Sequential changes in redox status and nitric oxide synthases expression in the liver after bile duct ligation

Bile duct ligation (BDL) in rats induces portal fibrosis. This process has been linked to changes in the oxidative state of the hepatic cells and in the production of nitric oxide. Our objective was to find possible temporal connections between hepatic redox state, NO synthesis and liver injury. In this work we have characterized hepatic lesions 17 and 31 days after BDL and determined changes in hepatic function, oxidative state, and NO production. We have also analyzed the expression and localization of inducible NO synthase (NOS2) and constitutive NO synthase (NOS3). After 17 and 31 days from ligature, lipid peroxidation is increased and both plasma concentration and biliary excretion of nitrite+nitrate are rised. 17 days after BDL both NOS2 and NOS3 are expressed intensely and in the same regions. 31 days after BDL, the expression of NOS2 remains elevated and is localized mostly in preserved hepatocytes in portal areas and in neighborhoods of centrolobulillar vein. NOS3 is localized in vascular regions of portal spaces and centrolobulillar veins and in preserved sinusoids and although its expression is greater than in control animals (34%), it is clearly lower (50%) than 17 days after BDL. The time after BDL is crucial in the study of NO production, intrahepatic localization of NOS isoforms expression, and cell type involved, since all these parameters change with time. BDL-induced, peroxidation and fibrosis are not ligated by a cause-effect relationship, but rather they both seem to be the consequence of common inductors.     

Role of nitric oxide and prostanoids in attenuation of rapid baroreceptor resetting

Because the regulation of vascular function involves complex mutual interactions between nitric oxide (NO) synthase (NOS) and cyclooxygenase (COX) products, we examined the contribution of NO and prostanoids derived from the COX pathway in modulating aortic baroreceptor resetting during an acute (30 min) increase in arterial pressure in anesthetized rats. Increase in pressure was induced either by administration of the nonselective NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) or aortic coarctation (COA) with or without treatment with the COX inhibitor indomethacin (INDO) or the selective neuronal NOS inhibitor 1-(2-trifluoromethylphenyl)imidazole (TRIM). The activity of the aortic depressor nerve and arterial pressure were simultaneously recorded, and the degree of resetting was determined by the shift of the pressure-nerve activity curve using the ratio [delta systolic pressure at 50% of maximum baroreceptor activity/delta systolic pressure] x 100. The magnitude of pressure rise was similar in the different groups (59 +/- 6, 53 +/- 5, 53 +/- 5, 45 +/- 5, 49 +/- 3, and 41 +/- 3 mmHg for COA, L-NAME, INDO+COA, INDO+L-NAME, TRIM+COA, and TRIM+INDO+COA, respectively, P = 0.27). The degree of resetting that occurred with L-NAME or COA combined with treatment with TRIM was attenuated compared with COA alone (7 +/- 4, 5 +/- 2, and 31 +/- 6%, respectively, P = 0.04). INDO failed to influence baroreceptor resetting to higher pressure but prevented L-NAME- and TRIM-induced effects (20 +/- 7, 21 +/- 8, and 32 +/- 6% for INDO+COA, INDO+L-NAME, and INDO+TRIM+COA, respectively; P = 0.38). Baroreceptor gain was affected only by l-NAME. These findings indicate that NO, probably from neuronal origin, may exert stimulatory influence on the degree of rapid baroreceptor resetting to hypertension that involves COX-derived prostanoids.     

Effects of Chronic Treatment With the CB1 Antagonist,Rimonabant on the Blood Pressure,and Vascular Reactivity of Obese Zucker Rats

