Downregulation of iNOS expression in rat mesangial cells by special extracts of Harpagophytum procumbens derives from harpagoside-dependent and independent effects.

Special extracts from the roots of Harpagophytum procumbens (Devil's Claw) are used in the supportive treatment of inflammatory diseases, and the iridoid derivative harpagoside is thought to be the active principle. To investigate, whether Harpagophytum extracts may also be useful therapeutics in the treatment of inflammatory kidney diseases, we studied the effects of two different extracts containing 8.9% (extract 1) and 27% harpagoside (extract 2), respectively, on IL-1beta-induced nitric oxide (NO) formation as well as transcriptional regulation of inducible NO synthase (iNOS) in rat renal mesangial cells. We observed a concentration-dependent suppression of nitrite formation by about 80%, which was due to an inhibition of iNOS expression. Moreover, a reduction of iNOS promoter activity and nuclear NF-kappaB translocation was observed, indicating that the extracts interfere with the transcriptional activation of iNOS. Three further Harpagophytum extracts containing about 2% harpagoside did not inhibit NO formation suggesting, that only extracts with a high harpagoside content elicit iNOS inhibition. However, pure harpagoside was only inhibitory at concentrations between 0.3 and 1 mg/ml, which is much higher than the harpagoside content present in an effective concentration of the total extracts. Moreover, a harpagoside-free extract 1 also markedly inhibited iNOS expression, indicating that other extract constituents are involved in this effect. Extract 1 exerted a strong antioxidative effect, whereas no such effect could be demonstrated for harpagoside. Together, these data show that special Harpagophytum extracts may represent potential antiinflammatory drugs in the treatment of glomerular inflammatory diseases.

The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells.

Immunosuppression correlates with the development and recurrence of cancer. Mycophenolate mofetil (MMF) has been shown to reduce adhesion molecule expression and leucocyte recruitment into the donor organ. We have hypothesized that MMF might also prevent receptor-dependent tumour dissemination. Therefore, we have investigated the effects of MMF on tumour cell adhesion to human umbilical vein endothelial cells (HUVEC) and compared them with the effects on T cell-endothelial cell interactions. Influence of MMF on cellular adhesion to HUVEC was analysed using isolated CD4+ and CD8+ T cells, or WiDr colon adenocarcinoma cells as the model tumour. HUVEC receptors ICAM-1, VCAM-1, E-selectin and P-selectin were detected by flow cytometry, Western blot or Northern blot analysis. Binding activity of T cells or WiDr cells in the presence of MMF were measured using immobilized receptor globulin chimeras. MMF potently blocked both T cell and WiDr cell binding to endothelium by 80%. Surface expression of the endothelial cell receptors was reduced by MMF in a dose-dependent manner. E-selectin mRNA was concurrently reduced with a maximum effect at 1 microm. Interestingly, MMF acted differently on T cells and WiDr cells. Maximum efficacy of MMF was reached at 10 and 1 microm, respectively. Furthermore, MMF specifically suppressed T cell attachment to ICAM-1, VCAM-1 and P-selectin. In contrast, MMF prevented WiDr cell attachment to E-selectin. In conclusion, our data reveal distinct effects of MMF on both T cell adhesion and tumour cell adhesion to endothelial cells. This suggests that MMF not only interferes with the invasion of alloactivated T cells, but might also be of value in managing post-transplantation malignancy.

Regulation of gene expression by nitric oxide.

Nitric oxide (NO) modulates transcription factors that bind specific cis-regulatory DNA responsible for coordinating the spatial and temporal patterns of gene expression that are initiated by a changing microenvironment. In this way NO helps to orchestrate gene transcription and forms the basis of functional cell responses to accommodate metabolic requirements and to coordinate endogenous defense mechanisms against a variety of stress and disease conditions. There is marked overlap between the signalling pathways triggered by NO, superoxide, and hypoxia. Understanding the redox-based regulation of signal transduction and gene expression will provide insights into how cell activities are constantly coordinated and how promising new therapies may be developed.

Glucocorticoids inhibit lipopolysaccharide-induced up-regulation of arginase in rat alveolar macrophages.

1. As arginase by limiting nitric oxide (NO) synthesis may play a role in airway hyperresponsiveness and glucocorticoids are known to induce the expression of arginase I in hepatic cells, glucocorticoid effects on arginase in alveolar macrophages (AM Phi) were studied. 2. Rat AM Phi were cultured in absence or presence of test substances. Thereafter, nitrite accumulation, arginase activity, and the expression pattern of inducible NO synthase, arginase I and II mRNA (RT - PCR) and proteins (immunoblotting) were determined. 3. Lipopolyssacharides (LPS, 20 h) caused an about 2 fold increase in arginase activity, whereas interferon-gamma (IFN-gamma), like LPS a strong inducer of NO synthesis, had no effect. 4. Dexamethasone decreased arginase activity by about 25% and prevented the LPS-induced increase. Mifepristone (RU-486) as partial glucocorticoid receptor agonist inhibited LPS-induced increase by 45% and antagonized the inhibitory effect of dexamethasone. 5. Two different inhibitors of the NF-kappa B-pathway also prevented LPS-induced increase in arginase activity. 6. Rat AM Phi expressed mRNA and protein of arginase I and II, but arginase I expression was stronger. Arginase I mRNA and protein was not affected by IFN-gamma, but increased by LPS and this effect was prevented by dexamethasone. Both, LPS and IFN-gamma enhanced the levels of arginase II mRNA and protein, effects also inhibited by dexamethasone. As IFN-gamma did not affect total arginase activity, arginase II may represent only a minor fraction of total arginase activity. 7. In rat AM Phi glucocorticoids inhibit LPS-induced up-regulation of arginase activity, an effect which may contribute to the beneficial effects of glucocorticoids in the treatment of inflammatory airway diseases.

Nitric oxide induces MIP-2 transcription in rat renal mesangial cells and in a rat model of glomerulonephritis.

Nitric oxide is a crucial mediator of several forms of glomerulonephritis. We examined the effects of NO on the mRNA expression pattern in glomerular mesangial cells by using a low-stringency reverse transcriptase-polymerase chain reaction method and detected a cDNA fragment that was induced by interleukin 1b (IL-1b) and further up-regulated by the NO donor diethylenetriamine-nitric oxide (DETA-NO). Each respective cDNA fragment was found to match with the cDNAs of rat macrophage inflammatory protein 2 (MIP-2) and GRO/cytokine-induced neutrophil chemoattractant 2b (CINC-2b). Further characterization of MIP-2 regulation by Northern blot analysis confirmed an NO- and IL-1b-dependent increase in MIP-2 mRNA levels. Moreover, inhibition of IL-1b-induced endogenous NO formation by the NO-synthase (NOS) inhibitor L-NMMA markedly attenuated MIP-2 protein expression. We cloned 770 bp of the 5'-flanking region of rat MIP-2 and fused this fragment to a luciferase reporter gene. Transfection of the construct into mesangial cells resulted in a 3.5-fold increase in luciferase activity in cells treated with DETA-NO when compared to controls, suggesting a transcriptional mechanism for NO-induced MIP-2 expression. Deletion and mutational analysis identified critical nuclear factor (NF)-kB and NF-IL-6 binding sites required for NO regulation of MIP-2. In vivo, inhibition of NO synthesis in the Thy-1.1 model of mesangioproliferative glomerulonephritis by the specific inducible-NOS inhibitor L-NIL resulted in a marked reduction of MIP-2 mRNA expression. Furthermore, infiltration of neutrophils into the glomerulus was dramatically attenuated in L-NIL-treated rats.

Downregulation of integrin-linked kinase mRNA expression by nitric oxide in rat glomerular mesangial cells.

