Measurement of NOS mRNA by northern blotting and the ribonuclease-protection assay.
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Animals↗
Biomedical subjects
L Boscá
Publications and source records attributed to L Boscá.
Differential regulation of nitric oxide synthase mRNA expression by lipopolysaccharide and pro-inflammatory cytokines in fetal hepatocytes treated with cycloheximide.
The effect of cycloheximide (CHX) on the mRNA expression of the cytokine-inducible, calcium-independent nitric oxide synthase (iNOS) was investigated in fetal hepatocytes stimulated with lipopolysaccharide (LPS) or pro-inflammatory cytokines. In the presence of CHX the LPS-dependent iNOS mRNA levels were reduced, whereas the response to pro-inflammatory cytokines was enhanced. Because iNOS transcription is highly dependent on the activation of nuclear factor kappaB (NF-kappaB), this factor was evaluated by electrophoretic mobility shift assays, and a close correlation between NF-kappaB activity and iNOS mRNA levels was observed. CHX itself potentiated the degradation of the IkappaB alpha and IkappaB beta inhibitory subunits (IkappaB is inhibitory kappaB) of the NF-kappaB complex, and therefore the loss of LPS-dependent iNOS mRNA expression cannot be attributed to a blockage in the activation of NF-kappaB. These results suggest the existence of a CHX-sensitive pathway in the expression of iNOS mediated by LPS, a mechanism that is not involved in the response to pro-inflammatory cytokines.
Animals↗
Nuclear factor kappaB is required for the transcriptional control of type II NO synthase in regenerating liver.
A concerted activation of transcription factors involved in the transactivation of type II NO synthase (iNOS) gene occurred after partial hepatectomy (PH), resulting in the transient expression of iNOS. The corresponding mRNA and protein levels of iNOS reached a maximum at 4 h and 8 h post-PH respectively. This induction was preceded by an early and transient activation of nuclear factor kappaB (NF-kappaB). Analysis of the kappaB inhibitory (I) proteins showed an important role for IkappaBalpha in the process of NF-kappaB activation, whereas the contribution of IkappaBbeta was less evident. Interferon regulatory factor 1, which has been described as an important activator of iNOS expression, was up-regulated after PH but failed to bind to the corresponding DNA binding sequences of the iNOS promoter. The transcriptional control of iNOS after PH, was compared with the events associated with the hepatic expression of this enzyme in animals challenged with lipopolysaccharide, showing a differential pattern of transcription-factor activation and IkappaB degradation between both models. Transfection of hepatoma cell lines with iNOS promoter constructs, followed by stimulation with post-PH sera, revealed the requirement of NF-kappaB activation for iNOS expression. These data suggest that there is an important role for the restricted NF-kappaB activation in the temporal pattern of iNOS expression in regenerating liver.
Animals↗
Rapid Up-regulation of IkappaBbeta and abrogation of NF-kappaB activity in peritoneal macrophages stimulated with lipopolysaccharide.
Lipopolysaccharide (LPS) administration to mice elicited the activation of nuclear factor kappaB (NF-kappaB) in several tissues including liver and macrophages. Maximal activation was observed 1 h after treatment but declined at 3 and 6 h. The levels of IkappaBalpha and IkappaBbeta were analyzed during this period in an attempt to correlate NF-kappaB activity with IkappaB resynthesis. Degradation of IkappaBalpha was very rapid and was followed by recovery 1 h after LPS administration. IkappaBbeta degradation, which has been associated with persistent NF-kappaB activation, was complete at 1 h. However, a rapid recovery of IkappaBbeta in these tissues was observed at 3 h in parallel with the abrogation of NF-kappaB activity. Immunolocalization of newly synthesized IkappaBbeta by confocal microscopy revealed its preferential accumulation in the cytosol. Analysis of IkappaBbeta by Western blot using high resolution polyacrylamide gel electrophoresis showed the presence of two bands in cytosolic extracts of LPS-treated macrophages at 3 h, but only one band with the same mobility as the control was detected at 6 h. Moreover, treatment of extracts of resynthesized IkappaBbeta with alkaline phosphatase resulted in the accumulation of the protein of slightly higher electrophoretic mobility, indicating the prevalence of a rapid phosphorylation of the newly synthesized IkappaBbeta. At the mRNA level, up-regulation of IkappaBbeta was observed in macrophages stimulated for 1 h with LPS. When the effect of pro-inflammatory cytokines was investigated, tumor necrosis factor alpha, but not interleukin-1 or interferon-gamma, promoted an important degradation of IkappaBbeta followed by an increase in the mRNA at 1 h. These results suggest the existence of LPS- and tumor necrosis factor alpha- specific pathways involved in a rapid IkappaBbeta degradation and resynthesis and might explain the transient period of activation of NF-kappaB in these tissues upon stimulation with these factors. This rapid control of NF-kappaB function may contribute to the attenuation of the inflammatory response of these cells.
