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Biomedical subjects

E Clementi

Publications and source records attributed to E Clementi.

At least 37 records · Page 2Linked to original sources
PubMed1999

NADH-Linked substrate-mediated enhancement of mitochondrial calcium accumulation and DNA single-strand breakage elicited by tert-butylhydroperoxide: the source of the cation is a ryanodine-sensitive calcium store.

We previously found that the membrane-permeant NADH-linked substrates pyruvate and beta-hydroxybutyrate enhance the formation of DNA single-strand breaks induced by tert-butylhydroperoxide (tB-OOH) in intact U937 cells. This effect is mediated by a process involving enforced mitochondrial calcium accumulation in the absence of discernible elevation in the cytosolic concentration of free calcium ions. We now show that the intracellular source of the cation is a ryanodine-sensitive calcium store. A high concentration of ryanodine, which suppressed the caffeine-mediated mobilization of calcium ions, also abolished the effects of the NADH-linked substrates on the mitochondrial accumulation of the cation as well as on the tB-OOH-induced genotoxic response. These data constitute a novel demonstration of a physiological mechanism with important pathological implications.

Caffeine↗
PubMed1999

On the mechanism by which vascular endothelial cells regulate their oxygen consumption.

Two enzymes, soluble guanylyl cyclase and cytochrome c oxidase, have been shown to be exquisitely sensitive to nitric oxide (NO) at low physiological concentrations. Activation of the soluble guanylyl cyclase by endogenous NO and the consequent increase in the second messenger cyclic GMP are now known to control a variety of biological functions. Cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, is inhibited by NO. However, it is not clear whether NO produced by the constitutive NO synthase interacts with cytochrome c oxidase, nor is it known what the biological consequences of such an interaction might be. We now show that NO generated by vascular endothelial cells under basal and stimulated conditions modulates the respiration of these cells in response to acute changes in oxygen concentration. This action occurs at the cytochrome c oxidase and depends on influx of calcium. Thus, NO plays a physiological role in adjusting the capacity of this enzyme to use oxygen, allowing endothelial cells to adapt to acute changes in their environment.

Animals↗
PubMed1999

Different signalling pathways mediate the opposite effects of endogenous versus exogenous nitric oxide on hydroperoxide toxicity in CHP100 neuroblastoma cells.

The results presented in this study indicate that the toxic response brought about by increasing concentrations of tert-butylhydroperoxide in CHP100 cells was mitigated significantly by exogenously added nitric oxide donors via a cyclic GMP-independent mechanism. In contrast with these results, endogenous nitric oxide generated by the Ca2+-mobilizing agent caffeine was found to increase hydroperoxide toxicity. Under these conditions, nitric oxide was not directly toxic to the cells. Rather, nitric oxide was found to promote the caffeine-mediated release of Ca2+ from ryanodine-sensitive Ca2+ stores via a cyclic GMP-independent mechanism. Release of the cation from ryanodine-sensitive Ca2+ stores was causally linked with the caffeine/nitric oxide-mediated enhancement of tert-butylhydroperoxide toxicity. It is concluded that endogenous and exogenous nitric oxide activate diverging signalling pathways independent of cyclic GMP formation and causing opposite effects on the toxic response evoked by tert-butylhydroperoxide in CHP100 cells.

Caffeine↗
PubMed1998

Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione.

Both reversible and irreversible inhibition of mitochondrial respiration have been reported following the generation of nitric oxide (NO) by cells. Using J774 cells, we have studied the effect of long-term exposure to NO on different enzymes of the respiratory chain. Our results show that, although NO inhibits complex IV in a way that is always reversible, prolonged exposure to NO results in a gradual and persistent inhibition of complex I that is concomitant with a reduction in the intracellular concentration of reduced glutathione. This inhibition appears to result from S-nitrosylation of critical thiols in the enzyme complex because it can be immediately reversed by exposing the cells to high intensity light or by replenishment of intracellular reduced glutathione. Furthermore, decreasing the concentration of reduced glutathione accelerates the process of persistent inhibition. Our results suggest that, although NO may regulate cell respiration physiologically by its action on complex IV, long-term exposure to NO leads to persistent inhibition of complex I and potentially to cell pathology.

Animals↗
PubMed1998

Role of nitric oxide and its intracellular signalling pathways in the control of Ca2+ homeostasis.

