Time course of nitric oxide production after endotoxin challenge in mice

Nitric oxide (NO) regulates numerous processes during endotoxemia and inflammation. However, the sequential changes in whole body (Wb) nitric oxide (NO) production during endotoxemia in vivo remain to be clarified. Male Swiss mice were injected intraperitoneally with saline (control group) or lipopolysaccharide (LPS group). After 0, 2, 4, 6, 9, 12, and 24 h, animals received a primed constant infusion of L-[guanidino-(15)N(2)-(2)H(2)]arginine, L-[ureido-(15)N]citrulline, L-[5-(15)N]glutamine, and L-[ring-(2)H(5)]phenylalanine in the jugular vein. Arterial blood was collected for plasma arginine (Arg), citrulline (Cit), glutamine (Gln), and phenylalanine (Phe) concentrations and tracer-to-tracee ratios. NO production was calculated as plasma Arg-to-Cit flux, Wb de novo Arg synthesis as plasma Cit-to-Arg flux, and Wb protein breakdown as plasma Phe flux. LPS reduced plasma Arg and Cit and increased Gln and Phe concentrations. Two peaks of NO production were observed at 4 and 12 h after LPS. Although LPS did not affect total Arg production, de novo Arg production decreased after 12 h. The second peak of NO production coincided with increased Wb Cit, Gln, and Phe production. In conclusion, the curve of NO production in both early and late phases of endotoxemia is not related to plasma Arg kinetics. However, because Wb Cit, Gln, and Phe fluxes increased concomitantly with the second peak of NO production, NO production is probably related to the catabolic phase of endotoxemia.     

Reduced citrulline availability by OTC deficiency in mice is related to reduced nitric oxide production

The amino acid arginine is the sole precursor for nitric oxide (NO) synthesis. We recently demonstrated that an acute reduction of circulating arginine does not compromise basal or LPS-inducible NO production in mice. In the present study, we investigated the importance of citrulline availability in ornithine transcarbamoylase-deficient spf(ash) (OTCD) mice on NO production, using stable isotope techniques and C57BL6/J (wild-type) mice controls. Plasma amino acids and tracer-to-tracee ratios were measured by LC-MS. NO production was measured as the in vivo conversion of l-[guanidino-(15)N(2)]arginine to l-[guanidine-(15)N]citrulline; de novo arginine production was measured as conversion of l-[ureido-(13)C-5,5-(2)H(2)]citrulline to l-[guanidino-(13)C-5,5-(2)H(2)]arginine. Protein metabolism was measured using l-[ring-(2)H(5)]phenylalanine and l-[ring-(2)H(2)]tyrosine. OTC deficiency caused a reduction of systemic citrulline concentration and production to 30-50% (P < 0.001), reduced de novo arginine production (P < 0.05), reduced whole-body NO production to 50% (P < 0.005), and increased net protein breakdown by a factor of 2-4 (P < 0.001). NO production was twofold higher in female than in male OTCD mice in agreement with the X-linked location of the OTC gene. In response to LPS treatment (10 mg/kg ip), circulating arginine increased in all groups (P < 0.001), and NO production was no longer affected by the OTC deficiency due to increased net protein breakdown as a source for arginine. Our study shows that reduced citrulline availability is related to reduced basal NO production via reduced de novo arginine production. Under basal conditions this is probably cNOS-mediated NO production. When sufficient arginine is available after LPS stimulated net protein breakdown, NO production is unaffected by OTC deficiency.     

NO production by cNOS and iNOS reflects blood pressure changes in LPS-challenged mice

