Search

The system of nitric oxide synthases (NOSs) is comprised of three isoforms: nNOS, iNOS, and eNOS. The roles of NOSs in respiratory diseases in vivo have been studied by using inhibitors of NOSs and NOS-knockout mice. Their exact roles remain uncertain, however, because of the non-specificity of inhibitors of NOSs and compensatory up-regulation of other NOSs in NOS-KO mice. We addressed this point in our triple-n/i/eNOSs-KO mice. Triple-n/i/eNOSs-KO mice spontaneously developed pulmonary emphysema and displayed exacerbation of bleomycin-induced pulmonary fibrosis as compared with wild-type (WT) mice. Triple-n/i/eNOSs-KO mice exhibited worsening of hypoxic pulmonary hypertension (PH), which was reversed by treatment with sodium nitrate, and WT mice that underwent triple-n/i/eNOSs-KO bone marrow transplantation (BMT) also showed aggravation of hypoxic PH compared with those that underwent WT BMT. Conversely, ovalbumin-evoked asthma was milder in triple-n/i/eNOSs-KO than WT mice. These results suggest that the roles of NOSs are different in different pathologic states, even in the same respiratory diseases, indicating the diversity of the roles of NOSs. In this review, we describe these previous studies and discuss the roles of NOSs in respiratory health and disease. We also explain the current state of development of inorganic nitrate as a new drug for respiratory diseases.

Food allergies (FAs) are caused by a failure of the immune system to regulate oral tolerance (OT). The use of soap containing hydrolyzed wheat overrides acquired OT to wheat through skin exposure. However, in mouse models, the experimental OT is robust, suggesting that acquired OT to allergens prevents the development of FAs. We aimed to analyze the mechanisms and developed a mouse model of FA that overrides acquired OT via skin exposure. Three murine FA models (intraperitoneal [IP], epicutaneous [EC], and intradermal [ID]) were compared to evaluate if allergies to ovalbumin (OVA) that had been previously tolerated orally could be induced. In the ID model, OT was overridden, and allergic reactions of severe anaphylaxis were developed. To analyze this effect in the ID model, we measured the migration of dendritic cells (DCs) into lymph nodes. The induction of OT promoted the migration of CD103+ dermal DCs; moreover, repeated percutaneous doses of OVA for sensitization gradually increased the migration of CD11b+ dermal DCs. The difference in the proportion of regulatory T cells between ID-sensitized groups at the first ID injection disappeared at the tenth injection. Although OT was robust in the IP model, ID sensitization was found to override OT.

Abstract The roles of endogenous nitric oxide (NO) derived from the entire NO synthases (NOSs) system have yet to be fully elucidated. We addressed this issue in mice in which all three NOS isoforms were deleted. Under basal conditions, the triple n/i/eNOSs−/− mice displayed significantly longer mean alveolar linear intercept length, increased alveolar destructive index, reduced lung elastic fiber content, lower lung field computed tomographic value, and greater end-expiratory lung volume as compared with wild-type (WT) mice. None of single NOS−/− or double NOSs−/− genotypes showed such features. These findings were observed in the triple n/i/eNOSs−/− mice as early as 4 weeks after birth. Cyclopaedic and quantitative comparisons of mRNA expression levels between the lungs of WT and triple n/i/eNOSs−/− mice by cap analysis of gene expression (CAGE) revealed that mRNA expression levels of three Wnt ligands and ten Wnt/β-catenin signaling components were significantly reduced in the lungs of triple n/i/eNOSs−/− mice. These results provide the first direct evidence that complete disruption of all three NOS genes results in spontaneous pulmonary emphysema in juvenile mice in vivo possibly through down-regulation of the Wnt/β-catenin signaling pathway, demonstrating a novel preventive role of the endogenous NO/NOS system in the occurrence of pulmonary emphysema.

Furuzono et al. identify DS20060511, a skeletal muscle cell-specific GLUT4 translocation enhancer. They find that DS20060511 have remarkable effects on lowering blood glucose and enhancing skeletal muscle glucose uptake via GLUT4, highlighting its potential as antidiabetic medicine.

