Nitric oxide-dependent down-regulation of angiotensin II type 2 receptors during experimental sepsis

OBJECTIVE: The systemic renin-angiotensin system is highly activated during septic shock. This has focused interest in regulation of the adrenal angiotensin II type 2 receptor (AT2) as the target thought to mediate angiotensin II-induced adrenal catecholamine release during experimental sepsis in vivo. In addition, the influence of typical endogenous mediators of sepsis, such as proinflammatory cytokines and nitric oxide, on AT2 receptor expression should be investigated in vitro. DESIGN: Prospective animal trial followed by a controlled cell culture study. SETTING: Laboratory of the Department of Anesthesiology. SUBJECTS: Male Sprague-Dawley rats weighing 200-250 g, PC12 cell line. INTERVENTIONS: Rats were injected with lipopolysaccharide to stimulate Gram-negative sepsis or lipoteichoic acid to stimulate Gram-positive sepsis. AT2 receptor expression, abundance of the proinflammatory cytokines (interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma), and nitric oxide synthase II expression have been determined in the adrenal gland. Rat adrenal pheochromocytoma cells were incubated with these cytokines or with the nitric oxide donors sodium nitroprusside or S-nitroso-N-acetylpenicillamine to investigate the regulation of AT2 receptors during severe inflammation on a cellular level. MEASUREMENTS AND MAIN RESULTS: In the adrenal gland, AT2 receptor expression was down-regulated in both models of sepsis, whereas tissue cytokine concentrations were elevated and nitric oxide synthase II expression was induced. Incubation of PC12 cells with proinflammatory cytokines resulted in a dose-dependent diminished expression of AT2 receptors, which was mimicked by incubation with nitric oxide donors. Blocking of cytokine-induced nitric oxide synthesis by co-incubation of PC12 cells with NG-nitro-l-arginine methyl ester prevented down-regulation of AT2 receptors. CONCLUSIONS: These findings show that in our model of sepsis, the expression of AT2 receptors in the adrenal gland is down-regulated in a nitric oxide-dependent manner. Because AT2 receptors are thought to be involved in adrenal catecholamine secretion in a stimulatory fashion, the diminished expression of AT2 receptors could play an important role in the pathogenesis of septic shock via impaired angiotensin II-induced adrenal catecholamine release, despite a strong activation of the systemic renin-angiotensin system.     

Endothelin-receptor gene-expression in rat endotoxemia

OBJECTIVE: The reduced vascular response to endothelin-1 has focused interest onto the regulation of the endothelin-receptor subtypes ET(A) and ET(B) during severe sepsis. DESIGN AND SETTING: Prospective animal trial followed by a controlled cell culture study in the laboratory of the Department of Anesthesiology. SUBJECTS: Male Sprague-Dawley rats weighing 200-250 g, aortic vascular smooth muscle cell line A7r5. INTERVENTIONS: Rats were injected with lipopolysaccharide to induce severe experimental endotoxemia. ET(A)/ET(B) receptor gene expression was investigated by specific RNase protection assay, and abundance of tumor necrosis factor alpha was determined in the lung and kidney. Aortic vascular smooth muscle cells were incubated with the proinflammatory cytokines interleukin-1beta, tumor necrosis factor alpha, and interferon gamma or with the nitric oxide donor S-nitroso- N-acetylpenicillamine to investigate the regulation of ET(A) receptor gene expression during severe inflammation. MEASUREMENTS AND RESULTS: ET(A)/ET(B) receptor gene expression was markedly downregulated in the lung but was unchanged in the kidney during endotoxemia. ET(A) receptor gene expression was downregulated in aortic vascular smooth muscle cells by tumor necrosis factor alpha but not by interleukin 1beta, interferon gamma, or nitric oxide. In vivo there seems to be a correlation between the tissue concentration of tumor necrosis factor alpha and gene expression of ET(A) receptors in the lung and kidney. CONCLUSIONS: Our data show that sepsis causes downregulation of ET(A) receptors at the level of gene expression, and provide correlative evidence that this effect can be mediated by tumor necrosis factor alpha. This downregulation of ET(A) receptors possibly contributes to the attenuated vascular response to endothelin-1 in the pulmonary circulation.     