Increased nitric oxide (NO) production is the cause of hypotension and shock during sepsis. In the present experiments, we have measured the contribution of endothelial (e) and inducible (i) nitric oxide synthase (NOS) to systemic NO production in mice under baseline conditions and upon LPS treatment (100 microg/10 g ip LPS). NO synthesis was measured by the rate of conversion of l-[guanidino-15N2]arginine to l-[ureido-15N]citrulline, and the contribution of the specific NOS isoforms was evaluated by comparing NO production in eNOS-deficient [(-/-)] and iNOS(-/-) mice with that in wild-type (WT) mice. Under baseline conditions, NO production was similar in WT and iNOS(-/-) mice but lower in eNOS(-/-) mice [WT: 1.2 +/- 0.2; iNOS(-/-): 1.2 +/- 0.2; eNOS(-/-): 0.6 +/- 0.3 nmol. 10 g body wt-1. min-1]. In response to the challenge with LPS (5 h), systemic NO production increased in WT and eNOS(-/-) mice but fell in iNOS(-/-) mice [WT: 2.7 +/- 0.3; eNOS(-/-): 2.2 +/- 0.6; iNOS(-/-): 0.7 +/- 0.1 nmol. 10 g body wt-1. min-1]. After 5 h of LPS treatment, blood pressure had dropped 14 mmHg in WT but not in iNOS(-/-) mice. The present findings provide firm evidence that, upon treatment with bacterial LPS, the increase of NO production is solely dependent on iNOS, whereas that mediated by cNOS is reduced. Furthermore, the data show that the LPS-induced blood pressure response is dependent on iNOS.     

Role of endogenous nitric oxide in endotoxin-induced alteration of hypoxic pulmonary vasoconstriction in mice

Pulmonary vasoconstriction in response to alveolar hypoxia (HPV) is frequently impaired in patients with sepsis or acute respiratory distress syndrome or in animal models of endotoxemia. Pulmonary vasodilation due to overproduction of nitric oxide (NO) by NO synthase 2 (NOS2) may be responsible for this impaired HPV after administration of endotoxin (LPS). We investigated the effects of acute nonspecific (N(G)-nitro-L-arginine methyl ester, L-NAME) and NOS2-specific [L-N6-(1-iminoethyl)lysine, L-NIL] NOS inhibition and congenital deficiency of NOS2 on impaired HPV during endotoxemia. The pulmonary vasoconstrictor response and pulmonary vascular pressure-flow (P-Q) relationship during normoxia and hypoxia were studied in isolated, perfused, and ventilated lungs from LPS-pretreated and untreated wild-type and NOS2-deficient mice with and without L-NAME or L-NIL added to the perfusate. Compared with lungs from untreated mice, lungs from LPS-challenged wild-type mice constricted less in response to hypoxia (69 +/- 17 vs. 3 +/- 7%, respectively, P < 0.001). Perfusion with L-NAME or L-NIL restored this blunted HPV response only in part. In contrast, LPS administration did not impair the vasoconstrictor response to hypoxia in NOS2-deficient mice. Analysis of the pulmonary vascular P-Q relationship suggested that the HPV response may consist of different components that are specifically NOS isoform modulated in untreated and LPS-treated mice. These results demonstrate in a murine model of endotoxemia that NOS2-derived NO production is critical for LPS-mediated development of impaired HPV. Furthermore, impaired HPV during endotoxemia may be at least in part mediated by mechanisms other than simply pulmonary vasodilation by NOS2-derived NO overproduction.     

NOS3 is involved in the increased protein and arginine metabolic response in muscle during early endotoxemia in mice

