Sonsoles Hortelano

Splenic B lymphocyte programmed cell death is prevented by nitric oxide release through mechanisms involving sustained Bcl-2 levels

Incubation of ex vivo cultured mature B cells in the presence of nitric oxide or nitric oxide-donor substances delays programmed cell death as determined by the appearance of DNA laddering in agarose gel electrophoresis or by flow-cytometry analysis of DNA. Nitric oxide also rescues B cells from antigen-induced apoptosis but fails to provide a co-stimulatory signal that converts the signal elicited by the antigen into a proliferative response. The protective effects of nitric oxide against programmed cell death can be reproduced by treatment of the cells with permeant analogues of cyclic GMP. Regarding the mechanisms by which nitric oxide prevents apoptosis in B cells, we have observed that nitric oxide release prevents the drop in the expression of the protooncogene bcl-2, both at the mRNA and protein levels, suggesting the existence of an unknown pathway that links nitric oxide signaling with Bcl-2 expression.

Nitric oxide induces apoptosis via triggering mitochondrial permeability transition

Nitric oxide (NO) induces apoptosis in thymocytes, peripheral T cells, myeloid cells and neurons. Here we show that NO is highly efficient in inducing mitochondrial permeability transition, thereby causing the liberation of apoptogenic factors from mitochondria which can induce nuclear apoptosis (DNA condensation and DNA fragmentation) in isolated nuclei in vitro. In intact thymocytes, NO triggers disruption of the mitochondrial transmembrane potential, followed by hypergeneration of reactive oxygen species, exposure of phosphatidyl serine on the outer plasma membrane leaflet, and nuclear apoptosis. Inhibitors of mitochondrial permeability transition such as bongkrekic acid and a cyclophilin D‐binding cyclosporin A derivative, N‐methyl‐Val‐4‐cyclosporin A, prevent the mitochondrial as well as all post‐mitochondrial signs of apoptosis induced by NO including nuclear DNA fragmentation and exposure of phosphatidylserine residues on the cell surface. These findings indicate that NO can cause apoptosis via triggering of permeability transition.

Inhibition of IκB Kinase and IκB Phosphorylation by 15-Deoxy-Δ12,14-Prostaglandin J2 in Activated Murine Macrophages

ABSTRACT Activation of the macrophage cell line RAW 264.7 with lipopolysaccharide (LPS) and gamma interferon (IFN-γ) induces the expression of gene products involved in host defense, among them type 2 nitric oxide synthase. Treatment of cells with 15-deoxy-Δ12,14-prostaglandin J2(15dPGJ2) inhibited the LPS- and IFN-γ-dependent synthesis of NO, a process that was not antagonized by similar concentrations of prostaglandin J2, prostaglandin E2, or rosiglitazone, a peroxisomal proliferator-activated receptor γ ligand. Incubation of activated macrophages with 15dPGJ2 inhibited the degradation of IκBα and IκBβ and increased their levels in the nuclei. NF-κB activity, as well as the transcription of NF-κB-dependent genes, such as those encoding type 2 nitric oxide synthase and cyclooxygenase 2, was impaired under these conditions. Analysis of the steps leading to IκB phosphorylation showed an inhibition of IκB kinase by 15dPGJ2 in cells treated with LPS and IFN-γ, resulting in an impaired phosphorylation of IκBα, at least in the serine 32 residue required for targeting and degradation of this protein. Incubation of partially purified activated IκB kinase with 2 μM 15dPGJ2 reduced by 83% the phosphorylation in serine 32 of IκBα, suggesting that this prostaglandin exerts direct inhibitory effects on the activity of the IκB kinase complex. These results show rapid actions of 15dPGJ2, independent of peroxisomal proliferator receptor γ activation, in macrophages challenged with low doses of LPS and IFN-γ.

Inhibition of IkappaB kinase and IkappaB phosphorylation by 15-deoxy-Delta(12,14)-prostaglandin J(2) in activated murine macrophages.

