia coli (Mulinari et al., 2007). This peptide displays potent insecticidal effects, but does not affect mice or neonate rats upon oral or intraperitoneal administration (Mulinari et al., 2007; Tomazetto et al., 2007; Staniscuaski et al., 2005). Recent evidence indicates that Jaburetox-2Ec also has deleterious effects in Rhodnius prolixus (Staniscuaski et al., 2009). Jack bean urease and the peptides released from it have great biotechnological potential for pest control. The mode of action of Jaburetox-2Ec in insects, as well as that of ureases, is still unclear. Ureases cause a decrease in post-meal weight loss in the blood-feeding insect, R. prolixus (Carlini et al., 1997). We have shown that both JBU and Jaburetox-2Ec inhibit serotonin-induced fluid secretion in isolated Malpighian tubules of R. prolixus, indicating that the intact protein also impairs physiological processes in this insect (Staniscuaski et al., 2009). In mammalian models, CNTX induces exocytosis in platelets, mast cells, brain synaptosomes, isolated pancreatic islets, neutrophils, and macrophages, but without altering the cell membrane Journal of Insect Physiology 56 (2010) 1078–1086 ARTICLE INFO Article history: Received 15 December 2009 Received in revised form 1 March 2010 Accepted 1 March 2010 Keywords: Canavalia ensiformis Fluid secretion Muscle contraction Prostaglandins ABSTRACT Urease isoforms from jack bean seeds are toxic to insects, and this entomotoxic effect is mostly due to the release of a peptide by insect digestive enzymes. We previously demonstrated that jack bean urease (JBU) has antidiuretic effects on Rhodnius prolixus Malpighian tubules, decreasing the serotonin- stimulated secretion of fluid. Now, we evaluate the toxicity of the intact JBU and its effect on R. prolixus anterior midgut, to further elucidate the mechanism of action of JBU in insects. JBU decreases the serotonin-induced fluid transport by the anterior midgut in vitro when injected into the lumen. A decrease in the levels of cAMP is observed in tissues treated with JBU (in the presence of serotonin). JBU also causes a dose-dependent increase in the frequency of serotonin-induced contractions in the anterior midgut, but does not alter the frequency of spontaneous contractions. The cyclooxygenase inhibitor indomethacin and the prostaglandin antagonist AH6809 block JBU’s potentiation of serotonin-induced contractions, indicating that prostaglandins might act as second messengers for JBU action. Prostaglandin E 2 (PGE 2 ) increases the frequency of serotonin-induced contractions, again supporting the role of prostaglandins as second messengers for JBU action. JBU and PGE 2 increase cGMP levels in the anterior midgut, indicating that this molecule might also be part of the JBU pathway. ß 2010 Elsevier Ltd. All rights reserved. * Corresponding author at: Department of Biophysics, Institute of Biosciences, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. Tel.: +55 51 3308 7600; fax: +55 51 3308 7606. E-mail address: fernanda.staniscuaski@ufrgs.br (F. Stanisc ¸uaski). 1 http://www.ufrgs.br/laprotox. Contents lists available at ScienceDirect Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys 0022-1910/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2010.03.002