Ca2+, a primary regulator of physiological functions in all cells, is involved in a variety of intracellular signalling pathways; control of Ca2+ homeostasis is, therefore, a fundamental cell activity. To this end, cells have developed a variety of mechanisms to ensure the buffering of Ca2+, its influx and extrusion from the plasma membrane, and its release/accumulation within specific intracellular storage compartments. Over the last few years, evidence gathered from a number of cell systems has indicated that one of the key messengers governing the overall control of Ca2+ homeostasis is nitric oxide (NO), which may be produced intracellularly or may originate from neighboring cells. The aim of the present commentary is to concentrate on the biochemical steps in Ca2+ homeostasis that are controlled by NO and to describe what is known thus far concerning the molecular mechanisms of its action. Particular attention will be given to the effects of NO on: (i) inositol 1,4,5-trisphosphate and cyclic ADP ribose generation; (ii) Ca2+ release from both inositol 1,4,5-trisphosphate-sensitive and ryanodine-sensitive Ca2+ stores; and (iii) Ca2+ influx via both store- and second messenger-operated Ca2+ channels. The evidence discussed here documents the complexity of the interactions between the Ca2+ and the NO signalling systems, which represent an extraordinary example of cross-talk operating at multiple sites and which are continuously active in the regulation of cytosolic Ca2+ (and NO) levels.

Animals↗
PubMed1998

The inositol 1,4,5-trisphosphate-generating agonist ATP enhances DNA cleavage induced by tert-butylhydroperoxide.

In this paper we present experimental evidence indicating that DNA cleavage induced by tert-butylhydroperoxide in U937 cells can be enhanced via ATP-mediated activation of membrane receptors coupled with hydrolysis of phosphatidylinositol 4,5-bisphosphate. The mechanism whereby ATP exerts this effect involves release of Ca2+ from the inositol 1,4,5-trisphosphate (IP3)-sensitive stores, further release of the cation from the ryanodine receptor, mitochondrial clearance of the fraction of Ca2+ derived from the ryanodine receptor, and Ca2(+)-dependent mitochondrial formation of DNA-damaging species. IP3-generating agonists must therefore be considered as potential modulators of the genotoxic effects of tert-butylhydroperoxide.

Adenosine Triphosphate↗
PubMed1998

Mycobacterium tuberculosis exploits the CD95/CD95 ligand system of gammadelta T cells to cause apoptosis.

Vgamma9/Vdelta2+ T cells specifically recognize Mycobacterium tuberculosis in vitro and are precociously recruited in early mycobacterial lesions. Even if gammadelta T cells are only fortuitously detected in granulomas or bronchoalveolar lavages of patients with active pulmonary tuberculosis, a role in shaping the mature alphabeta T cell response against M. tuberculosis is substantiated. Here we provide a molecular explanation for this paradox: the engagement of the gammadelta TCR by mycobacterial antigens induced the expression of CD95 ligand (CD95L) by chronically activated CD95+/CD95L- gammadelta T lymphocytes. The receptor was functional, as CD95/CD95L interaction triggered the bystander death of CD95+ cells by apoptosis. Cell death was abolished by CD95-blocking antibodies. The transient accumulation at the site of infection of CD95L+ gammadelta lymphocytes, capable of interacting with CD95+ leukocytes attracted by the response towards the pathogen, may determine the characteristics of the ensuing granulomatous disease.

Apoptosis↗
PubMed1998

Proinflammatory cytokines regulate antigen-independent T-cell activation by two separate calcium-signaling pathways in multiple sclerosis patients.

Central nervous system (CNS) lesions typical of multiple sclerosis (MS) are characterized by demyelinating inflammatory infiltrates that contain few CNS antigen-specific autoreactive T cells and a multitude of pathogenic non-antigen-specific mononuclear cells. Here, we report that in patients with MS the combined action of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNFalpha), interleukin (IL)-2, and IL-6 leads to the activation of most peripheral T cells (mainly CD4 memory) by promoting a persistent intracellular calcium increase via two independent signaling pathways. The activation of these pathways, one activated by IFNgamma and the other by the combination TNFalpha/IL-2/IL-6, is independent from myelin antigens and precedes by 2 weeks phases of disease activity (eg, clinical relapses and/or appearance of gadolinium-enhancing lesions on brain magnetic resonance imaging scans during 1 year of follow-up). Our results indicate that an appropriate combination of the four cytokines, three with a proinflammatory profile and one necessary for T-cell growth and differentiation, can activate in an antigen-independent fashion most peripheral T cells from MS patients. This mechanism is likely to contribute to the recruitment of nonspecific lymphocytes into the cellular activation processes leading to CNS demyelination and may represent a major target for immune intervention in MS.