Sepsis is a severe catabolic condition. The loss of skeletal muscle protein mass is characterized by enhanced release of the amino acids glutamine and arginine, which (in)directly affects interorgan arginine and the related nitric oxide (NO) synthesis. To establish whether changes in muscle amino acid and protein kinetics are regulated by NO synthesized by nitric oxide synthase-2 or -3 (NOS2 or NOS3), we studied C57BL6/J wild-type (WT), NOS2-deficient (NOS2-/-), and NOS3-deficient (NOS3-/-) mice under control (unstimulated) and lipopolysaccharide (LPS)-treated conditions. Muscle amino acid metabolism was studied across the hindquarter by infusing the stable isotopes L-[ring-2H5]phenylalanine, L-[ring-2H2]tyrosine, L-[guanidino-15N2]arginine, and L-[ureido-13C,2H2]citrulline. Muscle blood flow was measured using radioactive p-aminohippuric acid dilution. Under baseline conditions, muscle blood flow was halved in NOS2-/- mice (P < 0.1), with simultaneous reductions in muscle glutamine, glycine, alanine, arginine release and glutamic acid, citrulline, valine, and leucine uptake (P < 0.1). After LPS treatment, (net) muscle protein synthesis increased in WT and NOS2-/- mice [LPS vs. control: 13 +/- 3 vs. 8 +/- 1 (SE) nmol.10 g(-1).min(-1) (WT), 18 +/- 5 vs. 7 +/- 2 nmol.10 g(-1).min(-1) (NOS2-/-); P < 0.05 for LPS vs. control]. This response was absent in NOS3-/- mice (LPS vs. control: 11 +/- 4 vs. 10 +/- 2 nmol.10 g(-1).min(-1)). In agreement, the increase in muscle arginine turnover after LPS was also absent in NOS3-/- mice. In conclusion, disruption of the NOS2 gene compromises muscle glutamine release and muscle blood flow in control mice, but had only minor effects after LPS. NOS3 activity is crucial for the increase in muscle arginine and protein turnover during early endotoxemia.     

Despite minimal hemodynamic alterations endotoxemia modulates NOS and p38-MAPK phosphorylation via metalloendopeptidases

In the present study, we hypothesized that endotoxemia produces metalloendopeptidase (MEPD)-dependent generation of endothelin-1 (ET-1) and alters NOS expression correlating with p38-mitogen-activated protein kinase (MAPK) phosphorylation in thoracic aorta. Male Sprague-Dawley rats (350-400 g) were subjected to two groups randomly; sham-treated (N = 10) and lipopolysaccharide (LPS)-treated (N = 10) (E. coli LPS 2 mg/kg bolus + 2 mg/kg infusion for 30 min). The animals in each group were further subdivided into vehicle and MEPD inhibitor phosphoramidon (1 mg/kg bolus, PHOS)-treated groups. LPS produces a significant decrease in mean arterial pressure (MAP) at 2 h post endotoxemia that was blocked by PHOS. PHOS attenuated LPS-induced increase in tumor necrosis factor-alpha (TNF-alpha) concentration at 2- and 24 h post-LPS administration. LPS significantly elevated plasma concentrations of ET-1 at 2- and 24 h post endotoxemia. An upregulated preproET-1 expression following both LPS and MEPD inhibition was observed in thoracic aorta at 2 h post treatment. PHOS effectively blocked conversion of preproET-1 to ET-1 in thoracic aorta locally at 24 h post treatment in endotoxic rats. PHOS inhibited LPS-induced upregulation of inducible NOS (iNOS), downregulation of endothelial NOS (eNOS) and elevation of NO byproducts (NOx) in thoracic aorta. PHOS also blocked LPS-induced upregulated p38-MAPK phosphorylation in thoracic aorta at 24 h post endotoxemia. The data revealed that LPS induces MEPD-sensitive inflammatory response syndrome (SIRS) at 2- and 24 h post endotoxemia. We concluded that inhibition of MEPD not only decreases the levels of ET-1 but also simultaneously downregulates protein expression of iNOS and phosphorylated p38-MAPK while increasing eNOS in thoracic aorta during SIRS in endotoxemia. We suggest that MEPD-dependent ET-1 and NO mechanisms may be involved in endotoxemia-induced altered p38-MAPK phosphorylation.     

Renal cortical and medullary blood flow responses to L-NAME and ANG II in wild-type, nNOS null mutant, and eNOS null mutant mice