Rimonabant (RM) is a cannabinoid CB1 receptor antagonist useful in the treatment of obesity associated cardiovascular risk factors. Since cannabinoids are vasoactive compounds, the aim of this study is to evaluate the effect of chronic treatment with RM on systolic blood pressure (SBP), and endothelial and vascular reactivity. Obese Zucker rats (OZRs) and their lean counterparts were orally treated during 20 weeks with either RM (10 mg/kg/day). Endothelial and vascular function was assessed in aorta and small mesenteric arteries (SMAs) by concentration response curves to acetylcholine (ACh) and phenylephrine (Phe), respectively. Participation of nitric oxide (NO) was evaluated by incubation with the NO synthase (NOS) inhibitor N^G‐nitro‐l‐arginine methyl ester (L‐NAME) and cyclooxygenase (COX)‐derived products involvement was analyzed by incubation with indomethacin (INDO). Plasma lipid profile, insulin and adiponectin were also analyzed. Sympathetic activity was evaluated by urinary excretion of noradrenaline. As expected, RM decreased body weight gain and enhanced adiponectin concentration. Insulin resistance and sympathetic activity were also decreased. The increase in SBP observed in OZRs was reduced by treatment with RM. Aortae and SMAs from OZRs exhibited lower contractile response to Phe, being this effect prevented by RM administration. Although ACh‐induced response and NO participation remained unaltered with obesity, enhanced COX‐derived constrictor products were found in OZRs. RM treatment neither altered endothelium‐dependent relaxation nor L‐NAME‐sensitive component of the response. Nevertheless, it was able to regulate COX‐derived vasoactive products participation. Those effects may contribute to explain some of the cardiovascular protective actions elicited by this drug.     

Differential expression and localization of nitric oxide synthases in cirrhotic livers of bile duct-ligated rats.

Increased vascular nitric oxide (NO) production has been implicated in the pathogenesis of the hyperdynamic circulation in liver cirrhosis. This study investigated the expression of three isoforms of NO synthase (NOS) in rat cirrhotic livers. Cirrhosis was induced by chronic bile duct ligation (BDL). NOS enzyme activity was assessed by L-citrulline generation. Competitive RT-PCR was performed to detect the mRNA levels of NOS. In situ hybridization was done to localize NOS mRNA. Protein expression of NOS was evaluated by Western blotting and immunohistochemistry. The L-citrulline assay showed that constitutive NOS (cNOS) enzymatic activity was decreased, while inducible NOS (iNOS) activity was increased in BDL livers. Both endothelial NOS (eNOS) and neuronal NOS (nNOS) mRNA were detected in BDL and sham rats, but with enhanced expression in BDL rats. eNOS protein was redistributed with less expression in sinusoidal endothelial cells, but the total levels in liver were not changed. nNOS was induced in hepatocytes of BDL rats, in contrast to only a weak signal observed around some blood vessels in sham livers. Intense mRNA and protein expression of iNOS was induced in livers of BDL rats and was localized in hepatocytes, with no or a negligible amount in control livers. In conclusion, iNOS was induced in cirrhotic liver with its activity increased. In contrast, cNOS activity was impaired, regardless of unchanged eNOS protein levels and enhanced nNOS expression. These results suggest that all three types of NOS have a role in cirrhosis, but their expression and regulation are different.     

Effect of phosphodiesterase 5 inhibitor on alteration in vascular smooth muscle sensitivity and renal function in rats with liver cirrhosis

Previous studies suggested that increased activity of phosphodiesterase (PDE)5 in the kidneys of cirrhotic rats contributes to sodium retention. This study examined the role of PDE5 in the changes in vascular reactivity, hemodynamics, and sodium excretion in rats with liver cirrhosis. Four weeks after bile duct ligation (BDL) or sham operation (SO), in vitro reactivity of aortic rings to various agents and in vivo effects of a PDE5-selective inhibitor [1,3-dimethyl-6-(2-propoxy-5-methanesulfonylamidophenyl)pyrazolo[3,4d]-pyrimidin-4-(5H)-one, DMPPO] were studied. The vasodilator responses to nitroglycerin and S-nitroso-N-acetyl-penicillamine (SNAP) in phenylephrine-precontracted rings without endothelium were attenuated in BDL compared with SO rats. Pretreatment with DMPPO (0.1 microM) enhanced these responses and eliminated the differences between the two groups. Vasodilation to DMPPO itself was also less in BDL rats. The responses to phenylephrine were attenuated in endothelium-rich aorta from BDL relative to SO rats, but they were similar in endothelium-denuded aorta and remained similar despite preincubation with SNAP (0.1 microM) alone or with SNAP and DMPPO. In vivo, BDL rats were vasodilated relative to SO rats; DMPPO (5 mg/kg i.v.) decreased arterial pressure and vascular resistance in both groups equally and caused significant increase in sodium excretion in BDL rats only. In conclusion, the results are in accordance with a possible increase in PDE5 activity in aorta and kidney of cirrhotic rats that results in reduced responses to NO donors and contributes to the increase in sodium retention. PDE5 inhibitors may ameliorate sodium retention in cirrhosis but may worsen vasodilation. Examining the effect of PDE5 inhibitors after chronic administration will be more revealing.     

