Biomedical subjects

Letizia Palomba

Publications and source records attributed to Letizia Palomba.

11 recordsLinked to original sources

PubMed2006

TNF-alpha downregulates eNOS expression and mitochondrial biogenesis in fat and muscle of obese rodents.

Obesity is associated with chronic low-grade inflammation. Thus, at metabolically relevant sites, including adipose tissue and muscle, there is abnormal production of proinflammatory cytokines such as TNF-alpha. Here we demonstrate that eNOS expression was reduced, with a concomitant reduction of mitochondrial biogenesis and function, in white and brown adipose tissue and in the soleus muscle of 3 different animal models of obesity. The genetic deletion of TNF receptor 1 in obese mice restored eNOS expression and mitochondrial biogenesis in fat and muscle; this was associated with less body weight gain than in obese wild-type controls. Furthermore, TNF-alpha downregulated eNOS expression and mitochondrial biogenesis in cultured white and brown adipocytes and muscle satellite cells of mice. The NO donors DETA-NO and SNAP prevented the reduction of mitochondrial biogenesis observed with TNF-alpha. Our findings demonstrate that TNF-alpha impairs mitochondrial biogenesis and function in different tissues of obese rodents by downregulating eNOS expression and suggest a novel pathophysiological process that sustains obesity.

Adenosine Triphosphate↗

PubMed2006

5-Hydroxyeicosatetraenoic acid is a key intermediate of the arachidonate-dependent protective signaling in monocytes/macrophages exposed to peroxynitrite.

Endogenous generation of arachidonic acid via selective activation of cytosolic phospholipase A(2) has been implicated in the mechanism of monocytes/macrophage survival in the presence of peroxynitrite. In particular, the lipid messenger was shown to prevent the otherwise rapid onset of a mitochondrial permeability-transition (MPT)-dependent necrosis by causing the mitochondrial translocation of protein kinase Calpha (PKCalpha) and the ensuing cytosolic accumulation of the Bcl-2-antagonist of cell death (Bad), an event promoting the anti-MPT function of Bcl-2 (or Bcl-X(L)). Here, we show that the effects on PKCalpha are not mediated directly by arachidonate but rather, by downstream products of the enzyme 5-lipoxygenase (5-LO). Peroxynitrite elicited the nuclear membrane translocation of 5-LO and enhanced its enzymatic activity via a mechanism sensitive to low concentrations of inhibitors of 5-LO or the 5-LO-activating protein, as well as to genetic depletion of the latter enzyme. Inhibition of 5-LO activity was invariably associated with the cytosolic localization of PKCalpha, the mitochondrial accumulation of Bad, and a rapid MPT-dependent necrosis. All these events were prevented by nanomolar concentrations of the 5-LO product 5-hydroxyeicosatetraenoic acid.

Arachidonate 5-Lipoxygenase↗

PubMed2005

Peroxynitrite-induced mitochondrial translocation of PKCalpha causes U937 cell survival.

Our previous work has shown that non-toxic concentrations of peroxynitrite nevertheless commit U937 cells to mitochondrial permeability-transition (MPT)-dependent necrosis that is however prevented by a parallel survival signaling pathway involving cytosolic phospholipase A2 (cPLA2)-dependent arachidonic acid release and PKCalpha activation associated with the cytosolic translocation of Bad. The present study provides evidence of an early mitochondrial translocation of PKCalpha. Inhibition of the survival signaling at the level of either cPLA2, or PKC, was invariably associated with prevention of the mitochondrial localization of PKCalpha, with the mitochondrial translocation of Bad and Bax and with a very rapid lethal response. Collectively, the results presented in this study demonstrate that peroxynitrite, while committing U937 cells to necrosis, triggers a parallel signaling response leading to the cytosolic localization of two important members of the Bcl-2 family implicated in the onset of MPT.

Cell Survival↗

PubMed2005

Survival pathways triggered by peroxynitrite in cells belonging to the monocyte/macrophage lineage.

Peroxynitrite, a highly reactive nitrogen species, promotes in U937 cells (a promonocytic cell line) a mitochondrial permeability transition (MPT)-dependent necrosis. An initial event triggered by peroxynitrite (i.e., inhibition of complex III of the mitochondrial respiratory chain) is responsible for the time-dependent formation of H(2)O(2), essential for the occurrence of cell death. Otherwise non-toxic concentrations of peroxynitrite nevertheless commit cells to MPT-dependent necrosis, which is however prevented by a cytoprotective signaling driven by arachidonic acid (AA) released by the cytosolic PLA(2) isoform. Interestingly, the mechanism whereby delayed formation of H(2)O(2) promotes toxicity in cells exposed to intrinsically toxic concentrations of peroxynitrite is independent of the accumulation of additional damage. Cell death is in fact mediated by inhibition of the AA-dependent cytoprotective signaling. Exogenous AA, however, prevented toxicity also under these conditions. An additional point to be made is that the major findings obtained using U937 cells were reproduced in different cell types belonging to the monocyte/macrophage lineage. Hence, within the context of the inflammatory response, monocytes and macrophages may cope with peroxynitrite by using AA, a signaling molecule largely available at the inflammatory sites.

