Rene Oliveira Beleboni

Pharmacological and Biochemical Aspects of GABAergic Neurotransmission: Pathological and Neuropsychobiological Relationships

Abstract1. The GABAergic neurotransmission has been implicated in the modulation of many neural networks in forebrain, midbrain and hindbrain, as well as, in several neurological disorders.2. The complete comprehension of GABA system neurochemical properties and the search for approaches in identifying new targets for the treatment of neural diseases related to GABAergic pathway are of the extreme relevance.3. The present review will be focused on the pharmacology and biochemistry of the GABA metabolism, GABA receptors and transporters. In addition, the pathological and psychobiological implications related to GABAergic neurotransmission will be considered.

Enhancing glutamate transport: mechanism of action of Parawixin1, a neuroprotective compound from Parawixia bistriata spider venom

Previous studies have shown that a compound purified from the spider Parawixia bistriata venom stimulates the activity of glial glutamate transporters and can protect retinal tissue from ischemic damage. To understand the mechanism by which this compound enhances transport, we examined its effects on the functional properties of glutamate transporters after solubilization and reconstitution in liposomes and in transfected COS-7 cells. Here, we demonstrate in both systems that Parawixin1 promotes a direct and selective enhancement of glutamate influx by the EAAT2 transporter subtype through a mechanism that does not alter the apparent affinities for the cosubstrates glutamate or sodium. In liposomes, we observed maximal enhancement by Parawixin1 when extracellular sodium and intracellular potassium concentrations are within physiological ranges. Moreover, the compound does not enhance the reverse transport of glutamate under ionic conditions that favor efflux, when extracellular potassium is elevated and the sodium gradient is reduced, nor does it alter the exchange of glutamate in the absence of internal potassium. These observations suggest that Parawixin1 facilitates the reorientation of the potassium-bound transporter, the rate-limiting step in the transport cycle, a conclusion further supported by experiments showing that Parawixin1 does not stimulate uptake by an EAAT2 transport mutant (E405D) defective in the potassium-dependent reorientation step. Thus, Parawixin1 enhances transport through a novel mechanism targeting a step in the transport cycle distinct from substrate influx or efflux and provides a basis for the design of new drugs that act allosterically on transporters to increase glutamate clearance.

Purification of a neuroprotective component of Parawixia bistriata spider venom that enhances glutamate uptake

In this study, we examined the effects of crude venom from the spider Parawixia bistriata on glutamate and GABA uptake into synaptosomes prepared from rat cerebral cortex. Addition of venom to cortical synaptosomes stimulated glutamate uptake and inhibited GABA uptake in a concentration‐dependent manner. The venom was fractionated using reverse‐phase high‐performance liquid chromatography on a preparative column. The fraction that retained glutamate uptake‐stimulating activity was further purified on a reverse‐phase analytical column followed by ion‐exchange chromatography. The active fraction, referred to as PbTx1.2.3, stimulated glutamate uptake in synaptosomes without changing the KM value, and did not affect GABA uptake. Additional experiments showed that the enhancement of glutamate uptake by PbTx1.2.3 occurs when ionotropic glutamate receptors or voltage‐gated sodium and calcium channels are completely inhibited or when GABA receptors and potassium channels are activated, indicating that the compound may have a direct action on the transporters. In an experimental model for glaucoma in which rat retinas are subjected to ischemia followed by reperfusion, PbTx1.2.3 protected neurons from excitotoxic death in both outer and inner nuclear layers, and ganglion cell layers. This active spider venom component may serve as a basis for designing therapeutic drugs that increase glutamate clearance and limit neurodegeneration.

Convulsant activity and neurochemical alterations induced by a fraction obtained from fruit Averrhoa carambola (Oxalidaceae: Geraniales).