Acute-Phase Reaction↗
Requirement of nitric oxide and calcium mobilization for the induction of apoptosis in adrenal vascular endothelial cells.
Exposure of adrenal vascular endothelial cells (AVEC) to pharmacological nitric oxide (NO) donors, proinflammatory cytokines or lipopolysaccharide was unable to induce apoptosis as occurred when macrophages were treated under identical experimental conditions. However, when the intracellular Ca2+ concentration increased, AVEC displayed apoptotic features upon exposure to NO. This apoptosis was confirmed by the release of oligonucleosomes to the cytosol and by the characteristic DNA laddering observed after electrophoresis in agarose gels. Ca2+-mobilizing agents and interleukin-1beta (IL-1beta) also elicited an apoptotic response in these cells through a mechanism that required NO synthesis. The ability of NO and intracellular Ca2+ to promote apoptosis was dependent on the number of passages of the cells in culture, suggesting the loss of protective factors in the course of ex vivo cell culture. Because AVEC exhibit an important capacity to increase the intracellular Ca2+ concentration in response to a wide array of agonists, this condition might affect the integrity of the vascular system under pathological circumstances such as those prevailing in the course of septic shock.
Adrenal Glands↗
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.
Animals↗
Expression of the calcium-independent cytokine-inducible (iNOS) isoform of nitric oxide synthase in rat placenta.
The presence of the calcium-independent cytokine-inducible nitric oxide synthase (iNOS) has been investigated in rat placenta from day 19 of gestation till delivery. iNOS has been detected at the mRNA, enzyme activity and protein levels in complete placenta. Immunocytochemical detection of iNOS was heterogeneously distributed in control placenta. Intraperitoneal injection of pregnant rats at 21 days of gestation with lipopolysaccharide (LPS) increased the iNOS immunoreactivity in the decidua basalis of the placenta, and, when the mRNA levels and enzyme activity were measured in total tissue, a moderate increase (approx. 160%) was observed. A constitutive nuclear factor kappaB activity was observed in placenta from both control and LPS-treated animals. These results indicate constitutive expression of iNOS in rat placenta.
Animals↗
Macrophage triggering with cecropin A and melittin-derived peptides induces type II nitric oxide synthase expression.
Triggering of RAW 264.7 cells with a cecropin A-melittin hybrid peptide (CA(1-8)M(1-18)) promoted a rapid rise in the intracellular calcium concentration that was followed, after a lag period of 6 h, by nitric oxide synthesis through the expression of the cytokine-inducible form of nitric oxide synthase (type II NOS or iNOS). The maximal effect was obtained at peptide concentrations in the 2 to 5-microM range. Simultaneous incubation with the peptide and LPS abrogated the nitric oxide synthesis elicited after LPS treatment of the cells. CA(1-8)M(1-18) induced a rapid activation of nuclear factor kappaB as evidenced by the presence of p50/p65 heterodimers of the nuclear factor kappaB/c-Rel family in the nuclei of activated cells. This peptide also activated the reporter activity of cells transfected with a plasmid harboring a 1-kb fragment corresponding to the 5'-flanking region of the murine iNOS gene. CA(1-8)M(1-18) promoted apoptotic cell death at concentrations below 1 to 2 microM, whereas higher concentrations altered the plasma membrane integrity. These results suggest the involvement of multiple intracellular signaling pathways in the mechanism by which this peptide elicits macrophage triggering.
Animals↗
Linomide administration to mice attenuates the induction of nitric oxide synthase elicited by lipopolysaccharide-activated macrophages and prevents nephritis in MRL/Mp-lpr/lpr mice.
Nitric oxide is involved as a messenger molecule in a large number of physiologic and pathologic responses. Local generation of high nitric oxide output through the expression of the calcium-independent, cytokine-inducible form of nitric oxide synthase (iNOS) can result in either protective or damaging effects. The development of drugs that specifically suppress iNOS expression or inhibit its activity may therefore provide an excellent therapeutic tool for treatment of a diverse set of dysfunctions, including asthma, inflammatory processes, and autoimmune disease. We show compelling evidence that linomide, an immunomodulator known to ameliorate autoimmune diseases, prevents accumulation in the macrophages of mRNA encoding iNOS in mice injected with LPS. This effect is partially mediated by the blocking of TNF-alpha and IL-beta production by activated macrophages. Here, we also present evidence that kidneys from MRL/Mp-lpr/lpr mice express high iNOS levels when the mice develop glomerulonephritis. The administration of linomide to MRL/Mp-lpr/lpr mice significantly decreases iNOS mRNA levels and prevents the development of glomerulonephritis, extending the half-life of mice of this strain. This linomide effect is compatible with its role in preventing the development of autoimmune disease and extends its possible use to other pathologic manifestations associated with iNOS expression, such as the systemic lupus erythematosus-associated glomerulonephritis present in MRL/Mp-lpr/lpr mice.
