title: eNOS uncoupling in the cerebellum after BBB disruption by exposure to Phoneutria nigriventer spider venom.
abstract: Numerous studies have shown that the venom of Phoneutria nigriventer (PNV) armed-spider causes excitotoxic signals and blood-brain barrier breakdown (BBBb) in rats. Nitric oxide (NO) is a signaling molecule which has a role in endothelium homeostasis and vascular health. The present study investigated the relevance of endothelial NO synthase (eNOS) uncoupling to clinical neurotoxic evolution induced by PNV. eNOS immunoblotting of cerebellum lysates processed through low-temperature SDS-PAGE revealed significant increased monomerization of the enzyme at critical periods of severe envenoming (1-2 h), whereas eNOS dimerization reversal paralleled to amelioration of animals condition (5-72 h). Moreover, eNOS uncoupling was accompanied by increased expression in calcium-sensing calmodulin protein and calcium-binding calbindin-D28 protein in cerebellar neurons. It is known that greater eNOS monomers than dimers implies the inability of eNOS to produce NO leading to superoxide production and endothelial/vascular barrier dysfunction. We suggest that transient eNOS deactivation and disturbances in calcium handling reduce NO production and enhance production of free radicals thus contributing to endothelial dysfunction in the cerebellum of envenomed rats. In addition, eNOS uncoupling compromises the enzyme capacity to respond to shear stress contributing to perivascular edema and it is one of the mechanisms involved in the BBBb promoted by PNV.
authors.affiliation: Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas (UNICAMP), Zip Code 13 083-970, Campinas, SP, Brazil.
authors.forename: Edilene Siqueira
authors.initials: ES
authors.lastname: Soares
authors.affiliation: Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas (UNICAMP), Zip Code 13 083-970, Campinas, SP, Brazil; Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Zip Code 13 083-970, Campinas, SP, Brazil.
authors.forename: Monique Culturato Padilha
authors.initials: MC
authors.lastname: Mendonça
authors.affiliation: Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas (UNICAMP), Zip Code 13 083-970, Campinas, SP, Brazil; Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Zip Code 13 083-970, Campinas, SP, Brazil. Electronic address: hofling@unicamp.br.
authors.forename: Maria Alice
authors.initials: MA
authors.lastname: da Cruz-Höfling
mesh_terms: D000818:Animals; D001812:Blood-Brain Barrier / Q000187:drug effects* / Q000378:metabolism; D064092:Calbindin 1 / Q000235:genetics / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism; D002531:Cerebellum / Q000187:drug effects* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004727:Endothelium / Q000187:drug effects / Q000378:metabolism; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D001098:Spider Bites / Q000473:pathology; D013111:Spider Venoms / Q000633:toxicity*; D013112:Spiders / Q000378:metabolism*
chemical_list: D064092:Calbindin 1; D002147:Calmodulin; D013111:Spider Venoms; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat
keywords: Blood-brain barrier; Calbindin-D28; Calmodulin; Cerebral microvessels; Spider venom; eNOS dysfunction