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1.
Surg Endosc ; 21(8): 1354-8, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17180264

ABSTRACT

BACKGROUND: We have previously shown that mixing the S-nitrosylating agent ethyl nitrite with carbon dioxide can attenuate pneumoperitoneum-induced decreases in splanchnic blood flow, but it was unclear if this agent would alter gastric function. This question was answered using rats by assessing gastric emptying and gastrointestinal transit times following gavage with radioactive chromium. METHODS: There were five experimental groups: absolute control, anesthesia control, and carbon dioxide alone or with 100 or 300 parts per million ethyl nitrite. The period of insufflation was 1 h, and all animals were euthanized 6.5 h after chromium administration. RESULTS: The mean amount of radioactivity remaining in the stomach ranged between 16% and 27% of the total administered; these differences were not statistically significant (p > 0.05). Modest differences in chromium distribution were identified in the gastrointestinal tract, but for all treatments, the peak amount of radioactivity was located in the distal portion. Location of the peak, expressed as a percentage of total tract length, varied between 70% and 85% (p = 0.366). CONCLUSIONS: This study found no adverse effect of ethyl nitrite on postoperative gastric emptying or gastrointestinal transit time following pneumoperitoneum. The findings support continued assessment of the clinical utility of ethyl nitrite in the setting of laparoscopic surgery.


Subject(s)
Gastric Emptying/drug effects , Gastrointestinal Transit/drug effects , Nitrites/pharmacology , Pneumoperitoneum, Artificial , Animals , Carbon Dioxide/pharmacology , Chromium Radioisotopes , Gases , Male , Rats , Rats, Sprague-Dawley
5.
Proc Natl Acad Sci U S A ; 98(20): 11158-62, 2001 Sep 25.
Article in English | MEDLINE | ID: mdl-11562475

ABSTRACT

We have shown previously that at physiologically relevant oxygen tension (pO(2) approximately 10 mmHg), NO S-nitrosylates 1 of approximately 50 free cysteines per ryanodine receptor 1 (RyR1) subunit and transduces a calcium-sensitizing effect on the channel by means of calmodulin (CaM). It has been suggested that cysteine-3635 is part of a CaM-binding domain, and its reactivity is attenuated by CaM [Porter Moore, C., Zhang, J. Z., Hamilton, S. L. (1999) J. Biol. Chem. 274, 36831-36834]. Therefore, we tested the hypothesis that the effect of NO was mediated by C3635. The full-length RyR1 single-site C3635A mutant was generated and expressed in HEK293 cells. The mutation resulted in the loss of CaM-dependent NO modulation of channel activity and reduced S-nitrosylation by NO to background levels but did not affect NO-independent channel modulation by CaM or the redox sensitivity of the channel to O(2) and glutathione. Our results reveal that different cysteines within the channel have been adapted to serve in nitrosative and oxidative responses, and that S-nitrosylation of the cysteine-containing CaM-binding domain underlies the mechanism of CaM-dependent regulation of RyR1 by NO.


Subject(s)
Calmodulin/metabolism , Cysteine , Muscle, Skeletal/metabolism , Nitric Oxide/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Ryanodine/metabolism , Amino Acid Substitution , Animals , Binding Sites , Cell Line , Glutathione/pharmacology , Humans , Intracellular Membranes/metabolism , Microsomes/metabolism , Mutagenesis, Site-Directed , Nitric Oxide/pharmacology , Oxidation-Reduction , Oxygen/pharmacology , Rabbits , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Ryanodine Receptor Calcium Release Channel/chemistry , Ryanodine Receptor Calcium Release Channel/drug effects , Sarcoplasmic Reticulum/metabolism , Transfection
6.
Am J Physiol Cell Physiol ; 281(3): C849-56, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11502562

ABSTRACT

This study examined mRNA and protein expressions of neuronal (nNOS), inducible (iNOS), and endothelial nitric oxide synthases (eNOS) in peripheral nerve after ischemia-reperfusion (I/R). Sixty-six rats were divided into the ischemia only and I/R groups. One sciatic nerve of each animal was used as the experimental side and the opposite untreated nerve as the control. mRNA levels in the nerve were quantitatively measured by competitive PCR, and protein was determined by Western blotting and immunohistochemical staining. The results showed that, after ischemia (2 h), both nNOS and eNOS protein expressions decreased. After I/R (2 h of ischemia followed by 3 h of reperfusion), expression of both nNOS and eNOS mRNA and protein decreased further. In contrast, iNOS mRNA significantly increased after ischemia and was further upregulated (14-fold) after I/R, while iNOS protein was not detected. The results reveal the dynamic expression of individual NOS isoforms during the course of I/R injury. An understanding of this modulation on a cellular and molecular level may lead to understanding the mechanisms of I/R injury and to methods of ameliorating peripheral nerve injury.


