Acylated anthocyanins derived from purple carrot (Daucus carota L.) induce elevation of blood flow in rat cremaster arteriole.

Acylated anthocyanins are more stable than monomeric anthocyanins, but little is known about their physiological effects. We evaluated the hemodynamic effects of single intragastric doses of purple carrot (Daucus carota L.) anthocyanin (PCA) and two monomeric anthocyanins, cyanidin 3-O-glycoside (C3G) and delphinidin 3-O-ruthenoside (D3R). PCA, C3G, or D3R was administered orally to rat and blood flow in the cremaster artery was measured for 60 min using a laser Doppler blood flow meter. After measurements, the aorta of the animal was removed and the extent of phosphorylation of aortic epithelial nitric oxide synthase (eNOS) and Akt were determined by western blotting. PCA (10 mg kg-1) or C3G (1 mg kg-1) significantly increased rat cremaster arteriole blood flow and phosphorylation of eNOS and Akt; D3G (1 mg kg-1) only slightly altered cremaster arteriole blood flow and did not affect the phosphorylation of eNOS and Akt in the aorta. These results suggest that hemodynamic alterations depend more on the chemical structure of anthocyanins, particularly the aglycon, than on the glycoside. In addition, increase of blood flow by a single oral dose of PCA was practically reduced with treatment of carvedilol (CR), a non-specific adrenaline blocker. Blood concentrations of cyanidin or its glycoside 15, 30, or 60 min after the administration of 10 mg kg-1 PCA were below the limit of detection. These hemodynamic changes may have been associated with an indirect adrenergic action induced following a single dose of PCA.

What does "NO-Synthase" stand for ?

Mammalian NO-Synthases (NOSs) are the enzymatic sources of Nitric Oxide (NO°), a paradigmatic gasotransmitter involved in many (patho)-physiological processes. The increasing number of available genomes led to the identification of hundreds of new NOS proteins throughout the kingdoms of life, calling for a global investigation of this family of proteins. These new NOSs are commonly believed to share the same structure, functioning and role as mammalian NOSs. The scope of this article is to highlight the singularity of these NOSs and to describe their complex structural and functional diversity. NOS appears as a unique enzymatic machinery that exhibits a complex Structure - Activity - Function relationship. Its sophisticated redox mechanism and enzymatic regulation, coupled to the vast biological chemistry of reactive nitrogen species, leads to a specific cross-talk between NOS catalysis and its biological environment that implies a complex evolution of NOS function. This paper addresses the relationship between structure, function and evolution of NOS proteins using three NOS model families and advocates for an integrative and interdisciplinary approach that combines modelling studies, structural characterization, and in vitro/in vivo functional investigations.

Nitric oxide synthase in plants: Where do we stand?

Over the past twenty years, nitric oxide (NO) has emerged as an important player in various plant physiological processes. Although many advances in the understanding of NO functions have been made, the question of how NO is produced in plants is still challenging. It is now generally accepted that the endogenous production of NO is mainly accomplished through the reduction of nitrite via both enzymatic and non-enzymatic mechanisms which remain to be fully characterized. Furthermore, experimental arguments in favour of the existence of plant nitric oxide synthase (NOS)-like enzymes have been reported. However, recent investigations revealed that land plants do not possess animal NOS-like enzymes while few algal species do. Phylogenetic and structural analyses reveals interesting features specific to algal NOS-like proteins.

Occurrence, structure, and evolution of nitric oxide synthase-like proteins in the plant kingdom.

Nitric oxide (NO) signaling regulates various physiological processes in both animals and plants. In animals, NO synthesis is mainly catalyzed by NO synthase (NOS) enzymes. Although NOS-like activities that are sensitive to mammalian NOS inhibitors have been detected in plant extracts, few bona fide plant NOS enzymes have been identified. We searched the data set produced by the 1000 Plants (1KP) international consortium for the presence of transcripts encoding NOS-like proteins in over 1000 species of land plants and algae. We also searched for genes encoding NOS-like enzymes in 24 publicly available algal genomes. We identified no typical NOS sequences in 1087 sequenced transcriptomes of land plants. In contrast, we identified NOS-like sequences in 15 of the 265 algal species analyzed. Even if the presence of NOS enzymes assembled from multipolypeptides in plants cannot be conclusively discarded, the emerging data suggest that, instead of generating NO with evolutionarily conserved NOS enzymes, land plants have evolved finely regulated nitrate assimilation and reduction processes to synthesize NO through a mechanism different than that in animals.

Cellular, biochemical, and molecular characterization of nitric oxide synthase expressed in the nervous system of the prosobranch Stramonita haemastoma (Gastropoda, Neogastropoda).

Nitric oxide synthase (NOS) has been characterized in several opistobranchs and pulmonates but it was much less investigated in prosobranchs, which include more than 20,000 species and account for most of the gastropod diversity. Therefore, new data from this large group are needed for a better knowledge of the molecular evolution of NOS enzymes in molluscs. This study focused on NOS expressed in the nervous system of the prosobranch neogastropod Stramonita haemastoma. In this study we report compelling evidence on the expression of a constitutive Ca(2+) /CaM-dependent neuronal NOS in the central and peripheral nervous system. The prevailing neuronal localization of NADPHd activity was demonstrated by NADPHd histochemistry in both central and peripheral nervous system structures. L-arginine/citrulline assays suggested that Stramonita NOS is a constitutive enzyme which is both cytosolic and membrane-bound. Molecular cloning of the full-length Stramonita NOS (Sh-NOS) by reverse-transcription polymerase chain reaction (RT-PCR) followed by 5' and 3' RACE showed that Sh-NOS is a protein of 1,517 amino acids, containing a PDZ domain at the N-terminus and sharing similar regulatory domains to the mammalian neuronal NOS (nNOS). Regional expression of the Sh-NOS gene was evaluated by RT-PCR. This analysis showed different expression levels in the nerve ring, the osphradium, the cephalic tentacles, the buccal tissues, and the foot, whereas NOS expression was not found in the salivary glands and the gland of Leiblein. The present data provide a solid background for further studies addressing the specific functions of NO in neogastropods.

Evolution of the nitric oxide synthase family in metazoans.

Nitric oxide (NO) is essential to many physiological functions and operates in several signaling pathways. It is not understood how and when the different isoforms of nitric oxide synthase (NOS), the enzyme responsible for NO production, evolved in metazoans. This study investigates the number and structure of metazoan NOS enzymes by genome data mining and direct cloning of Nos genes from the lamprey. In total, 181 NOS proteins are analyzed from 33 invertebrate and 63 vertebrate species. Comparisons among protein and gene structures, combined with phylogenetic and syntenic studies, provide novel insights into how NOS isoforms arose and diverged. Protein domains and gene organization--that is, intron positions and phases--of animal NOS are remarkably conserved across all lineages, even in fast-evolving species. Phylogenetic and syntenic analyses support the view that a proto-NOS isoform was recurrently duplicated in different lineages, acquiring new structural configurations through gains and losses of protein motifs. We propose that in vertebrates a first duplication took place after the agnathan-gnathostome split followed by a paralog loss. A second duplication occurred during early tetrapod evolution, giving rise to the three isoforms--I, II, and III--in current mammals. Overall, NOS family evolution was the result of multiple gene and genome duplication events together with changes in protein architecture.

Early and prominent alterations in hemodynamics, signaling, and gene expression following renal ischemia in sickle cell disease.

Acute ischemic insults to the kidney are recognized complications of human sickle cell disease (SCD). The present study analyzed in a transgenic SCD murine model the early renal response to acute ischemia. Renal hemodynamics were profoundly impaired following ischemia in sickle mice compared with wild-type mice: glomerular filtration rate, along with renal plasma flow and blood flow rates, were markedly reduced, while renal vascular resistances were increased more than threefold in sickle mice following ischemia. In addition to these changes in renal hemodynamics, there were profound disturbances in renal signaling processes: phosphorylation of members of the MAPK and Akt signaling proteins occurred in the kidney in wild-type mice after ischemia, whereas such phosphorylation did not occur in the kidney in sickle mice after ischemia. ATP content in the postischemic kidney in sickle mice was less than half that observed in wild-type mice. Examination of the expression of candidate genes uncovered changes that may predispose to increased sensitivity of the kidney in sickle mice to ischemia: increased expression of inducible nitric oxide synthase and decreased expression of endothelial nitric oxide synthase, and increased expression of TNF-alpha. Inducibility of anti-inflammatory, cytoprotective genes, such as heme oxygenase-1 and IL-10, was not impaired in sickle mice after ischemia. We conclude that the kidney in SCD is remarkably vulnerable to acute ischemic insults. We speculate that such sensitivity of the kidney to ischemia in SCD may underlie the occurrence of acute kidney injury in patients with SCD and may set the stage for the emergence of chronic kidney disease in SCD.

Nitric oxide as an initiator of brain lesions during the development of Alzheimer disease.

Nitric oxide (NO) is an important regulatory molecule for the host defense that plays a fundamental role in the cardiovascular, immune, and nervous systems. NO is synthesized through the conversion of L-arginine to L-citrulline by the enzyme NO synthase (NOS), which is found in three isoforms classified as neuronal (nNOS), inducible (iNOS), and endothelial (eNOS). Recent evidence supports the theory that this bioactive molecule has an influential role in the disruption of normal brain and vascular homeostasis, a condition known to elucidate chronic hypoperfusion which ultimately causes the development of brain lesions and the pathology that typify Alzheimer disease (AD). In addition, vascular NO activity appears to be a major contributor to this pathology before any overexpression of NOS isoforms is observed in the neuron, glia, and microglia of the brain tree, where the overexpression the NOS isoforms causes the formation of a large amount of NO. We hypothesize that since an imbalance between the NOS isoforms and endothelin-1 (ET-1), a human gene that encodes for blood vessel constriction, can cause antioxidant system insufficiency; by using pharmacological intervention with NO donors and/or NO suppressors, the brain lesions and the downstream progression of brain pathology and dementia in AD should be delayed or minimized.

Role of shear stress on nitrite and NOS protein content in different size conduit arteries of swine.

AIM: Inherent fundamental difference exists among arteries of different sizes. The purpose of this study was to evaluate the relation between regional difference of wall shear stress (WSS) in various sizes arteries and contents of nitrite and NO synthase (NOS) isoforms. METHODS: Five different conduit arteries in a wide range of diameter (1-8 mm) were examined in the hind limbs of 13 pigs. Blood flow rate and outer diameter were measured in vivo to determine WSS. Arterial tissues were harvested for the measurement of nitrite and NOS protein contents. The concentration of nitrite, a product of NO synthesis, was determined by high-performance liquid chromatography method. Western blot analysis was used to assess the protein contents of endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS). RESULTS: Our data show that WSS increases with a decrease in artery diameter. Nitrite level increases with increasing WSS and hence decreases with artery diameter. The eNOS protein contents decrease with an increase in diameter. No significant difference for iNOS and nNOS protein contents was found with different artery diameter. A significant positive correlation between tissue nitrite and eNOS protein contents was also observed. Finally, the WSS-normalized eNOS is not significantly different in various size vessels. CONCLUSION: Regional difference in blood flow has no effect on iNOS and nNOS protein contents in these conduit arteries. Regional difference in eNOS expression and nitrite contents may be related to the WSS-induced NO by the endothelium under normal physiological conditions.

Endometrial nitric oxide production and nitric oxide synthases in the equine endometrium: Relationship with microvascular density during the estrous cycle.