Adjuvants, Immunologic↗
Regulation of rat liver S-adenosylmethionine synthetase during septic shock: role of nitric oxide.
We investigated the modulation of rat liver S-adenosylmethionine (SAM) synthetase in a model of acute sepsis. Our results show that animals treated with bacterial lipopolysaccharide experience a marked decrease in liver SAM synthetase activity. No changes were detected in the hepatic levels of SAM synthetase protein, suggesting that inactivation of the existing enzyme was the cause of the observed activity loss. Lipopolysaccharide treatment resulted in the expression of calcium-independent/cytokine-inducible nitric oxide (NO) synthase in liver and the accumulation in plasma of the NO-derived species nitrite and nitrate. NO implication in the in vivo regulation of SAM synthetase was evaluated in animals treated with the NO donor molecule 3-morpholinosydnonimine. The analysis of liver enzymatic activity, along with protein and messenger RNA levels yielded results similar to those obtained with lipopolysaccharide treatment. To assess directly the sensitivity of SAM synthetase to NO, the rat liver-purified high- and low-molecular weight forms of the enzyme were exposed to various doses of 3-morpholinosydnonimine and other NO donors such as S-nitroso-N-acetylpenicillamine, resulting in a dose-dependent inhibition of enzymatic activity. This effect was reversed by addition of the reducing agents beta-mercaptoethanol and glutathione. Finally, cysteine 121 was identified as the site of molecular interaction between NO and rat liver SAM synthetase that is responsible for the inhibition of the enzyme. To reach this conclusion, the 10 cysteine residues of the enzyme were changed to serine by site-directed mutagenesis, and the effect of NO on the various recombinant enzymes was measured.
Animals↗
Absence of NO synthase type II expression in fetal liver from pregnant rats under septic shock conditions.
Treatment of rats at 21 days' gestation with lipopolysaccharide (LPS) induced type II NO synthase (iNOS) expression in maternal liver but completely failed to elicit this response in the corresponding fetal tissue. The impaired response of fetal liver cannot be attributed to alterations in the sensitivity of this organ to LPS or to proinflammatory cytokines involved in iNOS expression (tumor necrosis factor alpha [TNF-alpha], interferon gamma [IFN-gamma]), because cultured fetal and adult hepatocytes similarly responded to these factors. Measurement of TNF-alpha and IFN-gamma levels in maternal and fetal serum showed a restricted permeability of placenta to these cytokines, results that were in agreement with the absence of iNOS expression in fetal liver. Moreover, analysis of nuclear factor kappaB, which is required for iNOS expression, showed an activation in nuclear extracts from livers of mothers under septic shock conditions but not in the corresponding fetal livers. However, cultured fetal hepatocytes prepared from mothers in septic shock displayed an anomalous pattern of iNOS expression, including a decreased response to LPS as well as a shift in sensitivity to TNF-alpha. These hepatocytes from LPS-treated mothers showed significant expression of iNOS in the absence of added stimuli. These results suggest that there is a priming of the fetal hepatocytes from mothers with septic shock. The characterization of the factors causing this priming process might provide additional clues understanding the control of iNOS expression in liver.
Animals↗
Involvement of nitric oxide synthesis in hepatic perturbations induced in rats by a necrogenic dose of thioacetamide.
