Juan Ignacio Ramos-Martinez

Nitric oxide release by hemocytes of the mussel Mytilus galloprovincialis Lmk was provoked by interleukin-2 but not by lipopolysaccharide

As other marine and land mollusks, mussels have special cells in charge of the immune function called hemocytes. The activation of these cells leads to a series of events that end up in phagocytosis and in secretion of digestive enzymes that eliminate the pathogen. The production of nitric oxide is among the early activation processes. Contrary to what happens in cells of vertebrates and of other species of mollusks, in hemocytes of Mytilus galloprovincialis, LPS did not induce secretion of NO to the medium. However, human IL-2 provoked an important increase in NO production. The maximal synthesis of NO was detected after the hemocytes were incubated with the cytokine for 24h. In both stimulated and non-stimulated cells, Western blotting showed the presence of a protein of 130kDa, recognized by anti-mouse iNOS. Therefore, the higher production of NO can only be explained as a direct action of some effector upon the nitric oxide synthetase. NO production decreased by the action of H-89, a powerful inhibitor of the cAMP-dependent protein kinase (PKA). This suggests the involvement of PKA in the pathway of NO synthesis.

Primary cultures of hemocytes from Mytilus galloprovincialis Lmk.: expression of IL-2Rα subunit

Abstract Hemocytes from the mollusk Mytilus galloprovincialis Lmk. were maintained for more than 20 days in Leibovitz L-15 medium supplemented with salts and antibiotics. Survival of cells cultured in foetal calf serum (FCS)-supplemented L-15 medium was similar to that of those cultured in inactivated hemolymph-supplemented L-15 medium. Cell activation by lipopolysaccharide (LPS) induces an increase of the number of α-subunits of the interleukin-2 (IL-2) receptor (IL-2Rα) in the cell membrane. The best response in the expression of IL-2Rα subunit was obtained after 3 days of culture. In cultured cells, as in fresh hemocytes, LPS induced a higher expression of IL-2Rα subunit than that induced by IL-2.

Purification and properties of glutathione reductase from hepatopancreas of Mytilus edulis L.

1. 1. The enzyme glutathione reductase (GR) from hepatopancreas of Mytilus edulis L. was purified 80-fold by extraction, fractionated precipitation with ammonium sulphate, ion-exchange chromatography on DEAE-Sephadex, absorption chromatography on hydroxyapatite and gel-filtration through Sephadex G-200. 2. 2. The optimal pH was 7.5 and the Km for GSSG and NADPH were respectively 0.065 and 0.0041 mM. 3. 3. Glutathione reductase is rapidly activated by preincubation at 40°C either alone or with 1 mM GSSG. After 2 hr incubation the activity is 150% of the initial value. At 60°C the enzyme is rapidly inactivated, whether incubated alone or in the presence of 0.1 mM NADP+. One mM GSSG protects the activity of the enzyme to some extent. At 20°C glutathione reductase is inactivated by NADPH and GSH. 4. 4. NaCl and KCl at concentrations close to those they have in seawater are activators. Contrary to the findings of other authors zinc sulphate is also a strong activator of enzyme at 0.2 mM concentration.

The IL-2 receptor in hemocytes of the sea mussel Mytilus galloprovincialis Lmk.

In the hemolymph of the sea mussel Mytilus galloprovincialis, two different cell types have been found. Rounded (RH) cells display a nucleus that is very large in relation to the cell size; spread (SH) cells have an expanded cytoplasm and multiple granules. We determined by flow cytometry that only the SH cell types express three interleukin‐2 receptor (IL‐2R) subunits. Mussel IL‐2Ralpha and IL‐2Rbeta subunits display a molecular weight similar to those in vertebrates tissues, whereas mussel IL‐2Rgamma is smaller than that in vertebrates. Both lipopolysaccharide and IL‐2 induce IL‐2Ralpha expression, and such induction depends on the concentration of both agonists.

In vitro effects of LPS, IL-2, PDGF and CRF on haemocytes of Mytilus galloprovincialis Lmk.

The cells in charge of the innate immune response in the marine mussel Mytilus galloprovincialis Lmk. are the haemocytes. These cells respond in different ways to agents such as lipopolysaccharide (LPS), interleukin-2 (IL-2), platelet-derived growth factor (PDGF) and corticotropin releasing factor (CRF). After stimulation of the haemocytes, the expression of molecules reactive with monoclonal antibodies raised to the alpha chain of the IL-2 receptor, present in their membrane, differed depending on the agent used. The same happened with regard to the levels of dopamine, adrenaline and noradrenaline released to the medium by the haemocytes. It should also be noted that no catecholamine release was detected and the level of expression of IL-2Ralpha showed no significant variation in cultured cells that had not been treated with inducers. These facts would indicate that most haemocytes were in the same starting condition at the moment that the stimulation was performed. Therefore, cultured haemocytes can be a highly reliable model in the study of the innate immune system.

