IL-1 Beta-induced Nitric Oxide Research Articles

Interleukin-1beta-induced nitric oxide production and inhibition of insulin secretion in rat islets of langerhans is dependent upon the nitric oxide synthase cofactor tetrahidrobiopterin.

Interleukin (IL)-1 beta-induced inhibition of glucose-stimulated insulin secretion in rat islets of Langerhans is mediated in part by nitric oxide (NO). The NO synthase cofactor 5,6,7,8-tetrahydrobiopterin (BH(4)) supports NO synthesis in many cell types and IL-1 beta-induced NO generation and inhibition of insulin secretion have been previously correlated with intracellular BH(4 )levels in rat insulinoma cells. Using rat islets and the beta cell line BRIN-BD11, we have investigated whether synthesis of BH(4) limits IL-1beta-induced NO generation and inhibition of glucose-induced insulin secretion. IL-1 beta-induced NO generation by BRIN cells and islets was reduced by 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of de novo BH(4) synthesis. Sepiapterin, the substrate for salvage pathway BH(4) synthesis, reversed this inhibitory effect of DAHP in islets but not BRIN cells. DAHP reversed IL-1 beta-induced inhibition of islet insulin secretion, an effect prevented by sepiapterin. We conclude that BH(4) generation is necessary for IL-1 beta-induced NO generation in rat islets and BRIN cells. While a contribution of non-NO mediators cannot be excluded, our results support the proposal that IL-1 beta-induced, NO-mediated inhibition of insulin secretion in rat islets is dependent on the NOS cofactor BH(4).

Inhibition by lysophosphatidylcholine of nitric oxide production in interleukin 1 beta-stimulated vascular smooth muscle cells.

In vascular smooth muscle cells (VSMC), inflammatory cytokines such as interleukin 1 beta(IL-1 beta) stimulated nitric oxide (NO) production via the expression of an inducible type of NO synthase (iNOS). Lysophosphatidylcholine (LPC) is a major phospholipid component of atherogenic oxidized low density lipoprotein (Ox-LDL). In this study, we examined the effect of LPC on IL-1 beta-stimulated NO production in cultured. VSMC. LPC by itself did not stimulate the production of nitrite, a stable metabolite of NO, but dose-dependently inhibited IL-1 beta-stimulated nitrite production. LPC inhibited IL-1 beta-stimulated iNOS protein expression, whereas LPC did not inhibit IL-1 beta-stimulated iNOS mRNA expression. Analysis of iNOS protein degradation rate revealed that LPC had no effect on degradation rate of iNOS protein, suggesting that LPC inhibited iNOS translation. Moreover, Ox-LDL inhibited IL-1 beta-stimulated NO production by inhibiting iNOS protein expression without affecting iNOS mRNA expression. These results indicate that Ox-LDL negatively modulates IL-1 beta-induced NO production through the action of LPC, probably by blocking translation of iNOS mRNA.

Differences in cellular responses to mitogens in arterial smooth muscle cells derived from patients with moyamoya disease.

Moyamoya disease is a progressive cerebrovascular occlusive disease affecting primarily children. The etiology remains unknown. We examined the chemotactic and proliferative activities of inflammatory cell products from arterial smooth muscle cells (SMCs) derived from moyamoya patients and compared them with those from control subjects. We used 12 SMC strains from moyamoya patients and eight from control subjects. SMC migration was examined in a micro chemotaxis chamber. DNA synthesis was measured by an immunoperoxidase technique. Platelet-derived growth factor (PDGF)-BB markedly stimulated cell migration and DNA synthesis in control SMCs. PDGF-AA stimulated only DNA synthesis in control SMCs. In moyamoya SMCs, PDGF-AA and PDGF-BB stimulated cell migration but not DNA synthesis. Basic fibroblast growth factor had little migratory activity but stimulated DNA synthesis in moyamoya SMCs and control SMCs. Conversely, hepatocyte growth factor stimulated cell migration but not DNA synthesis in moyamoya SMCs and control SMCs. In contrast, interleukin-1 beta (IL-1 beta) significantly stimulated the migration and DNA synthesis of control SMCs, while it inhibited moyamoya SMC migration. The levels of IL-1 beta-induced nitric oxide production did not differ between moyamoya SMCs and control SMCs, suggesting that IL-1 beta inhibits the migration of moyamoya SMCs through a nitric oxide-independent pathway. The differences in responses to PDGF and IL-1 in moyamoya SMCs are involved in the mechanism by which intimal thickening develops in moyamoya disease.

