RESUMO
The innate immune system of marine mussels (Mytilus galloprovincialis) is operated by phagocytic cells termed hemocytes. Lipopolysaccharide (LPS), interleukin-2 (IL-2), or platelet-derived growth factor (PDGF) increase biogenic amine synthesis in these cells, and the enzymes Ca(2+)-independent protein kinase C (PKC) (p105/108) and Ca(2+)-dependent PKC (p60) are involved in these processes. Stimulation by PDGF induces a down-regulation process affecting the form p108 of the Ca(2+)-independent PKC. In addition, PDGF produces the increase of expression of p60 in the membrane fraction. IL-2 induces the disappearance of p108 from the membrane but does not affect the presence of p60 in cytosol or membrane. For its part, LPS activates exclusively p60 by a down-regulation mechanism. The ensemble of results suggests that each agonist starts a pathway that implicates the PKC isoenzymes that mediate the regulation of the activities dopa decarboxylase, dopamine beta-hydroxilase, and phenyletanolamine N-methyltranferase, which lead to different actions related to biogenic amine synthesis.
Assuntos
Aminas Biogênicas/metabolismo , Hemócitos/efeitos dos fármacos , Interleucina-2/farmacologia , Lipopolissacarídeos/farmacologia , Mytilus/fisiologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteína Quinase C/fisiologia , Animais , Células Cultivadas , Dopa Descarboxilase/metabolismo , Dopamina beta-Hidroxilase/metabolismo , Regulação para Baixo/efeitos dos fármacos , Hemócitos/citologia , Hemócitos/enzimologia , Isoenzimas , Feniletanolamina N-Metiltransferase/metabolismoRESUMO
Previous works revealed the presence of a Ca(2+)-dependent protein kinase (p60) and a Ca(2+)-independent protein kinase (p105) in the mantle tissue from the sea mussel Mytilus galloprovincialis Lmk. The expression of both isoforms shows a balance between cytosolic and membrane fractions in mantle, gills, and hepatopancreas, whereas, in hemocytes, their expression is mainly cytosolic, as happens in muscle tissues with p60 alone. Both enzymatic forms contain phosphorylated serines, and no phosphorylation was detected in tyrosines. Only the form p105 mediates the PMA-induced activation of the hemocytes of M. galloprovincialis, and it does so by a process of down-regulation. The form p60 does not respond to the presence of the phorbol ester, suggesting structural differences related to the binding sites of the diacylglycerol.
Assuntos
Bivalves/enzimologia , Cálcio/metabolismo , Carcinógenos/farmacologia , Hemócitos/efeitos dos fármacos , Hemócitos/enzimologia , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Animais , Western Blotting , Isoenzimas , Proteína Quinase C/isolamento & purificação , Distribuição TecidualRESUMO
An enzyme that can be included into the so-called conventional PKCs has been purified to homogeneity from the mantle tissue of the sea mussel Mytilus galloprovincialis. This enzyme has a molecular weight of 60 kDa, which is DAG-dependent, PS-activated, and Ca2+-dependent. It was separated from a Ca2+-independent PKC (p105) (Mercado et al., Mol Cell Biochem 233:99-105, 2002) by means of an ionic exchange chromatography on DE-52 cellulose. The molecular weights and kinetic properties of both the enzymes are different. The protein p60 is broadly distributed among the tissues, which suggests that it may carry out specific functions, different from those performed by p105.
Assuntos
Cálcio/metabolismo , Mytilus/enzimologia , Proteína Quinase C/química , Animais , Cinética , Peso Molecular , Proteína Quinase C/isolamento & purificaçãoRESUMO
The exposure of organisms to stressing agents may affect the level and pattern of protein expression. Certain proteins with an important role in protein homeostasis and in the tolerance to stress, known as stress proteins, are especially affected. Different tissues and cells show a range of sensitivities to stress, depending on the habitat to which organisms have adapted. The response of different tissues and cells from the mussel Mytilus galloprovincialis Lmk. to heat shock has been studied in this work using different exposure times and temperatures. During the assays, protein expression was observed to vary depending on the tissue studied, the temperature or the exposure time used. But maybe the most prominent thing is the different response obtained from the cultured haemocytes and those freshly obtained from stressed mussels, which makes us think that the extraction procedure is the main cause of the response of non-cultured cells, although the haemolymph may contain components that modulate haemocyte response.