ABSTRACT Activation of the macrophage cell line RAW 264.7 with lipopolysaccharide (LPS) and gamma interferon (IFN-γ) induces the expression of gene products involved in host defense, among them type 2 nitric oxide synthase. Treatment of cells with 15-deoxy-Δ12,14-prostaglandin J2(15dPGJ2) inhibited the LPS- and IFN-γ-dependent synthesis of NO, a process that was not antagonized by similar concentrations of prostaglandin J2, prostaglandin E2, or rosiglitazone, a peroxisomal proliferator-activated receptor γ ligand. Incubation of activated macrophages with 15dPGJ2 inhibited the degradation of IκBα and IκBβ and increased their levels in the nuclei. NF-κB activity, as well as the transcription of NF-κB-dependent genes, such as those encoding type 2 nitric oxide synthase and cyclooxygenase 2, was impaired under these conditions. Analysis of the steps leading to IκB phosphorylation showed an inhibition of IκB kinase by 15dPGJ2 in cells treated with LPS and IFN-γ, resulting in an impaired phosphorylation of IκBα, at least in the serine 32 residue required for targeting and degradation of this protein. Incubation of partially purified activated IκB kinase with 2 μM 15dPGJ2 reduced by 83% the phosphorylation in serine 32 of IκBα, suggesting that this prostaglandin exerts direct inhibitory effects on the activity of the IκB kinase complex. These results show rapid actions of 15dPGJ2, independent of peroxisomal proliferator receptor γ activation, in macrophages challenged with low doses of LPS and IFN-γ.

Nitric oxide induces tyrosine nitration and release of cytochrome c preceding an increase of mitochondrial transmembrane potential in macrophages

Treatment of elicited peritoneal macrophages or the macrophage cell line RAW 264.7 with high concentrations of nitric oxide donors is followed by apoptotic cell death. Analysis of the changes in the mitochondrial transmembrane potential (ΔΨm) with specific fluorescent probes showed a rapid and persistent increase of ΔΨm, a potential that usually decreases in cells undergoing apoptosis through mitochondrial‐dependent mechanisms. Using confocal microscopy, the release of cytochrome c from the mitochondria to the cytosol was characterized as an early event preceding the rise of ΔΨm. The cytochrome c from cells treated with nitric oxide donors was modified chemically, probably through the formation of nitrotyrosine residues, suggesting the synthesis of peroxynitrite in the mitochondria. These results indicate that nitric oxide‐dependent apoptosis in macrophages occurs in the presence of a sustained increase of ΔΨm, and that the chemical modification and release of cytochrome c from the mitochondria precede the changes of ΔΨm.—Hortelano, S., Alvarez, A. M., Bosca, L. Nitric oxide induces tyrosine nitration and release of cytochrome c preceding an increase of mitochondrial transmembrane potential in macrophages. FASEB J. 13, 2311–2317 (1999)

Contribution of Cyclopentenone Prostaglandins to the Resolution of Inflammation Through the Potentiation of Apoptosis in Activated Macrophages

Activation of the macrophage cell line RAW 264.7 with LPS and IFN-γ induces apoptosis through the synthesis of high concentrations of NO due to the expression of NO synthase-2. In addition to NO, activated macrophages release other molecules involved in the inflammatory response, such as reactive oxygen intermediates and PGs. Treatment of macrophages with cyclopentenone PGs, which are synthesized late in the inflammatory onset, exerted a negative regulation on cell activation by impairing the expression of genes involved in host defense, among them NO synthase-2. However, despite the attenuation of NO synthesis, the percentage of apoptotic cells increased with respect to activated cells in the absence of cyclopentenone PGs. Analysis of the mechanisms by which these PGs enhanced apoptosis suggested a potentiation of superoxide anion synthesis that reacted with NO, leading to the formation of higher concentrations of peroxynitrite, a more reactive and proapoptotic molecule than the precursors. The effect of the cyclopentenone 15-deoxy-Δ12,14-PGJ2 on superoxide synthesis was dependent on p38 mitogen-activated protein kinase activity, but was independent of the interaction with peroxisomal proliferator-activated receptor γ. The potentiation of apoptosis induced by cyclopentenone PGs involved an increase in the release of cytochrome c from the mitochondria to the cytosol and in the nitration of this protein. These results suggest a role for cyclopentenone PGs in the resolution of inflammation by inducing apoptosis of activated cells.

