Protective role of MnSOD and redox regulation of neuronal cell survival.

Reactive oxygen species (ROS) play a central role in neuronal pathophysiology and in neurodegenerative disorders. However, recent evidence indicates that these molecules also operate as signaling intermediates in a variety of physiological settings, including cell protection from apoptosis. Data presented here strongly support such a dual role for oxidants in neuronal cell homeostasis. In rat pheocromocytoma cells, cell rescue by the nerve growth factor (NGF) is accompanied by a transient burst of ROS generated in the cytosol by a GTPase-dependent mechanism. Within the NGF signaling cascade, ROS lie upstream and are necessary for activation/phosphorylation of AKT/PKB and of the antiapoptotic transcription factor cAMP-responsive element-binding protein (CREB). Conversely, an increase in mitochondrial oxygen species heralds apoptosis of serum-deprived cells, and these events can be prevented by cell exposure to NGF or by treatment with the mitochondrially targeted antioxidant MitoQ. Importantly, NGF-mediated decrease of mitochondrial ROS is dependent on the transcriptional up-regulation of the manganese superoxide dismutase (MnSOD) by active CREB. These observations therefore outline a circuitry whereby cytosolic redox signaling promotes neuronal cell survival by increasing the mitochondrial antioxidant defenses.

Anticardiolipin antibodies in patients with primary antiphospholipid syndrome: a correlation between IgG titre and antibody-induced cell dysfunctions in neuronal cell cultures.

Anticardiolipin antibodies (aCL) of the immunoglobulin (Ig) G isotype have been significantly associated with neurological manifestations of antiphospholipid syndrome (APS). In a previous study we described the direct pathogenic effects of IgG aCL on living neurons in culture. Therefore, we studied the IgG aCL titre as a factor influencing the extent of this effect. Seventeen patients with a history of primary antiphospholipid syndrome were grouped according to their IgG aCL titre into low positive (GPL < or = 40), high positive (40< GPL <100) and very high positive (GPL >100). IgG from these patients were incubated with cerebellar neurons in primary culture for 24h and the effect was evaluated by using the tetrazolium salt (MTT) assay. We found that almost all patients' IgGs reduced cell viability in vitro, but the differences in the extent of the effect were statistically significant only for patients with >40 GPL. Our results reinforce the causal association between increasing level of IgG aCL and clinical features of aPS.

Excitatory amino acid stimulation of the survival of rat cerebellar granule cells in culture is associated with an increase in SMN, the spinal muscular atrophy disease gene product.

Excitatory amino acids which promote the survival of cerebellar granule cells in culture, also promote the expression of the survival of motor neuron (SMN) protein. Immunolocalization studies using SMN monoclonal antibody showed that SMN is decreased in cultures grown in low K+ or chemically defined medium with respect to cultures grown in high K+ medium and that an increase of SMN can be induced by treatment of low K+ cultures with glutamate or N-methyl-D-aspartate.

Antiphospholipid antibodies bind to rat cerebellar granule cells: the role of N-methyl-D-aspartate receptors.

IgGs from sera containing antiphospholipid antibodies (aPL), detected as antibodies to cardiolipin, or control sera were incubated with rat cerebellar granule cells in primary culture. Using a mitochondrial dehydrogenase activity assay (MTT test), aPL IgGs were shown to decrease MTT metabolism after 24 h incubation with the cells, and to cause non-toxic amounts of glutamate to become neurotoxic when added to the cells for 45 min. Acute and chronic aPL toxicity were prevented by MK-801. Sera containing aPL bound to intact cerebellar neurons, as revealed by an immunofluorescent technique. These results suggest that antiphospholipid antibodies interfere with excitatory pathways in glutamatergic cerebellar granule cells by a mechanism involving overactivation of the NMDA glutamate receptor.

Modulation by protein kinase C of nitric oxide and cyclic GMP poffation in cultured cerebellar granule cells.

The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated in primary cultures of rat cerebellar neurons. Incubation of the cells with L-arginine and nicotinamide-adenine dinucleotide phosphate (NADPH) produced detectable levels of NO, as quantified by photometric assay [0.14 +/- 0.03 nmol/h/dish (2.5 x 10(6) cells)]. The NO producing activity was paralleled by concomitant accumulation of cyclic GMP (cGMP) (0.12 +/- 0.02 pmol/dish). Downregulation of PKC by prolonged treatment with phorbol esters or inhibition of the kinase by treatment with 4taurosporine raised the basal levels of NO and cGMP five fold. When granule cells were incubated in the absence of extracellular Mg2+, N-methyl-D-aspartate and to a lesser extent, glutamate became effective in enhancing NO formation and cGMP accumulation with respect to the control. The NO and cGMP increases induced by the two agonists were almost doubled by treatment of the cells with staurosporine or depletion of PKC. Calphostin C. an inhibitor of the regulatory domain of PKC, was as effective as staurosporine in increasing the formation of NO in both resting and excited cells. These results indicate that downregulation or inhibition of PKC increase NOS activity in cerebellar neurons, and suggest that phosphorylation of NOS by PKC negatively modulates the catalytic activity of the enzyme in these cells.

