Uncoupling of nitric oxide synthase (NOS) secondary to redox signaling is a central mechanism in endothelial and macrophage activation. To date studies on the production of nitric oxide (NO) during the development of diabetic complications show paradoxical results. We previously showed that recoupling eNOS by increasing the eNOS cofactor tetrahydrobiopterin (BH4) could restore endothelial function and prevent kidney injury in experimental kidney transplantation. Here, we employed a diabetic mouse model to investigate the effects of diabetes on renal tissue NO bioavailability. For this, we used in vivo NO trapping, followed by electron paramagnetic resonance spectroscopy. In addition, we investigated whether coupling of NOS by supplying the cofactor BH4 could restore glomerular endothelial barrier function. Our data show that overall NO availability at the tissue level is not reduced sixteen weeks after the induction of diabetes in apoE knockout mice, despite the presence of factors that cause endothelial dysfunction, and the presence of the endogenous NOS inhibitor ADMA. Targeting uncoupled NOS with the BH4 precursor sepiapterin further increases NO availability, but did not modify renal glomerular injury. Notably, glomerular heparanase activity as a driver for loss of glomerular barrier function was not reduced, pointing towards NOS-independent mechanisms. This was confirmed by unaltered increased glomerular presence of cathepsin L, the protease that activates heparanase.

Atrasentan, a selective endothelin A receptor antagonist, has been shown to reduce albuminuria in type 2 diabetes. We previously showed that the structural integrity of a glomerular endothelial glycocalyx is required to prevent albuminuria. Therefore we tested the potential of atrasentan to stabilize the endothelial glycocalyx in diabetic apolipoprotein E (apoE)–deficient mice in relation to its antialbuminuric effects. Treatment with atrasentan (7.5 mg/kg/day) for 4 weeks reduced urinary albumin-to-creatinine ratios by 26.0 ± 6.5% (P < 0.01) in apoE knockout (KO) mice with streptozotocin-induced diabetes consuming an atherogenic diet, without changes in gross glomerular morphology, systemic blood pressure, and blood glucose concentration. Endothelial cationic ferritin surface coverage, investigated using large-scale digital transmission electron microscopy, revealed that atrasentan treatment increases glycocalyx coverage in diabetic apoE KO mice from 40.7 ± 3.2% to 81.0 ± 12.5% (P < 0.05). This restoration is accompanied by increased renal nitric oxide concentrations, reduced expression of glomerular heparanase, and a marked shift in the balance of M1 and M2 glomerular macrophages. In vitro experiments with endothelial cells exposed to laminar flow and cocultured with pericytes confirmed that atrasentan reduced endothelial heparanase expression and increased glycocalyx thickness in the presence of a diabetic milieu. Together these data point toward a role for the restoration of endothelial function and tissue homeostasis through the antialbuminuric effects of atrasentan, and they provide a mechanistic explanation for the clinical observations of reduced albuminuria with atrasentan in diabetic nephropathy.

•In ischemia copper dissociates from its carrier proteins, i.e., albumin.•Free Cu2+ ions are reduced to Cu1+ by ascorbate, Fe2+ and/or GSH.•Copper-catalyzed NO formation from nitrite exerts biological activity.•Cu1+-catalyzed NO formation represents a new protective principle to ischemic stress.

Human skin contains photolabile nitric oxide (NO) derivates such as nitrite and S-nitrosothiols, which upon UVA radiation decompose under high-output NO formation and exert NO-specific biological responses such as increased local blood flow or reduced blood pressure. To avoid the injurious effects of UVA radiation, we here investigated the mechanism and biological relevance of blue-light (420-453 nm)-induced nonenzymatic NO generation from photolabile nitric oxide derivates in human skin in vitro and in vivo. As quantified by chemiluminescence detection (CLD), at physiological pH blue light at 420 or 453 nm induced a significant NO formation from S-nitrosoalbumin and also from aqueous nitrite solutions by a to-date not entirely identified Cu(1+)-dependent mechanism. As detected by electron paramagnetic resonance spectrometry in vitro with human skin specimens, blue light irradiation significantly increased the intradermal levels of free NO. As detected by CLD in vivo in healthy volunteers, irradiation of human skin with blue light induced a significant emanation of NO from the irradiated skin area as well as a significant translocation of NO from the skin surface into the underlying tissue. In parallel, blue light irradiation caused a rapid and significant rise in local cutaneous blood flow as detected noninvasively by using micro-light-guide spectrophotometry. Irradiation of human skin with moderate doses of blue light caused a significant increase in enzyme-independent cutaneous NO formation as well as NO-dependent local biological responses, i.e., increased blood flow. The effects were attributed to blue-light-induced release of NO from cutaneous photolabile NO derivates. Thus, in contrast to UVA, blue-light-induced NO generation might be therapeutically used in the treatment of systemic and local hemodynamic disorders that are based on impaired physiological NO production or bioavailability.