Mechanisms of Nitric Oxide-Dependent Apoptosis: Involvement of Mitochondrial Mediators

Programmed cell death occurs in several physiopathological situations in multicellular organisms and constitutes a common mechanism of cell replacement, tissue remodelling and removal of altered cells. The effectors that induce apoptosis as well as the signalling pathways involved in the process are the subjects of current work. In addition to receptor-mediated apoptosis, highly reactive molecules, such as NO, influence cell viability either by acting as a protection against apoptogenic stimuli, or by inducing apoptosis when produced at elevated concentrations. The contribution to apoptosis of mediators released by the mitochondria and involved in the activation of caspases focused attention on the functional changes caused by NO in this organelle. NO induces mitochondrial permeability transition and promotes apoptosis in cell-free systems containing mitochondria and nuclei. Moreover, NO-dependent apoptosis can be blocked in most cases through the use of permeability transition or caspase inhibitors. The intracellular pathways activated in response to NO challenge and involved in the regulation of apoptosis are analysed.

Phorbol esters induce nitric oxide synthase and increase arginine influx in cultured peritoneal macrophages

Incubation of peritoneal macrophages with β‐phorbol 12,13‐dibutyrate promotes a time‐dependent release of NO to the incubation medium. This effect was antagonized by LPS, a well known inducer of nitric oxide synthase (NOS) expression in macrophages, and was inhibited by N G‐methyl‐l‐arginine and N ω‐nitro‐l‐arginine. An increase in intracellular cGMP and NOS activity was observed in parallel with NO release. The induction of NOS was accompanied by a stimulation of arginine influx within the cell. These results suggest that activation of protein kinase C by phorbol esters is sufficient to promote NOS induction in macrophages.

NITRIC OXIDE IMPLICATION IN THE CONTROL OF NEUROSECRETION BY CHROMAFFIN CELLS

Abstract: In this work, we have studied the effects of pure nitric oxide (NO) on the regulation of catecholamine (CA) secretion by chromaffin cells, as well as the possible presence of its synthesizing enzyme l‐arginine:NO synthase (NOS) in these cells. Our results show that NO produces a large stimulation of basal CA secretion. This effect was calcium‐ and concentration‐dependent (EC50 = 64 ± 8 µM) and was not due to nonspecific damage of the tissue by NO. NO also modulates the CA secretion evoked by nicotine in a dose‐dependent manner. Although it has a stimulatory effect on the CA secretion evoked by low doses of nicotine (<3 µM; EC50 = 16 ± 3 µM), it produces a dose‐dependent inhibition of the CA secretion induced by high doses of nicotine (≥30 µM; IC50 = 52 ± 6 µM). The mechanism by which NO modulates CA secretion seems to be through the increase in the cyclic GMP levels, because there was a close correlation between the CA secretion and the cyclic GMP levels. The presence of a specific activity of NOS in chromaffin cells has been demonstrated by two independent methods: release of [14C]citruiline from [14C]arginine and formation of an NO‐hemoglobin complex. NOS activity was about 0.5 pmol/min/mg of protein. It was calcium‐ and mainly calmodulin‐dependent and could be specifically blocked by the NOS inhibitor N‐methyl‐l‐arginine. These results suggest that NO could be an important intracellular messenger in the regulation of neurosecretion in chromaffin cells.

Protective effect of cyclosporin A and FK506 from nitric oxide-dependent apoptosis in activated macrophages.

Activation of macrophages with lipopolysaccharide (LPS) and low doses of interferon‐γ (IFN‐γ) induced apoptotic death through a nitric oxide‐dependent pathway. Treatment of cells with the immunosuppressors cyclosporin A (CsA) or FK506 inhibited the activation‐dependent apoptosis. These drugs decreased the up‐regulation of p53 and Bax characteristic of activated macrophages. Moreover, incubation of activated macrophages with CsA and FK506 contributed to maintain higher levels of Bcl‐2 than in LPS/IFN‐γ treated cells. The inhibition of apoptosis exerted by CsA and FK506 in macrophages was also observed when cell death was induced by treatment with chemical nitric oxide donors. Incubation of macrophages with LPS/IFN‐γ barely affected caspase‐1 but promoted an important activation of caspase‐3. Both CsA and FK506 inhibited pathways leading to caspase‐3 activation. Moreover, the cleavage of poly(ADP‐ribose) polymerase, a well established caspase substrate, was reduced by these immunosuppressive drugs. CsA and FK506 reduced the release of cytochrome c to the cytosol and the activation of caspase‐3 in cells treated with nitric oxide donors. These results indicate that CsA and FK506 protect macrophages from nitric oxide‐dependent apoptosis and suggest a contribution of the macrophage to innate immunity under conditions of immunosuppression of the host.