Experiments in wild-type (WT; C57BL/6J) mice, endothelial nitric oxide synthase null mutant [eNOS(-/-)] mice, and neuronal NOS null mutant [nNOS(-/-)] mice were performed to determine which NOS isoform regulates renal cortical and medullary blood flow under basal conditions and during the infusion of ANG II. Inhibition of NOS with N(omega)-nitro-l-arginine methyl ester (l-NAME; 50 mg/kg iv) in Inactin-anesthetized WT and nNOS(-/-) mice increased arterial blood pressure by 28-31 mmHg and significantly decreased blood flow in the renal cortex (18-24%) and the renal medulla (13-18%). In contrast, blood pressure and renal cortical and medullary blood flow were unaltered after l-NAME administration to eNOS(-/-) mice, indicating that NO derived from eNOS regulates baseline vascular resistance in mice. In subsequent experiments, intravenous ANG II (20 ng x kg(-1) x min(-1)) significantly decreased renal cortical blood flow (by 15-25%) in WT, eNOS(-/-), nNOS(-/-), and WT mice treated with l-NAME. The infusion of ANG II, however, led to a significant increase in medullary blood flow (12-15%) in WT and eNOS(-/-) mice. The increase in medullary blood flow following ANG II infusion was not observed in nNOS(-/-) mice, in WT or eNOS(-/-) mice pretreated with l-NAME, or in WT mice administered the nNOS inhibitor 5-(1-imino-3-butenyl)-l-ornithine (1 mg x kg(-1) x h(-1)). These data demonstrate that NO from eNOS regulates baseline blood flow in the mouse renal cortex and medulla, while NO produced by nNOS mediates an increase in medullary blood flow in response to ANG II.     

Muscarinic and beta-adrenergic regulation of heart rate, force of contraction and calcium current is preserved in mice lacking endothelial nitric oxide synthase

Nitric oxide (NO) is an ubiquitous signaling molecule produced from L-arginine by NO synthase (NOS). In the vasculature, NO mediates parasympathetic endothelium-dependent vasodilation. NO may also mediate the parasympathetic control of myocardial function. This is supported by the observations that NOS3, the endothelial constitutive NOS, is expressed in normal cardiac myocytes from rodents and human, and NOS and/or guanylyl cyclase inhibitors antagonize the effect of muscarinic agonists on heart rate, atrio-ventricular conduction, contractility and L-type calcium current. Here we examine the autonomic regulation of the heart in genetically engineered mice deficient in NOS3 (NOS3-KO). We show that the chronotropic and inotropic responses to both beta-adrenergic and muscarinic agonists were unaltered in isolated cardiac tissue preparations from NOS3-KO mice, although these mice have a defective parasympathetic regulation of vascular tone. Similarly, beta-adrenergic stimulation and muscarinic inhibition of the calcium current did not differ in cardiac myocytes from NOS3-KO mice and those from wild-type mice. RT-PCR did not demonstrate upregulation of other NOS isoforms. Similarly, Gi/Go proteins and muscarinic receptor density were unaltered. These data refute the idea that NOS3 is obligatory for the normal autonomic control of cardiac muscle function.     

Deficiency of mitogen-activated protein kinase phosphatase-1 results in iNOS-mediated hypotension in response to low-dose endotoxin

Mitogen-activated protein kinase phosphatase-1 (MKP-1) is essential in limiting the proinflammatory response to lipopolysaccharide (LPS). We hypothesized that Mkp-1(-/-) mice would respond to low-dose LPS with a fall in blood pressure due to augmented expression of inducible nitric oxide (NO) synthase (iNOS). To test this hypothesis, Mkp-1(-/-) mice and their wild-type littermates were treated with 10 microg/kg iv LPS, and mean arterial blood pressure (MAP) and exhaled NO production (exNO) were measured. Tissues were harvested for an assessment of iNOS protein levels. Wild-type mice had no change in MAP or exNO during the experimental period, whereas Mkp-1(-/-) mice had a fall (P < 0.005) in MAP [79 +/- 5% of baseline (BL)] and an increase (P < 0.01) in exNO (266 +/- 50% of BL) after 150 min. The tissue levels of iNOS were greater in Mkp-1(-/-) than in wild-type mice. In additional experiments, 60 min after LPS treatment, Mkp-1(-/-) and wild-type mice were given N(omega)-nitro-l-arginine methyl ester (l-NAME) or aminoguanidine, and MAP and exNO were monitored for 90 min. Treatment with l-NAME prevented the LPS-induced increase in exNO and decrease in MAP but resulted in decreased exNO and elevated MAP in wild-type mice. Aminoguanidine prevented the increase in exNO and the fall in MAP caused by LPS in Mkp-1(-/-) mice, without significantly affecting MAP or exNO in wild-type mice. These results demonstrate that a deficiency of MKP-1 results in an exaggerated hypotensive response to LPS mediated by augmented iNOS expression. We speculate that defects in the Mkp-1 gene may increase susceptibility for the development of septic shock.     