Nitric oxide (NO) plays an important role in angiogenesis and in the regulation of the blood flow. This study was carried out to investigate (i) the effects of endogenous estrogens and progestins and exogenous progesterone (P(4)) (5 ng/ml or 1 microg/ml) or estradiol 17beta (E(2)beta) (50 pg/ml or 1 microg/ml) on in vitro endometrial NO synthesis; (ii) the presence of different isoforms of NO synthase; (iii) and their relationship to microvascular density in the equine endometrium during the estrous cycle. NOS expression was also evaluated in the myometrium. Expression of endothelial and inducible forms of NOS in the uterus was assessed by Western blot and immunocytochemistry. Vascular density in endometrial tissue was determined on histologic sections. In the luteal phase, compared to the follicular phase, endometrial NO production increased without exogenous hormones and with exogenous E(2)beta (1 microg/ml). Although immunocytochemistry revealed iNOS and eNOS expression in the endometrium, no positive signal for iNOS was detected by Western blot. Endothelial NOS was observed in endometrial glands, endothelial cells, fibroblasts, blood and lymphatic vessels. Endometrial eNOS expression was the highest in the follicular and mid-luteal phases while it was found to be the lowest in the early luteal phase. In the follicular phase, hyperplasia of endometrial tissue with respect to myometrium was detected. No difference in vascular density was present between phases. All together, NO may play some roles in both proliferative and secretory phases of endometrial development in the mare.

Expression of endothelial nitric oxide synthase and vascular endothelial growth factor in oral squamous cell carcinoma: its correlation with angiogenesis and disease progression.

BACKGROUND: Angiogenesis is a crucial step in the successful growth, invasion, and metastasis of a tumor. It has been popularly accepted that vascular endothelial growth factor (VEGF) is the most potent angiogenic factor in tumor angiogenesis. As another possible star molecule responsible for tumor angiogenesis, the role of nitric oxide (NO) in tumor biology has gained much attention in recent years. The aim of this study was to investigate whether the expression of endothelial nitric oxide synthase (eNOS) and VEGF in oral squamous cell carcinoma (OSCC) is associated with angiogenesis. The present study also made a preliminary exploration of the possible cross-talking existing between eNOS and VEGF during tumor angiogenesis. METHODS: In this study, expression of eNOS and VEGF were studied immunohistochemically in tissue sections from 40 patients with OSCC and 20 normal controls. To exclude eNOS antibody cross-reactivity with inducible or neuronal nitric oxide (iNOS or nNOS), eNOS expression was confirmed by using an eNOS mRNA in situ hybridization kit. The intratumoral microvessels were highlighted by immunostaining with anti-factor VIII-related antigen monoclonal antibody and counted as well-established methods. Then, chi-square test or Student's t-test was performed to study the correlations between the expression of eNOS and VEGF, microvessel density (MVD), and various clinicopathologic factors. RESULTS: Both eNOS and VEGF expression significantly increased in OSCC tissues, with a positive rate of 47.5% and 50%, respectively. The average MVD in OSCC tissues was 23.45 per high-power field (HPF), showing an obvious association with lymph node metastasis and clinical stages of patients with OSCC. Either eNOS or VEGF positivity was correlated with vessel involvement and OSCC progression. The mean MVD was significantly higher in eNOS- or VEGF-positive tumors than in eNOS- or VEGF-negative tumors. An obvious, correlation was also seen between eNOS and VEGF expression in OSCC tissues in this study. CONCLUSIONS: Overexpression of eNOS and VEGF might make an important contribution to the tumor angiogenesis in OSCC. NO generation by eNOS might be implicated in the VEGF-associated angiogenic process. Further investigation of the possible cross-talking between eNOS and VEGF with respect to tumor angiogenesis is necessary.

NOS isoforms in adult human osteocytes: multiple pathways of NO regulation?

