Lidia Sautebin

Involvement of NF‐κB in the regulation of cyclooxygenase‐2 protein expression in LPS‐stimulated J774 macrophages

We investigated the involvement of NF‐κB in the regulation of COX‐2 protein expression and prostaglandin production in LPS‐stimulated J774 macrophages. Incubation of J774 cells with LPS (1 μg/ml) for 24 h caused an increase of COX‐2 protein expression and accumulation of both PGE2 and 6‐keto‐PGF1α in the cell culture medium. Ammonium pyrrolidinedithiocarbamate (APDC, 0.1, 1, 10 μM) and N‐α‐p‐tosyl‐l‐lysine chloromethylketone (TLCK, 1, 10, 100 μM), two inhibitors of NF‐κB activation, suppressed in a concentration‐dependent manner both LPS‐induced COX‐2 protein expression and prostanoid generation. Moreover, APDC and TLCK both inhibited the LPS‐induced increase of NF‐κB DNA binding activity and prevented IκB‐α degradation. Our results show for the first time that NF‐κB is involved in COX‐2 protein expression in LPS‐stimulated J774 macrophages and suggest that inhibitors of NF‐κB activation may represent a useful tool for the pharmacological control of inflammation.

Leptin increases serotonin turnover by inhibition of brain nitric oxide synthesis

Leptin administration inhibits diencephalic nitric oxide synthase (NOS) activity and increases brain serotonin (5-HT) metabolism in mice. We evaluated food intake, body-weight gain, diencephalic NOS activity, and diencephalic content of tryptophan (TRP), 5-HT, hydroxyindoleacetic acid (5-HIAA), and 5-HIAA/5-HT ratio after intracerebroventricular (ICV) or intraperitoneal (IP) leptin injection in mice. Five consecutive days of ICV or IP leptin injections induced a significant reduction in neuronal NOS (nNOS) activity, and caused a dose-dependent increase of 5-HT, 5-HIAA, and the 5-HIAA/5-HT ratio. Diencephalic 5-HT metabolism showed a significant increase in 5-HT, 5-HIAA, and the 5-HIAA/5-HT ratio 3 hours after a single leptin injection. This effect was maintained for 3 hours and had disappeared by 12 hours after injection. After a single IP leptin injection, the peak for 5-HT, 5-HIAA, and the 5-HIAA/5-HT ratio was achieved at 6 hours. Single injections of ICV or IP leptin significantly increased diencephalic 5-HT content. Leptin-induced 5-HT increase was antagonized by the coadministration of L-arginine only when the latter was ICV injected, whereas D-arginine did not influence leptin effects on brain 5-HT content. Finally, in nNOS-knockout mice, the appetite-suppressant activity of leptin was strongly reduced, and the leptin-induced increase in brain 5-HT metabolism was completely abolished. Our results indicate that the L-arginine/NO pathway is involved in mediating leptin effects on feeding behavior, and demonstrate that nNOS activity is required for the effects of leptin on brain 5-HT turnover.

Modulation of the induction of nitric oxide synthase by eicosanoids in the murine macrophage cell line J774

The effect of eicosanoids on the induction of nitric oxide synthase in the murine macrophage cell line J774 has been studied. We found that prostaglandin E2 (PGE2) and iloprost (a stable analogue of prostacyclin) both at nanomolar concentrations inhibited the lipopolysaccharide stimulated induction of NO synthase. In contrast PGF2α, U46619, a stable analogue of thromboxane A2, leukotrienes B4 and C4 had no effect. These data demonstrate that the l‐arginine: NO pathway in macrophages may be modulated by prostanoids.

Modulation by nitric oxide of prostaglandin biosynthesis in the rat.

