Cell-specific inhibition of inducible nitric oxide synthase activation by leflunomide

The influence of a novel immunomodulating drug, leflunomide, on iNOS-dependent nitric oxide (NO) production in rodent macrophages and fibroblasts was investigated. Leflunomide's active metabolite A77 1726 caused a dose-dependent decrease of NO production in IFN-gamma-treated L929 fibroblasts. The observed effect was cell-specific, as well as stimulus-specific, since A77 1726 did not affect NO production in IFN-gamma-stimulated murine peritoneal macrophages or db-cAMP-treated L929 cells. A77 1726 reduced expression of IFN-gamma-induced iNOS and IRF-1 mRNA in L929 cells, while iNOS enzymatic activity remained unchanged. Specific inhibitor of MAP kinase kinase (MEK), PD98059, but not unselective protein kinase inhibitor genistein, completely mimicked cell-type-specific and stimulus-specific NO-inhibitory action of leflunomide. Therefore, the recently described inhibition of MEK/MAP pathway by leflunomide could present a possible mechanism for its suppression of iNOS activation in L929 fibroblasts. Finally, a similar inhibitory effect of A77 1726 on both NO production and iNOS mRNA expression was observed also in IFN-gamma + LPS-activated murine and rat primary fibroblasts.     

Role of nitric oxide synthase isoforms in glucose-stimulated insulin release

The role of islet constitutive nitric oxide synthase (cNOS) in insulin-releasing mechanisms is controversial. By measuring enzyme activities and protein expression of NOS isoforms [i.e., cNOS and inducible NOS (iNOS)] in islets of Langerhans cells in relation to insulin secretion, we show that glucose dose-dependently stimulates islet activities of both cNOS and iNOS, that cNOS-derived nitric oxide (NO) strongly inhibits glucose-stimulated insulin release, and that short-term hyperglycemia in mice induces islet iNOS activity. Moreover, addition of NO gas or an NO donor inhibited glucose-stimulated insulin release, and different NOS inhibitors effected a potentiation. These effects were evident also in K+-depolarized islets in the presence of the ATP-sensitive K+ channel opener diazoxide. Furthermore, our results emphasize the necessity of measuring islet NOS activity when using NOS inhibitors, because certain concentrations of certain NOS inhibitors might unexpectedly stimulate islet NO production. This is shown by the observation that 0.5 mmol/l of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) stimulated cNOS activity in parallel with an inhibition of the first phase of glucose-stimulated insulin release in perifused rats islets, whereas 5.0 mmol/l of L-NMMA markedly suppressed cNOS activity concomitant with a great potentiation of the insulin secretory response. The data strongly suggest, but do not definitely prove, that glucose indeed has the ability to stimulate both cNOS and iNOS in the islets and that NO might serve as a negative feedback inhibitor of glucose-stimulated insulin release. The results also suggest that hyperglycemia-evoked islet NOS activity might be one of multiple factors involved in the impairment of glucose-stimulated insulin release in type II diabetes mellitus.     

Selective inhibition of inducible nitric oxide synthase exacerbates erosive joint disease

NO is an essential cytotoxic agent in host defense, yet can be autotoxic if overproduced, as evidenced in inflammatory lesions and tissue destruction in experimental arthritis models. Treatment of streptococcal cell wal1-induced arthritis in rats with N:(G)-monomethyl-L-arginine (L-NMMA), a competitive nonspecific inhibitor of both constitutive and inducible isoforms of NO synthase (NOS), prevents intraarticular accumulation of leukocytes, joint swelling, and bone erosion. Because increased inducible NOS (iNOS) expression and NO generation are associated with pathogenesis of chronic inflammation, we investigated whether a selective inhibitor of iNOS, N:-iminoethyl-L-lysine (L-NIL), would have more directed anti-arthritic properties. Whereas both L-NMMA and L-NIL inhibited nitrite production by streptococcal cell wall-stimulated rat mononuclear cells in vitro and systemic treatment of arthritic rats with L-NMMA ablated synovitis, surprisingly L-NIL did not mediate resolution of inflammatory joint lesions. On the contrary, daily administration of L-NIL failed to reduce the acute response and exacerbated the chronic inflammatory response, as reflected by profound tissue destruction and loss of bone and cartilage. Although the number of iNOS-positive cells within the synovium decreased after treatment with L-NIL, immunohistochemical analyses revealed a distinct pattern of endothelial and neuronal NOS expression in the arthritic synovium that was unaffected by the isoform-specific L-NIL treatment. These studies uncover a contribution of the constitutive isoforms of NOS to the evolution of acute and chronic inflammation pathology which may be important in the design of therapeutic agents.     