Immune dysfunction in endotoxicosis: role of nitric oxide produced by inducible nitric oxide synthase

OBJECTIVE: To investigate whether stimulation with lipopolysaccharide (LPS) alters cytokine production by splenocytes in mice and whether it changes the T-helper 1 (Th1)/Th2 balance. The role of nitric oxide in such immunologic changes was also explored using mice with genetic lack of inducible nitric oxide synthase (iNOS). DESIGN:: Prospective animal study with concurrent controls. SETTING: University research laboratory. SUBJECTS: iNOS knockout mice and wild-type littermates. INTERVENTIONS: iNOS knockout mice or wild-type mice were injected with LPS or saline with or without anti-interleukin (IL)-6 antibody, and survival was monitored for 7 days. MEASUREMENTS AND MAIN RESULTS: At 24 and 48 hrs after administration, blood samples and splenocytes were obtained to examine immunologic variables. Cell surface markers and cytokine expression of splenocytes were used to characterize the Th1/Th2 balance and were measured by flow cytometry. At 48 hrs after LPS administration, the Th1/Th2 balance shifted toward Th2 predominance in wild-type mice, irrespective of the IL-6 level, whereas it showed Th1 predominance in iNOS knockout mice, and the increase of IL-6 and IL-10 in response to LPS persisted in these animals. After LPS administration, the mortality rate was significantly higher in iNOS knockout mice than in wild-type mice, irrespective of the IL-6 level. CONCLUSIONS: These findings suggest that nitric oxide produced by iNOS during endotoxemia may be involved in down-regulation of Th1 cytokines and up-regulation of Th2 cytokines, whereas IL-6 has no such role. The increased lethality of LPS in iNOS knockout mice suggests that nitric oxide may be protective against proinflammatory cytokine-induced damage. Nitric oxide excess may increase susceptibility to nosocomial infections, so-called immunoparalysis.     

Cytokine-induced intestinal epithelial hyperpermeability: role of nitric oxide.

OBJECTIVE: Incubation of enterocytic monolayers with interferon (IFN)-gamma increases nitric oxide (NO) production and permeability, but NO synthesis inhibitors ameliorate the development of IFN-gamma-induced hyperpermeability. Induction of inducible nitric oxide synthase (iNOS), an isoform of the enzyme responsible for NO biosynthesis, is often enhanced by the synergistic effects of multiple cytokines. Moreover, many of the cytopathic effects of NO are mediated by peroxynitrite, which is produced by the reaction of NO with superoxide radical anion. In the present study, we sought to determine whether combinations of several proinflammatory cytokines, including IFN-gamma, interleukin-1beta, and tumor necrosis factor-alpha, have synergistic effects on the induction of iNOS expression and/or hyperpermeability to hydrophilic solutes in cultured enterocytic monolayers. We also assessed the effects of aminoguanidine (a relatively selective iNOS inhibitor), L-N(G)-monomethyl arginine (an isoform-nonselective NO synthase inhibitor), and Tiron (a superoxide radical anion scavenger) on the development of cytokine-induced hyperpermeability. DESIGN: Caco-2 monolayers were incubated under control conditions or with IFN-gamma, interleukin-1beta, or tumor necrosis factor-alpha alone, pairwise combinations of these cytokines, or all three cytokines together (cytomix; CM). iNOS messenger RNA (mRNA) expression was assessed using Northern blot analysis. The permeability of Caco-2 monolayers growing on permeable supports in bicameral chambers was assessed by measuring the apical-to-basolateral flux of fluorescein disulfonic acid. The concentration of nitrate plus nitrite in culture supernatants, an indirect measure of NO production, was determined using the Griess reaction. RESULTS: After 24 hrs of incubation, up-regulation of iNOS mRNA expression was evident only in cells exposed to IFN-gamma-containing formulations. Expression of iNOS mRNA was far greater in cells incubated with CM than in cells treated with IFN-gamma alone or either of the two-component IFN-gamma-containing cytokine combinations. Compared with IFN-gamma, CM resulted in a higher rate of NO production over 48 hrs of incubation. The permeability of Caco-2 monolayers increased by approximately six-fold and approximately 20-fold after incubation for 48 hrs with IFN-gamma alone and CM, respectively. The increase in permeability induced by incubation with CM was significantly ameliorated by the addition of aminoguanidine, L-N(G)-monomethyl arginine, or Tiron. CONCLUSIONS: IFN-gamma-containing combinations of cytokines are potent inducers of iNOS in cultured enterocytic monolayers. Peroxynitrite may be an important mediator of cytokine-induced gut epithelial hyperpermeability.     