Involvement of inducible isoforms of COX and NOS in streptozotocin-pancreatic damage in the rat: interactions between nitridergic and prostanoid pathway

Streptozotocin-induced pancreatic damage involves nitric oxide (NO) and prostaglandins (PGs) overproduction. In this work we aim to evaluate a putative relationship between the elevated NO levels and the altered prostanoid production in pancreatic tissue from streptozotocin-diabetic rats. Total NOS activity and nitrate/nitrite pancreatic levels in tissues from diabetic rats are decreased when the cyclooxygenase (COX) inhibitor indomethacin (INDO) is added to the incubating medium, while the addition of PGE(2)increases nitrate/nitrite production and NOS levels. INDO and PGE(2)selectively affect Ca(2+)-dependent NOS (iNOS) activity in diabetic tissues, and they have not been able to modify nitrate/nitrite levels, iNOS or Ca(2+)-dependent (cNOS) in control tissues. When the NOS inhibitor L-NMMA was present in the incubating medium, control pancreatic [(14)C]-Arachidonic Acid ([(14)C]-AA) conversion to 6-keto PGF(1 alpha)and to TXB(2)was lower, and PGF(2 alpha), PGE(2)and TXB(2)production from diabetic tissues diminished. The NO donors, spermine nonoate (SN) and SIN-1, enhanced TXB(2)levels in control tissues, while PGF(2 alpha), PGE(2)and TXB(2)levels from diabetic tissues were increased. PGE(2)production from control and diabetic tissues was assessed in the presence of the NO donor SN plus INDO or NS398, a specific PG synthase 2 inhibitor. When SN combined with INDO or NS398 was added, the increment of PGE(2)production was abolished by both inhibitors in equal amounts, indicating that the activating effect of nitric oxide is exerted on the inducible isoform of cyclooxygenase. In the diabetic rat, prostaglandins and NO seem to stimulate the generation of each other, suggesting a lack of regulatory mechanisms that control the levels of vasoactive substances in acute phase of beta-cell destruction.     

Ammonia reduction with ornithine phenylacetate restores brain eNOS activity via the DDAH-ADMA pathway in bile duct-ligated cirrhotic rats

Ammonia is central in the pathogenesis of hepatic encephalopathy, which is associated with dysfunction of the nitric oxide (NO) signaling pathway. Ornithine phenylacetate (OP) reduces hyperammonemia and brain water in cirrhotic animals. This study aimed to determine whether endothelial NO synthase activity is altered in the brain of cirrhotic animals, whether this is associated with changes in the endogenous inhibitor, asymmetric-dimethylarginine (ADMA) and its regulating enzyme, dimethylarginine-dimethylaminohydrolase (DDAH-1), and whether these abnormalities are restored by ammonia reduction using OP. Sprague-Dawley rats were studied 4-wk after bile duct ligation (BDL) (n = 16) or sham operation (n = 8) and treated with placebo or OP (0.6 g/kg). Arterial ammonia, brain water, TNF-α, plasma, and brain ADMA were measured using standard techniques. NOS activity was measured radiometrically, and protein expression for NOS enzymes, ADMA, DDAH-1, 4-hydroxynonenol ((4)HNE), and NADPH oxidase (NOX)-1 were measured by Western blotting. BDL significantly increased arterial ammonia (P < 0.0001), brain water (P < 0.05), and brain TNF-α (P < 0.01). These were reduced significantly by OP treatment. The estimated eNOS component of constitutive NOS activity was significantly lower (P < 0.05) in BDL rat, and this was significantly attenuated in OP-treated animals. Brain ADMA levels were significantly higher and brain DDAH-1 significantly lower in BDL compared with sham (P < 0.01) and restored toward normal following treatment with OP. Brain (4)HNE and NOX-1 protein expression were significantly increased in BDL rat brain, which were significantly decreased following OP administration. We show a marked abnormality of NO regulation in cirrhotic rat brains, which can be restored by reduction in ammonia concentration using OP.     

