Inhibition of glutathione reductase uncovers the activation of NADPH-inhibited glucose-6-phosphate dehydrogenase.

Eggleston and Krebs pointed to a paradox in glucose-6-phosphate dehydrogenase (G6PD) regulating process that has not yet been solved, and which originated the term "fine regulation" of G6PD and, therefore, of oxidative phase of pentose phosphate pathway (OPPP). The paradox is that, in basal-like conditions, the activity of G6PD evaluated "in vitro" is very low or nearly null because of the potent inhibiting effect exerted by NADPH, a coenzyme whose concentration in the cell is much higher than that of the substrate NADP+ . However, "in vivo," flow through OPPP occurs in basal conditions. Eggleston and Krebs speculated on the possible existence of a system that would reverse the inhibition by NADPH. Such system would involve oxidized glutathione and exclude the participation of glutathione reductase (GR). The present work confirms the experimental results obtained by Eggleston and Krebs and proves that oxidized glutathione (GSSG) in the absence of NADPH is a direct inhibitor of G6PD. In the presence of GSSG, the G6PD activity recovery system suggested can be observed when GR is previously inhibited by alkylating agents. An unknown element with a molecular weight ranging between 12 and 50 kDa has been found to reverse part of G6PD inhibition by NADPH.

The regulation of the pentose phosphate pathway: Remember Krebs.

The changes in gene expression and posttranslational modifications of enzymes are comprised in the concept of "coarse control" of the oxidative phase of the pentose phosphate pathway. However, these changes are slow in its implementation. The defensive mechanism against oxidative stress requires a most rapid response, impossible to achieve with coarse regulation systems. Recently, it has been suggested that a quick acceleration mechanism of G6PD activity could be produced by the reduction of NADPH-inhibition of G6PD. The hypothesis opens new ways on possible mechanisms for rapid modulation that could be in accordance with results obtained in the 70s by Krebs. These results seemed outdated in view of the subsequent research. However, they deserve to be re-assessed at present.

The regulation of the oxidative phase of the pentose phosphate pathway: new answers to old problems.

There is a paradox in the oxidizing phase of the phosphate pentose pathway that has not yet been solved. The flow through the pathway is reduced in basal conditions; however, it must rise notably when a NADPH supplement is required. The paradox consists of the strong inhibition that the NADPH exerts on the both dehydrogenases of the pathway, especially on the regulating enzyme glucose-6-phosphate dehydrogenase (G6PD). Theoretically, in anabolic situations, the increase of gene expression of G6PD and 6-phosphogluconate dehydrogenase can induce a rise in the production of NADPH, which would cause the immediate inhibition of the enzyme and a drastic flow reduction. However, increasing the flow through oxidative phase of the pentose phosphate pathway (OPPP) has been experimentally demonstrated in many physiological states. However, this situation will be resolved if the NADPH metabolized or otherwise sufficient NADPH is sequestered to relax the inhibition of the dehydrogenases of OPPP and to maintain high ratio of NADPH/NADP(+) needed to ensure the reducing environment of the cell cytoplasm and the contribution of NADPH for anabolic processes. In 1974, the presence of a protein capable of reversing the inhibition of G6PD by NADPH was detected; however, to date, this paradox remains undisclosed. This review deals with the possibility that such reverting action might be similar to the activity of a protein named HSCARG, which is responsible for the abduction of NADPH, thus keeping a portion of the coenzyme away from the catalytic action and, simultaneously, the immune response through the NF-κB (nuclear factor kappa light-chain enhancer of activated B cells) system. The model has many similarities with the hypothesis proposed some 40 years back on the reversion of G6PD inhibition by NADPH.

After the Prestige oil spill modifications in NO production and other parameters related to the immune response were detected in hemocytes of Mytilus galloprovincialis.