We obtained a neurotoxic fraction (AcTx) from star fruit (Averrhoa carambola) and studied its effects on GABAergic and glutamatergic transmission systems. AcTx had no effect on GABA/glutamate uptake or release, or on glutamate binding. However, it specifically inhibited GABA binding in a concentration-dependent manner (IC(50)=0.89muM). Video-electroencephalogram recordings demonstrated that following cortical administration of AcTx, animals showed behavioral changes, including tonic-clonic seizures, evolving into status epilepticus, accompanied by cortical epileptiform activity. Chemical characterization of AcTx showed that this compound is a nonproteic molecule with a molecular weight less than 500, differing from oxalic acid. This neurotoxic fraction of star fruit may be considered a new tool for neurochemical and neuroethological research.

Anticonvulsant profile of the alkaloids (+)-erythravine and (+)-11-α-hydroxy-erythravine isolated from the flowers of Erythrina mulungu Mart ex Benth (Leguminosae-Papilionaceae).

Neural mechanisms underlying the onset and maintenance of epileptic seizures involve alterations in inhibitory and/or excitatory neurotransmitter pathways. Thus, the prospecting of novel molecules from natural products that target both inhibition and excitation systems has deserved interest in the rational design of new anticonvulsants. We isolated the alkaloids (+)-erythravine and (+)-11-α-hydroxy-erythravine from the flowers of Erythrina mulungu and evaluated the action of these compounds against chemically induced seizures in rats. Our results showed that the administration of different doses of (+)-erythravine inhibited seizures evoked by bicuculline, pentylenetetrazole, and kainic acid at maximum of 80, 100, and 100%, respectively, whereas different doses of (+)-11-α-hydroxy-erythravine inhibited seizures at a maximum of 100% when induced by bicuculline, NMDA, and kainic acid, and, to a lesser extent, PTZ (60%). The analysis of mean latency to seizure onset of nonprotected animals, for specific doses of alkaloids, showed that (+)-erythravine increased latencies to seizures induced by bicuculline. Although (+)-erythravine exhibited very weak anticonvulsant action against seizures induced by NMDA, this alkaloid increased the latency in this assay. The increase in latency to onset of seizures promoted by (+)-11-α-hydroxy-erythravine reached a maximum of threefold in the bicuculline test. All animals were protected against death when treated with different doses of (+)-11-α-hydroxy-erythravine in the tests using the four chemical convulsants. Identical results were obtained when using (+)-erythravine in the tests of bicuculline, NMDA, and PTZ, and, to a lesser extent, kainic acid. Therefore, these data validate the anticonvulsant properties of the tested alkaloids, which is of relevance in consideration of the ethnopharmacological/biotechnological potential of E. mulungu.

Anticonvulsant and GABA Uptake Inhibition Properties of Venom Fractions from the Spiders Parawixia bistriata and Scaptocosa raptoria

In this article we describe an in vivo anticonvulsant effect from denatured crude venom and partially isolated fractions from two spiders: Parawixia bistriata and Scaptocosa raptoria. Intracerebroventricular injections of these venoms and fractions abolished rat convulsive tonic-clonic seizures induced by picrotoxin, bicuculline and pentylenetetrazole, and also, inhibited GABA uptake in synaptosomes of rat cerebral cortex. The venoms described in this work seems to be promising tools for the study of the GABAergic system, and may be a potential source for new anticonvulsant drugs.

Neurochemical characterization of a neuroprotective compound from Parawixia bistriata spider venom that inhibits synaptosomal uptake of GABA and glycine.

The major contribution of this work is the isolation of a neuroprotective compound referred to as 2-amino-5-ureidopentanamide (FrPbAII) (Mr = 174) from Parawixia bistriata spider venom and an investigation of its mode of action. FrPbAII inhibits synaptosomal GABA uptake in a dose-dependent manner and probably does not act on Na+, K+, and Ca2+ channels, GABAB receptors, or γ-aminobutyrate:α-ketoglutarate aminotransferase enzyme; therefore, it is not directly dependent on these structures for its action. Direct increase of GABA release and reverse transport are also ruled out as mechanisms of FrPbAII activities as well as unspecific actions on pore membrane formation. Moreover, FrPbAII is selective for GABA and glycine transporters, having slight or no effect on monoamines or glutamate transporters. According to our experimental glaucoma data in rat retina, FrPbAII is able to cross the blood-retina barrier and promote effective protection of retinal layers submitted to ischemic conditions. These studies are of relevance by providing a better understanding of neurochemical mechanisms involved in brain function and for possible development of new neuropharmacological and therapeutic tools.