Inflammatory glomerular diseases are accompanied by changes in the expression patterns of growth factors, mediators and matrix-associated proteins in mesangial cells and by the production of nitric oxide via the cytokine-inducible form of the nitric oxide synthase. Nitric oxide has been shown to act potently on gene transcription. To identify genes that are differentially expressed by endogenously produced nitric oxide, we forced rat mesangial cells to produce high amounts of nitric oxide by exposure to inflammatory cytokines and compared the mRNA expression patterns of cytokine-stimulated mesangial cells with cells that were additionally treated with the nitric oxide synthase inhibitor L-NMMA to block endogenous NO synthesis. We used a modification of a low stringency RT-PCR approach designated as RNA arbitrarily-primed polymerase chain reaction (RAP-PCR). In this way, we identified among others the integrin-linked kinase (ILK) as an NO-regulated gene. The NO-mediated changes in the mRNA and protein expression patterns of ILK were compared to that of "secreted protein acidic and rich in cystein" (SPARC), a gene that was identified as NO-regulated in the same set of experiments (Walpen et al., J. Am. Soc. Nephrol., 11, 468-476). ILK and SPARC mRNA levels by were downregulated by cytokines via endogenously produced nitric oxide in a comparable manner as verified by Northern blot analysis. In contrast, cytokine- induced NO production or administration of exogenous NO-donors strongly reduced SPARC protein levels without altering ILK protein content in mesangial cells over a period up to 72 hours. Blocking de novo protein synthesis showed a short halflife of SPARC (< 2 hours) whereas ILK-protein was stable over a period of 7 hours, indicating that NO-mediated reduction of ILK mRNA levels does not influence protein content of ILK in mesangial cells under the time limitations given under cell culture conditions. However, a role for cytokines/NO in ILK-long-term regulation in chronic inflammatory diseases that may influence phenotypic responses such as apoptosis or cell proliferation remains to be elucidated.

Amplification of IL-1 beta-induced matrix metalloproteinase-9 expression by superoxide in rat glomerular mesangial cells is mediated by increased activities of NF-kappa B and activating protein-1 and involves activation of the mitogen-activated protein kinase pathways.

The modulation of cell signaling by free radicals is important for the pathogenesis of inflammatory diseases. Recently, we have shown that NO reduces IL-1beta-induced matrix metalloproteinase (MMP-9) expression in glomerular mesangial cells (MC). Here we report that exogenously administrated superoxide, generated by the hypoxanthine/xanthine oxidase system (HXXO) or by the redox cycler 2, 3-dimethoxy-1,4-naphtoquinone, caused a marked amplification of IL-1beta-primed, steady state, MMP-9 mRNA level and an increase in gelatinolytic activity in the conditioned medium. Superoxide generators alone were ineffective. Cytokine-induced steady state mRNA levels of TIMP-1, an endogenous inhibitor of MMP-9, were affected similarly by HXXO. Transient transfection of rat mesangial cells with 0.6 kb of the 5'-flanking region of the rat MMP-9 gene proved a transcriptional regulation of MMP-9 expression by superoxide. HXXO augmented the IL-1beta-triggered nuclear translocation of p65 and c-Jun and, in parallel, increased DNA binding activities of NF-kappaB and AP-1. Mutation of either response element completely prevented MMP-9 promoter activation by IL-1beta. Moreover, specific inhibitors of the classical extracellular signal-regulated kinase (ERK) pathway and p38 mitogen-activated protein kinase (MAPK) cascade, partially reversed the HXXO-mediated effects on MMP-9 mRNA levels, thus demonstrating involvement of ERKs and p38 MAPKs in MMP-9 expression. Furthermore, IL-1beta-triggered phosphorylation of all three MAPKs, including p38-MAPK, c-Jun N-terminal kinase, and ERK, was substantially enhanced by superoxide. Our data identify superoxide as a costimulatory factor amplifying cytokine-induced MMP-9 expression by interfering with the signaling cascades leading to the activation of AP-1 and NF-kappaB.

Regulation and immunhistochemical localization of nitric oxide synthases and soluble guanylyl cyclase in mouse spinal cord following nociceptive stimulation.

The localization and regulation of neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS) and soluble guanylyl cyclase (sGC) were assessed in the spinal cord of mice stimulated by an intraplantar injection of zymosan. Both, nNOS and iNOS were upregulated in the dorsal horns of the spinal cord in response to the zymosan challenge. While nNOS was found in neurons of superficial laminae iNOS occurred in astrocytes. Thus, astrocytes might be involved in the processing of nociceptive stimuli. Expression of sGC was not affected by zymosan treatment. It was found exclusively in nerve fibers suggesting that it was predominantly localized to the presynaptic neuron. This supports the hypothesis that nitric oxide (NO) acts as retrograde messenger in spinal nociceptive processing.

Inhibition of NFkappaB-mediated pro-inflammatory gene expression in rat mesangial cells by the enolized 1,3-dioxane-4, 6-dione-5-carboxamide, CGP-43182.

1. CGP-43182 has been described as a potent inhibitor of group IIA secreted phospholipase A(2) (group IIA sPLA(2)) activity in vitro. In rat mesangial cells, inhibition of group IIA sPLA(2) activity by CGP-43182 results in a 70% reduction of cytokine-stimulated prostaglandin E(2) biosynthesis, suggesting that group IIA sPLA(2) participates in arachidonic acid release and eicosanoid formation. Under these conditions the cytosolic phospholipase A(2) is not affected. 2. In mesangial cells, in addition to inhibition of catalytic activity, the membrane-permeant CGP-43182 completely blocked interleukin 1beta (IL1beta)-stimulated group IIA sPLA(2) gene expression. 3. A further action of CGP-43182 was a complete inhibition of cyclo-oxygenase-2 gene expression, resulting in a drastic reduction of prostaglandin formation in mesangial cells. 4. Moreover, CGP-43182 completely blocked IL1beta-induced gene expression of the inducible nitric oxide synthase, leading to an inhibition of cytokine-stimulated nitric oxide formation. 5. In contrast, the stimulatory effect of the cell-permeant cyclic AMP-analogue, dibutyryl-cAMP, on the induction of these enzymes was not inhibited by CGP-43182. These data indicate that CGP-43182 interferes with IL1beta- but not cyclic AMP-activated transcriptional regulation. 6. By studying components of the upstream transcription machinery, we observed an inhibition of NFkappaB activation by CGP-43182 in IL1beta-treated cells. Moreover, we observed that CGP-43182 prevented the phosphorylation and proteolytic degradation of the endogenous NFkappaB inhibitor, IkappaB, a process necessary for NFkappaB activation. 7. From our data, we propose that CGP-43182 is a potent anti-inflammatory drug useful for preventing the consequences of a concerted action of cytokine-stimulated pro-inflammatory genes mediated by NFkappaB.

Nitric oxide modulates expression of matrix metalloproteinase-9 in rat mesangial cells.

UNLABELLED: Nitric oxide modulates expression of matrix metalloproteinase-9 in rat mesangial cells. BACKGROUND: High-output levels of nitric oxide (NO) are produced by rat mesangial cells (MCs) in response to proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) by the inducible isoform of NO synthase (iNOS). We tested modulatory effects of NO on the expression and activities of matrix metalloproteinases-9 and -2 (MMP-9 and MMP-2), respectively. Temporal and spatial expression of these MMPs and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), seems to be critical in the extensive extracellular matrix (ECM) remodeling that accompanies sclerotic processes of the mesangium. Methods and Results. Using the NO donors S-Nitroso-N-acetyl-D,L-penicillamine (SNAP) and DETA-NONOate, we found strong inhibitory effects of NO mainly on the IL-1beta-induced MMP-9 mRNA levels. NO on its own had only weak effects on the expression of MMP-9 and MMP-2. The addition of the NOS inhibitor NG-monomethyl L-arginine (L-NMMA) dose dependently increased steady-state mRNA levels of cytokine-induced MMP-9, suggesting that endogenously produced NO exerts tonic inhibition of MMP-9 expression. MMP-9 activity in conditioned media from MCs costimulated with IL-1beta and NO donor contained less gelatinolytic activity than media of cells treated with IL-1beta alone. Exogenously added NO did not alter gelatinolytic activity of MMP-9 in cell-free zymographs. The expression levels of TIMP-1 were affected by NO similarly to the expression of MMP-9. CONCLUSION: We conclude that NO modulates cytokine-mediated expression of MMP-9 and TIMP-1 in rat MCs in culture. Our results provide evidence that NO-mediated attenuation of MMP-9 gelatinolytic activity is primarily due to a reduced expression of MMP-9 mRNA, and not the result of direct inhibition of enzymatic activity.