Antigens↗
PubMed1998

Effects of nitric oxide on proliferation and differentiation of rat brown adipocytes in primary cultures.

1. In the present work, we study the effect of NO on the proliferation and differentiation of brown fat cells in primary cultures. 2. Brown fat precursor cells isolated from rat brown adipose tissue were cultured for 8 days until confluence and treated daily with the NO donating agents, S-nitroso-acetyl penicillamine (SNAP) or S-nitroso-L-glutathione (GSNO). Both agents (300 microM) decreased cell proliferation approximately 8 fold on day 8. The inhibitory effect of NO was unlikely to be due to cytotoxicity since (i) cells never completely lost their proliferation capacity even after 8 days of exposure to repeated additions of SNAP or GSNO, and (ii) the inhibitory effect was reversible after removal of the media containing NO donors. 3. Daily treatment with nitric oxide synthase inhibitors, such as NG-nitro-L-arginine methyl ester (L-NAME, 300 microM), led to the stimulation of cell proliferation by 44+/-5%, n=3, suggesting that NO, endogenously produced in brown adipocytes, may be involved in modulating cell growth. 4. Daily treatment with both SNAP or GSNO induced significant mitochondriogenesis, measured as the mitochondrial conversion of 3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyl tetrazolium bromide (MTT) to formazan, whilst daily treatment with L-NAME was without effect. 5. The inhibition of cell proliferation by NO donors was accompanied by the expression of two genes coding for peroxisome proliferator activated receptor-gamma and uncoupling protein-1, which are upregulated during differentiation. 6. Increasing cyclic GMP in cells by 8-bromo-cyclic GMP (100-1000 microM) did not reproduce the observed NO effects on either cell number or gene expression. On the other hand, chronic treatment with the inhibitor of the NO-stimulated guanylyl cyclase, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), reduced the expression of peroxisome proliferator activated receptor-gamma and uncoupling protein-1.

Adipose Tissue, Brown↗
PubMed1998

The mechanism of the nitric oxide-mediated enhancement of tert-butylhydroperoxide-induced DNA single strand breakage.

1. Caffeine (Cf) enhances the DNA cleavage induced by tert-butylhydroperoxide (tB-OOH) in U937 cells via a mechanism involving Ca2+-dependent mitochondrial formation of DNA-damaging species (Guidarelli et al., 1997b). Nitric oxide (NO) is not involved in this process since U937 cells do not express the constitutive nitric oxide synthase (cNOS). 2. Treatment with the NO donors S-nitroso-N-acetyl-penicillamine (SNAP, 10 microM), or S-nitrosoglutathione (GSNO, 300 microM), however, potentiated the DNA strand scission induced by 200 microM tB-OOH. The DNA lesions generated by tB-OOH alone, or combined with SNAP, were repaired with superimposable kinetics and were insensitive to anti-oxidants and peroxynitrite scavengers but suppressed by iron chelators. 3. SNAP or GSNO did not cause mitochondrial Ca2+ accumulation but their enhancing effects on the tB-OOH-induced DNA strand scission were prevented by ruthenium red, an inhibitor of the calcium uniporter of mitochondria. Furthermore, the enhancing effects of both SNAP and GSNO were identical to and not additive with those promoted by the Ca2+-mobilizing agents Cf or ATP. 4. The SNAP- or GSNO-mediated enhancement of the tB-OOH-induced DNA cleavage was abolished by the respiratory chain inhibitors rotenone and myxothiazol and was not apparent in respiration-deficient cells. 5. It is concluded that, in cells which do not express the enzyme cNOS, exogenous NO enhances the accumulation of DNA single strand breaks induced by tB-OOH via a mechanism involving inhibition of complex III.

Caffeine↗
PubMed1997

Mechanism of the antimycin A-mediated enhancement of t-butylhydroperoxide-induced single-strand breakage in DNA.

Inhibitors of complex III increased the DNA strand scission induced by t-butylhydroperoxide (tB-OOH) and cumene hydroperoxide but did not affect DNA damage induced by H2O2. The hypothesis that these effects are selectively linked to inhibition of the electron transport from cytochrome b to cytochrome c1 is validated by the following observations: (1) two complex III inhibitors, antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide, enhanced the tB-OOH-induced DNA cleavage over the same concentration range as that in which inhibition of oxygen consumption was observed; (2) the complex III inhibitor-mediated enhancement of tB-OOH-induced DNA damage was abolished by the complex I inhibitor rotenone or by glucose omission, and (3) the enhancing effects of antimycin A were not observed in respiration-deficient cells. The mechanism whereby the complex III inhibitors potentiate DNA cleavage promoted by tB-OOH was subsequently investigated with intact as well as permeabilized cells. H2O2, produced at the level of mitochondria via a Ca2+-dependent process, was found to account for the enhancing effects of antimycin A.