Nitric Oxide Release from the Liver Surface to the Intraabdominal Cavity during Acute Endotoxemia in Rats

Nitric oxide (NO) is produced by the liver during lipopolysaccharide (LPS)-induced endotoxemia. The aim of this study was to examine whether NO, which is produced in the liver, is released from the liver surface to the intraabdominal cavity during endotoxemia. NO was quantitatively determined by chemiluminescence and a newly developed gas purge technique was used to directly measure NO released from the liver surface and the intraabdominal cavity of rats before and after LPS (0.1 mg/kg, intraperitoneally) or saline administration. The expression of inducible NO synthase (iNOS) mRNA in the liver was detected by Northern blot analysis. NO levels from both the liver surface and in the intraabdominal cavity were elevated at 2 h after LPS injection and peaked at 10 h and both the time course of NO level were well correlated with each other. Both NO levels were below the detectable range before LPS and after saline administration. Inducible NOS mRNA in the liver exhibited a sharp increase to a maximum level at 4 h after LPS injection. The present study indicates that the hepatic NO, which might have been produced by iNOS in the liver, is released from the liver surface to the intraabdominal cavity during endotoxemia.     

Endogenous peroxynitrite mediates mitochondrial dysfunction in rat diaphragm during endotoxemia.

It has been shown that nitric oxide (NO), synthesized by the inducible NO synthase (iNOS) expressed in the diaphragm during endotoxemia, participates in the development of muscular contractile failure. The aim of the present study was to investigate whether this deleterious action of NO was related to its effects on cellular oxidative pathways. Rats were inoculated with E. coli lipopolysaccharide (LPS) or sterile saline solution (controls) and studied at 3 and 6 h after inoculation. iNOS protein and activity could be detected in the rat diaphragm as early as 3 h after LPS, with a sustained steady-state concentration of 0.5 microM NO in the muscle associated with increased detection of hydrogen peroxide (H(2)O(2)). In vitro, the same NO concentration produced a marked increase in H(2)O(2) production by isolated control diaphragm mitochondria, thus reflecting a higher intramitochondrial concentration of nondiffusible superoxide anion (O(2)(-.)). In a similar way, whole diaphragmatic muscle and diaphragm mitochondria from endotoxemic rats showed a progressive increase in H(2)O(2) production associated with uncoupling and decreased phosphorylating capacity. Simultaneous with the maximal impairment in respiration (6 h after LPS), nitration of mitochondrial proteins (a peroxynitrite footprint) was detected and diaphragmatic force was reduced. Functional mitochondrial abnormalities, nitration of mitochondrial proteins, and the decrease in force were significantly attenuated by administration of the NOS inhibitor L-NMMA. These results show that increased and sustained NO levels lead to a consecutive formation of O(2)(-.) that reacts with NO to form peroxynitrite, which in turn impairs mitochondrial function, which probably contributes to the impairment of muscle contractility. during endotoxemia.     

Fetal origins of adult vascular dysfunction in mice lacking endothelial nitric oxide synthase