Transformation linked decrease of pyruvate dehydrogenase complex in human epidermis.

Epidermis exhibits glycolytic features peculiar to cancer cells. The activity of pyruvate dehydrogenase complex, both active (PDHa) and total (PDHt) forms, has been investigated and compared in epidermis and epidermal carcinomas from human source. Low or undetectable PDHa is found in either normal and neoplastic tissue. PDHt is unchanged in human epidermis between the second and seventh decades of life but is dramatically decreased following neoplastic transformation (0.107 and 0.026 units/g fresh tissue for epidermis and epidermal carcinoma, respectively). As PDH plays a key role in mitochondrial carbohydrate metabolism, the decrease of total enzymic capacity found in tumors suggest that different mechanisms regulate PDH expression and, in turn, glycolytic mechanisms of epidermis and cancer cells.

Glutamate-induced protein phosphorylation in cerebellar granule cells: role of protein kinase C.

Protein phosphorylation in response to toxic doses of glutamate has been investigated in cerebellar granule cells. 32P-labelled cells have been stimulated with 100 microM glutamate for up to 20 min and analysed by one and two dimensional gel electrophoresis. A progressive incorporation of label is observed in two molecular species of about 80 and 43 kDa (PP80 and PP43) and acidic isoelectric point. Glutamate-stimulated phosphorylation is greatly reduced by antagonists of NMDA and non-NMDA glutamate receptors. The effect of glutamate is mimicked by phorbol esters and is markedly reduced by inhibitors of protein kinase C (PKC) such as staurosporine and calphostin C. PP80 has been identified by Western blot analysis as the PKC substrate MARCKS (myristoylated alanine-rich C kinase substrate), while antibody to GAP-43 (growth associated protein-43), the nervous tissue-specific substrate of PKC, failed to recognize PP43. Our results suggest that PKC is responsible for the early phosphorylative events induced by toxic doses of glutamate in cerebellar granule cells.

Differential involvement of protein kinase C in transmitter release and response to excitatory amino acids in cultured cerebellar neurons.

Cerebellar granule cells cultured in the presence of a differentiating factor isolated from rabbit serum exhibit, at variance with those cultured in fetal calf serum, an almost complete resistance to excitatory aminoacid (EAA)-induced cytotoxicity. We investigated the behaviour of protein kinase C (PKC), strongly implicated in EAA cytotoxicity, in the two types of culture. Phorbol esters, used to monitor the enzyme, enhanced the depolarization-evoked release of D-[3H]aspartate, but less effectively in factor-conditioned cells. EAAs increased phorbol esters binding in both cultures, but the effect was briefly lasting in factor-conditioned cells. The different behaviour of PKC is postulated to be causally related to different response to EAA of the cultures.

Cerebellar neurones: differentiation and modulation of sensitivity to excitotoxic treatment.

The neurite outgrowth and adhesion complex (NOAC), isolated from rabbit sera has been dissociated in its major components by reverse-phase chromatography in HPLC by using a C18 column. SDS-PAGE analysis of the active fractions revealed the presence of three major bands of approximately 100, 70 and 50 kDa. Studies on the biological activity of NOAC were carried out on rat cerebellar granule cells. NOAC-cultured cells exhibit a marked resistance to excitotoxic stimuli carried by glutamate.

Chronic myeloid leukemia with monoclonal gammopathy terminating in myeloid crisis and immunoblastic lymphoma.

A patient, with chronic myeloid leukemia and IgA monoclonal gammopathy, who contemporaneously developed myeloid blast crisis and immunoblastic lymphoma is reported. Cytogenetic studies showed complex chromosome abnormalities concerning chromosomes 8, 14 and 22, other than the Ph chromosome. A possible relationship between the emergence of immunoblasts from slow proliferating lymphoplasmacytoid cells, myeloid blasts crisis and chromosomal changes is discussed.

Oxidation of reduced cytosolic nicotinamide adenine dinucleotide by the malate-aspartate shuttle in the K-562 human leukemia cell line.

The activity of the malate-aspartate shuttle for the reoxidation of reduced cytosolic nicotinamide adenine dinucleotide (NADH) by mitochondria was studied in a line of human myeloid leukemia cells (K-562). The tumor cells showed mitochondrial reoxidation of cytosolic NADH, as evidenced by the accumulation of pyruvate, when incubated aerobically with L-lactate. The involvement of the respiratory chain in the reoxidation of cytosolic NADH was demonstrated by the action of rotenone, antimycin A, and oligomycin which strongly inhibited the formation of pyruvate from added L-lactate. Moreover, pyruvate production was greatly inhibited by the transaminase inhibitor, aminooxyacetate. Under glycolytic conditions, in the presence of aminooxyacetate, the rate of pyruvate production was also markedly inhibited, the rate of lactate accumulation was stimulated, and at 60 min the cytosolic NADH/nicotinamide adenine dinucleotide (NAD) ratio had increased progressively about 5-fold with respect to untreated cells. The maximal rate of the malate-aspartate shuttle has also been established by addition of arsenite to inhibit mitochondrial oxidation of the pyruvate formed from added L-lactate.

