cNOS-derived Nitric Oxide Research Articles

Dual Role of Nitric Oxide in Pancreatic β-Cells

An involvement of inducible nitric oxide (NO) synthase (NOS) in pancreatic β-cell degeneration during the process of type 1 diabetes has been well discussed. Recently, there is growing evidence for pivotal roles of constitutive NOS (cNOS) in β-cells; the presence of NOS1 and NOS3 in pancreatic β-cells and the effects of low-concentration NO, which is assumed to be derived from cNOS, on β-cell functions have been reported. However, the roles of cNOS-derived NO in β-cells are still under debate. One of the reasons seems to be that NO has multiple biological activities, which are dependent on its concentration. In β-cells, NO has been shown to exert positive and negative regulation of insulin secretion and anti- and pro-apoptotic activities, which is likely to be dependent on concentrations. In this review article, we will describe the current understanding of the roles of NO in pancreatic β-cells, especially focusing on cNOS-derived NO and its differential roles depending on concentrations.

Eicosapentaenoic acid-induced endothelium-dependent and -independent relaxation of sheep pulmonary artery

It is known that long chain polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), have beneficial effects on cardiovascular function including pulmonary hypertension. The purpose of the present study was to examine the mechanisms involved in EPA-induced relaxation of sheep isolated pulmonary artery by measuring isometric tension. Nitric oxide (NO) derived from constitutive nitric oxide synthase (cNOS) was measured by Greiss method in the presence of the inducible nitric oxide synthase (iNOS) selective inhibitor N-[[3-(aminomethyl) phenyl]methyl]-ethanimidamide, dihydrochloride (1400 W). EPA (10(-)(7)-10(-)(4)M) caused concentration-dependent relaxation of sheep pulmonary artery with a pD(2) of 5.56+/-0.09 and E(max) of 87.40+/-3.10% (n=9). N(G)-nitro-L-arginine methyl ester (L-NAME) 100 microM significantly attenuated (E(max) 41.95+/-6.70%; n=8) EPA-induced relaxation of endothelium intact arterial rings. Similarly, endothelium denudation markedly inhibited (E(max) 17.60+/-1.21%; n=4) EPA-induced relaxation. EPA (30 microM) significantly increased the cNOS-derived NO release (10.17+/-0.96; n=8 versus control 7.43+/-0.78 pmol/mg tissue wet wt./h; n=7) in endothelium intact vessels. However, EPA-stimulated NO release was markedly blunted by either 100 microM L-NAME (7.07+/-0.54 pmol/mg tissue wet wt./h; n=8) or endothelium removal (6.97+/-0.87 pmol/mg tissue wet wt./h; n=17). In endothelium-denuded K(+) (80 mM)-depolarized arterial rings, EPA (30 microM) significantly inhibited CaCl(2)-induced contractions (E(max) 42.77+/-5.90% versus control 94.78+/-9.82%; n=5). The fatty acid also inhibited nifedipine (1 microM)-insensitive 5-HT-induced contractions in this vessel (E(max) 70.57+/-4.88% versus control 161.50+/-17.46%; n=5). In conclusion, EPA relaxes sheep pulmonary artery primarily through endothelium-dependent NO release, and the residual endothelium-independent relaxation may result from inhibition of Ca(2+)-influx through L-type calcium channels, as well as 5-HT-stimulated intracellular Ca(2+) release.

Nitric oxide synthase isoenzyme activities in a premature piglet model of necrotizing enterocolitis: effects of nitrergic manipulation.