Epidemiological studies have shown increased incidence of hypertension and coronary artery disease in growth-restricted fetuses during their adult life. A novel animal model was used to test the hypothesis regarding the role of an abnormal uterine environment in fetal programming of adult vascular dysfunction. Mice lacking a functional endothelial nitric oxide synthase (NOS3-/-KO, where KO is knockout) and wild-type (WT) mice (NOS3+/+WT) were crossbred to produce homozygous NOS3-/-KO, maternally derived heterozygous (NOS3+/-mat, mother with NOS3 deficiency), paternally derived heterozygous (NOS3+/-pat, normal mother), and NOS3+/+WT litters. Number of fetuses per litter was smaller in NOS3-/-KO and NOS3+/-mat compared with NOS3+/-pat and NOS3+/+WT mice. Adult female mice from these litters (7-8 wk old) were killed, and ring preparations of carotid and mesenteric arteries were mounted in a wire myograph to evaluate the passive and reactive vascular characteristics. Slope of the length-tension plot (a measure of vascular compliance) was increased, and optimal diameter (as calculated by Laplace equation) was decreased in NOS3-/-KO and NOS3+/-mat compared with NOS3+/-pat and NOS3+/+WT mice. Acetylcholine caused vasorelaxation in NOS3+/-pat and NOS3+/+WT and contraction in NOS3-/-KO and NOS3+/-mat mice. Responses to phenylephrine and Ca2+ were increased in NOS3-/-KO and NOS3+/-mat compared with NOS3+/-pat and NOS3+/+WT mice. Relaxation to isoproterenol was decreased in NOS3-/-KO and NOS3+/-mat vs. NOS3+/-pat and NOS3+/+WT mice. Abnormalities in the passive and reactive in vitro vascular properties seen in NOS+/-mat that developed in a NOS3-deficient maternal/uterine environment compared with the genetically identical NOS3+/-pat mice that developed in a normal environment are the first direct evidence in support of a role for uterine environment in determining vascular function in later life.     

The spinal cord expression of neuronal and inducible nitric oxide synthases and their contribution in the maintenance of neuropathic pain in mice

Background

Nitric oxide generated by neuronal (NOS1), inducible (NOS2) or endothelial (NOS3) nitric oxide synthases contributes to pain processing, but the exact role of NOS1 and NOS2 in the maintenance of chronic peripheral neuropathic pain as well as the possible compensatory changes in their expression in the spinal cord of wild type (WT) and NOS knockout (KO) mice at 21 days after total sciatic nerve ligation remains unknown.

Methodology/Principal Findings

The mechanical and thermal allodynia as well as thermal hyperalgesia induced by sciatic nerve injury was evaluated in WT, NOS1-KO and NOS2-KO mice from 1 to 21 days after surgery. The mRNA and protein levels of NOS1, NOS2 and NOS3 in the spinal cord of WT and KO mice, at 21 days after surgery, were also assessed. Sciatic nerve injury led to a neuropathic syndrome in WT mice, in contrast to the abolished mechanical allodynia and thermal hyperalgesia as well as the decreased or suppressed thermal allodynia observed in NOS1-KO and NOS2-KO animals, respectively. Sciatic nerve injury also increases the spinal cord expression of NOS1 and NOS2 isoforms, but not of NOS3, in WT and NOS1-KO mice respectively. Moreover, the presence of NOS2 is required to increase the spinal cord expression of NOS1 whereas an increased NOS1 expression might avoid the up-regulation of NOS2 in the spinal cord of nerve injured WT mice.

Conclusions/Significance

These data suggest that the increased spinal cord expression of NOS1, regulated by NOS2, might be responsible for the maintenance of chronic peripheral neuropathic pain in mice and propose these enzymes as interesting therapeutic targets for their treatment.     

Urotensin-II activates L-arginine/nitric oxide pathway in isolated rat aortic adventitia