Selective suppression of M1 macrophages is involved in zinc inhibition of liver fibrosis in mice

Zinc deficiency is common in the liver of patients with chronic liver disease. Zinc supplementation suppresses the progression of liver fibrosis induced by bile duct ligation (BDL) in mice. The present study was undertaken to specifically investigate a possible mechanism by which zinc plays this role in the liver. Kunming mice were subjected to BDL for 4 weeks to induce liver fibrosis, and concomitantly treated with zinc sulfite or saline as control by gavage once a day. The results showed that zinc supplementation significantly suppressed liver fibrosis and inflammation along with inhibition of hepatic stellate cells activation induced by BDL. These inhibitory effects were accompanied by the reduction of collagen deposition and a significant reduction of macrophage infiltration affected livers. Importantly, zinc selectively inhibited M1 macrophage polarization and M1-related inflammatory cytokines. This inhibitory effect was further confirmed by the reduction of relevant biomarkers of M1 macrophages including inducible NO synthase (iNOS), monocyte chemotactic protein-1 (MCP-1/CCL2), and tumor necrosis factor-α in the zinc supplemented BDL livers. In addition, zinc inhibition of M1 macrophages was associated with a decrease of Notch1 expression. Taken together, these data indicated that zinc supplementation inhibited liver inflammation and fibrosis in BDL mice through selective suppression of M1 macrophages, which is associated with inhibition of Notch1 pathway in M1 macrophage polarization.     

Interaction between NO and COX pathways modulating hepatic endothelial cells from control and cirrhotic rats

Reduced intrahepatic nitric oxide (NO) bioavailability and increased cyclooxygenase-1 (COX-1)-derived vasoconstrictor prostanoids modulate the hepatic vascular tone in cirrhosis. We aimed at investigating the reciprocal interactions between NO and COX in the hepatic endothelium of control and cirrhotic rats. NO bioavailability (DAF-FM-DA staining), superoxide (O(2) (-) ) content (DHE staining), prostanoid production (PGI(2) and TXA(2) by enzyme immunoassays) as well as COX expression (Western Blot), were determined in hepatic endothelial cells (HEC) from control and cirrhotic rats submitted to different experimental conditions: COX activation, COX inhibition, NO activation and NO inhibition. In control and cirrhotic HEC, COX activation with arachidonic acid reduced NO bioavailability and increased O(2) (-) levels. These effects were abolished by pre-treating HEC with the COX inhibitor indomethacin. In control, but not in cirrhotic HEC, scavenging of O(2) (-) by superoxide dismutase (SOD) incubation partially restored the decrease in NO bioavailability promoted by COX activation. NO supplementation produced a significant and parallel reduction in PGI(2) and TXA(2) production in control HEC, whereas it only reduced TXA(2) production in cirrhotic HEC. By contrast, in control and cirrhotic HEC, NO inhibition did not modify COX expression or activity. Our results demonstrate that NO and COX systems are closely interrelated in HEC. This is especially relevant in cirrhotic HEC where COX inhibition increases NO bioavailability and NO supplementation induces a reduction in TXA(2) . These strategies may have beneficial effects ameliorating the vasoconstrictor/vasodilator imbalance of the intrahepatic circulation of cirrhotic livers.     

In vivo and in vitro effects of lysine clonixinate on nitric oxide synthase in LPS-treated and untreated rat lung preparations.