Effects of chronic inhibition of inducible nitric oxide synthase in hyperthyroid rats

We hypothesized that nitric oxide generated by inducible nitric oxide synthase (iNOS) may contribute to the homeostatic role of this agent in hyperthyroidism and may, therefore, participate in long-term control of blood pressure (BP). The effects of chronic iNOS inhibition by oral aminoguanidine (AG) administration on BP and morphological and renal variables in hyperthyroid rats were analyzed. The following four groups (n = 8 each) of male Wistar rats were used: control group and groups treated with AG (50 mg.kg(-1).day(-1), via drinking water), thyroxine (T4, 50 microg.rat(-1).day(-1)), or AG + T4. All treatments were maintained for 3 wk. Tail systolic BP and heart rate (HR) were recorded weekly. Finally, we measured BP (mmHg) and HR in conscious rats and morphological, plasma, and renal variables. T(4) administration produced a small BP (125 +/- 2, P < 0.05) increase vs. control (115 +/- 2) rats. AG administration to normal rats did not modify BP (109 +/- 3) or any other hemodynamic variable. However, coadministration of T4 and AG produced a marked increase in BP (140 +/- 3, P < 0.01 vs. T4). Pulse pressure and HR were increased in both T4- and T4 + AG -treated groups without differences between them. Plasma NOx (micromol/l) were increased in the T4 group (10.02 +/- 0.15, P < 0.05 vs. controls 6.1 +/- 0.10), and AG reduced this variable in T4-treated rats (6.81 +/- 0.14, P < 0.05 vs. T4) but not in normal rats (5.78 +/- 0.20). Renal and ventricular hypertrophy and proteinuria of hyperthyroid rats were unaffected by AG treatment. In conclusion, the results of the present paper indicate that iNOS activity may counterbalance the prohypertensive effects of T4.     

Increased expression of inducible nitric oxide synthase and cyclooxygenase-2 in pancreatic cancer

Despite recognition of the devastating malignant potential of the pancreatic ductal cancer, the exact pathophysiological events contributing to tumor growth remain to be elucidated. Expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were found to be frequently elevated in several types of human cancer and have also been directly linked to carcinogenesis. The purpose of this study was to determine the expression of COX-1, COX-2 and iNOS in human pancreatic cancer and matched normal adjacent tissue by the Western blot assay. Marked COX-2 expression was observed in cancer tissue compared with the normal surrounding tissue. The iNOS protein was markedly expressed only in pancreatic cancer while the expression of COX-1 was similar in both normal and cancerous tissue. Our findings indicate that COX-2 up-regulation and the expression of iNOS in pancreatic cancer, not seen in normal tissue, may play a role in the pathogenesis of human pancreatic adenocarcinomas. These observations suggest that COX-2 and iNOS may be a target for prevention or treatment of pancreatic carcinomas.     