Nitric oxide (NO) is a signal molecule and plays a critical role in the regulation of vascular tone, displays anti-platelet and anti-inflammatory properties. While our earlier and current studies found that low NO doses trigger a rapid heme insertion into immature heme-free soluble guanylyl cyclase β subunit (apo-sGCβ), resulting in a mature sGC-αβ heterodimer, more recent evidence suggests that low NO doses can also trigger heme-maturation of hemoglobin and myoglobin. This low NO phenomena was not only limited to sGC and the globins, but was also found to occur in all three nitric oxide synthases (iNOS, nNOS and eNOS) and Myeloperoxidase (MPO). Interestingly high NO doses were inhibitory to heme-insertion for these hemeproteins, suggesting that NO has a dose-dependent dual effect as it can act both ways to induce or inhibit heme-maturation of key hemeproteins. While low NO stimulated heme-insertion of globins required the presence of the NO-sGC-cGMP signal pathway, iNOS heme-maturation also required the presence of an active sGC. These effects of low NO were significantly diminished in the tissues of double (n/eNOS−/−) and triple (n/i/eNOS−/−) NOS knock out mice where lung sGC was found be heme-free and the myoglobin or hemoglobin from the heart/lungs were found be low in heme, suggesting that loss of endogenous NO globally impacts the whole animal and that this impact of low NO is both essential and physiologically relevant for hemeprotein maturation. Effects of low NO were also found to be protective against ischemia reperfusion injury on an ex vivo lung perfusion (EVLP) system prior to lung transplant, which further suggests that low NO levels are also therapeutic.

Under the dysfunction of mitochondria, cancer cells preferentially utilize both glycolytic and pentose phosphate pathways rather than electron transport chains to desperately generate adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH), classically recognized as the Warburg effect. Based on this background, the present study tested the hypothesis that anti-diabetic sodium-glucose cotransporter 2 (SGLT2) inhibitors would exert a tumor-suppressive impact on intractable human hematological malignancies via the modulation of glucose metabolism within cells and cell cycles. The level of mRNA for SGLT2 was remarkably elevated in leukemic cells from patients with adult T-cell leukemia (ATL), one of the most intractable blood cancers in humans, and as well as in two kinds of ATL cell lines (MT-1 and MT-2). Two kinds of SGLT2 inhibitors, Luseogliflozin and Tofogliflozin substantially suppressed the proliferation of MT-1 and MT-2 cells in both adherent and anchorage-independent culture conditions. Such a suppressive effect on tumor cell growth was reproduced by Luseogliflozin in leukemic cells in peripheral blood from patients with ATL. In MT-2 cells, both of SGLT2 inhibitors considerably attenuated glucose uptake, intracellular ATP levels, and NADPH production, resultantly enhancing cell cycle arrest at the G0/G1 phase. From the standpoint of metabolic oncology, the present study suggests that SGLT2 inhibitors would be a promising adjunctive option for the treatment of the most intractable human hematological malignancies like ATL.

Reactive sulfur species, or persulfides and polysulfides, such as cysteine hydropersulfide and glutathione persulfide, are endogenously produced in abundance in both prokaryotes and eukaryotes, including mammals. Various forms of reactive persulfides occur in both low-molecular-weight and protein-bound thiols. The chemical properties and great supply of these molecular species suggest a pivotal role for reactive persulfides/polysulfides in different cellular regulatory processes (e.g., energy metabolism and redox signaling). We demonstrated earlier that cysteinyl-tRNA synthetase (CARS) is a new cysteine persulfide synthase (CPERS) and is responsible for the in vivo production of most reactive persulfides (polysulfides). Some researchers continue to suggest that 3-mercaptopyruvate sulfurtransferase (3-MST), cystathionine β-synthase (CBS), and cystathionine γ-lyase (CSE) may also produce hydrogen sulfide and persulfides that may be generated during the transfer of sulfur from 3-mercaptopyruvate to the cysteine residues of 3-MST or direct synthesis from cysteine by CBS/CSE, respectively. We thus used integrated sulfur metabolome analysis, which we recently developed, with 3-MST knockout (KO) mice and CBS/CSE/3-MST triple-KO mice, to elucidate the possible contribution of 3-MST, CBS, and CSE to the production of reactive persulfides in vivo. We therefore quantified various sulfide metabolites in organs derived from these mutant mice and their wild-type littermates via this sulfur metabolome, which clearly revealed no significant difference between mutant mice and wild-type mice in terms of reactive persulfide production. This result indicates that 3-MST, CBS, and CSE are not major sources of endogenous reactive persulfide production; rather, CARS/CPERS is the principal enzyme that is actually involved in and even primarily responsible for the biosynthesis of reactive persulfides and polysulfides in vivo in mammals.

Food allergy is a refractory condition for which there are no standard effective therapies. Prebiotic supplementation such as oligosaccharides in infants was found to be associated with decreased risk of allergic diseases. Raffinose and stachyose are the major oligosaccharides identified in beans; these oligosaccharides have properties of regulating homeostasis. In this study, we explored the use of oligosaccharides as a means to prevent food allergy using an ovalbumin (OVA) sensitization and challenge model in which reductions in body temperature and diarrhea were evaluated. Raffinose and stachyose were administered ad libitum in drinking water for five weeks from one week prior the first sensitization through the final oral administration of OVA. Among our findings, treatment with stachyose suppressed allergic diarrhea and prevented elevations in OVA-specific immunoglobulin (Ig)G1. We hypothesize that suppression of these responses was associated with the actions of regulatory T cells and promoted by utilization of the oligosaccharides by intestinal microbiota. Taken together, our findings suggest that daily ingestion of oligosaccharides might be effective for the prevention of food allergy.

Extract of pine nodules (matsufushi) formed by bark proliferation on the surface of trees of Pinus tabulaeformis or Pinus massoniana has been used as an analgesic for joint pain, rheumatism, neuralgia, dysmenorrhea and other complaints in Chinese traditional medicine. Here we report the effects of matsufushi extract and its components on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. We found that matsufushi extract (0.0003–0.005%) and its component, SJ-2 (5-hydroxy-3-methoxy-trans-stilbene) (0.3–100 μM), but not the other three, concentration-dependently inhibited catecholamine secretion induced by acetylcholine, a physiological secretagogue. Matsufushi extract (0.0003–0.005%) and SJ-2 (0.3–100 μM) also inhibited 45Ca2+ influx induced by acetylcholine in a concentration-dependent manner, similar to its effect on catecholamine secretion. They also suppressed 14C-catecholamine synthesis and tyrosine hydroxylase activity induced by acetylcholine. In Xenopus oocytes expressing α3β4 nicotinic acetylcholine receptors, matsufushi extract (0.00003–0.001%) and SJ-2 (1–100 μM) directly inhibited the current evoked by acetylcholine. The present findings suggest that SJ-2, as well as matsufushi extract, inhibits acetylcholine-induced catecholamine secretion and synthesis by suppression of nicotinic acetylcholine receptor-ion channels in bovine adrenal medullary cells.

Recent epidemiological studies have demonstrated that coffee drinking is associated with reduced mortality of cardiovascular disease. However, its precise mechanisms remain to be clarified. In this study, we examined whether single ingestion of caffeine contained in a cup of coffee improves microvascular function in healthy subjects. A double-blind, placebo-controlled, crossover study was performed in 27 healthy volunteers. A cup of either caffeinated or decaffeinated coffee was drunk by the subjects, and reactive hyperemia of finger blood flow was assessed by laser Doppler flowmetry. In an interval of more than 2 days, the same experimental protocol was repeated with another coffee in a crossover manner. Caffeinated coffee intake slightly but significantly elevated blood pressure and decreased finger blood flow as compared with decaffeinated coffee intake. There was no significant difference in heart rate between caffeinated and decaffeinated coffee intake. Importantly, caffeinated coffee intake significantly enhanced post-occlusive reactive hyperemia of finger blood flow, an index of microvascular endothelial function, compared with decaffeinated coffee intake. These results provide the first evidence that caffeine contained in a cup of coffee enhances microvascular function in healthy individuals.

Nitric oxide (NO) is synthesized by three distinct NO synthases (neuronal, inducible, and endothelial NOSs), all of which are expressed in almost all tissues and organs in humans. The regulatory roles of NOSs in vivo have been investigated in pharmacological studies with non-selective NOS inhibitors. However, the specificity of the inhibitors continues to be an issue of debate, and the authentic significance of NOSs is still poorly understood. To address this issue, we generated mice in which all three NOS genes are completely disrupted. The triple NOSs null mice exhibited cardiovascular abnormalities, including hypertension, arteriosclerosis, myocardial infarction, cardiac hypertrophy, diastolic heart failure, and reduced EDHF responses, with a shorter survival. The triple NOSs null mice also displayed metabolic abnormalities, including metabolic syndrome and high-fat diet-induced severe dyslipidemia. Furthermore, the triple NOSs null mice showed renal abnormalities (nephrogenic diabetes insipidus and pathological renal remodeling), lung abnormalities (accelerated pulmonary fibrosis), and bone abnormalities (increased bone mineral density and bone turnover). These results provide evidence that NOSs play pivotal roles in the pathogenesis of a wide variety of disorders. This review summarizes the latest knowledge on the significance of NOSs in vivo, based on lessons learned from experiments with our triple mutant model.

Flavonoids are biologically active polyphenolic compounds widely distributed in plants. Recent research has focused on high dietary intake of flavonoids because of their potential to reduce the risks of diseases such as cardiovascular diseases, diabetes, and cancers. We report here the effects of plant flavonoids on catecholamine signaling in cultured bovine adrenal medullary cells used as a model of central and peripheral sympathetic neurons. Daidzein (0.01 – 1.0 μM), a soy isoflavone, stimulated 14C-catecholamine synthesis through plasma membrane estrogen receptors. Nobiletin (1.0 – 100 μM), a citrus polymethoxy flavone, enhanced 14C-catecholamine synthesis through the phosphorylation of Ser19 and Ser40 of tyrosine hydroxylase, which was associated with 45Ca2+ influx and catecholamine secretion. Treatment with genistein (0.01 – 10 μM), another isoflavone, but not daidzein, enhanced [3H]noradrenaline uptake by SK-N-SH cells, a human noradrenergic neuroblastoma cell line. Daidzein as well as nobiletin (≥ 1.0 μM) inhibited catecholamine synthesis and secretion induced by acetylcholine, a physiological secretagogue. The present review shows that plant flavonoids have various pharmacological potentials on the catecholamine system in adrenal medullary cells, and probably also in sympathetic neurons. Keywords:: adrenal medulla, catecholamine, flavonoid, membrane estrogen receptor, tyrosine hydroxylase

Abstract.: We previously reported the occurrence and function of plasma membrane estrogen receptors in cultured bovine adrenal medullary cells. Here we report the effects of raloxifene and tamoxifen, selective estrogen receptor modulators, on plasma membrane estrogen receptors and catecholamine synthesis and secretion in these cells. Raloxifene caused dual effects on the specific binding of [3H]17β-estradiol to the plasma membranes isolated from bovine adrenal medulla; that is, it had a stimulatory effect at 1.0 – 10 nM but an inhibitory effect at 1.0 – 10 μM, whereas tamoxifen (1.0 nM – 10 μM) increased binding at all concentrations (except for 100 nM). Tamoxifen at 100 nM caused a significant increase in basal 14C-catecholamine synthesis from [14C]tyrosine, whereas tamoxifen and raloxifene at higher concentrations attenuated basal and acetylcholine-induced 14C-catecholamine synthesis. Raloxifene (0.3, 1.0, and 3 – 100 μM) and tamoxifen (10 – 100 μM) also suppressed catecholamine secretion and 45Ca2+ and 22Na+ influx, respectively, induced by acetylcholine. Raloxifene (1.0 μM) inhibited Na+ current evoked by acetylcholine in Xenopus oocytes expressing α4β2 neuronal nicotinic acetylcholine receptors. The present findings suggest that raloxifene and tamoxifen at low concentrations allosterically modulate plasma membrane estrogen receptors and at high concentrations inhibit acetylcholine-induced catecholamine synthesis and secretion by inhibiting Na+ and Ca2+ influx in bovine adrenal medulla. Keywords:: adrenal medulla, catecholamine synthesis and secretion, plasma membrane estrogen receptor, raloxifene, selective estrogen receptor modulator

(±)-Pentazocine (PTZ), a non-narcotic analgesic, is used for the clinical management of moderate to severe pain. To study the effect of PTZ on the descending noradrenergic inhibitory system, in the present study we examined the effect of [3H]norepinephrine (NE) uptake by cultured bovine adrenal medullary cells and human neuroblastoma SK-N-SH cells. (−)-PTZ and (+)-PTZ inhibited [3H]NE uptake by adrenal medullary cells in a concentration-dependent (3 – 100 μM) manner. Eadie-Hofstee analysis of [3H]NE uptake showed that both PTZs caused a significant decrease in the Vmax with little change in the apparent Km, suggesting non-competitive inhibition. Nor-Binaltorphimine and BD-1047, κ-opioid and σ-receptor antagonists, respectively, did not affect the inhibition of [3H]NE uptake induced by (−)-PTZ and (+)-PTZ, respectively. PTZs suppressed specific [3H]nisoxetine binding to intact SK-N-SH cells, but not directly to the plasma membranes isolated from the bovine adrenal medulla. Scatchard analysis of [3H]nisoxetine binding to SK-N-SH cells revealed that PTZs reduced the Bmax without changing the apparent Kd. Western blot analysis showed a decrease in biotinylated cell-surface NE transporter (NET) expression after the treatment with (−)-PTZ. These findings suggest that PTZ inhibits the NET function by reducing the amount of NET in the cell surface membranes through an opioid and σ-receptor–independent pathway. Keywords:: adrenal medullary cell, descending noradrenergic inhibitory system, norepinephrine transporter, pentazocine, SK-N-SH cell

Abstract.: The role of nitric oxide (NO) derived from all three NO synthases (NOSs) in renal lesion formation remains to be fully elucidated. We addressed this point in mice lacking all NOSs. Renal injury was induced by unilateral ureteral obstruction (UUO). UUO caused significant renal lesion formation (tubular apoptosis, interstitial fibrosis, and glomerulosclerosis) in wild-type, singly, and triply NOS−/− mice. However, the extents of renal lesion formation were markedly and most accelerated in the triply NOS−/− genotype. UUO also elicited the infiltration of inflammatory macrophages, up-regulation of transforming growth factor (TGF)-β1, and induction of epithelial mesenchymal transition (EMT) in all of the genotypes; however, the extents were again largest by far in the triply NOS−/− genotype. Importantly, long-term treatment with the angiotensin II type 1 (AT1)-receptor blocker olmesartan significantly prevented the exacerbation of those renal structural changes after UUO in the triply NOS−/− genotype, along with amelioration of the macrophage infiltration, TGF-β1 levels, and EMT. These results provide the first evidence that the complete disruption of all NOS genes results in markedly accelerated renal lesion formation in response to UUO in mice in vivo through the AT1-receptor pathway, demonstrating the critical renoprotective role of all NOSs-derived NO against pathological renal remodeling. Keywords:: nitric oxide synthase, renal remodeling, unilateral ureteral obstruction, knockout mouse, angiotensin

In the present study, we investigated the direct effects of antiglaucoma drugs (timolol, betaxolol, pilocarpine, and latanoprost) on N-methyl-D-aspartate (NMDA)-receptor function using a Xenopus oocytes expression system and electrophysiological techniques. In oocytes expressing wild-type NMDA (NR1a/NR2A) receptors, timolol and betaxolol significantly inhibited glutamate-evoked currents, whereas less inhibition was obtained with pilocarpine, and latanoprost had few effects. Moreover, the effect of timolol and betaxolol was noncompetitive with respect to glutamate. Mutations that changed Asn616 of the NR1a subunit, a critical residue for Mg2+blocking of NMDA receptors, to Arg (N616R) or Gln (N616Q) almost eliminated the inhibitory effects of timolol and betaxolol, as well as the blocking effect of Mg2+. Experiments were also carried out to examine the protective effects of timolol and betaxolol against death of oocytes expressing NMDA receptors. During incubation of oocytes, especially in Mg2+-free medium, cell death was induced by addition of glutamate because of the continuous activation of the NMDA receptors expressed. Timolol and betaxolol significantly improved oocyte viability when they were added during the incubation period. These results suggest that timolol and betaxolol may have an additional role that they directly inhibit NMDA-receptor function, possibly via N616 of the NR1a subunit. Keywords:: glaucoma, β-antagonist, N-methyl-D-aspartate (NMDA) receptor, Xenopus oocyte, neuroprotection