Interactions between interleukin-1 beta, nitric oxide and prostaglandin E2 in the rat ovary: effects on steroidogenesis.

It has been reported that interleukin (IL)-1 beta induces the synthesis of both nitric oxide (NO) and prostaglandin (PG)E2 in cultures of dispersed ovarian cells and exerts cytotoxic effects on these cells. Since PGE2, NO and IL-1 beta have been implicated as modulators of steroidogenesis, experiments have been undertaken to determine how IL-1 beta-induced NO and PGE2 production may affect steroidogenesis in cultures of ovarian dispersates obtained from untreated adult oestrous rats and to compare the action of IL-1 beta in cultures of granulosa/luteal (GL) cell-only cultures and GL cells co-cultured with peritoneal macrophages. IL-1 beta significantly increased the production of NO (assessed by nitrite measured in the culture medium) and PGE2 in cultures of ovarian dispersates but had no effect on cultures of GL cells in which NO production was typically very low and PGE2 production was undetectable. In contrast both NO and PGE2 were high in co-cultures and were not significantly altered by the addition of IL-1 beta. The NO donor sodium nitroprusside inhibited steroidogenesis in cultures of ovarian cells in a dose-dependent manner while PGE2 had a stimulatory effect. Concomitant inhibition of NO production with aminoguanidine and PG production with indomethacin resulted in a significant enhancement of basal progesterone production in these cultures. Finally, IL-1 beta inhibited progesterone responses to forskolin and PGE2 in ovarian dispersates: an effect not observed in GL cell-only cultures nor in co-cultures in which forskolin-induced progesterone production is always inhibited. No cytotoxic effects of IL-1 beta during the 48 h period of culture were observed. A comparison of the steroidogenic NO and PGE2 responses to IL-1 beta in the three culture models suggests that (i) the response to IL-1 beta observed in ovarian dispersates could be due to cytokine activation of resident and infiltrating macrophages or that the action of IL-1 beta requires some other heterologous cell-cell contact, and (ii) IL-1 beta acts indirectly on signal transduction pathways which stimulate steroidogenesis.

Arginine vasopressin inhibits interleukin-1 beta-stimulated nitric oxide and cyclic guanosine monophosphate production via the V1 receptor in cultured rat vascular smooth muscle cells.

It has been reported that various vasoactive substance modulate cytokine stimulated nitric oxide (NO) production in many cell types. To examine the effects of arginine vasopressin (AVP) on the production of NO and cyclic GMP (cGMP), and on inducible nitric oxide synthase (INOS) in cultured rat vascular smooth muscle cells (VSMC). Because VSMC possess the V1 receptor which clauses vascular contraction and respond to various cytokines for producing NO, we used rat VSMC and selected interleukin-1 beta (IL-1 beta) as a potent stimulator of NO production among various cytokines. We also measured cGMP production, which is the final mediator of NO-induced vascular relaxation, in order to evaluate the physiologic meaning of the present study. VSMC were incubated with test agents for 24 h except for a time-course study. Nitrite as a stable end product of NO was measured in the medium. Intracellular cGMP contents were assayed by enzyme immunoassay. INOS messenger RNA expression was analyzed by Northern blotting. AVP inhibited IL-1 beta-induced nitrite production in a dose- and time-dependent manner with concomitant changes in intracellular cGMP contents. On the other hand, AVP did not affect nitrite and cGMP production in the absence of IL-1 beta. Inhibition of nitrite and cGMP production by AVP was reversed by administration of the specific V1 receptor antagonist [1-(beta-mercapto-beta,beta- cyclopentamethylene propionic acid), 2-(O-methyl)-tyrosine] -Arg8-vasopressin) but not by the oxytocin (OXT) receptor antagonist [d(CH2(5)), TyrMe2, Orn8]-Vasotocin. Administration of the V1 receptor antagonist or OXT receptor antagonist alone did not affect IL-1 beta-stimulated nitrite and cGMP production. Although administration of AVP inhibited IL-1 beta-induced INOS messenger RNA expression, administration of the V1 receptor antagonist but not of the OXT receptor antagonist reversed this inhibition. It is suggested that AVP inhibits IL-1 beta-induced NO and cGMP production via the V1 receptor but not via the OXT receptor in VSMC. AVP can cause vascular contraction not only through direct action but also through indirect action by inhibiting NO production under some inflammatory conditions.

Induction of guanosine triphosphate-cyclohydrolase by follicle-stimulating hormone enhances interleukin-1 beta-stimulated nitric oxide synthase activity in granulosa cells.

In cultured granulosa cells, interleukin-1 beta (IL-1 beta) induced a time-dependent (16-72 h) and dose-related (0.3-30 ng/ml) stimulation of nitric oxide (NO) synthase (NOS) activity, as determined by the catalytic conversion of [3H]arginine to [3H]citrulline and NO2- accumulation in the culture medium. Although FSH alone failed to stimulate NOS activity, concomitant treatment with the gonadotropin (200 ng/ ml) or the cell-permeant cAMP analog (Bu)2cAMP (0.5 mM) markedly enhanced IL-1 beta-induced NO generation in cultured granulosa cells. The effect of IL-1 beta on citrulline biosynthesis and NO2- accumulation was abrogated by the NOS inhibitor NG-methyl-L-arginine or the IL-1-receptor antagonist protein. In contrast bacterial endotoxin (lipopolysaccharide), interferon-gamma, or tumor necrosis factor-alpha, which are well known inducers of inducible NOS (iNOS) in a variety of immunocompetent and nonimmunocompetent cell types, failed to increase [3H]citrulline formation or NO2- accumulation in untreated or FSH-stimulated cells. As demonstrated by reverse transcriptase-PCR analysis, IL-1 beta-stimulated NO generation was accompanied by a time-dependent increase in messenger RNA levels for iNOS and GTP-cyclohydrolase (GTPCH), the rate-limiting step for de novo tetrahydrobiopterin (BH4) biosynthesis. Treatment with FSH augmented only GTPCH messenger RNA expression, and a more than additive GTPCH signal was observed when cells were simultaneously challenged with IL-1 beta and FSH. Treatment with the GTPCH inhibitor 2,4-diamino-6-hydroxypyrimidine prevented IL-1 beta-induced NOS activity in untreated or FSH-stimulated cells, and this inhibition was completely reversed by sepiapterin, a substrate for BH4 biosynthesis, via an alternative pterin salvage pathway present in many cell types. As BH4 is an essential cofactor for NOS catalytic activity, these observations strongly suggest that FSH-induced biosynthesis of endogenous BH4 is essential for full iNOS biosynthetic capacity in IL-1 beta-stimulated granulosa cells.

Cytokine-stimulated astrocytes damage human neurons via a nitric oxide mechanism.

Astrocytes have been reported to play a neuropathogenic role within the brain, although little is known about the mechanism underlying astrocyte-mediated neuronal injury. We investigated the hypothesis that cytokine-stimulated astrocytes adversely affect neuronal cell survival via generation of the free radical nitric oxide (NO). Primary human astrocytes produced substantial amounts of NO in response to interleukin (IL)-1 alpha or IL-1 beta, which was blocked by the NO synthase inhibitor NG-mono-methyl-L-arginine (NMMA). IL-1 beta-induced NO production was markedly potentiated by interferon (IFN)-gamma. IL-1 receptor agonist protein (IRAP) totally blocked NO generation by cytokine-stimulated astrocytes. Using reverse transcription-polymerase chain reaction and sequencing analyses of the astrocyte NO synthase gene, we found a single band encoding for a 615 bp product that was identical to the corresponding sequence reported for human hepatocytes. Treatment of human fetal brain cell cultures with IL-1 beta plus IFN-gamma resulted in marked neuronal loss, as assessed by microscopic analysis and measurement of lactate dehydrogenase release. This cytokine-induced neuronal damage was blocked by simultaneous treatment of the brain cell cultures with NMMA or IRAP, suggesting a critical role of IL-1. These findings indicate that cytokine-stimulated astrocytes are neurotoxic via a NO-mediated mechanism and point to potential new therapies for neurodegenerative disorders that involve cytokines and reactive astrocytes.

Nitric oxide: physiology and pharmacology.

Nitric oxide: physiology and pharmacology.

Nitric Oxide

Nitric Oxide

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

Nitric oxide (NO) plays an important role in immunological reactions as a host defense mechanism against tumor cells and invasive microorganisms, but it may also damage healthy tissue. The excessive formation of NO in IL-1 beta-stimulated renal mesangial cells not only alters glomerular filtration, but it may also cause tissue injury and thus contribute to the pathogenesis of certain forms of glomerulonephritis. We report here that, although NO alone has no evident effect on NO synthase expression, it potently augments IL-1 beta-stimulated NO synthase expression in mesangial cells. NO donors such as sodium nitroprusside and S-nitroso-N-acetyl-D,L-penicillamine markedly increase IL-1 beta-induced NO synthase mRNA and protein levels as well as enzyme activity. Nuclear run-on experiments suggest that NO acts to increase IL-1 beta-induced NO synthase gene expression at the transcriptional level. Furthermore, inhibition of NO synthesis by different pharmacological approaches reduces IL-1 beta-induced NO synthase expression, thus suggesting that NO functions in a positive feedback loop that speeds up and strengthens its own biosynthesis. We suggest that this potent amplification mechanism forms the basis for the excessive formation of NO in acute and chronic inflammatory diseases.

Two-dimensional gel electrophoresis of rat islet proteins. Interleukin 1 beta-induced changes in protein expression are reduced by L-arginine depletion and nicotinamide.

Interleukin (IL)-1 beta-mediated damage to beta-cells in isolated islets of Langerhans depends upon de novo synthesis of proteins that have not been fully identified. Further, IL-1 beta-induced and tumor necrosis factor alpha-induced islet damage partly depends on the intracellular production of the nitric oxide (NO) radical. IL-1 beta has also been reported to induce the synthesis of cellular defense proteins, e.g., heme-oxygenase and heat shock proteins 70 and 90. Nicotinamide, while in itself inactive, inhibited IL-1 beta-induced NO production in a time- and dose-dependent manner. To enable the identification of IL-1 beta-induced proteins with possible protective and deleterious effects, we characterized the effects of IL-1 beta, nicotinamide, and NO synthesis inhibition by L-arginine depletion on rat islet protein expression detected by high-resolution two-dimensional gel electrophoresis. More than 1,600 proteins were reproducibly detected in control rat islets. Incubation with IL-1 beta-, nicotinamide-, or L-arginine-depleted control medium upregulated 29, 3, and 1 protein, respectively, and downregulated 4, 0, and 1 protein, respectively. Addition of nicotinamide and L-arginine depletion reduced the upregulation of 16 and 20 IL-1 beta-induced proteins, respectively. The identity of these proteins is under study. The demonstrated changes in protein expression caused by IL-1 beta +/- nicotinamide and L-arginine depletion may form the basis for identification of proteins with possible protective and deleterious roles in the initial beta-cell destruction in insulin-dependent diabetes mellitus.

