Purification of a neuroprotective component of Parawixia bistriata spider venom that enhances glutamate uptake.
(1) 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. (2) 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. (3) The active fraction, referred to as PbTx1.2.3, stimulated glutamate uptake in synaptosomes without changing the K(M) 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. (4) 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. (5) This active spider venom component may serve as a basis for designing therapeutic drugs that increase glutamate clearance and limit neurodegeneration.
Fontana
Andréia Cristina Karklin
AC
Laboratory of Neurochemistry, Department of Biochemistry and Immunology, Ribeirão Preto School of Medicine, University of São Paulo, Brazil. fontanaa@ohsu.edu
Guizzo
Renato
R
de Oliveira Beleboni
Renê
R
Meirelles E Silva
Antonio Renato
AR
Coimbra
Norberto Cysne
NC
Amara
Susan G
SG
dos Santos
Wagner Ferreira
WF
Coutinho-Netto
Joaquim
J
D000818:Animals; D001938:Brazil; D002247:Carbon Isotopes; D002540:Cerebral Cortex / Q000166:cytology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D005901:Glaucoma / Q000188:drug therapy / Q000503:physiopathology; D018698:Glutamic Acid / Q000187:drug effects / Q000378:metabolism / Q000493:pharmacokinetics*; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism / Q000503:physiopathology; D008297:Male; D018696:Neuroprotective Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D018094:Receptors, Metabotropic Glutamate / Q000187:drug effects / Q000502:physiology; D012160:Retina / Q000187:drug effects / Q000473:pathology / Q000648:ultrastructure; D013111:Spider Venoms / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D013112:Spiders; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D014020:Tissue Extracts / Q000494:pharmacology*; D014316:Tritium; D005680:gamma-Aminobutyric Acid / Q000187:drug effects / Q000378:metabolism / Q000493:pharmacokinetics
D002247:Carbon Isotopes; D007473:Ion Channels; D018696:Neuroprotective Agents; D018094:Receptors, Metabotropic Glutamate; D013111:Spider Venoms; D014020:Tissue Extracts; D014316:Tritium; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid