ABO Blood Group Is Associated with Response to Inhaled Nitric Oxide in Neonates with Respiratory Failure

Background

Inhaled nitric oxide (iNO) reduces death or need for extracorporeal membrane oxygenation (ECMO) in infants with persistent pulmonary hypertension of the newborn (PPHN). However, the response to iNO is variable and only 50–60% of infants demonstrate a response to iNO. It is not known why only some infants respond to iNO. Adults and children with blood groups B or AB do not respond as well to iNO as those with blood groups O/A.

Methods/Principal Findings

To determine if blood group was associated with iNO response in newborn infants, a retrospective medical record review was done of infants admitted to a regional NICU from 2002-9 with a diagnosis of PPHN. Data were collected during the first twelve hours post-initiation of treatment. Of 86 infants diagnosed with PPHN, 23 infants had blood group A [18 received iNO], 21 had group B [18 with iNO], 40 had group O [36 with iNO], and 2 had group AB [both received iNO]. Change in PaO₂/FiO₂ was less in infants with blood group A, of whom less than half were responders (ΔPaO₂/FiO₂>20%) at 12 h versus 90% of infants with either O or B. Race, sex, birth weight, gestational age, Apgar scores at 1 and 5 minutes, and baseline PaO₂/FiO₂ were similar among groups. Outcomes including need for ECMO, death, length of ventilatory support, length of iNO use, and hospital stay were statistically not different by blood groups.

Conclusions/Significance

Our results indicate that blood group influences iNO response in neonates. We hypothesize that either there is genetic linkage of the ABO gene locus with vasoregulatory genes, or that blood group antigens directly affect vascular reactivity.     

Protein Kinase D Interacts with Neuronal Nitric Oxide Synthase and Phosphorylates the Activatory Residue Serine1412

Neuronal Nitric Oxide Synthase (nNOS) is the biosynthetic enzyme responsible for nitric oxide (·NO) production in muscles and in the nervous system. This constitutive enzyme, unlike its endothelial and inducible counterparts, presents an N-terminal PDZ domain known to display a preference for PDZ-binding motifs bearing acidic residues at -2 position. In a previous work, we discovered that the C-terminal end of two members of protein kinase D family (PKD1 and PKD2) constitutes a PDZ-ligand. PKD1 has been shown to regulate multiple cellular processes and, when activated, becomes autophosphorylated at Ser⁹¹⁶, a residue located at -2 position of its PDZ-binding motif. Since nNOS and PKD are spatially enriched in postsynaptic densities and dendrites, the main objective of our study was to determine whether PKD1 activation could result in a direct interaction with nNOS through their respective PDZ-ligand and PDZ domain, and to analyze the functional consequences of this interaction. Herein we demonstrate that PKD1 associates with nNOS in neurons and in transfected cells, and that kinase activation enhances PKD1-nNOS co-immunoprecipitation and subcellular colocalization. However, transfection of mammalian cells with PKD1 mutants and yeast two hybrid assays showed that the association of these two enzymes does not depend on PKD1 PDZ-ligand but its pleckstrin homology domain. Furthermore, this domain was able to pull-down nNOS from brain extracts and bind to purified nNOS, indicating that it mediates a direct PKD1-nNOS interaction. In addition, using mass spectrometry we demonstrate that PKD1 specifically phosphorylates nNOS in the activatory residue Ser¹⁴¹², and that this phosphorylation increases nNOS activity and ·NO production in living cells. In conclusion, these novel findings reveal a crucial role of PKD1 in the regulation of nNOS activation and synthesis of ·NO, a mediator involved in physiological neuronal signaling or neurotoxicity under pathological conditions such as ischemic stroke or neurodegeneration.     

糖皮质激素对机械通气大鼠肺组织iNOS、NO表达的影响

目的通过观察糖皮质激素对机械通气大鼠肺组织诱导型一氧化氮合酶（iNOS）及一氧化氮（NO）表达的影响,探讨糖皮质激素对呼吸机所致肺损伤（ventilator induced lung injury,VILI）的干预作用。方法 24只雄性Wistar大鼠随机分为对照组、机械通气组、地塞米松（DXM）干预组。用逆转录-聚合酶链反应（RT-PCR）法检测肺组织iNOS mRNA表达,用免疫组织化学染色法检测肺组织iNOS蛋白表达,用硝酸还原酶法测定肺组织和血浆NO含量。结果机械通气组和DXM干预组大鼠肺组织iNOS mRNA及其蛋白表达水平,以及血浆和肺组织NO含量均明显高于对照组（P〈0.01）;DXM干预组上述指标与机械通气组比较均明显降低（P〈0.01）。结论糖皮质激素可通过抑制肺组织iNOS的表达,减少NO的生成,对机械通气大鼠肺组织具有保护作用。     

Expression of Inducible Nitric Oxide Synthase mRNA and Nitric Oxide Production During the Development of Liver Abscess in Hamster Inoculated with Entamoeba histolytica

The present study analyzed iNOS and eNOS mRNA expression and NO production during development of hepatic abscess caused by Entamoeba histolytica trophozoites. One 374-bp sequence, which displayed 88% identity to mammalian iNOS protein, was isolated from LPS-stimulated peritoneal hamster macrophages. A separate 365-bp cDNA sequence showed 99% identity with eNOS protein. iNOS mRNA was detected in hamsters during formation of amoebic liver abscesses, but not in control hamsters. eNOS mRNA expression was not modified. Serum nitrite concentration in hamsters infected with E. histolytica was 33 ± 6 μM, in control hamsters was 20 ± 3 μM. The study shows that iNOS mRNA expression and NO production are induced by E. histolytica trophozoites during amoebic liver abscess formation. However, in spite of iNOS mRNA expression and NO production, E. histolytica trophozoites induced liver abscess formation in hamster.     

Interferon-Gamma and Nitric Oxide Synthase 2 Mediate the Aggregation of Resident Adherent Peritoneal Exudate Cells: Implications for the Host Response to Pathogens

Interferon-gamma (Ifnγ), a key macrophage activating cytokine, plays pleiotropic roles in host immunity. In this study, the ability of Ifnγ to induce the aggregation of resident mouse adherent peritoneal exudate cells (APECs), consisting primarily of macrophages, was investigated. Cell-cell interactions involve adhesion molecules and, upon addition of Ifnγ, CD11b re-localizes preferentially to the sites of interaction on APECs. A functional role of CD11b in enhancing aggregation is demonstrated using Reopro, a blocking reagent, and siRNA to Cd11b. Studies with NG-methyl-L-arginine (LNMA), an inhibitor of Nitric oxide synthase (Nos), NO donors, e.g., S-nitroso-N-acetyl-DL-penicillamine (SNAP) or Diethylenetriamine/nitric oxide adduct (DETA/NO), and Nos2^-/- mice identified Nitric oxide (NO) induced by Ifnγ as a key regulator of aggregation of APECs. Further studies with Nos2^-/- APECs revealed that some Ifnγ responses are independent of NO: induction of MHC class II and CD80. On the other hand, Nos2 derived NO is important for other functions: motility, phagocytosis, morphology and aggregation. Studies with cytoskeleton depolymerizing agents revealed that Ifnγ and NO mediate the cortical stabilization of Actin and Tubulin which contribute to aggregation of APECs. The biological relevance of aggregation of APECs was delineated using infection experiments with Salmonella Typhimurium (S. Typhimurium). APECs from orally infected, but not uninfected, mice produce high amounts of NO and aggregate upon ex vivo culture in a Nos2-dependent manner. Importantly, aggregated APECs induced by Ifnγ contain fewer intracellular S. Typhimurium compared to their single counterparts post infection. Further experiments with LNMA or Reopro revealed that both NO and CD11b are important for aggregation; in addition, NO is bactericidal. Overall, this study elucidates novel roles for Ifnγ and Nos2 in regulating Actin, Tubulin, CD11b, motility and morphology during the aggregation response of APECs. The implications of aggregation or “group behavior” of APECs are discussed in the context of host resistance to infectious organisms.     

Up-Regulation of the Neuronal Form of Nitric Oxide Synthase in Response to Prolonged Muscarinic M1 Receptor Stimulation

Abstract: It is generally believed that the neuronal form of nitric oxide synthase (nNOS) is constitutively expressed and that regulation of this enzyme's activity is mediated solely by changes in cytosolic calcium concentration. Serendipitously, however, we observed that pretreatment of Chinese hamster ovary (CHO) cells, which coexpress muscarinic M₁ receptors and nNOS, with 3.3 µ M or 1 m M carbachol (CCh) for 48 h resulted in marked enhancement of maximal muscarinic receptor-stimulated nNOS activity as determined by l -[³H]citrulline and cyclic [³H]GMP production. This was accompanied by a decrease in the potency of CCh. Muscarinic receptor density was reduced in the agonist-pretreated cells, as determined by specific [ N-methyl -³H]scopolamine methyl chloride binding, whereas competition binding studies revealed no changes in agonist affinity. Both receptor-stimulated inositol phosphate formation and elevation of intracellular calcium concentrations were found to be desensitized in agonist-pretreated cells in a manner dependent on CCh pretreatment concentration. It is interesting that ionomycin-stimulated nNOS activity was greater in CCh-pretreated cells. Also, western analysis revealed increased nNOS immunoreactivity in pretreated cells. A similar increase in nNOS immunoreactivity following agonist treatment was demonstrated in N1E-115 neuroblastoma cells, which endogenously express nNOS and muscarinic M₁ receptors. Thus, the enhancement of maximal receptor-stimulated nNOS activity following agonist pretreatment can be attributed to up-regulation of nNOS. It is interesting that this augmentation of the response takes place in spite of receptor down-regulation and desensitization of multiple steps involved in nNOS activation.     

The Severity of Murray Valley Encephalitis in Mice Is Linked to Neutrophil Infiltration and Inducible Nitric Oxide Synthase Activity in the Central Nervous System

A study of immunopathology in the central nervous system (CNS) during infection with a virulent strain of Murray Valley encephalitis virus (MVE) in weanling Swiss mice following peripheral inoculation is presented. It has previously been shown that virus enters the murine CNS 4 days after peripheral inoculation, spreads to the anterior olfactory nucleus, the pyriform cortex, and the hippocampal formation at 5 days postinfection (p.i.), and then spreads throughout the cerebral cortex, caudate putamen, thalamus, and brain stem between 6 and 9 days p.i. (P. C. McMinn, L. Dalgarno, and R. C. Weir, Virology 220:414-423, 1996). Here we show that the encephalitis which develops in MVE-infected mice from 5 days p.i. is associated with the development of a neutrophil inflammatory response in perivascular regions and in the CNS parenchyma. Infiltration of neutrophils into the CNS was preceded by increased expression of tumor necrosis factor alpha and the neutrophil-attracting chemokine N51/KC within the CNS. Depletion of neutrophils with a cytotoxic monoclonal antibody (RB6-8C5) resulted in prolonged survival and decreased mortality in MVE-infected mice. In addition, neutrophil infiltration and disease onset correlated with expression of the enzyme-inducible nitric oxide synthase (iNOS) within the CNS. Inhibition of iNOS by aminoguanidine resulted in prolonged survival and decreased mortality in MVE-infected mice. This study provides strong support for the hypothesis that Murray Valley encephalitis is primarily an immunopathological disease.     

Effects of Chronic Nitric Oxide Inhibition on the Renal Excretory Response to Leptin

Objective: Previous investigations have demonstrated that leptin promotes natriuresis with a renal tubular effect. However, the mechanisms involved in this response are unclear. The present study was designed to examine the hypothesis that the natriuretic response to leptin in normotensive Sprague‐Dawley rats is regulated by nitric oxide (NO). Research Methods and Procedures: The hemodynamic and renal excretory effects of intravenous bolus administration of pharmacological doses of synthetic murine leptin were examined in groups of control Sprague‐Dawley rats (n = 8), Sprague‐Dawley rats treated for 4 days with the NO synthase inhibitor Nω‐nitro‐l‐arginine methyl ester (l‐NAME) (n = 8), and Sprague‐Dawley rats treated for 4 days with l‐NAME followed by acute treatment with sodium nitroprusside (n = 8). Results: In the control group (n = 8), an intravenous bolus of leptin, 400 μg/kg body weight, increased urinary sodium excretion 4‐ to 6‐fold. In the Sprague‐Dawley rats chronically administered l‐NAME (n = 8), an intravenous bolus of 400 μg/kg of leptin did not increase sodium excretion. Acute sodium nitroprusside infusion to Sprague‐Dawley rats chronically treated with l‐NAME (n = 8) was associated with partial restoration of the sodium excretory response to leptin administration. Discussion: Collectively, these results are interpreted to suggest that the natriuretic and diuretic responses to leptin observed in the Sprague‐Dawley rat require a functional NO system.     

Gene Expression of Lupeol Synthase and Biosynthesis of Nitric Oxide in Cell Suspension Cultures of Betula platyphylla in Response to a Phomopsis Elicitor

The current work aimed to characterize the generation of nitric oxide (NO) and gene expression of lupeol synthase (LUS) in Betula platyphylla cells exposed to a Phomopsis elicitor. The effects of nitrate reductase (NR) and NO synthase (NOS), the two key enzymes responsible for endogenous NO biosynthesis in plants, were also investigated. NO production in B. platyphylla cell cultures exhibited a biphasic pattern, reaching the Wrst plateau within 1.0–10 h of exposure to the Phomopsis elicitor. LUS gene expression was found to increase abruptly 10 h after Phomopsis induction, reaching its highest level (18.08) at 24 h. The maximum levels of NOS and NR activities in elicitor-treated cells were found to be 1.7-fold and 6.9-fold those of untreated cells, respectively. Pharmacological experiments showed that Phomopsis elicitor-induced NO production and LUS gene expression level were significantly suppressed by the NOS inhibitor N^G-nitro-l-Arg methyl ester (l-NAME), the NR inhibitor sodium azide (NaN₃), and the NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). NaNO₂ and l-arginine (the substrates that produce NO via NR and NOS) and NO donor sodium nitroprusside (SNP) were found to increase both NO production and LUS gene expression. These results suggest that the increase in LUS gene expression due to fungal elicitor-induced NO may involve the NR and NOS biosynthetic pathways.     

Role of Nitric Oxide Synthase Pathways in the Effects of Proinflammatory Cytokines on the Respiratory Pattern and Hypoxic Ventilatory Response

Journal of Evolutionary Biochemistry and Physiology - Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) are the major proinflammatory cytokines. Cytokine receptors are...     

Protein kinase A-dependent Neuronal Nitric Oxide Synthase Activation Mediates the Enhancement of Baroreflex Response by Adrenomedullin in the Nucleus Tractus Solitarii of Rats

Background  

Adrenomedullin (ADM) exerts its biological functions through the receptor-mediated enzymatic mechanisms that involve protein kinase A (PKA), or neuronal nitric oxide synthase (nNOS). We previously demonstrated that the receptor-mediated cAMP/PKA pathway involves in ADM-enhanced baroreceptor reflex (BRR) response. It remains unclear whether ADM may enhance BRR response via activation of nNOS-dependent mechanism in the nucleus tractus solitarii (NTS).     