Nitric oxide (NO) plays a major role in gut mucosal protection and motility. Having demonstrated the protective effects of intravenous L-arginine (L-arg) and the NO donor, sodium nitroprusside (SNP), in an in-vivo premature piglet intraluminal model of necrotizing enterocolitis (NEC) that incorporates both mucosal damage and intestinal dysmotility, we measured the effects on NO synthase (NOS) isoenzyme activities during i.v. manipulation of the nitrergic system in the NEC-injured gut. In newborn premature Yorkshire piglets, NEC was induced in four groups by intraluminal injection of acidified casein solution in closed test loops of bowel separated by normal saline-injected control loops. Group 1 (n = 4) underwent no further treatment. Group 2 (n = 4) received concomitant continuous i.v. L-arg, a NO substrate. Group 3 (n = 6) received concomitant continuous i.v. SNP, a NO donor. Group 4 (n = 5) received concomitant continuous i.v. N-omega-nitro-L-arginine-methyl-ester (L-NAME), a non-selective NO inhibitor. Control and test gut specimens were harvested after 3 h. NO synthase activity in frozen gut segments was assessed using the (14)C-L-arg to (14)C-L-citrulline conversion assay. Total NOS (TNOS), constitutive NOS (cNOS), and inducible NOS (iNOS) activities were compared. The mean and standard error were calculated for each specimen. Group means were used to compare test and control gut enzyme activities in the different treatment groups. One-way analysis of variance and the Bonferroni post test were used to compare differences among groups. A P value of less than 0.05 was considered significant. In the L-NAME group, cNOS activity was lower than in the untreated NEC group. The SNP group had higher iNOS and TNOS activities than the L-arg group; cNOS was also higher in test and control loops in the SNP versus both L-arg and L-NAME groups. However, in L-arg control loops, cNOS activity was greater than in the L-NAME group. SNP and L-arg treatment of NEC did not significantly modify NOS isoenzyme activities. Thus, in this premature piglet 3-h model of NEC, i.v. L-NAME significantly decreases cNOS activity and correlates with our previously published histopathologic findings confirming the protective role of cNOS-derived NO in NEC-injured gut mucosa. In order to further elucidate the mechanisms involved in the mucosal protection afforded by i.v. L-arg and SNP in this NEC model, studies of a longer duration have been undertaken.

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.

Increased arginase activity underlies allergen-induced deficiency of cNOS-derived nitric oxide and airway hyperresponsiveness.

1. A deficiency of constitutive nitric oxide synthase (cNOS)-derived nitric oxide (NO), due to reduced availability of L-arginine, importantly contributes to allergen-induced airway hyperresponsiveness (AHR) after the early asthmatic reaction (EAR). Since cNOS and arginase use L-arginine as a common substrate, we hypothesized that increased arginase activity is involved in the allergen-induced NO deficiency and AHR. 2. Using a guinea-pig model of allergic asthma, we addressed this hypothesis by examining the effects of the specific arginase inhibitor N(omega)-hydroxy-nor-L-arginine (nor-NOHA) on the responsiveness to methacholine of isolated perfused tracheae from unchallenged control animals and from animals 6 h after ovalbumin challenge. Arginase activity in these preparations was investigated by measuring the conversion of L-[14C]arginine to [14C]urea. 3. Airways from allergen-challenged animals showed a 2 fold (P<0.001) increase in responsiveness to intraluminal (IL) administration of methacholine compared to controls. A similar hyperresponsiveness (1.8 fold, P<0.01) was observed in control airways incubated with the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 0.1 mM, IL), while L-NAME had no further effect on the airways from challenged animals. 4. Remarkably, 5 microM nor-NOHA (IL) normalized the hyperresponsiveness of challenged airways to basal control (P<0.001), and this effect was fully reversed again by 0.1 mM L-NAME (P<0.05). Moreover, arginase activity in homogenates of the hyperresponsive airways was 3.5 fold (P<0.001) enhanced compared to controls. 5. The results indicate that enhanced arginase activity contributes to allergen-induced deficiency of cNOS-derived NO and AHR after the EAR, presumably by competition with cNOS for the common substrate, L-arginine. This is the first demonstration that arginase is involved in the pathophysiology of asthma.

Morphine 6 glucuronide stimulates nitric oxide release in mussel neural tissues: evidence for a morphine 6 glucuronide opiate receptor subtype.

We have previously demonstrated that Mytilus edulis pedal ganglia contain opiate alkaloids, i.e., morphine and morphine 6 glucuronide (M6G), as well as mu opiate receptor subtype fragments exhibiting high sequence similarity to those found in mammals. Now we demonstrate that M6G stimulates pedal ganglia constitutive nitric oxide (NO) synthase (cNOS)-derived NO release at identical concentrations and to similar peak levels as morphine. However, the classic opiate antagonist, naloxone, only blocked the ability of morphine to stimulate cNOS-derived NO release and not that of M6G. CTOP, a mu-specific antagonist, blocked the ability of M6G to induce cNOS-derived NO release as well as that of morphine, suggesting that a novel mu opiate receptor was present and selective toward M6G. In examining a receptor displacement analysis, both opiate alkaloids displaced [3H]-dihydromorphine binding to the mu opiate receptor subtype. However, morphine exhibited a twofold higher affinity, again suggesting that a novel mu opiate receptor may be present.