Antimycin A↗
PubMed1997

Stimulation of oxygen consumption promotes mitochondrial calcium accumulation, a process associated with, and causally linked to, enhanced formation of tert-butylhydroperoxide-induced DNA single-strand breaks.

The NADH-linked substrates pyruvate, L-glutamine, and beta-hydroxybutyrate, while enhancing the rate of oxygen consumption, also increased the formation of DNA single-strand breaks induced by tert-butylhydroperoxide in intact U937 cells. A cause-effect relationship between these two parameters was established by showing that: (a) rotenone, an inhibitor of complex I, abolished respiration and prevented the enhancement of the DNA-damaging response under all the above circumstances; (b) the membrane-impermeant, complex I-activating substrate L-malate gave similar results in permeabilized cells; and (c) none of the NADH-linked substrates affected the DNA-damaging response to tert-butylhydroperoxide in respiration-deficient cells. Stimulation of electron transport potentiated the DNA-cleaving ability of tert-butylhydroperoxide via a process involving enforced mitochondrial calcium accumulation in the absence of a discernible elevation in the cytosolic concentration of free Ca2+. Finally, mitochondrial calcium was found to promote the mitochondrial formation of DNA-damaging levels of hydrogen peroxide. In conclusion, the data herein presented define a previously unexpected role of respiratory substrates in the control of the deleterious effects of an organic hydroperoxide at the level of genomic DNA. The enhanced DNA cleavage mediated by NADH-linked substrates in response to tert-butylhydroperoxide would appear to depend on a sequence of events involving stimulation of electron transport, mitochondrial accumulation of Ca2+, and mitochondrial formation of DNA-damaging levels of hydrogen peroxide via a Ca(2+)-dependent process.

3-Hydroxybutyric Acid↗
PubMed1997

Autocrine nitric oxide modulates CD95-induced apoptosis in gammadelta T lymphocytes.

Gammadelta T lymphocytes play an important early role in the defense against pathogens. Their function is terminated by acquisition of susceptibility to CD95-triggered apoptosis. Here we show that the regulation of this process depends on the activity of the endothelial NO synthase expressed by gammadelta T lymphocytes, which is modulated in an activation-dependent way. The effects of nitric oxide thus generated, mediated via cGMP generation, are exerted at at least two sites along the CD95 signaling cascade: one at, or upstream, and the other downstream of ceramide generation. At either site, nitric oxide/cGMP action is sufficient for protection from apoptosis. The effect of NO is selective for apoptosis induced by CD95 cross-linking, since it does not affect apoptotic program triggered by other stimuli. The evidence here reported demonstrates a new physiological role for nitric oxide, acting as a survival factor for T lymphocytes.

Apoptosis↗
PubMed1997

Nitric oxide effects on cell growth: GMP-dependent stimulation of the AP-1 transcription complex and cyclic GMP-independent slowing of cell cycling.

1. The role of nitric oxide (NO) in the control of cell growth is controversial since both stimulation and (more often) inhibition have been demonstrated in various cell types. In order to reinvestigate the problem and identify the sites of NO action, we have employed murine NIH-3T3 fibroblasts overexpressing epidermal growth factor (EGF) receptors. 2. The effects of four structurally-unrelated NO donors: S-nitroso-N-acetyl penicillamine, S-nitroso-L-glutathione, 3-morpholinosydnonimine and isosorbide dinitrate (0.01-3 mM) on EGF (10 nM)-stimulated cell growth were estimated by both thymidine incorporation and the colorimetric MTT assay, while those of a messenger generated in response to NO, cyclic GMP, were revealed by the use of 8-Br cyclic GMP (0.01-3 mM) as well as of blockers of guanylyl cyclase and cyclic GMP-dependent kinase I. 3. Studies were focused on: (i) multiple signalling events, including receptor-induced tyrosine phosphorylations, phosphorylation of mitogen-activated protein kinase, activation of the AP-1 transcription complex and deoxyribonucleotide synthesis; (ii) the progression through the cell cycle, dissected out by the use of staurosporine (1 nM), lovastatin (10 microM), mimosine (200 microM), hydroxyurea (1 mM) and nocodazole (1.5 microM). 4. NO was found to have no effects on the phosphorylation events of the growth factor cascade. In contrast, later processes were modified by the messenger but with opposite effects. 5. A cyclic GMP-dependent stimulation of growth was shown to be sustained in part by the activation of the AP-1 transcription complex, while a predominant, cyclic GMP-independent inhibition was found to be mediated by both the negative regulation of ribonucleotide reductase and the marked slowing down of the cell cycle occurring at early and late G1 and during the S phase. 6. Although multiple and apparently conflicting, the effects of NO here described could work coordinately in a general programme of cell growth regulation. In particular, the cyclic GMP-dependent actions might function as rapid modulatory events, while the effects on cell cycle might operate collectively as a multi-switch process whenever growth inhibition is required.