1. The biological actions of nitric oxide (NO), a highly diffusible and short-lived radical, range from signal transduction to cytotoxicity. The present study investigated whether NO is released in the course of liver necrosis and regeneration induced by a single necrogenic dose of thioacetamide (6.6 mmol kg-1 body wt) to rats. Samples of liver were obtained at 0, 3, 12, 24, 48, 72 and 96 h after thioacetamide administration. 2. Inducible nitric oxide synthase (iNOS) activity was determined in purified liver homogenates and a sharp 6 fold increase (P < 0.001) in iNOS activity was recorded at 48 h of intoxication, followed by a slight but progressive increase at 72 and 96 h. Changes in the expression of iNOS, as detected by its mRNA levels, were parallel to the NOS enzyme activity. Hepatocyte NO synthesis showed a progressive increase at 24, 48 and 72 h, to 8 (P < 0.001), 13 (P < 0.001) and 13 (P < 0.001) times the initial values, respectively. 3. In isolated Kupffer cells, where initial NO release was ten fold higher than in hepatocytes, a progressive increase was detected from 48 h which reached two fold of initial at 72 h of intoxication (192%; P < 0.001). Hepatic cyclic GMP concentration did not change significantly. However, mitochondrial aconitase activity decreased markedly at 12 and 24 h of intoxication showing a sharp increase towards normal values at 48 h which was maintained at 72 and 96 h. 4. As protein kinase C (PKC) is one of the likely candidates to mediate iNOS expression, translocation (activation) of PKC was assayed in hepatocytes, and a significant two fold increase (P < 0.001) between 48 and 96 h after thioacetamide intoxication was observed. When peritoneal macrophages from control rats were incubated with serum from thioacetamide-treated rats, a sharp increase in NO release was detected with serum obtained at 48 h, reaching at 96 h a value four fold (P < 0.001) that of the control. 5. These results suggest that iNOS activity and NO release play a role in the pathophysiological mechanisms that trigger post-necrotic hepatocellular regeneration following thioacetamide administration.
Aconitate Hydratase↗
6-Mercaptopurine decreases the Bcl-2/Bax ratio and induces apoptosis in activated splenic B lymphocytes.
6-Mercaptopurine and related purine antimetabolites are used in the treatment of several B cell disorders. These drugs inhibited the proliferation of mature splenic B cells after being triggered with polyclonal mitogens. In addition to the antiproliferative effects, 6-mercaptopurine, 2-mercaptopurine, and aminoguanidine evoked a rapid apoptotic cell death in activated B cells that started at 6 hr after drug treatment and therefore preceded DNA synthesis. Incubation of activated B lymphocytes with 6-mercaptopurine blocked the low but sustained nitric oxide release observed in these cells that contributes to the prevention of apoptotic cell death; the addition of chemical nitric oxide donors significantly antagonized the apoptosis elicited by these drugs. The inhibition of nitric oxide synthesis elicited by mercaptopurines correlated with a decrease in the release of nitric oxide-derived species to the culture medium and in the intracellular levels of cGMP. The ratio between the amounts of Bcl-2 and Bax, two proteins involved in the control of apoptosis in mature B cells, markedly decreased as result of mercaptopurine treatment.
Animals↗
Up-regulation of protein kinase C-epsilon promotes the expression of cytokine-inducible nitric oxide synthase in RAW 264.7 cells.
Stimulation of the murine macrophage RAW 264.7 cell line with phorbol esters fails to promote nitric oxide synthesis as occurs in rat hepatocytes or peritoneal macrophages. Transfection of RAW 264.7 cells with plasmids harboring protein kinase C (PKC) -epsilon isotype but not with PKC-alpha, -beta1, -delta, or constitutively active -alpha and -beta1 isotypes resulted in the expression of nitric oxide synthase type II (iNOS), as reflected by the synthesis of nitric oxide measured in the culture medium of transfected cells. cotransfection of RAW 264.7 cells with the -1592 to +121-base pair promoter region of the murine iNOS gene and PKC isotypes specifically induced the transactivation of this promoter in the case of the plasmids containing the PKC-epsilon isotype. The mechanism by which PKC-epsilon induced iNOS expression involved the activation of nuclear factor binding to kappaB sites (NF-kappaB) as deduced by the suppressive effect of pyrrolidine dithiocarbamate on nitric oxide synthesis, an inhibitor of NF-kappaB activation, and by the activation of kappaB sites in cells transfected with a vector containing a kappaB motif linked to a chloramphenicol acetyltransferase reporter gene. These results suggest that PKC-epsilon can regulate a pathway that promotes iNOS expression in macrophages in response to phorbol ester activation.
Animals↗
Evidence for common mechanisms in the transcriptional control of type II nitric oxide synthase in isolated hepatocytes. Requirement of NF-kappaB activation after stimulation with bacterial cell wall products and phorbol esters.