Effect of the nitric oxide inhibitor, L-N(G)-monomethylarginine, on accumulation of interleukin-6 and interleukin-8, and nuclear factor-kappaB activity in a human endothelial cell line.

OBJECTIVE: To determine the effect of the nitric oxide synthase inhibitor, L-N(G)-monomethylarginine, on interleukin-6 and interleukin-8 accumulation, and nuclear factor-kappaB expression in an endothelial cell model of sepsis. DESIGN: Controlled cell culture experiments examining the immunomodulatory effects of nitric oxide synthase inhibition. SUBJECTS: A human endothelial cell line (EA.hy926). MEASUREMENTS AND RESULTS: Cells were incubated with tumor necrosis factor-alpha and interleukin (IL)-1beta in the presence of L-N(G)-monomethylarginine (L-NMMA). IL-6 and IL-8 were measured in culture supernatants using enzyme immunoassay. Nuclear factor-kappaB was measured using electrophoretic mobility shift assay and was quantified using phosphorimaging. IL-6 accumulation was decreased (p < .05) and IL-8 accumulation increased (p < .01) with L-NMMA. Increased nuclear factor-kappaB expression in stimulated cells was unaltered on exposure to L-NMMA. Cell viability was unaffected. CONCLUSIONS: Excessive production of nitric oxide has been implicated in septic shock, and the use of nitric oxide synthase inhibitors has been suggested. The immunoregulatory actions of nitric oxide synthase inhibitors affects the profile of cytokine release. This effect is not mediated through modulation of nuclear factor-kappaB. These findings have implications for the use of nitric oxide synthase inhibiting agents in septic shock.     

Effect of 2,4-diamino-6-hydroxy-pyrimidine on postburn Staphylococcus aureus sepsis in rats