1 Modulation of prostaglandin biosynthesis in vivo by either exogenous or endogenous nitric oxide (NO) has been studied in the rat using arachidonic acid (AA)‐induced paw oedema and measuring both the foot volume and the amount of 6‐keto‐prostaglandin F1α (6‐keto‐PGF1α), the stable metabolite of prostacyclin (PGI2), in the oedematous fluid recovered from inflamed paws. 2 Paw injections of 150 or 300 nmol of AA were virtually inactive whereas 600 nmol produced a moderate oedema which was greatly reduced by the NO synthase inhibitor l‐NG‐nitro arginine methyl ester (l‐NAME, 100 nmol/paw) and the NO scavenger haemoglobin (Hb, 30 μmol/paw), but unaffected by the inhibitor of the soluble guanylate cyclase, methylene blue (Mb, 3 μmol/paw) and L‐arginine (15 μmol/paw). 3 The NO‐donors (10 μmol/paw) 3‐morpholino‐sydnonimine‐hydrochloride (SIN‐1), S‐nitroso‐N‐acetyl‐d, l‐penicillamine (SNAP) and sodium nitroprusside (SNP) significantly potentiated the paw oedema induced by AA (300 nmol/paw). 4 SIN‐1 (2.5, 5 and 10 μmol/paw) produced a significant dose‐dependent increase of the oedema induced by AA which was correlated with increased amounts of 6‐keto‐PGF1α in the fluid recovered from inflamed paws. 5 Both oedema and prostaglandin biosynthesis induced by the combination AA + SIN‐1 were greatly suppressed by either Hb (30 μmol/paw) or indomethacin (3 μmol/paw or 5 mg kg−1 s.c.) but unaffected by Mb (3 μmol/paw). 6 In LPS‐treated rats (6 mg kg−1, i.p.) doses of AA inactive in normal animals produced a remarkable oedema which was reduced by l‐NAME or Hb, unaffected by Mb and increased by l‐arginine. 7 These results demonstrate that NO increases prostaglandin biosynthesis in vivo through a guanosine 3′:5′‐cyclic monophosphate (cyclic GMP)‐independent mechanism and suggest that the interaction between NO synthase and cyclo‐oxygenase (COX) pathways may represent an important mechanism for the modulation of the inflammatory response.

Protective Effects of Anthocyanins from Blackberry in a Rat Model of Acute Lung Inflammation

Anthocyanins are a group of naturally occuring phenolic compounds related to the coloring of plants, flowers and fruits. These pigments are important as quality indicators, as chemotaxonomic markers and for their antioxidant activities. Here, we have investigated the therapeutic efficacy of anthocyanins contained in blackberry extract (cyanidin-3-O-glucoside represents about 80% of the total anthocyanin contents) in an experimental model of lung inflammation induced by carrageenan in rats. Injection of carrageenan into the pleural cavity elicited an acute inflammatory response characterized by fluid accumulation which contained a large number of neutrophils as well as an infiltration of polymorphonuclear leukocytes in lung tissues and subsequent lipid peroxidation, and increased production of nitrite/nitrate (NOx) and prostaglandin E2 (PGE2). All parameters of inflammation were attenuated in a dose-dependent manner by anthocyanins (10, 30 mg kg-1 30 min before carrageenan). Furthermore, carrageenan induced an upregulation of the adhesion molecule ICAM-1, nitrotyrosine and poly (ADP-ribose) synthetase (PARS) as determined by immunohistochemical analysis of lung tissues. The degree of staining was lowered by anthocyanins treatment. Thus, the anthocyanins contained in the blackberry extract exert multiple protective effects in carrageenan-induced pleurisy.

ERK-mediated regulation of leukotriene biosynthesis by androgens: A molecular basis for gender differences in inflammation and asthma

5-Lipoxygenase initiates the biosynthesis of leukotrienes, lipid mediators involved in normal host defense and in inflammatory and allergic disorders. Despite an obvious gender bias in leukotriene-related diseases (e.g., asthma), gender aspects have been neglected in studies on leukotrienes and 5-lipoxygenase. Here, we show that leukotriene formation in stimulated whole blood or neutrophils from males is substantially lower compared with females, accompanied by changed 5-lipoxygenase trafficking. This is due to gender-specific differential activation of extracellular signal-regulated kinases (ERKs). The differences are directly related to variant male/female testosterone plus 5α-dihydrotestosterone levels, and addition of 5α-dihydrotestosterone to female blood or neutrophils reduced the high (female) LT biosynthesis capacity to low (male) levels. In conclusion, regulation of ERKs and leukotriene formation by androgens constitutes a molecular basis for gender differences in the inflammatory response, and in inflammatory diseases such as asthma.