Tyrosine kinase inhibitors suppress endotoxin- and IL-1 beta-induced NO synthesis in aortic smooth muscle cells.

Nitric oxide (NO) formation via the expression of an endotoxin- and cytokine-inducible NO synthase (iNOS) within the vascular smooth muscle is thought to be responsible for the cardiovascular collapse that occurs during septic shock and antitumor therapy with cytokines. Because the molecular mechanisms that underlie induction of iNOS are still unclear and because tyrosine kinases are implicated in interleukin-1 beta (IL-1 beta)-induced prostaglandin synthesis in mesangial cells and in NO generation by an insulinoma cell line, we investigated the influence of tyrosine kinase inhibitors on iNOS induction in cultured rat aortic smooth muscle cells (RASMC). The production of biologically active NO was demonstrated by L-arginine-dependent guanosine 3',5'-cyclic monophosphate (cGMP) accumulation after a 3-h exposure to either IL-1 beta or lipopolysaccharide (LPS). Pretreatment of RASMC for 30 min with the tyrosine kinase inhibitor genistein prevented both IL-1 beta- and LPS-elicited cGMP accumulation in a concentration-dependent manner. Geldanamycin, a chemically different tyrosine kinase inhibitor, also blocked cGMP formation in response to both LPS and IL-1 beta at nanomolar concentrations. Genistein and geldanamycin inhibited cGMP accumulation even when added 90 min after LPS exposure, but no inhibition was observed when they were included at later time points (120-180 min), suggesting that the inhibitors had no direct effect on iNOS activity after its induction. Formation of cGMP in response to sodium nitroprusside and to NO released from bovine aortic endothelial cells remained virtually unaffected by genistein and geldanamycin.(ABSTRACT TRUNCATED AT 250 WORDS)

Exogenous nitric oxide (NO) generation or IL-1 beta-induced intracellular NO production stimulates inhibitory auto-ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase in RINm5F cells.

Nitric oxide (NO) stimulates the auto-ADP-ribosylation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which results in the inhibition of enzyme activity. In the present work we show that addition of exogenous NO or IL-1 beta-induced intracellular NO generation cause GAPDH ADP-ribosylation and inhibition of enzyme activity. Incubation of RINm5F cells with sodium nitroprusside (SNP) for 18 h caused a time- and dose-dependent inhibition of GAPDH activity. Half-maximal inhibition of GAPDH activity was observed with 80 microM of the NO donor, with maximal inhibition after roughly 6 h of incubation. In parallel, SNP induced endogenous ADP-ribosylation of GAPDH measured by a decreased incorporation of [32P]ADP-ribose from [32P]NAD+ in the cytosol of the SNP-treated cells. Stimulation of endogenous NO production by inducing the NO synthase by exposure to the cytokine IL-1 beta results in decreased GAPDH activity. IL-1 beta (10(-9) M) inhibited GAPDH activity about 55%, compared with control values. Production of nitrite and inhibition of GAPDH was reversed by the NO synthase inhibitor NG-monomethyl-L-arginine, indicating that endogenous generated NO was the effective molecule. Again, GAPDH inhibition was associated with NO-stimulated endogenous ADP-ribosylation of the enzyme. Western blot analysis of GAPDH excluded degradation of GAPDH by NO. NO-stimulated auto-ADP-ribosylation resulted in inhibition of the glycolytic enzyme GAPDH and may be relevant as a cytotoxic effect of NO. In concert with its inhibitory actions on iron-sulfur enzymes like aconitase and electron transport proteins of the respiratory chain NO may mediate autocytotoxic effect in beta-cells.

Interleukin 1 beta induces the formation of nitric oxide by beta-cells purified from rodent islets of Langerhans. Evidence for the beta-cell as a source and site of action of nitric oxide.