OBJECTIVE: Guanosine triphosphate-cyclohydrolase I (GTP-CHI) is the first and rate-limiting enzyme for the de novo biosynthesis of biopterin. The objective of present study was to observe the effect of 2,4-diamino-6-hydroxy-pyrimidine (DAHP), an inhibitor of GTP-CHI, on the development of postburn Staphylococcus aureus sepsis. DESIGN: A prospective, controlled animal study. SETTING: A research laboratory in a hospital. SUBJECTS: Male Wistar rats. INTERVENTIONS: Fifty-six male Wistar rats were randomly divided into four groups as follows: normal control group (n = 10), scald control group (n = 10), postburn sepsis group (n = 20), and DAHP treatment group (n = 16). In the scald control group, rats were subjected to a 20% total body surface area third-degree scald injury and then were killed at 24 hrs. In the postburn sepsis group (n = 20), rats were inflicted with 20% total body surface area third-degree scald followed by Staphylococcus aureus challenge, and they were further divided into 2- and 6-hr groups. In the DAHP treatment group (n = 16), animals were intraperitoneally injected with a dose of 1 g/kg DAHP before Staphylococcus aureus challenge and then were further divided into 2- and 6-hr groups. Tissue samples from liver, kidneys, lungs, and heart were collected to determine GTP-CHI, inducible nitric oxide synthase, and tumor necrosis factor-alpha messenger RNA expression. Meanwhile, biopterin and nitric oxide concentrations in these tissues were also measured. MEASUREMENTS AND MAIN RESULTS: After the scald injury followed by Staphylococcus aureus challenge, GTP-CHI messenger RNA expression and biopterin concentrations were significantly elevated in various tissues such as liver, heart, kidneys, and lungs, as were the values of inducible nitric oxide synthase messenger RNA expression and nitric oxide formation (p <.01). Pretreatment with DAHP significantly reduced GTP-CHI/biopterin induction (p <.05-.01), and the up-regulation of inducible nitric oxide synthase/nitric oxide was also suppressed. Furthermore, DAHP administration inhibited the gene expression of tumor necrosis factor-alpha. Two hours after septic challenge, tumor necrosis factor-alpha messenger RNA expression in liver, kidneys, and lungs in the DAHP-treated group was 35.7%, 37.3%, and 33.0% of that in the postburn septic group, respectively. Additionally, in animals without DAHP treatment, the 6-hr mortality rate was 55.6% (20 of 36), whereas it was only 25.0% in DAHP-treated animals (4 of 16, p =.08). CONCLUSIONS: Early treatment with DAHP might be a potential strategy to prevent the development of postburn Staphylococcal sepsis, which appears to be associated with down-regulation of biopterin and nitric oxide formation by DAHP.     

Sesame oil protects against lead-plus-lipopolysaccharide-induced acute hepatic injury

Lead (Pb) increases lipopolysaccharide (LPS)-induced tumor necrosis factor alpha, which causes liver damage. In this study, we investigated the effect of sesame oil on Pb-plus-LPS (Pb + LPS)-induced acute liver damage in mice. Mice were given sesame oil (8 mL/kg orally) just after Pb acetate (10 mmol/kg i.p.) plus LPS (5 mg/kg i.p.). Aspartate aminotransferase, alanine aminotransferase, tumor necrosis factor-alpha, interleukin-1beta, nitric oxide, and inducible nitric oxide synthase levels were examined. Sesame oil significantly decreased serum aspartate aminotransferase and alanine aminotransferase levels in Pb + LPS-stimulated mice. Sesame oil reduced Pb + LPS-induced tumor necrosis factor-alpha, interleukin-1beta, and nitric oxide production in serum and liver tissue. Furthermore, sesame oil decreased inducible nitric oxide synthase expression in leukocytes and liver tissue in Pb + LPS-treated mice. We hypothesize that the inhibition of proinflammatory cytokines and nitric oxide might be involved in sesame oil-associated protection against Pb + LPS-induced acute hepatic injury in mice.     

The angiotensin II AT1 receptor-associated protein Arap1 is involved in sepsis-induced hypotension

Introduction

Hypotension in septic patients results from hypovolemia, vasodilatation and hyporeactivity to vasoconstrictors, such as angiotensin II. The AT1 receptor-associated protein 1 (Arap1) is expressed in vascular smooth muscle cells and increases the surface expression of the AT1-receptor in vitro. We hypothesized that dysregulation of Arap1 may contribute to vascular hyporeactivity to angiotensin II during endotoxemia.

Methods

Arap1-deficient mice were used to assess the role of Arap1 in sepsis-induced hypotension. The isolated perfused kidney was used as an in vitro model to determine the relevance of Arap1 for vascular resistance and sensitivity to angiotensin II.

Results

During endotoxemia, mean arterial blood pressure (MAP) decreased in both genotypes, with the time course of sepsis-induced hypotension being markedly accelerated in Arap1-/- compared to +/+ mice. However, baseline MAP was similar in Arap1-/- and wildtype mice (102 ± 2 vs.103 ± 2 mmHg; telemetry measurements; n = 10; P = 0.66). Following lipopolysaccharide (LPS) injections (3 mg/kg), Arap1 expression was successively down-regulated in the wildtype mice, reaching levels below 10% of baseline expression. The endotoxemia-related decline in Arap1 expression could be recapitulated in cultured mesangial cells by incubation with pro-inflammatory cytokines, such as tumor necrosis factor α and interferon γ. Plasma renin concentration was increased in Arap1-/- mice compared to wildtype mice (66 ± 6 vs. 41 ± 4 ng AngI/ml/h; n = 23; P = 0.001), presumably contributing to preserved MAP under baseline conditions. The sensitivity of the vasculature to angiotensin II was reduced in Arap1-/- compared to +/+ mice, as determined in the isolated perfused kidney.

Conclusions

Our data suggest that down-regulation of Arap1 expression during sepsis contributes to the development of hypotension by causing reduced vascular sensitivity to angiotensin II.     

Inhibition of NF-kappaB by IkappaB prevents cytokine-induced NO production and promotes enterocyte apoptosis in vitro

Nuclear factor-kappaB (N-kappaB) plays a key role in gut inflammation. NF-kappaB up-regulates proinflammatory genes encoding cytokines, adhesion molecules, and inducible nitric oxide synthase (iNOS). However, NF-kappaB has also been shown to up-regulate protective or anti-apoptotic factors. We utilized an adenoviral vector carrying a super-repressor form of the inhibitor of NF-kappaB, IkappaB, to examine the effects of NF-kappaB inhibition on cytokine-induced nitric oxide production and apoptosis in rat small intestinal epithelial cells (IEC-6). Chemical inhibitors of NF-kappaB, including pyrrolidine dithiocarbamate (PDTC), tosyl-lysine-chloromethylketone (TLCK), genistein, and N-acetyl-leu-leu-norleucinal (n-LLnL) were also utilized. Treatment of AdIkappaB-transfected cells with cytomix [1000 U/mL IFN-gamma, 1 nM IL-1beta, and 10 ng/mL tumor necrosis factor alpha (TNFalpha)] or TNFalpha-containing cytokine combinations resulted in inhibition of cytokine-induced nitrite production and a marked increase in apoptosis compared to control cells. Apoptosis occurred independently of nitric oxide (NO) production since exogenous sources of NO did not inhibit apoptosis. Inducible NOS and clAP were down-regulated in AdIkappaB-transfected cells treated with cytomix. TLCK and LLnL treatment also induced apoptosis in cytomix-treated cells, while PDTC and genistein did not. Thus, although NF-kappaB up-regulates various pro-inflammatory genes, it may also have protective or anti-apoptotic effects in enterocytes. NF-kappaB appears necessary for upregulating cIAP in IEC-6 cells upon cytokine exposure.     

Myometrial maturation and labour

The increased expression of contraction-associated proteins, including oxytocin receptors, connexin-43, and prostaglandin F2alpha receptors, in term pregnant myometrium is classically considered to be the concrete expression of myometrial activation. However, the decrease in prostaglandin E2 receptor subtype EP2 on one hand and the down-regulation of the nitric oxide (NO) pathway and various vasorelaxing peptides on the other hand probably also play a key role in the loss of quiescence, and, with the above-mentioned activation, in the maturation of the myometrium. Decidual activation and production of interleukin-1, tumour necrosis factor-alpha and epidermal growth factor enhance prostaglandin production in both the amnion and chorion, and also in the myometrium. A substantial increase of eicosanoids concentration in myometrial tissue is probably an important condition for the success of the ultimate step of myometrial stimulation and the onset of labour. During labour, prostaglandins and oxytocin seem to act in synergy, perhaps along with endothelin-1, to trigger contractility through an increase in intracellular Ca2+ concentration. An overall view of these phenomena in which myometrial cells are the common targets of uterorelaxant and uterotonic agents appears essential for a rational use of tocolytic therapies and labour inductors.     

Effect of endogenous nitric oxide on hyperoxia and tumor necrosis factor-alpha-induced leukosequestration and proinflammatory cytokine release in rat airways

OBJECTIVE: To investigate the effects of endogenous nitric oxide on hyperoxia and tumor necrosis factor-alpha-induced leukosequestration and proinflammatory cytokine release in rat airways. DESIGN: Prospective, randomized, controlled animal study. SETTING: Experimental laboratory. SUBJECTS: Male Sprague-Dawley rats weighing 350-500 g. INTERVENTIONS: The rats were pretreated with N(G)-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg) or saline intravenously 4-6 mins before intratracheal administration of tumor necrosis factor-alpha, 95% oxygen, or both, when the vasopressor effect of L-NAME had reached a plateau. MEASUREMENTS AND MAIN RESULTS: Bronchoalveolar lavage fluid was recovered from the airway of rats after exposure to 95% oxygen and tumor necrosis factor-alpha for 6 hrs under ventilator support. Neutrophils in lavage fluid were isolated and examined for the inducible nitric oxide synthase expression by flow-cytometric assay. Tumor necrosis factor-alpha and interleukin-1 beta in lavage fluid were measured by enzyme-linked immunosorbent assay. The percentage of neutrophils in bronchoalveolar fluid was significantly higher in rats exposed to hyperoxia + tumor necrosis factor-alpha (29.7 +/- 12.5%) compared with rats with hyperoxia (16.3 +/- 1.2%), tumor necrosis factor-alpha (4.2 +/- 1.1%), or room air (5.0 +/- 1.8%) alone (p <.05). Rats exposed to hyperoxia + tumor necrosis factor-alpha had significantly higher concentrations of inducible nitric oxide synthase of neutrophils (350.1 +/- 75.7 mean fluorescence intensity), compared with rats with hyperoxia (64.9 +/- 1.6 mean fluorescence intensity), tumor necrosis factor-alpha (102.6 +/- 15.3 mean fluorescence intensity), or room air (111.2 +/- 25.8 mean fluorescence intensity) alone (p <.05). Rats exposed to hyperoxia + tumor necrosis factor-alpha significantly produced higher concentrations of tumor necrosis factor-alpha and interleukin-1 beta, compared with rats with tumor necrosis factor-alpha, hyperoxia, or room air alone. Hyperoxia + tumor necrosis factor-alpha also significantly increased growth-related oncogene/cytokine-induced neutrophil chemoattractant (GRO/CINC)-1 in bronchoalveolar fluid, compared with those receiving tumor necrosis factor-alpha alone, hyperoxia alone, or room air alone. L-NAME significantly enhanced the percentage of neutrophil recovery and the production of tumor necrosis factor-alpha, interleukin-1 beta, and GRO/CINC-1 in airways compared with the corresponding hyperoxia + tumor necrosis factor-alpha treatment alone. CONCLUSIONS: Endogenous nitric oxide may be an important endogenous inhibitor of hyperoxia + tumor necrosis factor-alpha-induced leukocyte recruitment and subsequently tumor necrosis factor-alpha, interleukin-1 beta, and GRO/CINC-1 release.     

L-arginine supplementation in hyperdynamic endotoxemic pigs: effect on nitric oxide synthesis by the different organs

OBJECTIVES: Under septic conditions, the protective role of nitric oxide in the organs may become compromised at a time of increased demand as a result of decreased availability of L-arginine. It remains unknown whether supplementation with L-arginine, as a substrate, can modulate organ nitric oxide production. DESIGN: Controlled study with laboratory animals. SETTING: University research laboratory. SUBJECTS: Female crossbred pigs. INTERVENTION: Pigs were challenged with Escherichia coli endotoxin (intravenously) and received intravenous fluid resuscitation for 24 hrs to reproduce a model of long-lasting hyperdynamic endotoxemia. Pigs were infused with either L-arginine or L-alanine intravenously during endotoxin and via the intragastric route after cessation of endotoxin infusion. The effects of L-arginine supplementation on nitric oxide synthesis and the relationships with arginine metabolism were determined with a stable isotope infusion protocol. Also, organ nitrite plus nitrate fluxes were measured. Implantation of multiple catheters enabled in vivo measurements across the hindquarter muscle, the portal-drained viscera, the liver, and the kidneys. MEASUREMENTS AND RESULTS: The isotope conversion method showed that L-arginine intervention significantly increased nitric oxide production by the portal-drained viscera, liver, and kidneys, resulting in elevated whole-body nitric oxide synthesis under endotoxemic and postendotoxemic conditions. Organ nitrite plus nitrate fluxes only tended to increase because of high variance among data. CONCLUSIONS: In this endotoxemia model, supplemental use of L-arginine favored nitric oxide synthesis in various organs.     