Until now, eNOS has been considered to be the predominant osteocytic nitric oxide synthase (NOS) isoform in bone. We previously studied the distribution of eNOS protein expression in the human femoral neck because of its possible involvement in the response to load. Studies in rat and human fracture callus have shown that nNOS mRNA is expressed sometime after fracture, but no study has yet immunolocalized NOS isoforms in mature adult human bone. In this study, we have examined the distribution of NOS isoforms in iliac osteocytes. Frozen sections (10 microm) were cut from transiliac biopsies from 8 female osteoporotic patients (range, 56-80 years) and from 7 female postmortem femoral neck biopsies (range, 65-90 years). Sections were incubated overnight in antiserum for eNOS, nNOS, or iNOS followed by peroxidase/VIP substrate detection. We used eNOS and iNOS antisera directed against the C-terminus. For nNOS, three different antisera were used, two binding to different C-terminal epitopes and one binding to N-terminal epitope. Sections were then incubated in propidium iodide or methyl green to detect all osteocytes. eNOS antibody was able to detect eNOS epitopes in osteocytes. All three nNOS antibodies detected nNOS epitopes in osteocytes, but those directed against the C-terminus had higher detection rates. iNOS was rarely seen. In the iliac crest, the percentage of osteocytes positive for nNOS was higher than that for eNOS (cortical: nNOS 84.04%, eNOS 61.78%, P < 0.05; cancellous: nNOS 82.33%, eNOS 65.21%, P < 0.05). In the femoral neck, the percentage of osteocytes positive for nNOS (60.98%) was also higher than that for eNOS (40.41%), although this difference was not statistically significant. In conclusion, both eNOS and nNOS isoforms are present in osteocytes in the iliac crest and femoral neck.

Nitric oxide synthase expression and enzymatic activity in human brain tumors.

Nitric oxide (NO) is synthesized by NO synthases (NOS), existing in 3 isoforms. NO influences a great variety of vital functions including vascular tone and neurotransmission. Under conditions of excessive formation, NO emerges as an important mediator of neurotoxicity in a variety of disorders of the central nervous system (CNS). Inhibitors of NOS are available that may modify the activity of all isoforms, which may be of clinical relevance. The expression of the 3 NOS isoforms nNOS, iNOS and eNOS and NOS enzymatic activity was examined in 40 patients with primary CNS tumors (gliomas WHO grades I - IV and meningeomas WHO grades I - III) and in 13 patients with metastases from adenocarcinomas or malignant melanomas. A polyclonal antibody directed against nNOS and monoclonal antibodies directed against iNOS and eNOS were used for immunohistochemical staining. NOS enzymatic activity, measured by labeled arginine to citrulline conversion, was assessed in tissue specimens obtained from the same tumors. NOS data were compared with clinical variables and the degree of edema as judged from MR scanning. nNOS expression was increased in tumor cells of glial neoplasms and most pronounced in high-grade tumors, WHO grades III and IV, and in the carcinoma and melanoma metastases. Low-grade gliomas, WHO grades I and II and meningeomas expressed no or only little nNOS. iNOS was only expressed in a few tumors. eNOS was expressed sporadically in the tumor cells while the expression was increased in vascular endothelial cells in both the tumor itself and the peritumoral area of glial neoplasms, and in metastases. eNOS expression was sporadic in endothelial cells of meningeomas. NOS enzymatic activities were heterogeneous among tumor types (0 - 13.8 pmol/min/mg of protein) without correlation to the NOS expression found by immunohistochemical techniques. Likewise, NOS activity and expression was not correlated to the clinical scores or brain edema. In conclusion, nNOS expression may be a putative useful indicator of brain tumor differentiation and malignancy. The enhanced expression of eNOS in vascular endothelial cells of glial neoplasms and metastases raises the possibility that NO production in tumor endothelial cells may contribute to tumor blood flow regulation and possibly brain edema.

Nitric oxide system and protein nitration are modified by an acute hypobaric hypoxia in the adult rat hippocampus.

Changes in the nitric oxide system of the hippocampus from rats submitted to hypobaric hypoxia were investigated. Adult rats were exposed to a simulated altitude of 8,325 m (27,000 ft) for 7 h and killed after 0 h, 1, 3, 5, 10 and 20 days of reoxygenation. The number of neuronal nitric oxide synthase immunoreactive neurons and their dendritic plexus, as well as neuronal nitric oxide synthase immunoblotting densitometry and calcium-dependent activity increased from 0 h to 3 days of reoxygenation. In addition, endothelial nitric oxide synthase immunoreactivity peaked after 7 h of hypobaric hypoxia. Nitrotyrosine immunoreactivity showed an increase in the pyramidal cells of CA2-CA3 and in glial cells surrounding the blood vessels after 0 h, 1 and 3 days of reoxygenation. Immunoblotting densitometry of 1 of the 2 nitrotyrosine-immunoreactive bands detected also increased after 0 h and 1 day of reoxygenation. Inducible nitric oxide synthase immunoreactivity was found only in some blood vessels after 0 h, 1 and 3 days of reoxygenation, but no changes in inducible nitric oxide synthase activity or immunoblotting were detected. We conclude that transient activation of the nitric oxide system constitutes a hippocampal response to hypobaric hypoxia.

