RESUMO
BACKGROUND: Plants have developed a vast range of mechanisms to compete with phytophagous insects, including entomotoxic proteins such as ureases. The legume Canavalia ensiformis produces several urease isoforms, of which the more abundant is called Jack Bean Urease (JBU). Previews work has demonstrated the potential insecticidal effects of JBU, by mechanisms so far not entirely elucidated. In this work, we investigated the mechanisms involved in the JBU-induced activity upon neurotransmitter release on insect neuromuscular junctions. METHODS: Electrophysiological recordings of nerve and muscle action potentials, and calcium imaging bioassays were employed. RESULTS AND CONCLUSION: JBU (0.28â¯mg/animal/day) in Locusta migratoria 2nd instar through feeding and injection did not induce lethality, although it did result in a reduction of 20% in the weight gain at the end of 168â¯h (nâ¯=â¯9, pâ¯≤â¯0.05). JBU (0.014 and 0.14â¯mg) injected direct into the locust hind leg induced a dose and time-dependent decrease in the amplitude of muscle action potentials, with a maximum decrease of 70% in the amplitude at the highest dose (nâ¯=â¯5, pâ¯≤â¯0.05). At the same doses JBU did not alter the amplitude of action potentials evoked from motor neurons. Using Drosophila 3rd instar larvae neuromuscular preparations, JBU (10-7â¯M) increased the occurrence of miniature Excitatory Junctional Potentials (mEJPs) in the presence of 1â¯mM CaCl2 (nâ¯=â¯5, pâ¯≤â¯0.05). In low calcium (0.4â¯mM) assays, JBU (10-7â¯M) was not able to modulate the occurrence of the events. In Ca2+-free conditions, with EGTA or CoCl2, JBU induced a significant decrease in the occurrence of mEPJs (nâ¯=â¯5, pâ¯≤â¯0.05). Injected into the 3rd abdominal ganglion of Nauphoeta cinerea cockroaches, JBU (1⯵M) induced a significant increase in Ca2+ influx (nâ¯=â¯7, pâ¯≤â¯0.01), similar to that seen for high KCl (35â¯mM) condition. Taken together the results confirm a direct action of JBU upon insect neuromuscular junctions and possibly central synapses, probably by disrupting the calcium machinery in the pre-synaptic region of the neurons.
Assuntos
Acetilcolinesterase/genética , Lepidópteros/genética , Mutação , Animais , EspanhaRESUMO
Quercetin is a flavonol widely distributed in plants and has various described biological functions. Several studies have reported on its ability to restore neuronal function in a wide variety of disease models, including animal models of neurodegenerative disorders such as Parkinson's disease. Quercetin per se can act as a neuroprotector/neuromodulator, especially in diseases related to impaired dopaminergic neurotransmission. However, little is known about how quercetin interacts with the dopaminergic machinery. Here we employed the nematode Caenorhabditis elegans to study this putative interaction. After observing behavioral modulation, mutant analysis and gene expression in C. elegans upon exposure to quercetin at a concentration that does not protect against MPTP, we constructed a homology-based dopamine transporter protein model to conduct a docking study. This led to suggestive evidence on how quercetin may act as a dopaminergic modulator by interacting with C. elegans' dopamine transporter and alter the nematode's exploratory behavior. Consistent with this model, quercetin controls C. elegans behavior in a way dependent on the presence of both the dopamine transporter (dat-1), which is up-regulated upon quercetin exposure, and the dopamine receptor 2 (dop-2), which appears to be mandatory for dat-1 up-regulation. Our data propose an interaction with the dopaminergic machinery that may help to establish the effects of quercetin as a neuromodulator.
Assuntos
Dopamina , Quercetina , Transmissão Sináptica , Animais , Caenorhabditis elegans , Quercetina/farmacologia , Dopamina/metabolismo , Proteínas de Caenorhabditis elegans , Fármacos Neuroprotetores/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Neurônios/metabolismo , Estresse Oxidativo , Transmissão Sináptica/efeitos dos fármacos , Receptores de Dopamina D2/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-HidropiridinaRESUMO
BACKGROUND: Ureases of Canavalia ensiformis are natural insecticides with a still elusive entomotoxic mode of action. We have investigated the mechanisms involved in the neurotoxicity induced by Jack Bean Urease (JBU) in Nauphoeta cinerea (Olivier). METHODS: To carry out this study we have employed biochemical and neurophysiological analysis of different cockroach organ systems. RESULTS AND CONCLUSIONS: The injection of the insects with JBU (0.75-6µg/g animal), although not lethal within 24h, caused significant inhibition of the brain acetylcholinesterase activity (60±5%, p<0.05, n=6). JBU (1.5µg/200µL), acetylcholine (0.3µg/200µL) or neostigmine (0.22µg/200µL), induced a positive cardiac chronotropism (â¼25%) in the cockroaches (p<0.05, n=9). JBU (6µg/g) increased the insects' grooming activity (137±7%), similarly to octopamine (15µg/g) (p<0.05, n=30, respectively). Pretreating the insects with phentolamine (0.1µg/g) prevented the JBU- or octopamine-induced increase of grooming activity. JBU (6µg/g) caused 65±9% neuromuscular blockade in the cockroaches, an effect prevented by bicuculline (5µg/g) (p<0.05, n=6). JBU (6µg/g) decreased the frequency whilst increasing the amplitude of the spontaneous neural compound action potentials (1425±52.60min-1, controls 1.102±0.032mV, p<0.05, n=6, respectively). Altogether the results indicate that JBU induces behavioral alterations in Nauphoeta cinerea cockroaches probably by interfering with the cholinergic neurotransmission. The neuromuscular blocking activity of JBU suggests an interplay between acetylcholine and GABA signaling. GENERAL SIGNIFICANCE: The search for novel natural molecules with insecticide potential has become a necessity more than an alternative. Understanding the mode of action of candidate molecules is a crucial step towards the development of new bioinsecticides. The present study focused on the neurotoxicity of Canavalia ensiformis urease, a natural insecticide, in cockroaches and revealed interferences on the cholinergic, octopaminergic and GABA-ergic pathways as part of its entomotoxic mode of action.