Tumor necrosis factor alpha activates soluble guanylate cyclase in bovine glomerular mesangial cells via an L-arginine-dependent mechanism

Endothelium-derived nitric oxide (NO) causes vasodilatation by activating soluble guanylate cyclase, and glomerular mesangial cells respond to NO with elevations of intracellular guanosine 3',5'-cyclic monophosphate (cGMP). We explored whether mesangial cells can be stimulated to produce NO and whether NO modulates mesangial cell function in an autocrine or paracrine fashion. Tumor necrosis factor alpha (TNF-alpha) raised mesangial cell cGMP levels in a time- and concentration-dependent manner (threshold dose 1 ng/ml, IC50 13.8 ng/ml, maximal response 100 ng/ml). TNF-alpha-induced increases in mesangial cGMP content were evident at 8 h and maximal at 18-24 h. The TNF-alpha-induced stimulation of mesangial cell cGMP production was abrogated by actinomycin D or cycloheximide suggesting dependence on new RNA or protein synthesis. Hemoglobin and methylene blue, both known to inhibit NO action, dramatically reduced TNF-alpha-induced mesangial cell cGMP production. Superoxide dismutase, known to potentiate NO action, augmented the TNF-alpha-induced effect. Ng-monomethyl-L-arginine (L-NMMA) decreased cGMP levels in TNF-alpha-treated, but not vehicle-treated mesangial cells in a concentration-dependent manner (IC50 53 microM). L-arginine had no effect on cGMP levels in control or TNF-alpha-treated mesangial cells but reversed L-NMMA-induced inhibition. Interleukin 1 beta and lipopolysaccharide (LPS), but not interferon gamma, also increased mesangial cell cGMP content. Transforming growth factor beta 1 blunted the mesangial cell response to TNF-alpha. TNF-alpha-induced L-arginine-dependent increases in cGMP were also evident in bovine renal artery vascular smooth muscle cells, COS-1 cells, and 1502 human fibroblasts. These findings suggest that TNF-alpha induces expression in mesangial cell of an enzyme(s) involved in the formation of L-arginine-derived NO. Moreover, the data indicate that NO acts in an autocrine and paracrine fashion to activate mesangial cell soluble guanylate cyclase. Cytokine-induced formation of NO in mesangial and vascular smooth muscle cells may be implicated in the pathogenesis of septic shock.     

Aspirin inhibits inducible nitric oxide synthase expression and tumour necrosis factor-alpha release by cultured smooth muscle cells

BACKGROUND: Inflammatory related cardiovascular disease, i.e. cardiac allograft rejection, myocarditis, septic shock, are accompanied by cytokine production, which stimulates the expression of inducible nitric oxide (iNOS). MATERIALS AND METHODS: The aim of the present study was to examine whether anti-inflammatory doses of acetylsalicylic acid (aspirin) could regulate iNOS protein expression in bovine vascular smooth muscle cells (BVSMCs) in culture. RESULTS: Interleukin 1 beta (IL-1 beta, 0.03 U mL-1) induced nitric oxide release by BVSMCs. Aspirin inhibited nitric oxide release from IL-1 beta-stimulated BVSMCs in a dose-dependent manner. In addition, aspirin significantly inhibited iNOS protein expression in BVSMCs and reduced the translocation of the nuclear factor-kappa B (NF-kappa B). Furthermore, aspirin and the blockade of NO generation by BVSMCs reduced the production of tumour necrosis factor alpha (TNF-alpha) by these cells. CONCLUSION: High doses of aspirin inhibited iNOS protein expression in BVSMCs and decreased NF-kappa B mobilization. The inhibition of iNOS expression by aspirin was further associated with a reduced ability of BVSMCs to produce TNF-alpha. This study could provide new mechanisms of action for aspirin in the treatment of the inflammation-related cardiovascular diseases.     