Ghrelin activates neuronal constitutive nitric oxide synthase in pancreatic islet cells while inhibiting insulin release and stimulating glucagon release

In view of our previous data, showing that ghrelin and nitric oxide (NO) display apparently parallel effects on insulin secretion (inhibitory) and glucagon secretion (stimulatory), we have now investigated the effect of ghrelin on islet hormone secretion in relation to its effect on NO synthase (NOS) isoenzymes in isolated rat pancreatic islets. Dose-response studies revealed that ghrelin at concentrations of 0.01-1 micromol l-1 inhibited insulin secretion stimulated by 8.3 mmol l-1 glucose, while ghrelin at concentrations lower than the physiological range (0.01 pmol l-1 to 1 nmol l-1) were without effect. In contrast, glucagon secretion was stimulated by 1.0 nmol l-1 to 1 micromol l-1 ghrelin. These effects of ghrelin on insulin and glucagon secretion were accompanied by increased NO production through activation of neuronal constitutive NOS (ncNOS). Ghrelin had no appreciable effect on the activity of inducible NOS (iNOS) in the islets. Addition of an NO scavenger (cPTIO) or the NOS inhibitor L-NAME to the incubation medium prevented the effects of ghrelin on hormone secretion from isolated islets. The present results confirm our previous data showing that ghrelin inhibits insulin and stimulates glucagon secretion from pancreatic islets of the mouse and we now show similar effects in rat islets. The effects of ghrelin were accompanied by an increased rate of NO production. Conceivably, ncNOS activation partly accounts for to the inhibitory effect of ghrelin on insulin secretion and the stimulatory effect of ghrelin on glucagon secretion.     

What sense lies in antisense inhibition of inducible nitric oxide synthase expression?

The impact of nitric oxide (NO) synthesized after activation by proinflammatory cytokines and/or bacterial products by an inducible NO synthase (iNOS) is still contradictory. Expression of iNOS in inflammatory reactions is often found predominantly in cells of epithelial origin, and in these cases NO may serve as a protective agent limiting pathogen spreading, downregulating local inflammatory reactions by inducing production of Th2-like responses in a classical feedback circle, or limiting tissue damage during stress conditions. However, an abundant amount of data on chronic human disorders with predominant proinflammatory Th1-like reactions points to a destructive role of iNOS activity calling for a specific inhibition. Various methods to inhibit iNOS have been established to elucidate a protective versus a destructive role of NO during various stresses. In this review, we focus on antisense (AS)-mediated gene knock-down as a relatively new method to inhibit NO production and summarize the techniques applied and their successes. At least in theory, it provides a specific, rapid, and potentially high-throughput method for inhibiting gene expression and function. We here discuss the opportunities of iNOS-directed AS-ODN, and extensively deal with limitations and experimental problems.     

Non-quantal acetylcholine release is increased after nitric oxide synthase inhibition

After anticholinesterase treatment, depolarization of the postsynaptic muscle membrane by about 5 mV develops due to non-quantally released acetylcholine from the motor nerve terminal and can be revealed as hyperpolarization by the addition of curare (H-effect). The H-effect increases significantly to 8.7 mV after inhibition of NO-synthase by L-nitroarginine methylester (L-NAME) whilst no changes in the amplitude and frequency of quantal miniature endplate potentials are observed.     

Mechanism of inducible nitric oxide synthase exclusion from mycobacterial phagosomes

Mycobacterium tuberculosis is sensitive to nitric oxide generated by inducible nitric oxide synthase (iNOS). Consequently, to ensure its survival in macrophages, M. tuberculosis inhibits iNOS recruitment to its phagosome by an unknown mechanism. Here we report the mechanism underlying this process, whereby mycobacteria affect the scaffolding protein EBP50, which normally binds to iNOS and links it to the actin cytoskeleton. Phagosomes harboring live mycobacteria showed reduced capacity to retain EBP50, consistent with lower iNOS recruitment. EBP50 was found on purified phagosomes, and its expression increased upon macrophage activation, paralleling expression changes seen with iNOS. Overexpression of EBP50 increased while EBP50 knockdown decreased iNOS recruitment to phagosomes. Knockdown of EBP50 enhanced mycobacterial survival in activated macrophages. We tested another actin organizer, coronin-1, implicated in mycobacterium-macrophage interaction for contribution to iNOS exclusion. A knockdown of coronin-1 resulted in increased iNOS recruitment to model latex bead phagosomes but did not increase iNOS recruitment to phagosomes with live mycobacteria and did not affect mycobacterial survival. Our findings are consistent with a model for the block in iNOS association with mycobacterial phagosomes as a mechanism dependent primarily on reduced EBP50 recruitment.     