Seasonal variations in glycolysis in Mytilus galloprovincialis Lmk.

1. 1. In the hepatopancreas and mantle of the sea mussel Mytilus galloprovincialis Lmk glycolysis shows seasonal variations. 2. 2. In both these tissues, glycolytic activity shows a maximum in winter and a minimum in summer. 3. 3. Smaller fluctuations also take place in the posterior adductor muscle. 4. 4. In all three tissues, Fru 2,6-P2 indicates the direction and strength of glycolysis, correlating well with other parameters such as energy charge or the phosphoenolpyruvate/pyruvate ratio. 5. 5. In the posterior adductor muscle, however, Fru 2,6-P2 does not correlate with the MAR/Keq ratio from phosphofructokinase owing to the synergic activation of the enzyme by both Fru 2,6-P2 and AMP.

Regulation of the futile cycle of fructose phosphate in sea mussel

Carbohydrate metabolism in mussels shows two phases separated seasonally. During summer and linked to food supply, carbohydrates, mainly glycogen, are accumulated in the mantle tissue. During winter, mantle glycogen decreases concomitantly with an increase in triglyceride synthesis. In spring, after spawning, the animals go in to metabolic rest until the beginning of a new cycle. This cycle is regulated by the futile cycle of fructose phosphate that implicates PFK-1 and FBPase-1 activities. These enzymes and the bifunctional PFK-2/FBPase-2 that regulates the Fru-2,6-P2 levels, are seasonally modulated by covalent phosphorylation/dephosphorylation mechanisms, as a response to unknown factors. The futile cycle of the fructose phosphates also controls the transition from physiological aerobiosis to hypoxia. The process is independent of the phosphorylation state. In this sense, a pH decrease triggers a small Pasteur effect during the first 24 h of aerial exposure. Variations in the concentration of Fru-2,6-P2 and AMP are the sole factor responsible for this effect. Longer periods of hypoxia induce a metabolic depression characterized by a decrease in Fru-2,6-P2 which is hydrolyzed by drop in the pH. In this review, the authors speculate on the two regulation processes.

Purification of a lipid-activated and Ca2+-independent protein kinase from the mantle tissue of Mytilus galloprovincialis Lmk.

A phospholipid-sensitive Ca2+-independent protein kinase (p105) was purified to homogeneity from mantle tissue of the mussel Mytilus galloprovincialis Lmk., employing consecutively DE-52 cellulose, Sephacryl S-200 and Biogel HTP chromatographies. The purified enzyme appeared as a single band on 10% SDS-PAGE, and had a molecular weight of 105 kDa.The positive Western blotting of the purified eluate for anti-human-PKCδ and PKCε suggests that the enzyme from mussel mantle may be an ancestral nPKC isoform, with the kinetic properties of the enzyme very close to those of PKCε isoform of vertebrates.Western blotting of samples from different steps of purification using specific mouse anti-p105, showed two protein bands in samples from the initial steps. However, only one band was detected in the Biogel-HTP eluate, the most purified fraction. The purification steps did not affect the presence of P-serine in p105. No P-tyrosine peptides were detected in any of the purification steps. These results open a new field of work on the study of several molecular processes related to energetic metabolism and reproduction in molluscs, whose regulation is associated with the activation of protein kinases.

Implication of PKC in the seasonal variation of the immune response of the hemocytes of Mytilus galloprovincialis Lmk. and its role in interleukin‐2‐induced nitric oxide synthesis

The hemocytes are the cells responsible for the immune response in marine mollusks. The role of NO in processes related to the activation of the hemocytes has turned out evident over the late years. In the case of the mussel Mytilus galloprovincialis Lmk., hemocyte NO basal production varies throughout the year, showing a maximum in summer and a minimum in winter. IL‐2 reverts the low winter NO basal production through a process mediated by cAMP‐dependent protein kinase and by an apparent side effect of protein kinase C. The seasonal variation of NO production in the presence of the PKC inhibitor bisindolylmaleimide (BSM) allows suggesting a model in which PKC would modulate the activity of the enzymes responsible for nitric oxide production. IUBMB Life, 59: 659‐663, 2007

Phorbol esters induce translocation of the nPKC p105 to membrane in mussel hemocytes

Previous works revealed the presence of the nPKC enzyme p105 in hemocytes of M. galloprovincialis Lmk. Specific mussel antibodies were obtained from mouse and used in confocal microscopy and Western blotting. These techniques allowed the observation of p105 cytosol-to-membrane translocation induced by TPA for the first time in hemocytes of molluscs. The incubation of mussel immune cells with TPA for longer than 30 min also triggered a down-regulation process. Mussel hemocytes are an excellent model to study the molecular processes of innate immunity.