Cross-talk between group IIA-phospholipase A2 and inducible NO-synthase in rat renal mesangial cells.

Features of glomerulonephritis are expression of the inducible form of NO synthase (iNOS) as well as expression of the secretory group IIA-phospholipase A2 (sPLA2) in mesangial cells. Interleukin 1beta (IL-1beta) induces both enzymes with a similar time course resulting in an increase in nitrite production and sPLA2-IIA activity. In this study we investigated the relationship between the formation of NO and sPLA2-IIA induction in rat renal mesangial cells. Incubation of mesangial cells with the NO-donor, spermine-NONOate, for 24 h induced sPLA2-IIA mRNA expression and activity, whereas S-nitroso glutathione alone had only a small stimulatory effect. Stimulation of cells with IL-1beta caused a marked increase in sPLA2-IIA mRNA and activity that were potentiated 3 fold by both NO donors. Coincubation of cells with IL-1beta and the NOS inhibitor, L-N(G) monomethylarginine (L-NMMA), caused a dose-dependent inhibition of cytokine-induced sPLA2-IIA mRNA expression and activity. sPLA2-IIA activity was not stimulated by 8-bromo-cyclic GMP indicating that NO-induced sPLA2-IIA induction is independent of cyclic GMP-mediated signal transduction. These data show that NO contributes to the expression by cytokines of sPLA2-IIA and establishes a novel type of interaction between iNOS and sPLA2-IIA in mesangial cells. This cross-talk between inflammatory mediators may help to promote and sustain an inflammatory state in the kidney.

Inducible NO synthase: role in cellular signalling.

The discovery of endothelium-derived relaxing factor and its identification as nitric oxide (NO) was one of the most exciting discoveries of biomedical research in the 1980s. Besides its potent vasodilatory effects, NO was found under certain circumstances to be responsible for the killing of microorganisms and tumour cells by activated macrophages and to act as a novel, unconventional type of neurotransmitter. In 1992, Science picked NO as the 'Molecule of the Year', and over the past years NO has become established as a universal intercellular messenger that acutely affects important signalling pathways and, on a more long-term scale, modulates gene expression in target cells. These actions will form the focus of the present review.

Potentiation of nitric oxide synthase expression by superoxide in interleukin 1 beta-stimulated rat mesangial cells.

Exposure of mesangial cells to superoxide, generated by the hypoxanthine/xanthine oxidase system or by the redox cycler 2,3-dimethoxy-1,4-naphthoquinone caused a concentration-dependent amplification of interleukin (IL)-1beta-stimulated nitrite production. The effect of superoxide was accompanied by an increase in inducible nitric oxide synthase (iNOS) protein and iNOS mRNA levels. Incubation of mesangial cells with superoxide alone did not induce iNOS expression. To elucidate whether the increase of iNOS expression is due to transcriptional upregulation we fused a 4.5-kb genomic iNOS fragment that contains the transcriptional start site of the rat iNOS gene to a luciferase reporter gene. In transient transfection studies, superoxide caused a 10-fold augmentation of iNOS promoter activity in IL-1beta-challenged mesangial cells. Our data identify superoxide as a co-stimulatory factor amplifying cytokine-induced iNOS gene expression and subsequent nitric oxide (NO) synthesis.

Cooperative binding of NF-Y and Sp1 at the DNase I-hypersensitive site, fatty acid synthase insulin-responsive element 1, located at -500 in the rat fatty acid synthase promoter.