Expression of islet inducible nitric oxide synthase and inhibition of glucose-stimulated insulin release after long-term lipid infusion in the rat is counteracted by PACAP27

Chronic exposure of pancreatic islets to elevated plasma lipids (lipotoxicity) can lead to beta-cell dysfunction, with overtime becoming irreversible. We examined, by confocal microscopy and biochemistry, whether the expression of islet inducible nitric oxide synthase (iNOS) and the concomitant inhibition of glucose-stimulated insulin release seen after lipid infusion in rats was modulated by the islet neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP)27. Lipid infusion for 8 days induced a strong expression of islet iNOS, which was mainly confined to beta-cells and was still evident after incubating islets at 8.3 mmol/l glucose. This was accompanied by a high iNOS-derived NO generation, a decreased insulin release, and increased cyclic GMP accumulation. No iNOS expression was found in control islets. Addition of PACAP27 to incubated islets from lipid-infused rats resulted in loss of iNOS protein expression, increased cyclic AMP, decreased cyclic GMP, and suppression of the activities of neuronal constitutive (nc)NOS and iNOS and increased glucose-stimulated insulin response. These effects were reversed by the PKA inhibitor H-89. The suppression of islet iNOS expression induced by PACAP27 was not affected by the proteasome inhibitor MG-132, which by itself induced the loss of iNOS protein, making a direct proteasomal involvement less likely. Our results suggest that PACAP27 through its cyclic AMP- and PKA-stimulating capacity strongly suppresses not only ncNOS but, importantly, also the lipid-induced stimulation of iNOS expression, possibly by a nonproteasomal mechanism. Thus PACAP27 restores the impairment of glucose-stimulated insulin release and additionally might induce cytoprotection against deleterious actions of iNOS-derived NO in beta-cells.     

Acute pancreatitis,expression of inducible nitric oxide synthase and defective insulin secretion

A high level of nitric oxide (NO) produced by inducible NO synthase (iNOS) is involved in pancreatic beta-cell dysfunction and apoptosis. In the present study, we examined whether iNOS is also expressed in beta cells after induction of acute pancreatitis (AP) in the rat. Pancreatic islets taken from AP animals and incubated for 60 min in the presence of 20.0 mmol/l glucose showed a decreased insulin secretory response to glucose. The basal insulin release at 1.0 mmol/l glucose was also moderately reduced. Experiments on the dynamics of insulin secretion from perfused pancreas revealed an impairment of both first and second phase of glucose-stimulated insulin release after the induction of AP. Confocal microscopy demonstrated that most of the beta cells in pancreas of rat with AP expressed strong immunoreactivity for iNOS. This was further confirmed by biochemical and Western blot analysis that showed a marked increase in iNOS protein expression and enzyme activity concomitant with a modest reduction in the cNOS protein and activity. Although the mechanisms underlying the defective insulin secretory response of beta cells seen during the early stage of AP are complex, the present finding suggests that the expression of iNOS and a marked iNOS-derived NO production in the beta cells may play at least a contributory role in the impairment of beta-cell function.This study was supported by the Swedish Medical Research Council (14X-4286), the Swedish Diabetes Association, and the Crafoord, Albert Påhlsson and Magnus Bergvall Foundations     

Targeted disruption of inducible nitric oxide synthase protects against obesity-linked insulin resistance in muscle

Inducible nitric oxide synthase (iNOS) is induced by inflammatory cytokines in skeletal muscle and fat. It has been proposed that chronic iNOS induction may cause muscle insulin resistance. Here we show that iNOS expression is increased in muscle and fat of genetic and dietary models of obesity. Moreover, mice in which the gene encoding iNOS was disrupted (Nos2-/- mice) are protected from high-fat-induced insulin resistance. Whereas both wild-type and Nos2-/- mice developed obesity on the high-fat diet, obese Nos2-/- mice exhibited improved glucose tolerance, normal insulin sensitivity in vivo and normal insulin-stimulated glucose uptake in muscles. iNOS induction in obese wild-type mice was associated with impairments in phosphatidylinositol 3-kinase and Akt activation by insulin in muscle. These defects were fully prevented in obese Nos2-/- mice. These findings provide genetic evidence that iNOS is involved in the development of muscle insulin resistance in diet-induced obesity.     