Recent studies have shown that some nonsteroidal antiinflammatory drugs (NSAIDS) inhibited the inducible NO synthase (iNOS) without direct effect on the catalytic activity of this enzyme. This study was conducted to investigate the in vitro and in vivo effects of lysine clonixinate (LC) and indomethacin (INDO) on NOS activity in rat lung preparation. LC is a drug with antiinflammatory, antipyretic, and analgesic action. In the in vitro experiments, rats were injected with saline or lipopolysaccharide (LPS) and killed 6 h after treatment. Lung preparations were incubated with LC at 2.3 x 10(-5) M or 3.8 x 10(-5) M. The minimum concentration did not modify NOS activity in control or LPS-treated rats but the maximum dose inhibited increased NO production induced by LPS. Furthermore, INDO at 10(-6) M had no effect on enzymatic activity in control or LPS-treated rats. In the in vivo experiments, 40 mg/kg of LC were injected ip. Such a dose did not affect basal production of NO. When LC and LPS were injected simultaneously 6 h before sacrifice, a significant decrease in LPS-induced NOS activity was observed. INDO 10 mg/kg injected in control animals had no effect on NOS activity and did not block LPS induced stimulation of NO production when injected simultaneously. Finally, when LC (40 mg/kg) was injected 3 h after LPS, the enzymatic activity remained unchanged. Expression of iNOS was detected by Western blotting in rats treated with LPS plus 4, 10, 20, and 40 mg/kg of LC. The lowest dose was the only one showing no effect on LPS-induced increase of iNOS. In short, LC is a NSAID with inhibitory action on the expression of LPS-induced NOS, effect that was not seen with INDO in our experimental conditions.     

Regulation of hepatic eNOS by caveolin and calmodulin after bile duct ligation in rats

In carbon tetrachloride-induced liver cirrhosis, diminution of hepatic endothelial nitric oxide synthase (eNOS) activity may contribute to impaired hepatic vasodilation and portal hypertension. The mechanisms responsible for these events remain unknown; however, a role for the NOS-associated proteins caveolin and calmodulin has been postulated. The purpose of this study is to characterize the expression and cellular localization of the NOS inhibitory protein caveolin-1 in normal rat liver and to then examine the role of caveolin in conjunction with calmodulin in regulation of NOS activity in cholestatic portal hypertension. In normal liver, caveolin protein is expressed preferentially in nonparenchymal cells compared with hepatocytes as assessed by Western blot analysis of isolated cell preparations. Additionally, within the nonparenchymal cell populations, caveolin expression is detected within both liver endothelial cells and hepatic stellate cells. Next, studies were performed 4 wk after bile duct ligation (BDL), a model of portal hypertension characterized by prominent cholestasis, as evidenced by a significant increase in serum cholesterol in BDL animals. After BDL, caveolin protein levels from detergent-soluble liver lysates are significantly increased as assessed by Western blot analysis. Immunoperoxidase staining demonstrates that this increase is most prominent within sinusoids and venules. Additionally, caveolin-1 upregulation is associated with a significant reduction in NOS catalytic activity in BDL liver lysates, an event that is corrected with provision of excess calmodulin, a protein that competitively binds eNOS from caveolin. We conclude that, in cholestatic portal hypertension, caveolin may negatively regulate NOS activity in a manner that is reversible by excess calmodulin.     

Hemodynamic effects of Salvia miltiorrhiza on cirrhotic rats

Salvia miltiorrhiza (Sm) administration has been shown to reduce hepatic fibrosis in rats. We investigated the hemodynamic effects of Sm on bile duct ligated (BDL) rats. Hemodynamic, histological, and vascular contractile studies were conducted in rats 4 weeks after bile duct ligation. An aqueous extract of Sm (0.2 g twice per day) or vehicle was administered for 4 weeks to BDL rats. Sm treatment in BDL rats significantly reduced histological grades of fibrosis and ameliorated the portal hypertensive state (including portal venous pressure, superior mesenteric artery blood flow, cardiac index, and total peripheral resistance) as compared with vehicle treatment. Moreover, Sm treatment enhanced the vascular sensitivity of mesenteric arteries to phenylephrine in BDL rats. Sm treatment had no effect on plasma biochemical profiles of either BDL or normal rats. Our results suggest that 4-week Sm treatment ameliorates the portal hypertensive state in BDL rats.     

Ghrelin alleviates biliary obstruction-induced chronic hepatic injury in rats

BACKGROUND: Reactive oxygen species and oxidative stress are implicated in hepatic stellate cell activation and liver fibrosis, which are initiated by recruitment of inflammatory cells and by activation of cytokines. OBJECTIVE: The possible anti-oxidant and anti-inflammatory effects of ghrelin were evaluated in a hepatic fibrosis model in rats with bile duct ligation (BDL). METHODS: Under anesthesia, bile ducts of Sprague Dawley rats were ligated, and half of the rats were subcutaneously administered with ghrelin (10 ng/kg/day) and the rest with saline for 28 days. Sham-operated control groups were administered saline or ghrelin. On the 28th day of the study, rats were decapitated and malondialdehyde (MDA) content--an index of lipid peroxidation, and myeloperoxidase (MPO) activity--an index of neutrophil infiltration--were determined in the liver tissues. Oxidant-induced tissue fibrosis was determined by collagen contents, while the hepatic injury was analyzed microscopically. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) levels and lactate dehydrogenase (LDH) levels were determined to assess liver function and tissue damage, respectively. Pro-inflammatory cytokines; TNF-alpha, IL-1beta and IL-6 were also assayed in plasma samples. RESULTS: In the saline-treated BDL group, hepatic MDA levels, MPO activity and collagen content were increased (p<0.001), suggesting oxidative organ damage, as confirmed histologically. In the ghrelin-treated BDL group, however, all of the oxidant responses were reversed significantly (p<0.05-p<0.001). Serum AST, ALT, LDH levels, and cytokines were elevated in the BDL group as compared to the control group, while this increase was significantly decreased by ghrelin treatment. CONCLUSION: Owing to the anti-inflammatory and anti-oxidant effect as demonstrated in our study, it is possible to speculate that exogenously administered ghrelin may possess an antifibrotic effect against biliary obstruction-induced liver fibrosis. Thus, it seems likely that ghrelin may be of potential therapeutic value in protecting the liver fibrosis and oxidative injury due to biliary obstruction.     

内源性一氧化氮在高血压心肌肥厚中的作用

目的和方法：本实验用Ｌ精氨酸和一氧化氮合酶（ＮＯＳ）抑制剂ＬＮＡＭＥ观察内源性一氧化氮（ＮＯ）在高血压性心肌肥厚中的作用。结果：腹主动脉缩窄引起大鼠动脉血压显著升高,左心室重量／体重比值显著增加,左心室ＮＯ含量显著下降;Ｌ精氨酸不影响主动脉缩窄大鼠动脉血压,但减轻左心室重量／体重比值,明显升高左心室ＮＯ含量,加入ＬＮＡＭＥ可消除Ｌ精氨酸的上述作用;主动脉缩窄大鼠给予ＬＮＡＭＥ,动脉血压和左心室／体重比值并没有进一步增加;假手术大鼠给予ＬＮＡＭＥ,血压明显升高,左心室重量／体重比值轻度增加;主动脉缩窄大鼠不论是服用Ｌ精氨酸还是ＬＮＡＭＥ,左心室ｃＧＭＰ含量都明显增加。结论：口服Ｌ精氨酸可减轻主动脉缩窄大鼠心肌肥厚但不影响动脉血压,此作用可能是通过Ｌ精氨酸ＮＯ途径实现的,与ｃＧＭＰ机制无关。     

Hemodynamic Effects of the Non-Peptidic Angiotensin-(1-7) Agonist AVE0991 in Liver Cirrhosis

Background & Aims

Although in cirrhosis with portal hypertension levels of the vasoconstrictor angiotensin II are increased, this is accompanied by increased production of angiotensin (Ang)-(1–7), the endogenous ligand of the Mas receptor (MasR), which blunts hepatic fibrosis and decreases hepatic vascular resistance. Therefore, we investigated the effects of the non-peptidic Ang-(1–7) agonist, AVE0991, in experimental cirrhosis.

Methods

Cirrhosis was induced by bile duct ligation (BDL) or carbon tetrachloride (CCl₄) intoxication. The coloured microsphere technique assessed portal and systemic hemodynamic effects of AVE0991 in vivo. Hepatic expression of eNOS, p-eNOS, iNOS, JAK2, ROCK and p-Moesin were analyzed by western blots. Activities of ACE and ACE2 were investigated fluorometrically. Moreover, fibrosis was assessed in BDL rats receiving AVE0991.

Results

In vivo, AVE0991 decreased portal pressure (PP) in both rat models of cirrhosis. Importantly, systemic effects were not observed. The hepatic effects of AVE0991 were based on upregulation of vasodilating pathways involving p-eNOS and iNOS, as well as by downregulation of the vasoconstrictive pathways (ROCK, p-Moesin). Short-term treatment with AVE0991 decreased the activity of ACE2, long-term treatment did not affect hepatic fibrosis in BDL rats.

Conclusions

The non-peptidic agonist of Ang-(1–7), AVE0991, decreases portal pressure without influencing systemic pressure. Thus, although it does not inhibit fibrosis, AVE0991 may represent a promising new therapeutic strategy for lowering portal pressure.     

Lipopolysaccharide-induced increase of prostaglandin E(2) is mediated by inducible nitric oxide synthase activation of the constitutive cyclooxygenase and induction of membrane-associated prostaglandin E synthase

NO produced by the inducible NO synthase (NOS2) and prostanoids generated by the cyclooxygenase (COX) isoforms and terminal prostanoid synthases are major components of the host innate immune and inflammatory response. Evidence exists that pharmacological manipulation of one pathway could result in cross-modulation of the other, but the sense, amplitude, and relevance of these interactions are controversial, especially in vivo. Administration of 6 mg/kg LPS to rats i.p. resulted 6 h later in induction of NOS2 and the membrane-associated PGE synthase (mPGES) expression, and decreased constitutive COX (COX-1) expression. Low level inducible COX (COX-2) mRNA with absent COX-2 protein expression was observed. The NOS2 inhibitor aminoguanidine (50 and 100 mg/kg i.p.) dose dependently decreased both NO and prostanoid production. The LPS-induced increase in PGE(2) concentration was mediated by NOS2-derived NO-dependent activation of COX-1 pathway and by induction of mPGES. Despite absent COX-2 protein, SC-236, a putative COX-2-specific inhibitor, decreased mPGES RNA expression and PGE(2) concentration. Ketoprofen, a nonspecific COX inhibitor, and SC-236 had no effect on the NOS2 pathway. Our results suggest that in a model of systemic inflammation characterized by the absence of COX-2 protein expression, NOS2-derived NO activates COX-1 pathway, and inhibitors of COX isoforms have no effect on NOS2 or NOS3 (endothelial NOS) pathways. These results could explain, at least in part, the deleterious effects of NOS2 inhibitors in some experimental and clinical settings, and could imply that there is a major conceptual limitation to the use of NOS2 inhibitors during systemic inflammation.     

Modulation of nerve growth factor-induced activation of MAP kinase in PC12 cells by inhibitors of nitric oxide synthase

Nerve growth factor (NGF) increases expression of nitric oxide synthase (NOS) isozymes leading to enhanced production of nitric oxide (NO). NOS inhibitors attenuate NGF-mediated increases in cholinergic gene expression and neurite outgrowth. Mechanisms underlying this are unknown, but the mitogen-activated protein (MAP) kinase pathway plays an important role in NGF signaling. Like NGF, NO donors activate Ras leading to phosphorylation of MAP kinase. The present study investigated the role of NO in NGF-mediated activation of MAP kinase in PC12 cells. Cells were treated with 50 ng/mL NGF to establish the temporal pattern for rapid and sustained activation phases of MAP kinase kinase (MEK)-1/2 and p42/p44-MAP kinase. Subsequently, cells were pretreated with NOS inhibitors Nomega-nitro-L-arginine methylester and s-methylisothiourea and exposed to NGF for up to 24 h. NGF-induced activation of MEK-1/2 and p42/p44-MAP kinase was not dependent on NO, but sustained phosphorylation of MAP kinase was modulated by NO. This modulation did not occur at the level of Ras-Raf-MEK signaling or require activation of cGMP/PKG pathway. NOS inhibitors did not affect NGF-mediated phosphorylation of MEK. Expression of constitutively active-MEKK1 in cells led to phosphorylation of p42/p44-MAP kinase and robust neurite outgrowth; constitutively active-MKK1 also caused differentiation with neurite extension. NOS inhibitor treatment of cells expressing constitutively active kinases did not affect MAP kinase activation, but neurite outgrowth was attenuated. NOS inhibitors did not alter NGF-mediated nuclear translocation of phospho-MAP kinase, but phosphorylated kinases disappeared more rapidly from NOS inhibitor-treated cells suggesting greater phosphatase activity and termination of sustained activation of MAP kinase.     

Twenty‐Four‐Hour Variation of L‐Arginine/Nitric Oxide/Cyclic Guanosine Monophosphate Pathway Demonstrated by the Mouse Visceral Pain Model

The L‐arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway is known to be involved in central and peripheral nociceptive processes. This study evaluated the rhythmic pattern of the L‐arginine/NO/cGMP pathway using the mouse visceral pain model. Experiments were performed at six different times (1, 5, 9, 13, 17, and 21 h after light on) per day in male mice synchronized to a 12 h:12 h light‐dark cycle. Animals were injected s.c. with saline, 2 mg/kg L‐arginine (a NO precursor), 75 mg/kg L‐N^G‐nitroarginine methyl ester (L‐NAME, a NOS inhibitor), 40 mg/kg methylene blue (a soluble guanylyl cyclase and/or NOS inhibitor), or 0.1 mg/kg sodium nitroprusside (a nonenzymatic NO donor) 15 min before counting 2.5 mg/kg (i.p.) p‐benzoquinone (PBQ)‐induced abdominal constrictions for 15 min. Blood samples were collected after the test, and the nitrite concentration was determined in serum samples. L‐arginine or L‐NAME caused both antinociception and nociception, depending on the circadian time of their injection. The analgesic effect of methylene blue or sodium nitroprusside exhibited significant biological time‐dependent differences in PBQ‐induced abdominal constrictions. Serum nitrite levels also displayed a significant 24 h variation in mice injected with PBQ, L‐NAME, methylene blue, or sodium nitroprusside, but not saline or L‐arginine. These results suggest that components of L‐arginine/NO/cGMP pathway exhibit biological time‐dependent effects on visceral nociceptive process.     

The anabolic action of intermittent parathyroid hormone on cortical bone depends partly on its ability to induce nitric oxide‐mediated vasorelaxation in BALB/c mice

There is strong evidence that vasodilatory nitric oxide (NO) donors have anabolic effects on bone in humans. Parathyroid hormone (PTH), the only osteoanabolic drug currently approved, is also a vasodilator. We investigated whether the NO synthase inhibitor L‐NAME might alter the effect of PTH on bone by blocking its vasodilatory effect. BALB/c mice received 28 daily injections of PTH[1–34] (80 µg/kg/day) or L‐NAME (30 mg/kg/day), alone or in combination. Hindlimb blood perfusion was measured by laser Doppler imaging. Bone architecture, turnover and mechanical properties in the femur were analysed respectively by micro‐CT, histomorphometry and three‐point bending. PTH increased hindlimb blood flow by >30% within 10 min of injection (P < 0.001). Co‐treatment with L‐NAME blocked the action of PTH on blood flow, whereas L‐NAME alone had no effect. PTH treatment increased femoral cortical bone volume and formation rate by 20% and 110%, respectively (P < 0.001). PTH had no effect on trabecular bone volume in the femoral metaphysis although trabecular thickness and number were increased and decreased by 25%, respectively. Co‐treatment with L‐NAME restricted the PTH‐stimulated increase in cortical bone formation but had no clear‐cut effects in trabecular bone. Co‐treatment with L‐NAME did not affect the mechanical strength in femurs induced by iPTH. These results suggest that NO‐mediated vasorelaxation plays partly a role in the anabolic action of PTH on cortical bone. © 2016 The Authors. Cell Biochemistry and Function published by John Wiley & Sons, Ltd.