Animals↗

PubMed2005

U937 cell necrosis mediated by peroxynitrite is not caused by depletion of ATP and is prevented by arachidonate via an ATP-dependent mechanism.

Exposure of U937 cells to an otherwise nontoxic concentration of peroxynitrite promotes a rapid necrotic response in the presence of pharmacological inhibitors of phospholipase A2. A 12-fold higher concentration of the oxidant, in the absence of additional treatments, caused remarkably greater DNA single-strand breakage, delayed formation of H2O2, and depletion of reduced glutathione but an identical level of toxicity. Cell death was prevented in both circumstances by nanomolar levels of arachidonic acid or by cyclosporin A via mechanisms unrelated to elimination of the above effects and was causally linked to prevention of mitochondrial permeability transition. Treatment with a high dose of peroxynitrite for 30 min caused an approximately 40% decline in ATP, both in the absence and presence of arachidonate, whereas only a small, arachidonic acid-sensitive reduction of the ATP pool was detected in cells treated with the low dose of peroxynitrite and the phospholipase A2 inhibitor. ATP-predepleted cells, however, were hypersensitive to peroxynitrite, and under these conditions, toxicity was not prevented by arachidonate. The above findings were reproduced in another promonocytic cell line, THP-1 cells. We concluded that the rapid necrotic response triggered by peroxynitrite in monocytes is mediated by a regulated process, not by ATP depletion, associated with reduced arachidonate availability. Supplementation of exogenous arachidonic acid always rescued cells via an ATP-dependent survival pathway.

Adenosine Triphosphate↗

PubMed2004

Mitochondrial biogenesis by NO yields functionally active mitochondria in mammals.

We recently found that long-term exposure to nitric oxide (NO) triggers mitochondrial biogenesis in mammalian cells and tissues by activation of guanylate cyclase and generation of cGMP. Here, we report that the NO/cGMP-dependent mitochondrial biogenesis is associated with enhanced coupled respiration and content of ATP in U937, L6, and PC12 cells. The observed increase in ATP content depended entirely on oxidative phosphorylation, because ATP formation by glycolysis was unchanged. Brain, kidney, liver, heart, and gastrocnemius muscle from endothelial NO synthase null mutant mice displayed markedly reduced mitochondrial content associated with significantly lower oxygen consumption and ATP content. In these tissues, ultrastructural analyses revealed significantly smaller mitochondria. Furthermore, a significant reduction in the number of mitochondria was observed in the subsarcolemmal region of the gastrocnemius muscle. We conclude that NO/cGMP stimulates mitochondrial biogenesis, both in vitro and in vivo, and that this stimulation is associated with increased mitochondrial function, resulting in enhanced formation of ATP.

Adenosine Triphosphate↗

PubMed2004

Inhibition of nitric-oxide synthase-I (NOS-I)-dependent nitric oxide production by lipopolysaccharide plus interferon-gamma is mediated by arachidonic acid. Effects on NFkappaB activation and late inducible NOS expression.

Previous results have indicated that lipopolysaccharide (LPS) plus interferon-gamma (IFNgamma) inhibits nitric-oxide synthase (NOS)-I activity in glial cells. We report here that arachidonic acid (AA) plays a pivotal role in this response, which was consistently reproduced in different glial cell lines and in primary rat astrocytes. This notion was established using pharmacological inhibitors of phospholipase A2 (PLA2), cytosolic PLA2 (cPLA2) antisense oligonucleotides, and AA add-back experiments. This approach not only allowed the demonstration that AA promotes inhibition of NOS-I activity but also produced novel experimental evidence that LPS/IFNgamma itself is a potential stimulus for NOS-I. Indeed, LPS/IFNgamma fails to generate nitric oxide (NO) via NOS-I activation simply because it activates the AA-dependent signal that impedes NOS-I activity. Otherwise, LPS/IFNgamma promotes NO formation, sensitive to exogenous AA, in cells in which cPLA2 is pharmacologically inhibited or genetically depleted. Because NO suppresses the NFkappaB-dependent NOS-II expression, inactivation of NOS-I by the LPS/IFNgamma-induced AA pathway provides optimal conditions for NFkappaB activation and subsequent NOS-II expression. Inhibition of cPLA2 activity, while reducing the availability of AA, consistently inhibited NFkappaB activation and NOS-II mRNA induction and delayed NO formation. These responses were promptly reestablished by addition of exogenous AA. Finally, we have demonstrated that the LPS/IFNgamma-dependent tyrosine phosphorylation of NOS-I and inhibition of its activity are mediated by endogenous AA.