Urotensin-II (U-II), a cyclic peptide widely expressed in blood vessels, has diverse vascular actions that range from potent vasoconstriction to vasodilation. Although, U-II-induced vasodilation has been shown to be partially dependent on nitric oxide (NO), the involvement of vascular adventitia-derived NO, remains unknown. The present study aimed to elucidate the activation of U-II on L-arginine/NO pathway in isolated rat aortic adventitia. In adventitia of thoracic and abdominal aortas, the l-arginine/NO pathway was similarly characterized: the uptake of l-[(3)H]arginine was Na(+)-independent, with the peak occurring over around 40 min incubation; the total NO synthase (NOS) activity was mostly calcium-independent (>90%), and significantly inhibited by a specific iNOS inhibitor AMT; the production of NO metabolites nitrate and nitrite (NO(x)) was stimulated by L-arginine but not by D-arginine. In aortic adventitia exposed to rat U-II (10(-9) and 10(-8)M) for 6 h, the V(max) of l-[(3)H]arginine uptake over 40 min incubation was significantly increased, while the K(m) of l-[(3)H]arginine uptake showed no significant change. Besides, the iNOS mRNA level was up-regulated, the total NOS activity, largely calcium-independent, was significantly induced, and the NO(x) production was significantly stimulated by U-II. According to the same protocol as U-II, the positive control lipopolysaccharide (LPS, 10 microg/ml), which had been established to activate adventitial L-arginine/NO pathway, increased l-[(3)H]arginine uptake, iNOS activity and NO(x) production to a greater extent than U-II. In addition, the total NOS activities induced by 3 and 6h incubation of U-II and LPS were significantly inhibited by a specific inhibitor of protein synthesis, actinomycin D. In conclusion, the results showed that rat U-II activated L-arginine/NOS/NO pathway in rat aortic adventitia, suggesting a potential contributive role of adventitia-derived NO in the vasodilator response of U-II.     

Evaluation of islet heme oxygenase-CO and nitric oxide synthase-NO pathways during acute endotoxemia

We investigated, by acombined in vivo and in vitro approach, the temporal changes of isletnitric oxide synthase (NOS)-derived nitric oxide (NO) and hemeoxygenase (HO)-derived carbon monoxide (CO) production in relation toinsulin and glucagon secretion during acute endotoxemia induced bylipopolysaccharide (LPS) in mice. Basal plasma glucagon, islet cAMP andcGMP content after in vitro incubation, the insulin response to glucosein vivo and in vitro, and the insulin and glucagon responses to theadenylate cyclase activator forskolin were greatly increased after LPS. Immunoblots demonstrated expression of inducible NOS (iNOS), inducible HO (HO-1), and an increased expression of constitutive HO (HO-2) inislet tissue. Immunocytochemistry revealed a marked expression of iNOSin many -cells, but only in single -cells after LPS. Moreover,biochemical analysis showed a time dependent and markedly increasedproduction of NO and CO in these islets. Addition of a NOS inhibitor tosuch islets evoked a marked potentiation of glucose-stimulated insulinrelease. Finally, after incubation in vitro, a marked suppression of NOproduction by both exogenous CO and glucagon was observed in controlislets. This effect occurred independently of a concomitant inhibitionof guanylyl cyclase. We suggest that the impairing effect of increasedproduction of islet NO on insulin secretion during acute endotoxemia isantagonized by increased activities of the islet cAMP and HO-COsystems, constituting important compensatory mechanisms against thenoxious and diabetogenic actions of NO in endocrine pancreas.

     

Autologous nitric oxide protects mouse and human keratinocytes from ultraviolet B radiation-induced apoptosis

Nitric oxide (NO) can either prevent or promote apoptosis, depending on cell type. In the present study, we tested the hypothesis that NO suppresses ultraviolet B radiation (UVB)-induced keratinocyte apoptosis both in vitro and in vivo. Irradiation with UVB or addition of the NO synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) increased apoptosis in the human keratinocyte cell line CCD 1106 KERTr, and apoptosis was greater when the two agents were given in combination. Addition of the chemical NO donor S-nitroso-N-acetyl-penicillamine (SNAP) immediately after UVB completely abrogated the rise in apoptosis induced by l-NAME. An adenoviral vector expressing human inducible NOS (AdiNOS) also reduced keratinocyte death after UVB. Caspase-3 activity, an indicator of apoptosis, doubled in keratinocytes incubated with l-NAME compared with the inactive isomer, d-NAME, and was reduced by SNAP. Apoptosis was also increased on addition of 1,H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase. Mice null for endothelial NOS (eNOS) exhibited significantly higher apoptosis than wild-type mice both in the dermis and epidermis, whereas mice null for inducible NOS (iNOS) exhibited more apoptosis than wild-type mice only in the dermis. These results demonstrate an antiapoptotic role for NO in keratinocytes, mediated by cGMP, and indicate an antiapoptotic role for both eNOS and iNOS in skin damage induced by UVB.     

