RESUMO
Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, is the most toxic extracellular enzyme produced by this bacteria and is essential for virulence in gas gangrene. At lytic concentrations, CpPLC causes membrane disruption, whereas at sublytic concentrations this toxin causes oxidative stress and activates the MEK/ERK pathway, which contributes to its cytotoxic and myotoxic effects. In the present work, the role of PKC, ERK 1/2 and NFκB signalling pathways in ROS generation induced by CpPLC and their contribution to CpPLC-induced cytotoxicity was evaluated. The results demonstrate that CpPLC induces ROS production through PKC, MEK/ERK and NFκB pathways, the latter being activated by the MEK/ERK signalling cascade. Inhibition of either of these signalling pathways prevents CpPLC's cytotoxic effect. In addition, it was demonstrated that NFκB inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of CpPLC in mice. Understanding the role of these signalling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridialmyonecrosis.
Assuntos
Toxinas Bacterianas/farmacologia , Proteínas de Ligação ao Cálcio/farmacologia , MAP Quinase Quinase 1/metabolismo , Melanoma/patologia , Músculo Esquelético/patologia , NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fosfolipases Tipo C/farmacologia , Animais , Western Blotting , Células CHO , Proliferação de Células/efeitos dos fármacos , Cricetulus , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Transdução de Sinais , Células Tumorais CultivadasRESUMO
S100B is a calcium binding protein expressed and secreted by astrocytes. Extracellular S100B stimulates the proliferation of astroglial cells and the survival of neurons. Extracellular signal regulated kinases (ERK) are involved in the transduction of proliferating signals in astrocytes. Here we report that S100B significantly increases the activity of ERK in primary cultures of astrocytes, a result which may be related to previous observations of the effect of this protein on glial proliferation. We further confirm that conversion of S100B to its covalent dimer by oxidation of cysteine residues increases its extracellular activity. Although we cannot exclude S100B involvement in other mechanisms of signal transduction, these results suggest that ERK activity in astrocytes is modulated by extracellular S100B.
Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/farmacologia , Espaço Extracelular/metabolismo , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/farmacologia , Proteínas S100 , Animais , Animais Recém-Nascidos , Técnicas de Cultura de Células , Ratos , Subunidade beta da Proteína Ligante de Cálcio S100 , Fatores de TempoRESUMO
Adult male Wistar rats were bilaterally implanted with indwelling cannulae in the hippocampus. Forty-eight hours after surgery, animals were habituated to an open-field box during 2 min, being tested 24 h later; next they were trained in a step-down inhibitory avoidance task (3.0 s, 0.4 mA foot-shock), being tested again 24 h later. Immediately after the training session of each task, animals received a 0.5-microl infusion of calcium-phosphate-buffered saline (PBS) and S100B (20, 200, 2000, or 20,000 nM). In the inhibitory avoidance task, animals infused with the two highest concentrations of S100B, 2 and 20 microM, obtained higher scores of retention relative to controls in the test session (p<0.05), and a trend toward an increase was observed in animals infused with 200 nM (p<0. 10). In both sessions of the habituation task, groups were not different regarding crossings, rearings, and time for leaving the first square (p>0.10). These results indicate that, in rats, post-training increased hippocampal levels of S100B right after training facilitate, in a dose-dependent way, long-term memory for an inhibitory avoidance task, but not for an open-field habituation.
Assuntos
Aprendizagem da Esquiva/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/farmacologia , Habituação Psicofisiológica/efeitos dos fármacos , Hipocampo/fisiologia , Memória/efeitos dos fármacos , Proteínas S100/farmacologia , Animais , Proteínas de Ligação ao Cálcio/administração & dosagem , Relação Dose-Resposta a Droga , Injeções , Masculino , Fatores de Crescimento Neural , Ratos , Ratos Wistar , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/administração & dosagemRESUMO
We describe a Triton-insoluble cytoskeletal fraction extracted from cerebral cortex of young rats retaining an endogenous Ca(2+)-mediated mechanism acting in vitro on Ca2+/calmodulin-dependent protein kinase II (CaM-KII) activity and on phosphorylation and proteolysis of the 150 kDa neurofilament subunit (NF-M), alpha and beta tubulin. Exogenous Ca2+ induced a 70% decrease in the in vitro phosphorylation of the NF-M and tubulins and a 30-50% decrease in the total amount of these proteins. However, when calpastatin was added basal phosphorylation and NF-M and tubulin content were recovered. Furthermore, exogenous Ca2+/calmodulin induced increased in vitro phosphorylation of the cytoskeletal proteins and CaM-KII activity only in the presence of calpastatin, suggesting the presence of Ca(2+)-induced calpain-mediated proteolysis. This fraction could be an interesting model to further studies concerning the in vitro effects of Ca(2+)-mediated protein kinases and proteases associated with the cytoskeletal fraction.
Assuntos
Cálcio/farmacologia , Córtex Cerebral/ultraestrutura , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Endopeptidases/metabolismo , Proteínas Quinases/metabolismo , Animais , Proteínas de Ligação ao Cálcio/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Neurofilamentos/metabolismo , Fosforilação , Ratos , Ratos Wistar , Tubulina (Proteína)/metabolismoRESUMO
The effects of beta and alpha-adrenergic stimulation in amphibian superfused hearts and ventricular strips were studied. Superfusion with 3 x 10(-8) M isoproterenol produced a positive inotropic effect, as detected by a 92 +/- 24% increase in the maximal rate of contraction (+T) and a positive lusitropic effect characterized by a decrease in both the ratio +T/-T (23 +/- 5%) and the half relaxation time (t1/2) (19 +/- 4%). The mechanical behavior induced by the beta-agonist was associated with an increase in the intracellular cAMP levels from control values of 173 +/- 19 to 329 +/- 28 nmol/mg wet tissue. Hearts superfused with 32P in the presence of isoproterenol showed a significant increase in Tn 1 phosphorylation (from 151 +/- 13 to 240 +/- 44 pmol 32P/mg MF protein) without consistent changes in phosphorylation of C-protein. In sarcoplasmic reticulum membrane vesicles, no phospholamban phosphorylation was detected either by beta-adrenergic stimulation of superfused hearts or when phosphorylation conditions were optimized by direct treatment of the vesicles with cAMP-dependent protein kinase (PKA) and [gamma 32P] ATP. The effect of alpha-adrenergic stimulation on ventricular strips was studied at 30 and 22 degrees C. At 30 degrees C, the effects of 10(-5) to 10(-4) M phenylephrine on myocardial contraction and relaxation were diminished to non significant levels by addition of propranolol. At 22 degrees C, blockage with propranolol left a remanent positive inotropic effect (10% of the total effect of phenylephrine) and changed the phenylephrine-induced positive lusitropic effect into a negative lusitropic action.(ABSTRACT TRUNCATED AT 250 WORDS)