Nitric oxide (NO) is produced in almost all tissues and organs, exerting multiple biological actions under both physiological and pathological conditions. NO is synthesized by three different isoforms of NO synthase (NOS): neuronal, inducible, and endothelial NOSs. Due to the substantial compensatory interactions among the NOS isoforms, the ultimate roles of endogenous NO in our body still remain to be fully elucidated. To address this point, we have successfully developed mice in which all three NOS genes are completely disrupted. NOS expression and activities were totally absent in the triply n/i/eNOS−/− mice before and after treatment with lipopolysaccharide. While the triply n/i/eNOS−/− mice were viable, their survival and fertility rates were markedly reduced as compared with wild-type mice. The phenotypes of those mice that we first noticed were polyuria, polydipsia, and renal unresponsiveness to vasopressin, characteristics consistent with nephrogenic diabetes insipidus. We subsequently observed that in those mice, arteriosclerosis is spontaneously developed with a clustering of cardiovascular risk factors. These results provide the first evidence that the systemic deletion of all three NOSs causes a variety of cardiovascular diseases in mice, demonstrating a critical role of the endogenous NOSs system in maintaining cardiovascular homeostasis. Keywords:: nitric oxide synthase, knockout mouse, cardiovascular disease, nephrogenic diabetes insipidus, metabolic syndrome

AIMS: Mechanisms regulating adiponectin expression have not been fully clarified. MicroRNAs (miRNAs), small non-coding RNAs that regulate gene expression, are involved in biological processes, including obesity and insulin resistance. We evaluated whether the miRNA-378 pathway is involved in regulating adiponectin expression. METHODS AND RESULTS: First, we determined a putative target site for miRNA-378 in the 3 prime untranslated region (3'UTR) of the adiponectin gene by in silico analysis. The levels of adiponectin mRNA and protein were decreased in 3T3-L1 cells overexpressing the mimic of miRNA-378. Luminescence activity in HEK293T cells expressing a renilla-luciferase-adiponectin-3'UTR sequence was inhibited by overexpressing the mimic of miRNA-378, and the decrease was reversed by adding the inhibitor of miRNA-378. Moreover, we confirmed the inhibitory effects of the mimic were cancelled in a deleted mutant of the miR-378 3'-UTR binding site. Addition of tumor necrosis factor-α (TNFα) led a upregulation of miR-378 and downregulation of adiponectin at mRNA and protein levels in 3T3-L1 cells. Level of miR-378 was higher and mRNA level of adiponectin was lower in diabetic ob/ob mice than those of normal C57BL/6 mice and levels of miR378 and adiponectin were negatively well correlated (r = -0.624, p = 0.004). CONCLUSIONS: We found that levels of miRNA-378 could modulate adiponectin expression via the 3'UTR sequence-binding site. Our findings warrant further investigations into the role of miRNAs in regulating the adiponectin expression.

The role of the nitric oxide synthases (NOSs) system in inter-organ communication is not fully understood. We addressed this point in our mice lacking all three NOS isoforms (triple n/i/eNOSs-/- mice). We previously reported that the triple n/i/eNOSs-/- mice spontaneously develop myocardial infarction. However, it takes a long time (approximately 1 year) to develop myocardial infarction. We then revealed that 2/3-nephrectomized triple n/i/eNOSs-/- mice suddenly die due to early onset of myocardial infarction, succeeding in developing an experimentally useful model of myocardial infarction. These results suggest the protective role of NOSs in reno-cardiac communication. We next studied the role of NOSs in bone marrow cells (BM) in vascular lesion formation. Constrictive vascular remodeling and neointimal formation at 14 days after carotid artery ligation were markedly accelerated in wild-type (WT) mice transplanted with triple n/i/eNOSs-/- BM as compared with those with WT BM. These results suggest the protective role of NOSs in BM-vascular communication. We then investigated the role of NOSs in BM in pulmonary hypertension (PH). The extents of PH at 3 weeks after hypoxic exposure were markedly exacerbated in WT mice transplanted with triple n/i/eNOSs-/- BM as compared with those with WT BM, suggesting the protective role of NOSs in BM-lung communication. These lines of evidence indicate that systemic and BM NOSs may be a novel therapeutic target in MI, vascular disease, and PH. [ABSTRACT FROM AUTHOR]