Alpha-adrenergic responses in asthma

Medication-free subjects with asthma who had normal pulmonary function were evaluated for alpha 1-adrenergic sensitivity by determination of the dose of topical phenylephrine required for mydriasis and for alpha 2-adrenergic responsiveness by determination of the inhibition by norepinephrine of adenosine 3',5'-cyclic monophosphate synthesis in platelets. Asthmatic subjects required significantly lower concentrations of phenylephrine for mydriasis than normal individuals (1.20 +/- 0.38% vs. 1.53 +/- 0.57%, p less than 0.05), indicating increased sensitivity. However, platelet alpha 2-adrenergic responses were normal in these same asthmatic subjects, as were alpha 2-receptor number and antagonist affinity determined by Scatchard analysis of equilibrium binding of [3H]-yohimbine to platelet particulates. These data suggest that the autonomic aberrations associated with asthma do not extend to the alpha 2-adrenergic system of the platelet, and that the alpha 1-adrenergic hypersensitivity cannot be attributed to medication use or pulmonary dysfunction, but might be an intrinsic property of the disease.     

Nitric oxide modifies the sarcoplasmic reticular calcium release channel in endotoxemia by both guanosine-3',5' (cyclic) phosphate-dependent and independent pathways

OBJECTIVES: a) To determine whether decreased sarcoplasmic calcium release channel (CRC) activity is a mechanism by which myocardial contractility is reduced in endotoxemia; b) to determine whether nitric oxide modulates CRC activity in endotoxemia; and c) to examine two nitric oxide signaling pathways in relation to CRC function in endotoxemia. DESIGN: Randomized, prospective using a rat model of endotoxemia. SETTING:: Research laboratory. SUBJECTS: Sprague-Dawley rats. INTERVENTIONS: Endotoxemia was induced by lipopolysaccharide administration. The effects of nitric oxide were studied using the highly selective inducible nitric oxide synthase inhibitor N-(3-(aminomethyl)benzyl)acetamidine dihydrochloride (1400W) and the specific guanylyl cyclase inhibitor 1-H (1, 2, 4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). MEASUREMENTS AND MAIN RESULTS: We assessed myocardial contractility, myocardial nitric oxide content, and guanosine-3',5' (cyclic) phosphate (cGMP) content. We determined CRC activity by calcium release and ryanodine binding assays. We followed these variables at four time points through the course of endotoxemia. We found that myocardial contractility and CRC activity were decreased in late but not in early endotoxemia. Furthermore, inducible nitric oxide synthase inhibition with 1400W restored contractility and CRC activity in late endotoxemia but paradoxically worsened these variables in early endotoxemia. Through the use of the guanylyl cyclase inhibitor ODQ, we demonstrate that nitric oxide acts through cGMP-mediated mechanisms in early and late endotoxemia. We investigated cGMP-independent pathways by assessing the oxidative status of the CRC. We found that in late endotoxemia, nitric oxide decreased the number of free thiols, demonstrating that nitric oxide also acts through cGMP-independent pathways. CONCLUSIONS: Nitric oxide has a dual effect on the CRC in endotoxemia. At low concentrations, as measured in early endotoxemia, nitric oxide stabilizes the CRC through cGMP-mediated mechanisms. In late endotoxemia, high nitric oxide concentrations decrease channel activity through both cGMP-dependent and cGMP-independent mechanisms.