Differential biological effects of products of nitric oxide (NO) synthase: it is not enough to say NO.

The radical gas nitric oxide (NO) is implicated in an enormous number of biological function both in physiological and pathological conditions. Often it is not clear if it plays a deleterious or beneficial role. Here briefly, are analyzed some of the reasons of this multitude of effects. Emphasis is given to factors influencing NO formation and to the type and quantity of radicals formed by nitric oxide synthase. In particular, a comparison between the biological effects of nitroxyl anion (HNO/NO(-)) and nitric oxide NO(.) is considered. These redox siblings often exhibit orthogonal behavior in physiological and pathological conditions. In the light of the multitude of effects of NO, the role of this gas, their siblings and their derivatives in cardiac ischemic preconditioning scenario is more extensively analyzed.

Role of nitric-oxide synthase isoforms in nitrous oxide antinociception in mice.

Exposure of mice to the anesthetic gas N2O evokes a prominent antinociceptive effect that is sensitive to antagonism by nonselective nitric-oxide synthase (NOS) inhibitors. The present study was conducted to identify whether a specific NOS isoform is implicated in N2O antinociception in mice. In the abdominal constriction test, exposure of mice to 25, 50, and 70% N2O resulted in a concentration-dependent antinociceptive effect that persisted for up to 6 min following removal of the mice from the N2O atmosphere into room air. This N2O antinociceptive effect was antagonized by pretreatment with S-methyl-l-thiocitrulline (SMTC) and higher doses of l-N5-(1-iminoethyl)-ornithine (l-NIO), which reportedly inhibit the neuronal and endothelial isoforms of NOS, respectively. Nevertheless, the N2O-induced antinociception was unaffected by pretreatment with low doses of either SMTC or l-NIO or by pretreatment with 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), which selectively inhibits inducible NOS. The s.c. pretreatment with SMTC and l-NIO reduced brain NOS activity in a dose-dependent manner, whereas AMT had no such effect. Moreover, in blood pressure experiments, SMTC increased SBP in dose-unrelated fashion, whereas l-NIO showed an appreciably weaker but dose-related increase in SBP. The i.c.v. pretreatment with SMTC also reduced N2O antinociception and brain NOS activity without increasing of SBP. These results suggest that it is the neuronal isoform of NOS that is involved in mediation of the antinociceptive effect of N2O in the mice.

Óxido nítrico exhalado y asma / Exhaled nitric oxide and asthma

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The dynamics of cellular injury: transformation into neuronal and vascular protection.

Despite the immediate event, such as cerebral trauma, cardiac arrest, or stroke that may result in neuronal or vascular injury, specific cellular signal transduction pathways in the central nervous system ultimately influence the extent of cellular injury. Yet, it is a cascade of mechanisms, rather than a single cellular pathway, which determine cellular survival during toxic insults. Although neuronal injury associated with several disease entities, such as Alzheimer's disease, Parkinson's disease, and cerebrovascular disease was initially believed to be irreversible, it has become increasingly evident that either acute or chronic modulation of the cellular and molecular environment within the brain can prevent or even reverse cellular injury. In order to develop rational, efficacious, and safe therapy against neurodegenerative disorders, it becomes vital to elucidate the cellular and molecular mechanisms that control neuronal and vascular injury. These include the pathways of free radical injury, the independent mechanisms of programmed cell death, and the downstream signal transduction pathways of endonuclease activation, intracellular pH, cysteine proteases, the cell cycle, and tyrosine phosphatase activity. Employing the knowledge gained from investigations into these pathways will hopefully further efforts to successfully develop effective treatments against central nervous system disorders.