Endothelial nitric oxide synthase (NOS)3-derived nitric oxide (NO) modulates inotropic response and diastolic interval for optimal cardiac performance under non-inflammatory conditions. In sepsis, excessive NO production plays a key role in severe hypotension and myocardial dysfunction. We aimed to determine the role of NOS3 on myocardial performance, NO production, and time course of sepsis development. NOS3(-/-) and C57BL/6 wildtype mice were rendered septic by cecum ligation and puncture (CLP). Cardiac function was analyzed by serial echocardiography, in vivo pressure and isolated heart measurements. Cardiac output (CO) increased to 160 % of baseline at 10 h after sepsis induction followed by a decline to 63 % of baseline after 18 h in wildtype mice. CO was unaltered in septic NOS3(-/-) mice. Despite the hyperdynamic state, cardiac function and mean arterial pressure were impaired in septic wildtype as early as 6 h post CLP. At 12 h, cardiac function in septic wildtype was refractory to catecholamines in vivo and respective isolated hearts showed impaired pressure development and limited coronary flow reserve. Hemodynamics remained stable in NOS3(-/-) mice leading to significant survival benefit. Unselective NOS inhibition in septic NOS3(-/-) mice diminished this survival benefit. Plasma NOx- and local myocardial NOx- and NO levels (via NO spin trapping) demonstrated enhanced NOx- and bioactive NO levels in septic wildtype as compared to NOS3(-/-) mice. Significant contribution by inducible NOS (NOS2) during this early phase of sepsis was excluded. Our data suggest that NOS3 relevantly contributes to bioactive NO pool in developing sepsis resulting in impaired cardiac contractility.

Nitric oxide (NO) has been implicated in matrix metallopeptidase 9 (MMP9)-dependent mobilization of hematopoietic stem and progenitor cells from bone marrow (BM). However, direct measurement of NO in the BM remained elusive due to its low in situ concentration and short lifetime. Using NO spin trapping and electron paramagnetic resonance (EPR) spectroscopy we give the first experimental confirmation of free NO radicals in rodent BM. NO production was quantified and attributed to enzymatic activity of NO synthases (NOS). Although endothelial NOS (eNOS) accounts for most (66%) of basal NO, we identified a significant contribution (23%) from inducible NOS (iNOS). Basal NO levels closely correlate with MMP9 bioavailability in BM of both hypertensive and control rats. Our observations support the hypothesis that inadequate mobilization of BM-derived stem and progenitor cells in hypertension results from impaired NOS/NO/MMP9 signalling in BM, a condition that may be corrected with pharmacological intervention.

Many local hemodynamic and vascular disorders may be the result of impaired bioavailability of nitric oxide (NO). Previous findings point to a therapeutic potential of dermal NO application in the treatment of hemodynamic disorders, but no reliable data are available on the mechanisms, kinetics, or biological responses relating to cutaneous exposure to NO in humans in vivo. Here we show that, owing to its excellent diffusion capacity, cutaneously applied NO rapidly penetrates the epidermal barrier in significant amounts, strongly enriching skin tissue and blood plasma with its vasoactive derivates. In parallel, it significantly increased vasodilatation and blood flow and reduced thrombocyte aggregation capacity. Data presented here for the first time show that, in humans, dermal application of NO has strong potential for use in the therapy of local hemodynamic disorders arising from insufficient availability of NO or its bioactive derivates.

Vascular ischemic diseases, hypertension, and other systemic hemodynamic and vascular disorders may be the result of impaired bioavailability of nitric oxide (NO). NO but also its active derivates like nitrite or nitroso compounds are important effector and signal molecules with vasodilating properties. Our previous findings point to a therapeutical potential of cutaneous administration of NO in the treatment of systemic hemodynamic disorders. Unfortunately, no reliable data are available on the mechanisms, kinetics and biological responses of dermal application of nitric oxide in humans in vivo. The aim of the study was to close this gap and to explore the therapeutical potential of dermal nitric oxide application. We characterized with human skin in vitro and in vivo the capacity of NO, applied in a NO-releasing acidified form of nitrite-containing liniments, to penetrate the epidermis and to influence local as well as systemic hemodynamic parameters. We found that dermal application of NO led to a very rapid and significant transepidermal translocation of NO into the underlying tissue. Depending on the size of treated skin area, this translocation manifests itself through a significant systemic increase of the NO derivates nitrite and nitroso compounds, respectively. In parallel, this translocation was accompanied by an increased systemic vasodilatation and blood flow as well as reduced blood pressure. We here give evidence that in humans dermal application of NO has a therapeutic potential for systemic hemodynamic disorders that might arise from local or systemic insufficient availability of NO or its bio-active NO derivates, respectively.