Pyruvate dehydrogenase levels in Morris hepatomas with different growth rate.

Pyruvate dehydrogenase (PDH) activity has been evaluated with respect to normal liver in 3 lines of Morris hepatomas (H), i.e. the highly differentiated H 9618A, the well differentiated H 44 and the poorly differentiated H 3924A. Assays of both total (PDHt) and active (PDHa) form show a progressive decrease of enzyme activity going from liver to the H 3924A. PDHa better correlates with the degree of hepatoma differentiation than does PDHt. Further enzyme analysis has been achieved in partially purified extracts from liver and H 3924A. The possible implications of such an enzymatic variation are discussed.

Inhibition of mitochondrial oxidative metabolism by SKF-525A in intact cells and isolated mitochondria.

We have examined the effects of various concentrations of SKF-525A (beta-diethylaminoethyldiphenylpropyl acetate X HCl) on the energy metabolism of liver slices, isolated liver mitochondria, and two types of ascites tumor cells, as well as on ion transport in liver slices. In liver slices, 0.2 to 1.0 mM SKF-525A caused an initial stimulation of O2 uptake which was followed, at 0.5 to 1.0 mM, by a progressive inhibition of O2 consumption, a fall of slice ATP content, and a reduced transport of K+, Na+ and Ca2+. In isolated mitochondria, we studied the effects of SKF-525A on the rate of respiration and on the oxidation-reduction responses of NAD(P)+ and cytochrome b in the presence of various substrates. The results suggest that SKF-525A had three distinct actions on liver mitochondria, viz. an uncoupling action at low concentrations (0.02 to 0.17 mM); at higher concentrations (0.2 to 0.5 mM) an inhibition of the oxidation of NAD(P)+-linked substrates, exerted close to the substrate level; also at 0.2 to 0.5 mM, a less effective inhibition of electron transfer at a point between cytochrome b and O2 in the electron-transfer chain. Experiments on O2 consumption and cytochrome b oxidation-reduction changes in ascites cells showed only the first two of these effects in the intact tumor cells. We conclude that inhibition of mitochondrial energy-conserving reactions by SKF-525A can have a marked influence on energy-requiring aspects of liver-cell metabolism, one example of which is inhibition of cation active transport.

Evidence for the occurrence of the malate-citrate shuttle in intact Ehrlich ascites tumor cells.

A possible activity of the malate-citrate shuttle has been investigated in Ehrlich ascites cells by testing the effects of 1,2,3-benzenetricarboxylic acid, an inhibitor of the malate-citrate exchange, and (-)-hydroxycitrate, an inhibitor of the citrate cleavage enzyme, on the glucose-dependent oxidation-reduction rates of pyridine nucleotides and cytochrome b as well as two inhibitors glycolyzing cells. Moreover, to quantitate such an activity, the effects of these two inhibitors have been compared with those induced under the same experimental conditions by aminooxyacetate, an inhibitor of the malate-aspartate shuttle which is known to operate in this strain of ascites tumor. Both benzenetricarboxylic acid and hydroxycitrate are able to increase the reduction of pyridine nucleotides, which follows glucose addition to whole cells, to about the same extent. A much more pronounced effect is elicited by aminooxyacetate under the same condition. When n-butylmalonate is added to slow down the flux of glycolytic reducing equivalents to the respiratory chain via the malate-aspartate shuttle, benzenetricarboxylic acid or hydroxycitrate promotes an ATP-driven reversal of electron transfer. Indeed, the glucose induced reduction of cytochrome b becomes sensitive to oligomycin and the ATP level is raised significantly with respect to the value of uninhibited cells. It is concluded that the malate-citrate shuttle operates in Ehrlich ascites cells, although with a substantially lower activity with respect to the malate-aspartate shuttle.

Calcium transport and translocation of adenine nucleotides in mitochondria from Morris hepatoma 3924A.

The interaction of Ca2+ with Morris hepatoma 3924A mitochondria and its effect on the adenine nucleotide translocation have been studied. The characteristics of the Ca2+ transport process in mitochondria from Morris hepatoma are not significantly different from those of normal liver mitochondria. The Km for Ca2+ is 2 to 3 microM, and the rate versus concentration curve exhibits hyperbolic kinetics. A lower activity of the adenine nucleotide translocation was found, probably due to the high endogenous Ca2+ content of Morris hepatoma mitochondria (123 +/- 15 nmol Ca2+ per mg protein). No further inhibition of the translocase activity was observed after isolated mitochondria had accumulated extra amounts of Ca2+. The total amount of adenine nucleotides in tumor mitochondria is one-half those present in control liver, and a significantly lower percentage of the pool is present as adenosine 5'-monophosphate.

Pyruvate transport in tumour-cell mitochondria.

Tumour-cell mitochondria contain a pyruvate-transporting system exhibiting the same general properties as those described in rat liver mitochondria. The Km for net pyruvate uptake in tumour-cell mitochondria is practically similar to that measured in rat liver mitochondria but the V is lower. This difference is also shown by swelling experiments. The possible implication of these observations in the context of lactate accumulation in tumour-cell is discussed.