Anticonvulsant and anxiolytic activity of frPbaII, a novel GABA uptake inhibitor isolated from the venom of the social spider Parawixia bistriata (Araneidae: Araneae)

This study was aimed at determining the effects of FrPbAII (174 Da), a novel isolated component from Parawixia bistriata spider venom, in the CNS of Wistar rats. Considering that FrPbAII inhibits the high affinity GABAergic uptake in a dose-dependent manner, its anxiolytic and anticonvulsant effects were analyzed in well-established animal models. Injection of FrPbAII in the rat hippocampus induced a marked anxiolytic effect, increasing the occupancy in the open arms of the elevated plus maze (EC(50)=0.09 microg/microl) and increasing the time spent in the lit area of the light-dark apparatus (EC(50)=0.03 microg/microl). Anxiolytic effects were also observed considering the number of entries in the open arms of the EPM and in the lit compartment of the light-dark box. Interestingly, when microinjected bilaterally in the SNPr of freely moving rats, FrPbAII (0.6 microg/microl) effectively prevented seizures induced by the unilateral GABAergic blockade of Area tempestas (bicuculline, 0.75 microg/microl). This anticonvulsant effect was similar to that evoked by muscimol (0.1 microg/microl) and baclofen (0.6 microg/microl), but differed from that of the specific GAT1 inhibitor, nipecotic acid (0.7 microg/microl). This difference could be accounted either for the parallel action of FrPbAII over glycinergic transporters or to an inspecific activity on GABAergic transporters. Data from the present investigation might be pointing to a novel compound with interesting and yet unexplored pharmacological potential.

Erysothrine, an alkaloid extracted from flowers of Erythrina mulungu Mart. ex Benth: Evaluating its anticonvulsant and anxiolytic potential

In this study, we isolated the alkaloid erysothrine from the hydroalcoholic extract of flowers from E. mulungu and screened for its anticonvulsant and anxiolytic actions based on neuroethological and neurochemical experiments. Our results showed that the administration of erysothrine inhibited seizures evoked by bicuculline, PTZ, NMDA and most remarkably, kainic acid. Also, erysothrine induced an increase in the number of entries but not in the time spent in the open arms of the EPM. However, we did not notice any alterations in the light-dark choice or in the open-field tests. In preliminary neurochemistry tests, we also showed that erysothrine (0.001-10 μg/mL) did not alter the GABA or glutamate synaptossomal uptake and binding. Altogether, our results describe an alkaloid with anticonvulsant activity and mild anxiolytic activity that might be considered well tolerated as it does not alter the general behavior of the animals in the used doses.

Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest

Chalcones are precursors of flavonoids that exhibit structural heterogeneity and potential antitumor activity. The objective of this study was to characterize the cytotoxicity of trans-chalcone and licochalcone A (LicoA1) against a breast cancer cell line (MCF-7) and normal murine fibroblasts (3T3). Also the mechanisms of the anti-cancer activity of these two compounds were studied. The alkaline comet assay revealed dose-dependent genotoxicity, which was more responsive against the tumor cell line, compared to the 3T3 mouse fibroblast cell line. Flow cytometry showed that the two chalcones caused the cell cycle arrest in the G1 phase and induced apoptosis in MCF-7 cells. Using PCR Array, we found that trans-chalcone and LicoA trigger apoptosis mediated by the intrinsic pathway as demonstrated by the inhibition of Bcl-2 and induction of Bax. In western blot assay, the two chalcones reduced the expression of cell death-related proteins such as Bcl-2 and cyclin D1 and promoted the cleavage of PARP. However, only trans-chalcone induced the expression of the CIDEA gene and protein in these two experiments. Furthermore, transient transfections of MCF-7 using a construction of a promoter-luciferase vector showed that trans-chalcone induced the expression of the CIDEA promoter activity in 24 and 48h. In conclusion, the results showed that trans-chalcone promoted high induction of the CIDEA promoter gene and protein, which is related to DNA fragmentation during apoptosis.