Background Impaired microcirculation during endotoxemia correlates with a disturbed arginine-nitric oxide (NO) metabolism and is associated with deteriorating organ function. Improving the organ perfusion in endotoxemia, as often seen in patients with severe infection or systemic inflammatory response syndrome (SIRS) is, therefore, an important therapeutic target. We hypothesized that supplementation of the arginine precursor citrulline rather than arginine would specifically increase eNOS-induced intracellular NO production and thereby improve the microcirculation during endotoxemia. Methodology/Principal Findings To study the effects of L-Citrulline and L-Arginine supplementation on jejunal microcirculation, intracellular arginine availability and NO production in a non-lethal prolonged endotoxemia model in mice. C57/Bl6 mice received an 18 hrs intravenous infusion of endotoxin (LPS, 0.4 µg•g bodyweight−1•h−1), combined with either L-Citrulline (6.25 mg•h-1), L-Arginine (6.25 mg•h−1), or L-Alanine (isonitrogenous control; 12.5 mg•h−1) during the last 6 hrs. The control group received an 18 hrs sterile saline infusion combined with L-Alanine or L-Citrulline during the last 6 hrs. The microcirculation was evaluated at the end of the infusion period using sidestream dark-field imaging of jejunal villi. Plasma and jejunal tissue amino-acid concentrations were measured by HPLC, NO tissue concentrations by electron-spin resonance spectroscopy and NOS protein concentrations using Western blot. Conclusion/Significance L-Citrulline supplementation during endotoxemia positively influenced the intestinal microvascular perfusion compared to L-Arginine-supplemented and control endotoxemic mice. L-Citrulline supplementation increased plasma and tissue concentrations of arginine and citrulline, and restored intracellular NO production in the intestine. L-Arginine supplementation did not increase the intracellular arginine availability. Jejunal tissues in the L-Citrulline-supplemented group showed, compared to the endotoxemic and L-Arginine-supplemented endotoxemic group, an increase in degree of phosphorylation of eNOS (Ser 1177) and a decrease in iNOS protein level. In conclusion, L-Citrulline supplementation during endotoxemia and not L-Arginine reduced intestinal microcirculatory dysfunction and increased intracellular NO production, likely via increased intracellular citrulline and arginine availability.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionORIGINAL ARTICLEThis notice is a correctionCorrection to NCN-Pincer Metal Complexes (Ti, Cr, V, Zr, Hf, and Nb) of the Phebox Ligand (S,S)-2,6-Bis(4′-isopropyl-2′-oxazolinyl)phenylAlexey V. Chuchuryukin, Rubin Huang, Martin Lutz, John C. Chadwick, Ernst E. van Faassen, Anthony L. Spek, and Gerard van Koten*Cite this: Organometallics 2011, 30, 24, 6782Publication Date (Web):November 23, 2011Publication History Published online23 November 2011Published inissue 26 December 2011https://pubs.acs.org/doi/10.1021/om201138phttps://doi.org/10.1021/om201138pcorrectionACS PublicationsCopyright © 2011 American Chemical Society. This publication is available under these Terms of Use. Request reuse permissions This publication is free to access through this site. Learn MoreArticle Views655Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail PDF (65 KB) Get e-Alertsclose Get e-Alerts

Transmetallation of 4,4'-bis{(2,6-bis[(dimethylamino)methyl]phenylgold)diphenyl-phosphino}biphenyl (3) with MCl(4) (M = Ti, NbCl, V) in benzene gave the corresponding transition metal pincer complexes (4) and insoluble 4,4'-bis[P-(chloro gold(I))diphenylphosphino]biphenyl (2), which can be quantitatively recovered and recycled. Interestingly, 3 did not react with TiCl(3). However, reaction of 2,6-bis[(dimethylamino)methyl]phenyllithium (1) with TiCl(3) resulted in formation of the novel diaryltitanium(IV) compound 5 (16% yield), comprising one N,C,N-mer bound NCN-pincer ligand and a second NCN-pincer ligand that is rearranged from a 1,2,6-isomer to a 1,2,4 one. The latter NCN-ligand is dianionic and is bidentate bonded; one of the CH(2)NMe(2) substituents (para to C'(ipso)) is non-coordinated, while the second CH(2)NMe(2) group, after C-H activation of one of the Me groups, is η(2)-C,N-bonded to the titanium centre trans to C(ipso) of the mer-NCN ligand. The new NCN-pincer metal complexes 2,6-bis[(dimethylamino)methyl]phenylTiCl(3) (4a) and 2,6-bis[(dimethylamino)methyl]-phenylVCl(2) (4d) gave, after immobilization on MgCl(2)-based supports, very high activity in ethene polymerisation.