Subject(s)
Gene Expression Regulation, Enzymologic/physiology , Ischemia/enzymology , Nitric Oxide Synthase/genetics , Sciatic Nerve/blood supply , Sciatic Nerve/enzymology , Animals , Blotting, Western , DNA Primers , Immunohistochemistry , In Vitro Techniques , Ischemia/genetics , Male , Nitric Oxide Synthase/analysis , Nitric Oxide Synthase Type I , Nitric Oxide Synthase Type II , Nitric Oxide Synthase Type III , Polymerase Chain Reaction , Protein Biosynthesis , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Reperfusion , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic
7.
Proc Natl Acad Sci U S A ; 98(18): 10108-12, 2001 Aug 28.
Article in English | MEDLINE | ID: mdl-11517313

ABSTRACT

We have previously reported that bacterial flavohemoglobin (HMP) catalyzes both a rapid reaction of heme-bound O(2) with nitric oxide (NO) to form nitrate [HMP-Fe(II)O(2) + NO --> HMP-Fe(III) + NO(3)(-)] and, under anaerobic conditions, a slower reduction of heme-bound NO to an NO(-) equivalent (followed by the formation of N(2)O), thereby protecting against nitrosative stress under both aerobic and anaerobic conditions, and rationalizing our finding that NO is rapidly consumed across a wide range of O(2) concentrations. It has been alternatively suggested that HMP activity is inhibited at low pO(2) because the enzyme is then in the relatively inactive nitrosyl form [k(off)/k(on) for NO (0.000008 microM) k(off)/k(on) for O(2) (0.012 microM) and K(M) for O(2) = 30-100 microM]. To resolve this discrepancy, we have directly measured heme-ligand turnover and NADH consumption under various O(2)/NO concentrations. We find that, at biologically relevant O(2) concentrations, HMP preferentially binds NO (not O(2)), which it then reacts with oxygen to form nitrate (in essence NO(-) + O(2) --> NO(3)(-)). During steady-state turnover, the enzyme can be found in the ferric (FeIII) state. The formation of a heme-bound nitroxyl equivalent and its subsequent oxidation is a novel enzymatic function, and one that dominates the oxygenase activity under biologically relevant conditions. These data unify the mechanism of HMP/NO interaction with those recently described for the nematode Ascaris and mammalian hemoglobins, and more generally suggest that the peroxidase (FeIII)-like properties of globins have evolved for handling of NO.


Subject(s)
Bacterial Proteins/metabolism , Dihydropteridine Reductase , Escherichia coli Proteins , Hemeproteins/metabolism , Hemoglobins/metabolism , NADH, NADPH Oxidoreductases , Oxygenases/metabolism , Binding, Competitive , Hemeproteins/genetics , Kinetics , Ligands , NAD/metabolism , Nitrogen Oxides/metabolism , Oxygen/metabolism , Oxygenases/genetics , Substrate Specificity
8.
Biochemistry ; 40(6): 1688-93, 2001 Feb 13.
Article in English | MEDLINE | ID: mdl-11327828

ABSTRACT

It is not clear if redox regulation of transcription is the consequence of direct redox-related modifications of transcription factors, or if it occurs at some other redox-sensitive step. One obstacle has been the inability to demonstrate redox-related modifications of transcription factors in vivo. The redox-sensitive transcriptional activator NF-kappaB (p50-p65) is a case in point. Its activity in vitro can be inhibited by S-nitrosylation of a critical thiol in the DNA-interacting p50 subunit, but modulation of NF-kappaB activity by nitric oxide synthase (NOS) has been attributed to other mechanisms. Herein we show that cellular NF-kappaB activity is in fact regulated by S-nitrosylation. We observed that both S-nitrosocysteine and cytokine-activated NOS2 inhibited NF-kappaB in human respiratory cells or murine macrophages. This inhibition was reversed by addition of the denitrosylating agent dithiothreitol to cellular extracts, whereas NO bioactivity did not affect the TNFalpha-induced degradation of IkappaBalpha or the nuclear translocation of p65. Recapitulation of these conditions in vitro resulted in S-nitrosylation of recombinant p50, thereby inhibiting its binding to DNA, and this effect was reversed by dithiothreitol. Further, an increase in S-nitrosylated p50 was detected in cells, and the level was modulated by TNFalpha. Taken together, these data suggest that S-nitrosylation of p50 is a physiological mechanism of NF-kappaB regulation.