Pharmacological inhibition of inducible nitric oxide synthase attenuates the development of seizures in mice

The present study has been designed to pharmacologically expound the significance of inducible nitric oxide synthase in the pathophysiological progression of seizures using mouse models of chemically induced kindled epilepsy and status epilepticus induced spontaneous recurrent seizures. Pentylenetetrazole (40 mg kg⁻¹) (PTZ) administration every second day for a period of 15 days was used to elicit kindled seizure activity in mice. Severity of kindled seizures was assessed in terms of a composite kindled seizure severity score (KSSS). Pilocarpine (100 mg kg⁻¹) was injected every 20 min until the onset of status epilepticus. A spontaneous recurrent seizure severity score (SRSSS) was recorded as a measure of quantitative assessment of the progressive development of spontaneous recurrent seizures induced after pilocarpine status epilepticus. Sub-acute PTZ administration induced the development of severe form of kindled seizures in mice. Further, pharmacological status epilepticus elicited a progressive evolution of spontaneous recurrent seizures in the animals. However, treatment of aminoguanidine, a relatively selective inhibitor of inducible nitric oxide synthase, markedly and dose dependently suppressed the development of both PTZ induced kindled seizures as well as pilocarpine induced spontaneous recurrent seizures. Therefore inducible nitric oxide synthase may be implicated in the development of seizures.     

Antihypertensive effects of inducible nitric oxide synthase inhibition in experimental pre‐eclampsia

Upregulation of inducible nitric oxide synthase (iNOS) has been reported in both experimental and clinical hypertension. However, although pro‐inflammatory cytokines that up‐regulate iNOS contribute to pre‐eclampsia, no previous study has tested the hypothesis that a selective iNOS inhibitor (1400 W) could exert antihypertensive effects associated with decreased iNOS expression and nitrosative stress in pre‐eclampsia. This study examined the effects of 1400 W in the reduced uteroplacental perfusion pressure (RUPP) placental ischaemia animal model and in normal pregnant rats. Sham‐operated and RUPP rats were treated with daily vehicle or 1 mg/kg/day N‐[3‐(Aminomethyl) benzyl] acetamidine (1400 W) subcutaneously for 5 days. Plasma 8‐isoprostane levels, aortic reactive oxygen species (ROS) levels and nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent ROS production were evaluated by ELISA, dihydroethidium fluorescence microscopy and lucigenin chemiluminescence respectively. Inducible nitric oxide synthase expression was assessed by western blotting analysis and aortic nitrotyrosine was evaluated by immunohistochemistry. Mean arterial blood pressure increased by ~30 mmHg in RUPP rats, and 1400 W attenuated this increase by ~50% (P < 0.05). While RUPP increased plasma 8‐isoprostane levels, aortic ROS levels, and NADPH‐dependent ROS production (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). Moreover, while RUPP increased iNOS expression and aortic nitrotyrosine levels (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). These results clearly implicate iNOS in the hypertension associated with RUPP. Our findings may suggest that iNOS inhibitors could be clinically useful in the therapy of pre‐eclampsia, especially in particular groups of patients genetically more prone to express higher levels of iNOS. This issue deserves further confirmation.     