3T3 Cells↗
PubMed1997

Calcium-dependent mitochondrial formation of species mediating DNA single strand breakage in U937 cells exposed to sublethal concentrations of tert-butylhydroperoxide.

Treatment of U937 cells with a sublethal albeit DNA-damaging concentration of tert-butylhydroperoxide (tB-OOH) enhanced mitochondrial Ca++ uptake and ruthenium red (RR), a polycation that inhibits the calcium uniporter of mitochondria, significantly reduced the extent of DNA cleavage generated by the hydroperoxide. Release of Ca++ from the ryanodine(Ry)/caffeine(Cf)-sensitive stores further increased mitochondrial Ca++ uptake and elicited a parallel enhancement in DNA strand scission induced by tB-OOH that was prevented by both Ry and RR. DNA damage caused by tB-OOH alone or associated with either Cf or RR was prevented by iron chelators, insensitive to antioxidants and repaired with kinetics superimposable with those observed after treatment with H2O2. Cf enhanced the DNA-damaging effects of tB-OOH in permeabilized cells as well, and similar effects were observed upon addition of CaCl2. Cf did not further increase the formation of DNA lesions elicited by tB-OOH in the presence of CaCl2. The enhancing effects of Cf were prevented by RR and ryanodine, whereas those mediated by exogenous calcium were prevented only by RR. DNA strand scission caused by tB-OOH alone or associated with Cf in the permeabilized cell system was severely inhibited by ethylene glycol-bis(beta-aminoethyl ether)-N, N,N',N'-tetraacetic acid. The mechanism(s) whereby Ca++ promotes the mitochondrial formation of species that will ultimately result in the formation of DNA lesions was subsequently analyzed using intact as well as permeabilized cells. Hydrogen peroxide was identified to be one of these species.

Caffeine↗
PubMed1996

Overall lack of regulated secretion in a PC12 variant cell clone.

A stable clone of PC12 neuroendocrine cells, named 27, known from previous studies to exhibit a defect of regulated secretion (lack of regulated secretory proteins, of synaptophysin, of dense granules and of catecholamine uptake and release; Clementi, E., Racchetti, G., Zacchetti, D., Panzeri, M. C., and Meldolesi, J. (1992) Eur. J. Neurosci. 4, 944-953) was characterized in detail to clarify the nature of its phenotype and the mechanisms of its establishment. The neuroendocrine nature of the PC12-27 phenotype was documented by specific markers: synapsins, neurofilament subunit H, neuronal kinesin, and alpha-latrotoxin receptor. Moreover, various intracellular membrane systems of PC12-27, including the endoplasmic reticulum and the Golgi complex, appeared similar to control PC12 in both morphology and marker expression. In contrast, all the investigated markers located either in dense granules (dopamine-beta-hydroxylase), in synaptic-like microvesicles (the acetylcholine transporter) or in both these regulated secretory organelles (VAMP2/synaptobrevin-2, synaptotagmin) were missing in PC12-27 cells, and the same was true also for the cytosolic and plasmalemma proteins involved in regulated exocytosis (Rab3, SNAP25, syntaxin). Pulse labeling and in vitro translation experiments revealed the defect to consist in a protein synthesis blockade that mRNA studies (reverse transcription-polymerase chain reaction, Northern blotting, and actinomycin D experiments) revealed to take place primarily at the transcriptional level. The secretion defect of PC12-27 cells was modified neither by various types of long term stimulation nor by nerve growth factor treatment. Moreover, when one of the missing regulated secretory proteins, chromogranin B, was expressed by cDNA transfection, it was secreted, however via the constitutive pathway. Our results demonstrate that PC12-27 cells are fully incompetent for both branches of regulated secretion, those of dense granules and synaptic-like microvesicles, possibly because of the impairment of a general expression control system that appears to operate independently of neuroendocrine cell differentiation.

Animals↗