Animals↗

PubMed2004

Downregulation of nitric oxide formation by cytosolic phospholipase A2-released arachidonic acid.

Exposure of PC12 cells to A23187 or thapsigargin caused a concentration-dependent release of arachidonic acid (AA) mediated by cytosolic phospholipase A2 (PLA2). Under the same conditions, however, analysis of nitric oxide (NO) formation revealed that activation of NO synthase (NOS) is best described by a bell-shaped curve. Reduced detection of NO observed at increasing A23187 or thapsigargin concentrations was not due to formation of peroxynitrite or to activation of NO-consuming processes, but rather to AA-dependent inhibition of NOS activity. Furthermore, NO formation observed under optimal conditions for NOS activity was suppressed by AA as well as by the PLA2 activator melittin. Finally, the effects of AA were not the consequence of direct enzyme inhibition, because this lipid messenger failed to inhibit formation of NO by purified neuronal NOS, but were mediated by an AA-dependent signaling and not by downstream products of the cyclooxygenase and lipoxygenase pathways. In conclusion, the present study underscores a novel mechanism whereby endogenous, or exogenous, AA promotes inhibition of NOS activity. Because AA is generated in response to various agonists acting on membrane receptors and extensively released in inflammatory conditions, these findings have important physiopathological implications.

Animals↗

PubMed2003

The Raf/MEK inhibitor PD98059 enhances ERK1/2 phosphorylation mediated by peroxynitrite via enforced mitochondrial formation of reactive oxygen species.

Exposure of PC12 cells to 100 microM peroxynitrite promotes phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) sensitive to PD98059 or U0126. At higher concentrations, however, ERK1/2 phosphorylation was prevented by U0126 and increased by PD98059 via a U0126-sensitive mechanism. PD98059, unlike U0126, enhanced the peroxynitrite-dependent formation of reactive oxygen species (ROS). These results, along with others obtained using respiratory chain inhibitors and respiration-deficient cells, lead to the conclusion that PD98059, while effectively inhibiting the peroxynitrite-induced Raf/MEK signaling leading to ERK1/2 phosphorylation, promotes an enforced mitochondrial formation of ROS inducing ERK1/2 phosphorylation via a Raf-1-independent/MEK-dependent mechanism.

Adrenal Gland Neoplasms↗

PubMed2002

Beta-amyloid inhibits NOS activity by subtracting NADPH availability.

The amyloid peptides Abeta1-42 and Abeta25-35 strongly inhibited the activity of constitutive neuronal and endothelial nitric oxide synthases (i.e., NOS-I and NOS-III, respectively) in cell-free assays. The molecular mechanism of NOS inhibition by Ab fragments was studied in detail with Abeta25-35. The inhibitory ability was mostly NADPH-dependent and specific for the soluble form of Abeta25-35. Optical, fluorescence, and NMR spectroscopy showed that the soluble, but not aggregated, Abeta25-35 interacted with NADPH, thus suggesting that a direct recruitment of NADPH may result in diminished availability of the redox cofactor for NOS functioning. To assess the physiological relevance of our findings, rat neuronal-like PC12 and glioma C6 cell lines were used as cellular models. After Abeta25-35 internalization into cells was verified, the activity of constitutive NOS was measured using the DAF-2DA detection system and found to be severely impaired upon Abeta25-35 uptake. Consistent with previous results on the molecular cross-talk between NOS isoforms, repression of constitutive NOS by Abeta25-35 resulted in enhanced expression of inducible NOS (NOS-II) mRNA in C6 cells. Our results represent the first evidence that amyloid fragments impair constitutive NOS activity in cell-free and cellular systems, providing a possible molecular mechanism for the onset and/or maintenance of Alzheimer's disease.

Amyloid beta-Peptides↗

PubMed2002

Cell signaling and cytotoxicity by peroxynitrite.

Reactive nitrogen species are now considered to play an important role in various pathologies. Although the pathological significance of these molecules, peroxynitrite in particular, has long been attributed to their abilities to react with any component of the cells, including lipids, proteins, and DNA, a paradigm shift has recently been occurring whereby reactive nitrogen species are appreciated as signaling molecules. The question therefore arises as to whether nitrosative stress is indeed the result of a random (stochastic) process of cell damage, as it has traditionally been viewed, or rather is a consequence of the specific activation of a cascade of signaling events. The above considerations have provided the bases for the research work performed in our laboratory, and the results obtained are illustrated in the present article. In particular, our results indicate that some effects of peroxynitrite are not directly mediated by the oxidant; rather, it appears that peroxynitrite triggers a signaling pathway that finally leads to cytotoxicity.

Apoptosis↗