Release of Phosphorylated HSP27 (HSPB1) from Platelets Is Accompanied with the Acceleration of Aggregation in Diabetic Patients

We investigated the relationship between HSP27 phosphorylation and collagen-stimulated activation of platelets in patients with diabetes mellitus (DM). Platelet-rich plasma was prepared from blood of type 2 DM patients. The platelet aggregation was analyzed in size of aggregates by an aggregometer using a laser scattering method. The protein phosphorylation was analyzed by Western blotting. Phosphorylated-HSP27 and PDGF-AB released from platelets were measured by ELISA. The phosphorylated-HSP27 levels at Ser-78 and Ser-82 induced by collagen were directly proportional to the platelet aggregation. Total HSP27 levels in platelets were decreased concomitantly with the phosphorylation. The released HSP27 levels were significantly correlated with the phosphorylated levels of HSP27 in the platelets stimulated by 0.3 μg/ml collagen. The low dose collagen-stimulated release of HSP27 was detected but relatively small in healthy donors. The released levels of PDGF-AB were in parallel with the levels of released HSP27. Area under the curve (AUC) of small aggregation (9-25 μm) induced by 0.3 μg/ml collagen was inversely proportional to the levels of released HSP27. AUC of large aggregation (50-70 μm) was directly proportional to the levels of released HSP27. Exogenous recombinant phosphorylated- HSP27 hardly affected the aggregation or the released levels of PDGF-AB induced by collagen. These results strongly suggest that HSP27 is released from human platelets accompanied with its phosphorylation induced by collagen, which is correlated with the acceleration of platelet aggregation in type 2 DM patients.     

Development of NADPH-Diaphorase in the Avian Retina: Regulation by Calcium Ions and Relation to Nitric Oxide Synthase

Abstract: Nitric oxide plays an important role as an intercellular messenger in the CNS. In the present work we measured NADPH-diaphorase activity, which is considered to be a marker of cells producing nitric oxide, in homogenates of the developing chick retina. The enzyme activity can be detected beginning in 8-day-old embryonic retinas with no further quantitative variations throughout development. Arginine analogues inhibit ∼65% of the activity in embryonic retinas and 50% in posthatched retinas. The enzyme is stimulated 50% by 2 m M calcium chloride in retinas from 8 to 14 embryonic days, but this effect decreases to 20% in 17-day embryonic retinas and practically disappears in posthatched animals. The stimulation by calcium is completely blocked by arginine analogues. The decrease in enzyme activity at posthatched retinas is not due to stimulation by endogenous calcium or the presence of insufficient amounts of calmodulin, because addition of EGTA or calmodulin, respectively, did not restore the stimulation to levels observed at embryonic stages. Inhibition of NADPH-diaphorase activity by N ^G-nitro- l -arginine or l - N ^G-(iminoethyl)ornithine is concentration dependent with IC₅₀ values of ∼1 m M at all stages studied. However, in the presence of calcium, the inhibition by both analogues is shifted to the left and is apparently biphasic at all developmental stages, including in posthatched animals, with IC₅₀ values in the low micromolar range. NADPH-diaphorase was also detected by histochemistry in specific groups of cells in the early embryonic retina and in subsets of amacrine and ganglion cells, as well as in photoreceptors, in more developed retinas. The results indicate that different isoforms of nitric oxide synthase are present in the chick retina and that a calcium-dependent isoform is predominant in early periods of development.     

Pathological Lesions and Inducible Nitric Oxide Synthase Expressions in the Liver of Mice Experimentally Infected with Clonorchis sinensis

The nitric oxide (NO) formation and intrinsic nitrosation may be involved in the possible mechanisms of liver fluke-associated carcinogenesis. We still do not know much about the responses of inducible NO synthase (iNOS) induced by Clonorchis sinensis infection. This study was conducted to explore the pathological lesions and iNOS expressions in the liver of mice with different infection intensity levels of C. sinensis. Extensive periductal inflammatory cell infiltration, bile duct hyperplasia, and fibrosis were commonly observed during the infection. The different pathological responses in liver tissues strongly correlated with the infection intensity of C. sinensis. Massive acute spotty necrosis occurred in the liver parenchyma after a severe infection. The iNOS activity in liver tissues increased, and iNOS-expressing cells with morphological differences were observed after a moderate or severe infection. The iNOS-expressing cells in liver tissues had multiple origins.