In marine mollusks, many physiologic functions are regulated seasonally depending on such factors as the reproductive cycle or the presence of food. The synthesis of nitric oxide by hemocytes of Mytilus galloprovincialis is among the multiple physiologic actions in the immune response, and it is also affected by season. The maximal basal production of NO by hemocytes of M. galloprovincialis was detected in summer, whereas the minimum values were detected in winter. In winter, the presence of IL-2 induced an increase in NO production that was not detected in summer. Three months after the Prestige oil spill (November 2002), basal NO production by the hemocytes of mussels in the Galician coast showed a progressive decrease and stopping, both in summer and in winter. The characteristic increase of NO synthesis induced by IL-2 in winter also disappeared all through 2003 and 2004. The two different nitric oxide synthases previously identified by immunoblotting between 1999 and 2002 were undetectable in both 2003 and 2004. When comparing the data obtained during 2003 and 2004 to those obtained in previous years, an increase in the proportion of SH cells was detected. Also, these cells showed a higher sensitivity to apoptosis- and necrosis-inducing agents than in earlier years.

In hemocytes from Mytilus galloprovincialis Lmk., treatment with corticotropin or growth factors conditions catecholamine release.

The cells in charge of the innate immune response in the sea mussel Mytilus galloprovincialis Lmk. are the hemocytes, which have the capacity to release catecholamines when subjected to stressing conditions. Hemocytes were kept in culture before stimulation. That is, their behaviour was not studied immediately after extraction from the mollusc, as happens in most studies. This avoids the interference and variability caused by the conditions in which mussels may be when collected. This work describes the great variability found in the pattern of catecholamine release when the hemocytes are stimulated with either corticotropins or growth factors. Dopamine, adrenaline and noradrenaline release differs with each of the inducers assayed, with stimulation time and with the season of hemocyte collection. One of the results presented is particularly remarkable; such is the great amount of adrenaline and noradrenaline released to the medium when the hemocytes obtained in summer are stimulated with transforming growth factor-beta1 (TGF-beta1) for 60 min.

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.

Nitric oxide production by haemocytes from Mytilus galloprovincialis shows seasonal variations.

Nitric oxide (NO) has been identified as an important physiological modulator, with evidence of its role as a signalling molecule throughout the whole phylogenetic scale. In marine molluscs, it intervenes in processes related to the immune function of haemocytes. The presented results indicate that basal NO production by haemocytes of Mytilus galloprovincialis shows seasonal variations, with summer values statistically higher than those of winter. The presence of IL-2 increased NO production in winter. In summer, incubating the haemocytes with TNF-alpha for 6h slightly increased NO production. LPS, TGF-beta1 or PDGF did not induce significant effects on NO production by the haemocytes. Immunoblotting experiments detected two proteins that bind to vertebrate iNOS and eNOS antibodies, with different seasonal expression: the protein that binds to anti-iNOS antibody was expressed throughout the year, whereas the anti-eNOS antibody bound with a protein that was only detected in winter. IL-2 is suggested to start a signalling system dependent on the seasonal presence of winter protein. Such a system would activate the enzyme, thus favouring the higher NO production detected in winter.

Implication of PKA and PKC in the activation of the haemocytes of Mytilus galloprovincialis Lmk by LPS and IL-2.

LPS and IL-2 play an essential role in the generation of the immune response in diverse eukaryotic species, as they provoke the activation of several pathways of signal transduction in macrophages. Among the kinases related to these pathways, PKA and the PKCs are some of the most important. In the haemolymph of the marine mussel Mytilus galloprovincialis Lmk, the cells responsible for the defence reactions are the haemocytes. These act as active phagocytes, and are able also to secrete humoral factors. The effect of the stimulation of the haemocytes with either LPS or IL-2 on the expression of both a Ca(2+)-independent PKC (p105) and a regulatory subunit (RII) of PKA found in mussel tissues are studied in this work. Also, the effect of inhibitors specific for these kinases on their expression and on their release of catecholamines is reported.

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.

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.

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.

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-PKCdelta and PKCepsilon 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 PKCepsilon 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.