In vitro DNase I footprint analysis of the rat fatty acid synthase (FAS) promoter from -568 to -468 revealed four protein binding sites: A, B, and C boxes and the FAS insulin-responsive element 1 (FIRE1). As demonstrated by gel mobility shift analysis and supershift experiments, FIRE1, located between -516 and -498, is responsible for binding NF-Y. The C box located downstream of FIRE1 was shown by in vitro footprinting to be a Sp1 binding site, and furthermore, competition with Sp1 also abolished FIRE1 binding. Since the half-life of the Sp1.NF-Y.DNA complex is significantly longer than the half-lives of the Sp1.DNA or NF-Y.DNA complexes, the two transcription factors are deemed to bind cooperatively in the FAS promoter at -500. It is unusual that NF-Y binds at this distance from the start site of transcription. NF-Y binding sites are found in the promoters of at least three other FAS genes, viz. goose, chicken, and man. A second NF-Y binding site is located in the FAS promoter at the more usual position of -103 to -87, and it too has a neighboring Sp1 site. CTF/NF-1 competes for proteins binding to the B box. The A box binds Sp1 and contains a 12/13 match of the inverted repeat sequence responsible for binding the nuclear factor EF-C/RFX-1 in the enhancer regions of hepatitis B virus and the major histocompatibility complex class II antigen promoter. The same relative positions of NF-Y and Sp1 binding sites in the promoters of FAS genes of goose, rat, chicken, and man emphasize the involvement of these transcription factors in the diet and hormonal regulation of FAS.

Cytotoxic effect of autocrine and macrophage-derived nitric oxide on cultured rat mesangial cells.

Expression of the inducible form of nitric oxide synthase (iNOS) has been found to be up-regulated in cytokine-stimulated mesangial cells (MC) and in experimental glomerulonephritis. Since direct toxicity of nitric oxide (NO) has been implicated in damage of bacteria, neoplastic and intact pancreatic cells, we investigated whether NO is cytotoxic to cultured MC, which may be relevant to pathogenesis of glomerular injury. MC isolated from rat glomeruli generated substantial amounts of nitrite, the stable NO end-product, when cells were stimulated with IL-1beta and tumour necrosis factor-alpha (TNF-alpha). Total DNA synthesis was significantly reduced in the presence of IL-1beta and TNF-alpha, and this effect was completely reversed by N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of iNOS. Stimulation of MC with IL-1beta and TNF-alpha caused remarkable toxicity to these cells, measured by the MTT test (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide cleavage, specific cytotoxicity 41.5 +/- 20.3%), and much less prominent MC lysis (3H-thymidine release, specific cytolysis 11.5 +/- 5.3%). Toxic effects of cytokines were fully reversible by the iNOS inhibitor. Lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), but not IL-1beta and TNF-alpha, induced rat peritoneal macrophages to produce large amounts of nitrite. In co-culture, such prestimulated macrophages had significantly cytotoxic (MTT test 62.9 +/- 19.9%) and cytolytic (3H-thymidine release 57.9 +/- 13.8%) effects on MC. Again, this toxicity was totally inhibited in the presence of L-NMMA. We conclude from these results that cytokine-stimulated generation of NO by MC or macrophages is directly toxic to MC, and may play a role in pathogenesis of glomerular injury involving mesangiolysis.

NF-Y binds to the inverted CCAAT box, an essential element for cAMP-dependent regulation of the rat fatty acid synthase (FAS) gene.

Using EMSA competition experiments together with supershifts and in vitro transcription/translation we show that the basal transcription factor NF-Y or a related factor binds to the cAMP-responsive inverted CCAAT box recently identified in the rat fatty acid synthase (FAS) gene from nucleotide -99 to -92 relative to the transcription start site of the FAS mRNA. This result indicates a putative novel role for NF-Y in the cAMP-dependent gene regulation in a small class of genes such as FAS and tryptophan hydroxylase. Since NF-Y is a constitutively produced factor, not surprisingly, no differences in the specific DNA/protein complex with the CCAAT(FAS) box and nuclear proteins from H4IIE cells treated with cAMP and/or insulin or not could be observed. This implies that NF-Y might be modified in response to cAMP or might interact with another factor whose properties are altered by cAMP.

Cloning and sequencing of the proximal promoter of the rat iNOS gene: activation of NFkappaB is not sufficient for transcription of the iNOS gene in rat mesangial cells.