The selective inhibition of inducible nitric oxide synthase prevents intestinal ischemia-reperfusion injury in mice.

Nitric oxide (NO) involvement in intestinal ischemia-reperfusion (I/R) injury has been widely suggested but its protective or detrimental role remains still question of debate. Here, we examine the impact of supplementation or inhibition of NO availability on intestinal dysmotility and inflammation caused by mesenteric I/R in mice. Ischemia 45min and reperfusion 24h were performed by superior mesenteric artery occlusion in female Swiss mice. Saline-treated sham-operated (S) or normal mice without surgery (N) served as controls. Drugs were subcutaneously injected 0, 4, 8, and 18 h after ischemia. Upper gastrointestinal transit (GIT, estimated through black marker gavage), intestinal myeloperoxidase activity (MPO), intestinal malondialdehyde levels (MDA), Evans blue extravasation (EB), intestinal histological damage, and mean arterial pressure (MAP) were considered. In I/R mice, GIT was significantly delayed compared to S and N groups; MPO activity and EB extravasation enhanced, whereas MDA levels did not change. Compared to N and S groups, in I/R mice selective iNOS inhibitor P-BIT significantly prevented motor, MPO and EB changes; putative iNOS inhibitor aminoguanidine significantly counteracted GIT delay but not neutrophil recruitment and the increase in vascular permeability; NOS inhibitor l-NAME and NO precursor l-arginine were scarcely or no effective. Furthermore, in S mice aminoguanidine caused a significant increase of MPO activity reverted by H(1) histamine receptor antagonist pre-treatment. Unlike P-BIT, aminoguanidine and l-NAME injection increased MAP. These findings confirm a detrimental role for iNOS-derived NO overproduction during reperfusion. Aminoguanidine-associated neutrophil recruitment suggests that this drug could act through mechanisms additional to iNOS inhibition involving both eNOS blockade, as indicated by its hemodynamic effects, and indirect activation of H(1) histamine receptors.     

Carbon monoxide reduces neuropathic pain and spinal microglial activation by inhibiting nitric oxide synthesis in mice

Background

Carbon monoxide (CO) synthesized by heme oxygenase 1 (HO-1) exerts antinociceptive effects during inflammation but its role during neuropathic pain remains unknown. Our objective is to investigate the exact contribution of CO derived from HO-1 in the modulation of neuropathic pain and the mechanisms implicated.

Methodology/Principal Findings

We evaluated the antiallodynic and antihyperalgesic effects of CO following sciatic nerve injury in wild type (WT) or inducible nitric oxide synthase knockout (NOS2-KO) mice using two carbon monoxide-releasing molecules (CORM-2 and CORM-3) and an HO-1 inducer (cobalt protoporphyrin IX, CoPP) daily administered from days 10 to 20 after injury. The effects of CORM-2 and CoPP on the expression of HO-1, heme oxygenase 2 (HO-2), neuronal nitric oxide synthase (NOS1) and NOS2 as well as a microglial marker (CD11b/c) were also assessed at day 20 after surgery in WT and NOS2-KO mice. In WT mice, the main neuropathic pain symptoms induced by nerve injury were significantly reduced in a time-dependent manner by treatment with CO-RMs or CoPP. Both CORM-2 and CoPP treatments increased HO-1 expression in WT mice, but only CoPP stimulated HO-1 in NOS2-KO animals. The increased expression of HO-2 induced by nerve injury in WT, but not in NOS2-KO mice, remains unaltered by CORM-2 or CoPP treatments. In contrast, the over-expression of CD11b/c, NOS1 and NOS2 induced by nerve injury in WT, but not in NOS2-KO mice, were significantly decreased by both CORM-2 and CoPP treatments. These data indicate that CO alleviates neuropathic pain through the reduction of spinal microglial activation and NOS1/NOS2 over-expression.

Conclusions/Significance

This study reports that an interaction between the CO and nitric oxide (NO) systems is taking place following sciatic nerve injury and reveals that increasing the exogenous (CO-RMs) or endogenous (CoPP) production of CO may represent a novel strategy for the treatment of neuropathic pain.