Prostaglandins prevent inducible nitric oxide synthase protein expression by inhibiting nuclear factor-κB activation in J774 macrophages

We investigated the effect of PGE2 and iloprost (a prostacyclin analogue) on inducible nitric oxide synthase (iNOS) protein expression and nuclear factor‐κB (NF‐κB) activation in lipopolysaccharide (LPS)‐stimulated J774 macrophages. Incubation of J774 cells with LPS (10 μg/ml) caused an increase of iNOS protein expression which was prevented in a concentration‐dependent fashion by PGE2 (0.1, 1, 10 μM) and iloprost (0.01, 0.1, 1 μM). Electrophoretic mobility shift assay indicated that both prostanoids blocked the activation of NF‐κB, a transcription factor necessary for NO synthase induction. PGE2 and iloprost also blocked disappearance of IκB‐α from cytosolic fraction and nuclear translocation of NF‐κB subunits p50 and p65. These results show for the first time that PGE2 and iloprost down‐regulate iNOS protein expression by inhibiting NF‐κB activation and suggest a negative feed‐back mechanism that may be important for limiting excessive or prolonged NO production in pathological events.

Food deprivation increases brain nitric oxide synthase and depresses brain serotonin levels in rats

We studied nitric oxide (NO) synthase activity and serotonin content in the diencephalon of 24 hr food deprived rats. NO synthase activity was significantly increased whereas serotonin levels together with those of tryptophan and 5-hydroxyindoleacetic acid (5-HIAA) were reduced in food deprived rats when compared to control rats. NG-Nitro-L-arginine (L-NO Arg), an inhibitor of NO synthase, was used as a tool to study the role of NO in food deprivation. Twenty-four hr food deprived male Sprague-Dawley rats were intraperitoneally (i.p.) administered L-NO Arg (12.5, 25 and 50 mg/kg) before food presentation. Control rats received a NaCl (0.9%) solution. Food consumption was monitored 1 and 2 hr after food presentation. L-NO Arg administration produced a dose-dependent reduction in food intake. Pretreatment with metergoline (2 mg/kg) but not with ritanserin (1 mg/kg) antagonized the anorectic effect of L-NO Arg. Moreover, in the diencephalon L-NO Arg significantly reduced NO synthase activity whereas it increased serotonin levels. Our data indicate that NO might have a physiological role in the regulation of food intake and suggest that brain NO may modulate the central serotoninergic system.

Prostaglandins and nitric oxide as molecular targets for anti-inflammatory therapy

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most used drugs worldwide, in spite of their renal and gastric side effects. Medicinal plants may represent a useful source of new effective therapeutic agents, particularly considering the new findings concerning the mediators of inflammation, such as prostaglandins and nitric oxide. In fact, the discovery of two isoforms of the enzyme cyclooxygenase, which catalyzes the conversion of arachidonic acid to prostaglandins, has opened new interesting perspectives in the treatment of inflammatory diseases. As cyclooxygenase, also nitric oxide synthase, the enzyme which converts L-arginine to nitric oxide, exists in two isoforms. It appears that the constitutive isoforms of both enzymes (cyclooxygenase-1 and constitutive nitric oxide synthase) have a regulatory-physiological role, whereas the inducible isoforms (cyclooxygenase-2 and inducible nitric oxide synthase) are involved in inflammation. A number of medicinal plants have been screened for their ability to inhibit cyclooxygenase-2 and/or inducible nitric oxide synthase activity and/or expression.

17β-Estradiol Antiinflammatory Activity in Carrageenan-Induced Pleurisy

We have recently demonstrated that 17β-estradiol (E2) opposes cytokine-dependent increase of inducible nitric oxide synthase (iNOS) activity in rat smooth muscle cells and proposed that this effect might be associated to an antiinflammatory activity of this hormone. In the present study, we examine the E2 effects on a well-known in vivo model of inflammation. We show that, in carrageenan treatment of ovariectomized rats, prior exposure to E2 significantly attenuated inflammatory response as measured by histological examination and exudate production. The effect was visible with a single injection of a physiological dose of E2 1 h before the carrageenan treatment and was blocked by coadministration of the estrogen receptor antagonists ICI 182,780 or tamoxifen. This latter observation suggests that the effect is receptor mediated. The mechanisms by which estradiol has beneficial effects in this model of inflammation are unclear: we show that in hormonally treated rats there is a decrease in polymorphonuclea...