Glucose-induced release of nitric oxide from mouse pancreatic islets as detected with nitric oxide-selective glass microelectrodes

Nitric oxide (NO) is believed to play an important role in pancreatic islet physiology and pathophysiology. Research in this area has been hampered, however, by the use of indirect methods to measure islet NO. To investigate the role of NO in islet function, we positioned NO-sensitive, recessed-tip microelectrodes in close proximity to individual islets and monitored oxidation current to detect subnanomolar NO in the bath. NO release from islets consisted of a series of rapid bursts lasting several seconds and/or slow oscillations with a period of approximately 100-300 s. Average baseline NO near the islets in 2.8 mM glucose was 524+/-59 nM (n=12). Raising glucose from 2.8 to 11.1 mM augmented NO release by 429+/-133 nM (n=12, P<0.05), an effect blocked by the NO synthase inhibitor L-NAME (n=3). We also observed that glucose-stimulated increases in NO release were contemporaneous with changes in NAD(P)H and O2 but occurred well before increases in calcium associated with glucose-stimulated insulin secretion. In summary, we demonstrate that NO release from islets is oscillatory and rapidly augmented by glucose, suggesting that NO release occurs early following an increase in glucose metabolism and may contribute to the stimulated insulin secretion triggered by suprathreshold glucose.     

Efficient formation of nitric oxide from selective oxidation of N-aryl N'-hydroxyguanidines by inducible nitric oxide synthase

Inducible nitric oxide synthase (NOS II) efficiently catalyzes the oxidation of N-(4-chlorophenyl)N'-hydroxyguanidine 1 by NADPH and O2, with concomitant formation of the corresponding urea and NO. The characteristics of this reaction are very similar to those of the NOS-dependent oxidation of endogenous Nomega-hydroxy-L-arginine (NOHA), i.e., (i) the formation of products resulting from an oxidation of the substrate C=N(OH) bond, the corresponding urea and NO, in a 1:1 molar ratio, (ii) the absolute requirement of the tetrahydrobiopterin (BH4) cofactor for NO formation, and (iii) the strong inhibitory effects of L-arginine (L-arg) and classical inhibitors of NOSs. N-Hydroxyguanidine 1 is not as good a substrate for NOS II as is NOHA (Km = 500 microM versus 15 microM for NOHA). However, it leads to relatively high rates of NO formation which are only 4-fold lower than those obtained with NOHA (Vm = 390 +/- 50 nmol NO min-1 mg protein-1, corresponding roughly to 100 turnovers min-1). Preliminary results indicate that some other N-aryl N'-hydroxyguanidines exhibit a similar behavior. These results show for the first time that simple exogenous compounds may act as NO donors after oxidative activation by NOSs. They also suggest a possible implication of NOSs in the oxidative metabolism of certain classes of xenobiotics.     

Antifibrotic role of inducible nitric oxide synthase.

Long-term treatment in rats with l-NAME, an isoform-non-specific inhibitor of nitric oxide synthase (NOS), leads to fibrosis of the heart and kidney, suggesting that nitric oxide (NO) may play a role in preventing tissue fibrosis. In this process, a likely target of NO is the quenching of reactive oxygen species (ROS) through peroxynitrite formation, and one possible source for this NO is inducible NOS (iNOS). Using Peyronie's disease (PD) tissue from both human specimens and from a rat model of PD as the source of fibrotic tissue, we investigated if NO derived from iNOS could act as such an antifibrogenic defense mechanism by determining whether: (a) tunical ROS and iNOS are increased in PD; and (b) the long-term inhibition of iNOS activity decreases the NO/ROS balance in the tunica albuginea thereby promoting collagen deposition. It was determined that in the human PD plaque, iNOS mRNA and protein, ROS, collagen, and the peroxynitrite marker, nitrotyrosine, were all increased in comparison to the normal tunica. In the rat model of PD, the fibrotic plaque also showed significant increases in iNOS mRNA and protein, nitrotyrosine, ROS as measured by heme oxygenase-1, and collagen when compared with the normal control tunica. When a selective inhibitor of iNOS, L-NIL, was given to rats with the PD-like plaque, this resulted in a decrease in nitrotyrosine levels but intensified ROS levels and collagen deposition. These data demonstrate that: (a) iNOS induction occurs in both the human and rat PD fibrotic plaque; and (b) that the NO derived from iNOS appears to counteract ROS formation and collagen deposition. Because the inhibition of iNOS activity leads to a decrease in the NO/ROS ratio, thereby favoring the development of fibrosis, it is proposed that iNOS induction in this tissue may be a protective mechanism against fibrosis and abnormal wound healing.