It has previously been shown that expression of the inducible form of NO synthase (EC 1.14.23) is controlled at the transcriptional level and that induction of iNOS transcription is dependent on activation of transcription factors of the NFkappaB family. TNF-alpha and IL-1beta synergistically stimulate iNOS transcription in rat glomerular mesangial cells. We have recently reported that endothelin-1 completely blocks cytokine-induced iNOS expression at the transcriptional level. To further investigate the molecular mechanisms and the role of NFkappaB in cytokine-elicited iNOS transcription, we cloned a 661 bp genomic rat DNA fragment, which contains 497 bp of the proximal iNOS promoter. An NFkappaB-binding site identical to that described for the murine sequence was identified and used for electrophoretic mobility shift experiments. We found that binding of NFkappaB is strongly induced in mesangial cells by both IL-1beta and TNF-alpha. While endothelin-1 blocks cytokine-induced iNOS expression, it has no influence on the binding pattern of NFkappaB. We conclude from these data that transcription of iNOS in mesangial cells requires additional signals besides activation of NFkappaB.

Endothelin-1 inhibits cytokine-stimulated transcription of inducible nitric oxide synthase in glomerular mesangial cells.

Endothelin-1 (ET-1) is a potent vasoconstrictor while nitric oxide (NO) has strong vasodilatory effects. Recent studies have indicated that vasoconstrictors and NO may mutually modulate their production and/or activity, thus regulating each other in the context of microcirculatory maintenance. We examined the question whether ET-1 may affect NO formation by controlling the expression of the inducible isoform of the NO synthase (iNOS) in cultured rat glomerular mesangial cells (MCs), as induced by the inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) plus interleukin-1 beta (IL-1 beta). We found that ET-1 in MCs markedly reduced cytokine-induced NO production (measured as stable NO2-) and inhibited the expression of iNOS mRNA (Northern blot analysis) and of iNOS protein (Western blotting). Inhibition of cytokine-stimulated iNOS mRNA expression by ET-1 was almost complete at the level of gene transcription while post-transcriptional effects were not detected. The ETA receptor antagonist BQ-123 blocked the inhibitory effect of ET1. The ETA agonist sarafotoxin 6b (S6b) inhibited, while the ETB agonist-sarafotoxin 6c (S6c) did not inhibit cytokine-initiated iNOS transcription in MCs. The results demonstrate that ET-1 can strongly inhibit cytokine induction of iNOS and formation of NO in cultured MCs, and that this action is mediated via the ETA receptor. While the precise mechanism(s) and biological relevance of this ET-1 effect are presently unclear, it is conceivable that down-regulation of iNOS by the vasopressor ET-1 may serve in vivo to prevent massive NO build-up and subsequent vasomotor collapse in the glomerular capillary tuft. This could help to maintain glomerular ultrafiltration in states of endotoxin excess as well as during glomerular formation and action of TNF-alpha and IL-1 beta causing iNOS induction and subsequent overproduction of NO.

Insulin-responsive regions of the rat fatty acid synthase gene promoter.

Here we show that insulin may play a role in the diet-induced regulation of the rat fatty acid synthase (FAS; EC 2.3.1.85). Transient transfection of human and rat hepatoma cell lines with successively deleted FAS/CAT promoter fusion plasmids was used to determine the effect of insulin on FAS promoter activity. Our results indicate the existence of cis-acting insulin-responsive elements in the FAS promoter; the position of one of these is coincident with the position of a previously determined diet-induced DNAse I hypersensitive site (HSi-1) at approximately -500 bp relative to the transcription start site of FAS mRNA.

Phosphoribosylpyrophosphate synthetase (PRS): a new gene family in Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains at least four PRS genes, all of which have been cloned and sequenced. Each of the four derived amino acid sequences have more than 60% similarity to the corresponding polypeptides of man, rat, Escherichia coli and Salmonella typhimurium. The PRS1 gene maps on chromosome XI, PRS2 on chromosome V, PRS3 on chromosome VIII and PRS4 on chromosome II. One member of this gene family, PRS1, contains a region of non-homology (NHR) shown by cDNA cloning and sequencing not to be an intron. The results presented here suggest that the presence of this NHR is not detrimental to the function of the gene. To date the possibility of protein splicing can be neither proven nor disputed.