title: Bothropstoxin-I reduces evoked acetylcholine release from rat motor nerve terminals: radiochemical and real-time video-microscopy studies.
abstract: Understanding the biological activity profile of the snake venom components is fundamental for improving the treatment of snakebite envenomings and may also contribute for the development of new potential therapeutic agents. In this work, we tested the effects of BthTX-I, a Lys49 PLA(2) homologue from the Bothrops jararacussu snake venom. While this toxin induces conspicuous myonecrosis by a catalytically independent mechanism, a series of in vitro studies support the hypothesis that BthTX-I might also exert a neuromuscular blocking activity due to its ability to alter the integrity of muscle cell membranes. To gain insight into the mechanisms of this inhibitory neuromuscular effect, for the first time, the influence of BthTX-I on nerve-evoked ACh release was directly quantified by radiochemical and real-time video-microscopy methods. Our results show that the neuromuscular blockade produced by in vitro exposure to BthTX-I (1 μM) results from the summation of both pre- and postsynaptic effects. Modifications affecting the presynaptic apparatus were revealed by the significant reduction of nerve-evoked [(3)H]-ACh release; real-time measurements of transmitter exocytosis using the FM4-64 fluorescent dye fully supported radiochemical data. The postsynaptic effect of BthTX-I was characterized by typical histological alterations in the architecture of skeletal muscle fibers, increase in the outflow of the intracellular lactate dehydrogenase enzyme and progressive depolarization of the muscle resting membrane potential. In conclusion, these findings suggest that the neuromuscular blockade produced by BthTX-I results from transient depolarization of skeletal muscle fibers, consequent to its general membrane-destabilizing effect, and subsequent decrease of evoked ACh release from motor nerve terminals.
authors.affiliation: Instituto de Ciências Biomédicas de Abel Salazar, UMIB, Univ. Porto, 4099-003 Porto, Portugal.
authors.forename: Paulo
authors.initials: P
authors.lastname: Correia-de-Sá
authors.forename: José B
authors.initials: JB
authors.lastname: Noronha-Matos
authors.forename: Maria A
authors.initials: MA
authors.lastname: Timóteo
authors.forename: Fátima
authors.initials: F
authors.lastname: Ferreirinha
authors.forename: Patrícia
authors.initials: P
authors.lastname: Marques
authors.forename: Andreimar M
authors.initials: AM
authors.lastname: Soares
authors.forename: Cicilia
authors.initials: C
authors.lastname: Carvalho
authors.forename: Walter L G
authors.initials: WL
authors.lastname: Cavalcante
authors.forename: Márcia
authors.initials: M
authors.lastname: Gallacci
mesh_terms: D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D017837:Bothrops; D003435:Crotalid Venoms / Q000494:pharmacology*; D003964:Diaphragm / Q000187:drug effects; D005089:Exocytosis / Q000187:drug effects; D005260:Female; D005456:Fluorescent Dyes; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D008839:Microelectrodes; D018715:Microscopy, Video; D009046:Motor Neurons / Q000187:drug effects / Q000378:metabolism*; D009119:Muscle Contraction / Q000187:drug effects; D009213:Myography; D054467:Phospholipases A2 / Q000494:pharmacology; D010791:Phrenic Nerve / Q000187:drug effects; D017729:Presynaptic Terminals / Q000187:drug effects / Q000378:metabolism*; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D051381:Rats; D017208:Rats, Wistar
chemical_list: D003435:Crotalid Venoms; C092350:FM 4-64; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; C078097:bothropstoxin; D007770:L-Lactate Dehydrogenase; D054467:Phospholipases A2; D000109:Acetylcholine