Nitric oxide has recently been implicated as the effector molecule that mediates IL-1 beta-induced inhibition of glucose-stimulated insulin secretion and beta-cell specific destruction. The pancreatic islet represents a heterogeneous cell population containing both endocrine cells (beta-[insulin], alpha-]glucagon], gamma[somatostatin], and PP-[polypeptide] secreting cells) and non-endocrine cells (fibroblast, macrophage, endothelial, and dendritic cells). The purpose of this investigation was to determine if the beta-cell, which is selectively destroyed during insulin-dependent diabetes mellitus, is both a source of IL-1 beta-induced nitric oxide production and also a site of action of this free radical. Pretreatment of beta-cells, purified by FACS with IL-1 beta results in a 40% inhibition of glucose-stimulated insulin secretion that is prevented by the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (NMMA). IL-1 beta induces the formation of nitric oxide by purified beta-cells as evidenced by the accumulation of cGMP, which is blocked by NMMA. IL-1 beta also induces the accumulation of cGMP by the insulinoma cell line Rin-m5F, and both NMMA as well as the protein synthesis inhibitor cycloheximide prevent this cGMP accumulation. Iron-sulfur proteins appear to be intracellular targets of nitric oxide. IL-1 beta induces the formation of an iron-dinitrosyl complex by Rin-m5F cells indicating that nitric oxide mediates the destruction of iron-sulfur clusters of iron containing enzymes. This is further demonstrated by IL-1 beta-induced inhibition of glucose oxidation by purified beta-cells, mitochondrial aconitase activity of dispersed islet cells, and mitochondrial aconitase activity of Rin-m5F cells, all of which are prevented by NMMA. IL-1 beta does not appear to affect FACS-purified alpha-cell metabolic activity or intracellular cGMP levels, suggesting that IL-1 beta does not exert any effect on alpha-cells. These results demonstrate that the islet beta-cell is a source of IL-1 beta-induced nitric oxide production, and that beta-cell mitochondrial iron-sulfur containing enzymes are one site of action of nitric oxide.

Interleukin-lβ-Induced Nitric Oxide Production in Isolated Rat Pancreatic Islets Requires Gene Transcription and May Lead to Inhibition of the Krebs Cycle Enzyme Aconitase*

The aim of this study was to characterize the dynamics and functional relevance of interleukin-1 beta (IL-1 beta)-induced nitric oxide production in isolated pancreatic islets. Thus, islets were isolated from adult rats, precultured for 3-5 days in medium RPMI-1640 plus 10% fetal calf serum, and then exposed to IL-1 beta for different time periods, after which islet nitrite production and aconitase activity were determined. IL-1 beta (5 ng/ml) did not increase islet nitrite production during the first hour of incubation. Moreover, the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (Meth-arg; 5 mM) failed to prevent the initial (90 min) IL-1 beta-induced increase in islet insulin release. After 4, 7, and 24 h, however, nitrite production was increased by 50%, 93%, and 139%, respectively. Islet aconitase activity and glucose oxidation rates were decreased by 70% after incubation for 24 h with IL-1 beta. Both Meth-arg and N alpha-p-tosyl-L-lysine chloromethyl ketone (0.1 mM), a protease inhibitor, could completely counteract the IL-1 beta-induced increases in nitrite production and inhibition of aconitase activity and glucose oxidation rates. In a separate series of experiments, islets were incubated for 60 min with or without IL-1 beta and the RNA synthesis inhibitor actinomycin-D (5 micrograms/ml) and subsequently incubated for another 9 h without any additions. The presence of actinomycin-D during the 1-h IL-1 beta incubation period prevented the IL-1 beta-induced rise in nitrite production and the IL-1 beta-induced inhibition of aconitase activity and insulin release. It is concluded that IL-1 beta-induced nitric oxide production is a late event which requires gene transcription and does not mediate the initial stimulatory effects of IL-1 beta on beta-cell function. However, the gradually augmented rate of nitric oxide production may inhibit the enzyme aconitase, leading to a suppressed mitochondrial activity and a defective insulin release in response to nutrient secretagogues.