Peroxynitrite generated from constitutive nitric oxide synthase mediates the early biochemical injury in short-term cultured hepatocytes

Early loss of P450 in rat hepatocyte cultures appears directly related to nitric oxide (NO) overproduction. This study provides experimental evidence for the induction – shortly after isolation through the classical procedure – of strong oxidative stress that involves both oxygen-derived and NO-derived species. NO formation at this stage is due to the early activation of liver constitutive NO synthase (cNOS). Immunodetection of nitrated proteins provides direct evidence of endogenous peroxynitrite (PN) formation upon hepatocyte isolation. On the basis of the combined use of dihydrorhodamine 123 and NOS inhibitors, the analysis of the amount, time course and nature of the species involved supports the view that PN generated from cNOS-derived NO, while not affecting cell viability and hepatocyte monolayer development, is the main species likely responsible for the early biochemical injury commonly observed in hepatocyte cultures.

ＮＯと消化管の病態生理

Nitric oxide (NO) has been shown to elicit both favorable and unfavourable effects on gastrointestinal (GI) system. NO is generated from L-arginine by two different types of NO synthase (NOS) . One type of NOS, constitutive NOS (cNOS), is con-stitutively expressed by endothelial cells (eNOS) and by neural cells (nNOS) . cNOS-derived NO causes a variety of physiogical effects by stimulating guanylate cyclase-cylic GMP system, effects genereally beneficial to GI system. For example, nNOS-derived NO modulates GI motility. eNOS-derived NO increases mucosal blood flow, and stimulates production of mucus and HCO3-, all of which enhance mucosal defense and repair system. Based on these background, several attemps have been made to use NO as a therapeutic drugs. For example, tetraprenylacetone (teprenone) has been shown to enhance gastric mucosal restitution by upregulating cNOS activity. NO-releasing NSAIDs have been developed to mitigate NSAIDs induced mucosal injury by protection afforded by NO. In contrast, other type of NOS, inducible NOS (iNOS), is transiently expressed in macrophages in response to stimuli, such as bacterial invasion. iNOS generates huge amount of NO, that pre-vents invasion of bacteria, but causes serious injury in host tissues. For example, Helicobacter pylori upregulates iNOS expression in gastric mucosa, and enhances release of NO, thereby accelerating epithelial cell apoptosis. In chronic inflammatory bowel diseases, inceased expression of mucosal iNOS is closely associated with developement of mucosal injury. However, it should be noted that in certain circum-stances iNOS affords mucosal protection against noxious stimuli, and promotes mucosal repair after injury.

Role of nitric oxide in the development and partial reversal of allergen-induced airway hyperreactivity in conscious, unrestrained guinea-pigs.

1. Using a conscious, unrestrained guinea-pig model of allergic asthma, we investigated the role of endogenous nitric oxide (NO) in the regulation of airway (hyper)reactivity to histamine before and after the allergen-induced early and late asthmatic reactions, by examining the effect of inhalation of the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 12 mM, 15 min) on the histamine-induced airway obstruction of ovalbumin-sensitized guinea-pigs before, and at 5.5 h and 23.5 h after allergen challenge. 2. Before allergen challenge, inhaled L-NAME caused a significant 2.02+/-0.25 fold increase (P<0.01) in airway reactivity to histamine; this effect was reversed within 2.5 to 6 h after administration. 3. After the allergen-induced early asthmatic reaction at 5 h after ovalbumin provocation, a significant 3.73+/-0.67 fold increase (P<0.01) of the airway reactivity to histamine was observed; subsequent inhalation of L-NAME at 5.5 h had no effect on the airway hyperreactivity, reassessed at 6 h. 4. After the late asthmatic reaction, at 23 h after ovalbumin provocation, a reduced, but still significant airway hyperreactivity to histamine (2.18+/-0.40 fold; P<0.05) was observed. Subsequent inhalation of L-NAME now significantly potentiated the partially reduced airway hyperreactivity 1.57+/-0.19 fold (P<0.05) to the level observed after the early asthmatic reaction. 5. When administered 30 min before allergen exposure, L-NAME significantly enhanced the allergen-induced early asthmatic reaction. However, when administered at 5.5 h after allergen provocation, L-NAME did not affect the subsequent late asthmatic reaction. 6. These results indicate that endogenous NO is involved the regulation of histamine- and allergen-induced bronchoconstriction and that a deficiency of cNOS-derived NO contributes to the allergen-induced airway hyperreactivity to histamine after the early asthmatic reaction, while a recovery of NO deficiency may account for the partial reversal of the allergen-induced airway hyperreactivity after the late asthmatic reaction.