Inativação precoce de canais de sódio voltagem dependentes e atividade neurotóxica induzida pelo peptídeo jaburetox (jbtx) em baratas nauphoeta cinerea e periplaneta americana

Abstract

There is the urgent need to increase world food production, making the use of pesticides a recurring necessity, even if these compounds exibit several undesirable effects on the environmentl. Therefore, the development of new natural compounds with entomotoxic potential became not only an alternative. Several natural molecules with enomotoxic potential have as main target ionic channels, standing out the voltage gated sodium channel (Nav). Nav is a transmembrane protein complex, with a pore filled with water, where sodium ions diffuse through electrochemical gradient. The pharmacological diversity of the Navs is remarkable and can be observed through the multiplicity of toxic substances with specificity to these channels. Thus, the investigation of the interaction between natural molecules and this channel is pertinent. Ureases are metalloenzymes witch hydrolyze urea into ammonia. These compounds are synthesized by several organisms and have been characterized as natural insecticides. The entomotoxic activity of Jack Bean Urease (JBU) is directly correlated with the release of a toxic peptide named Jaburetox (Jbtx), derived from the cleavage by cathepsins inside insect digestive system. Jbtx exhibits fungitoxic, entomotoxic, antimicrobial neurotoxic effects and its mode of action still awaits elucidation. The objective of this work was to investigate the mechanism of neurotoxicity induced by Jaburetox, on the central and peripheral nervous system of Nauphoeta cinerea and Periplaneta americana cockroaches, using different electrophysiological techniques. Adult males Nauphoeta cinerea and Periplaneta americana cockroaches were reared under laboratory conditions. Ex situ recordings of the action potentials of Nauphoeta cinerea leg were performed. Also, we recorded the spontaneous activity of DUM neurons on terminal abdominal ganglion and the action potentials through the voltage / current clamp mode using the Double Oil Gap technique in the ventral nervous chord of Periplaneta americana. Data were expressed as mean ± S.E.M. The experimental groups were analyzed by one-way ANOVA / two-way regression, followed by the Dunnet test as post hoc. The effect of Jbtx (16 and 32 μg / g animal) on N. cinerea leg SNCAPs was characterized by significant time and dose-dependent decrease in the number of events (n = 10). The effect of Jbtx (20μg / 300μl) on the spontaneous activity of DUM cells was a progressive decrease in the frequency of potentials after 25 minutes of recording (n = 3). After 40 minutes, there was complete inactivation of spontaneous DUM cell activity. Treatment with phentolamine (5μg / 300μl) induced a delay in the effect of Jbtx (20μg / 300μl) on the spontaneous potentials of DUM cells (8 ± 0.34% spikes / 200ms up to 40 min, complete inhibition after 55 min (n = P <0.05) from the Double Oil Gap technique, the 12 voltage and current clamp records demonstrate that Jbtx (20μg / 300μl) induced the decrease of Na + peaks from the 25 minutes of recording. of resting membrane potentials caused a recovery to 76 ± 3% and 73 ± 3% of the mean amplitude of the current and voltage clamp registers, respectively. The decrease in the number of events in SNCAP is consistent with the neuromuscular block previously demonstrated by our group, and where voltage and current clamp records reveal that Jbtx affect sodium currents. The delay on DUM cell silencing, induced by the application of phentolamine, suggests that Jbtx is ambiguously modulates the release of octopamine. The results suggest that the interaction of Jbtx with Navs promotes physiological changes responsible for behavioral modulation observed in previous studies.