Incubation of primary cultures of rat hepatocytes with lipopolysaccharide (LPS), S-[2,3-bis(palmitoyloxy)-(2-R, S)-propyl]-N-palmitoyl-(R)-Cys-Ser-Lys4 (TPP), a synthetic lipopeptide present in bacterial cell wall lipoproteins, or with phorbol 12,13-dibutyrate (PDBu) induced an increase in nitric oxide synthesis through the expression of type II nitric oxide synthase (iNOS). Transfection of hepatocytes with a HindII fragment corresponding to the promoter region of the murine iNOS gene (from nucleotide -1588 to +165) resulted in the expression of the reporter gene when cells were stimulated with these factors. The transcription factors activated by these stimuli involved an increase in the nuclear content of proteins that bind to kappaB, AP-1, GAS, and SIE sequences. Inhibition of NF-kappaB activation with pyrrolidine dithiocarbamate eliminated the expression of iNOS in hepatocytes stimulated with LPS, TPP, or PDBu. In addition to this, transfection of hepatocytes with promoter mutants in which a sequential 2-base pair change within the kappaB sites was introduced (position -971 to -961 and -85 to -75, respectively), resulted in approximately 17 and 35%, respectively, of the activity of the naive promoter. Simultaneous mutation of both kappaB sites abolished the promoter activity. Analysis of the proteins involved in kappaB binding showed the presence of p50/p65 dimers in the nuclei of activated cells at the time that an important decrease of IkappaB-alpha was observed soon after cell stimulation with LPS, TPP, or PDBu. However, only LPS was able to decrease the amount of IkappaB-beta. These results suggest that LPS, TPP, and PDBu, although activating different signal transduction pathways, use a common mechanism mediating iNOS expression in cultured hepatocytes.
Animals↗
CD53 antigen and epidermal growth factor induce similar changes in the pattern of phorbol ester binding in a B cell lymphoma.
The CD53 antigen is a prototype member of the transmembrane-4 superfamily which includes several tumor antigens. In this report we have studied the changes in the cellular binding of phorbol esters after stimulation with monoclonal antibody (mAb) MRC OX-44 (anti-CD53) and epidermal growth factor (EGF) using a fluorochrome-phorbol ester binding assay. Incubation of a rat B cell lymphoma cell line with this mAb or EGF induces the appearance of high- and low-affinity phorbol ester binding sites and changes the total number of binding sites. Four binding sites with different characteristics have been detected. The binding data suggest that two structurally different receptors, CD53 antigen and EGF receptor, induce a similar change in the functional protein kinase C expressed in the cell which might be implicated in the responses elicited after cell stimulation.
Animals↗
Bacterial lipopolysaccharide antagonizes transforming growth factor beta 1-induced apoptosis in primary cultures of hepatocytes.
Incubation of primary cultures of fetal hepatocytes with lipopolysaccharide (LPS) elicited the expression of nitric oxide (NO) synthetase as well as antagonized the apoptotic cell death evoked by treating the cells with transforming growth factor beta 1 (TGF-beta 1). In addition to LPS, exposure of the cells to chemical NO donors also protected against apoptotic cell death when assayed at concentrations in the low micromolar range. Treatment of hepatocytes with large concentrations of NO donors promoted both apoptotic and necrotic cell death. These results suggest that NO synthesis by hepatocytes might be involved in the protection against apoptotic death.
Animals↗
Differential expression pattern of S-adenosylmethionine synthetase isoenzymes during rat liver development.
The pattern of expression of liver-specific and extrahepatic S-adenosylmethionine (SAM) synthetase in developing rat liver was established by determining steady-state levels of the respective messenger RNAs (mRNAs) and protein content. Levels of liver-specific SAM synthetase mRNA increased progressively from day 20 of gestation, increased 10-fold immediately after birth, and reached a peak at 10 days of age, decreasing slightly by adulthood. Conversely, mRNA levels of extrahepatic isoenzyme decreased toward birth, increased threefold in the newborn, and decreased further in the postnatal life, reaching a minimum in the adult. Similar expression profiles were observed in isolated hepatocytes, indicating that both mRNAs are differentially regulated in the same cell type. Western blot analysis showed that levels of immunoreactive liver-specific isoenzyme followed a trend similar to the mRNA, indicating that developmental regulation of this enzyme is mediated at the mRNA level. Developmental patterns of expression of albumin and alpha-fetoprotein (AFP) mRNAs were closely related to those for liver-specific and extrahepatic isoenzymes, respectively. Therefore, it is suggested that liver-specific SAM synthetase may be a marker for hepatocyte differentiation. Incubation of primary cultures of hepatocytes from 21-day-old fetuses with permeant cyclic adenosine monophosphate (cAMP) analogues elicited an up-regulation of the mRNA for the liver-specific isoenzyme with a concomitant down-regulation of the extrahepatic message, suggesting a physiological role for the increased postnatal glucagonemia in the control of this isoenzyme switching. In contrast with the isoenzyme expression profiles, the levels of SAM, the product of SAM synthetase reaction, were determined to be greater during gestation than in immediate postnatal periods. These results indicate that synthesis and utilization of SAM may be regulated differentially in fetal and adult hepatocytes.
Albumins↗