Nitric oxide (NO) in human skin has been under investigation since first reports of NOS expression in skin tissue in 1992 [1]. NO plays a key role in the dermal response to external stimuli such as heat, ultraviolet (UV) light, or infection, and in healing of abrasions, lesions or burns. Recently, a range of non-enzymatic pathways for NO release has been identified, mostly in the context of systemic blood flow. In this article we consider the non-enzymatic formation of NO in human skin tissues. Significant quantities of NO are continuously released from human skin into the ambient air. This release can be significantly enhanced by photolysis of endogenous NO stores under UVA. In addition, we give the first estimate of the basal enzymatic NO production in healthy human skin.

Exogenous gaseous nitric oxide (gNO) is an FDA approved drug for treatment of a variety of human pathologies like Persistent Pulmonary Hypertension in neonates and premature babies, skin lesions and fungal dermatophyte infections. Substantial disadvantages of current gNO-based therapies are the high therapy costs, high storage costs of the gas cylinders, and the rapid contamination of compressed NO gases with various decomposition products. Here we describe a new, very simple, and inexpensive photolytic generator of uncontaminated NO-containing gas mixtures at therapeutic concentrations. The new method bases on UVA-induced and redox-assisted decomposition of nitrite ions in aqueous solutions. NO formation via UVA-induced photolysis of nitrite is accompanied by an OH radical-dependent production of NO(2) that beside its toxic character additionally strongly reduces the NO yield by consuming NO in its reaction to N(2)O(3). During the UVA-induced photodecomposition process both, inhibition of NO(2) formation or NO(2) depletion by antioxidants hinders the NO-consuming reaction with NO(2) and ensured a maximal purity and maximal yield of NO-containing gas mixtures. Therefore, NO-containing gas mixtures generated by the described method are suitable for medical applications like inhalation or gassing of chronic non-healing wounds. Control of temperature, UVA intensity and composition of the reaction mixture allows facile control over the final NO level in the carrier gas over a wide concentration range. We demonstrate the sustained and stable release of NO over a wide dynamic range (10-5000 ppm NO) for many hours. The method avoids contamination-prone long time storage of NO gas. As such, it appears particularly relevant for applications involving the additional presence of oxygen (e.g. inhalation).

Mutations in ATP8B1 cause familial intrahepatic cholestasis type 1, a spectrum of disorders characterized by intrahepatic cholestasis, reduced growth, deafness, and diarrhea. ATP8B1 belongs to the P4 P-type adenosine triphosphatase (ATPase) family of putative aminophospholipid translocases, and loss of aminophospholipid asymmetry in the canalicular membranes of ATP8B1-deficient liver cells has been proposed as the primary cause of impaired bile salt excretion. To explore the origin of the hepatic and extrahepatic symptoms associated with ATP8B1 deficiency, we investigated the impact of ATP8B1 depletion on the domain-specific aminophospholipid translocase activities and polarized organization of polarized epithelial Caco-2 cells. Caco-2 cells were stably transfected with short hairpin RNA constructs to block ATP8B1 expression. Aminophospholipid translocase activity was assessed using spin-labeled phospholipids. The polarized organization of these cells was determined by pulse-chase analysis, cell-fractionation, immunocytochemistry, and transmission electron microscopy. ATP8B1 was abundantly expressed in the apical membrane of Caco-2 cells, and its expression was markedly induced during differentiation and polarization. Blocking ATP8B1 expression by RNA interference (RNAi) affected neither aminophospholipid transport nor the asymmetrical distribution of aminophospholipids across the apical bilayer. Nonetheless, ATP8B1-depleted Caco-2 cells displayed profound perturbations in apical membrane organization, including a disorganized apical actin cytoskeleton, a loss in microvilli, and a posttranscriptional defect in apical protein expression. Conclusion: Our findings point to a critical role of ATP8B1 in apical membrane organization that is unrelated to its presumed aminophospholipid translocase activity, yet potentially relevant for the development of cholestasis and the manifestation of extrahepatic features associated with ATP8B1 deficiency. (HEPATOLOGY 2010)