RESUMO
Phagocytosis, an essential process for host defense, requires the coordination of a variety of signaling reactions. MT-II, an enzymatically inactive Lys49 phospholipase A2 (PLA2) homolog, and MT-III, a catalytically-active Asp49 PLA2, are known to activate phagocytosis in macrophages. In this study, the signaling pathways mediating phagocytosis, focusing on protein kinases, were investigated. Macrophages from male Swiss mice peritoneum were obtained 96 h after intraperitoneal thioglycolate injection. Phagocytosis was evaluated using non-opsonized zymosan particles in the presence or absence of specific inhibitors, as well as PKC and PKC-α localization by confocal microscopy. Moreover, protein kinase C (PKC) activity was assessed by γP32 ATP in macrophages stimulated by both PLA2s. Data showed that both sPLA2s increased phagocytosis. Cytochalasin D, staurosporine/H7, wortmannin, and herbimycin, inhibitors of actin polymerization, PKC, phosphoinositide 3-kinase (PI3K), and protein tyrosine kinase (PTK), respectively, significantly reduced phagocytosis induced by both PLA2s. PKC activity was increased in macrophages stimulated by both PLA2s. Actin polymerization and talin were evidenced by immunofluorescence and talin was recruited 5 min after both PLA2s stimulation. PKC and PKC-α localization within the cell were increased after 60 min of MT-II and MT-III stimulation. These data suggest that the effect of both PLA2s depends on actin cytoskeleton rearrangements and the activation of PKC, PI3K, and PTK signaling events required for phagocytosis.
Assuntos
Fagocitose , Proteína Quinase C-alfa , Transdução de Sinais , Animais , Fagocitose/efeitos dos fármacos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Masculino , Proteína Quinase C-alfa/metabolismo , Macrófagos/efeitos dos fármacos , Fosfolipases A2 Secretórias/metabolismo , Venenos de Serpentes/toxicidade , Rifabutina/análogos & derivados , Rifabutina/farmacologiaRESUMO
1. O veneno de C. d. terrificus foi fracionado de forma a obter da mesma preparaçäo: 5'-nucleotidase, fosfodiesterase, enzima tipo trombínico, fosfolipase A2, crotapotina, convulxina e giroxina. Todas estas proteínas foram purificadas até a homogeneidade a eletroforese em acrilamina na presença de dodecil-sulfato de sódio. 2. Em outras fraçöes foram identificadas a presença de L-amino oxidase, atividade de kalikreina tipo tissular e NAD-hidrolase. 3. Uma das fraçöes homogeneas apresentou uma atividade tóxica produzindo sintomas diferentes dos das toxinas conhecidas. Tem uma peso molecular de 8.600 Daltons. 4. A atividade da fosfolipase A2 do veneno e das fraçöes foi completamente inibida pela crotapotina, e esta inibiçäo é específica para a fosfolipase do veneno do Crotalus, e nao ocorre com a atividade de fosfolipase A2 do veneno da Bothrops jararaca, Bothrops moojeni e Tityus bahiensis