Subject(s)
Cysteine/analogs & derivatives , Cysteine/metabolism , I-kappa B Proteins , Mercaptoethanol , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Nitroso Compounds/metabolism , S-Nitrosothiols , Active Transport, Cell Nucleus/drug effects , Animals , Cell Line , Cysteine/pharmacology , Cytokines/pharmacology , DNA/antagonists & inhibitors , DNA/metabolism , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Down-Regulation/drug effects , Enzyme Inhibitors/pharmacology , Humans , Mice , NF-KappaB Inhibitor alpha , NF-kappa B/genetics , NF-kappa B/isolation & purification , NF-kappa B/physiology , NF-kappa B p50 Subunit , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Nitrosation/drug effects , Nitroso Compounds/pharmacology , Protein Binding/drug effects , Transcription, Genetic/drug effects , Transfection
9.
Proc Natl Acad Sci U S A ; 98(10): 5792-7, 2001 May 08.
Article in English | MEDLINE | ID: mdl-11320213

ABSTRACT

NO synthases are widely distributed in the lung and are extensively involved in the control of airway and vascular homeostasis. It is recognized, however, that the O(2)-rich environment of the lung may predispose NO toward toxicity. These Janus faces of NO are manifest in recent clinical trials with inhaled NO gas, which has shown therapeutic benefit in some patient populations but increased morbidity in others. In the airways and circulation of humans, most NO bioactivity is packaged in the form of S-nitrosothiols (SNOs), which are relatively resistant to toxic reactions with O(2)/O(2)(-). This finding has led to the proposition that channeling of NO into SNOs may provide a natural defense against lung toxicity. The means to selectively manipulate the SNO pool, however, has not been previously possible. Here we report on a gas, O-nitrosoethanol (ENO), which does not react with O(2) or release NO and which markedly increases the concentration of indigenous species of SNO within airway lining fluid. Inhalation of ENO provided immediate relief from hypoxic pulmonary vasoconstriction without affecting systemic hemodynamics. Further, in a porcine model of lung injury, there was no rebound in cardiopulmonary hemodynamics or fall in oxygenation on stopping the drug (as seen with NO gas), and additionally ENO protected against a decline in cardiac output. Our data suggest that SNOs within the lung serve in matching ventilation to perfusion, and can be manipulated for therapeutic gain. Thus, ENO may be of particular benefit to patients with pulmonary hypertension, hypoxemia, and/or right heart failure, and may offer a new therapeutic approach in disorders such as asthma and cystic fibrosis, where the airways may be depleted of SNOs.


Subject(s)
Lung/physiology , Mercaptoethanol , Nitric Oxide/administration & dosage , Nitroso Compounds/metabolism , S-Nitrosothiols , Administration, Inhalation , Animals , Animals, Newborn , Gas Chromatography-Mass Spectrometry , Hypertension, Pulmonary/chemically induced , Respiratory Function Tests , Swine
10.
Biochemistry ; 40(16): 4904-10, 2001 Apr 24.
Article in English | MEDLINE | ID: mdl-11305905

ABSTRACT

Nitric oxide (NO) and related molecules play important roles in vascular biology. NO modifies proteins through nitrosylation of free cysteine residues, and such modifications are important in mediating NO's biologic activity. Tissue transglutaminase (tTG) is a sulfhydryl rich protein that is expressed by endothelial cells and secreted into the extracellular matrix (ECM) where it is bound to fibronectin. Tissue TG exhibits a Ca(2+)-dependent transglutaminase activity (TGase) that cross-links proteins involved in wound healing, tissue remodeling, and ECM stabilization. Since tTG is in proximity to sites of NO production, has 18 free cysteine residues, and utilizes a cysteine for catalysis, we investigated the factors that regulated NO binding and tTG activity. We report that TGase activity is regulated by NO through a unique Ca(2+)-dependent mechanism. Tissue TG can be poly-S-nitrosylated by the NO carrier, S-nitrosocysteine (CysNO). In the absence of Ca(2+), up to eight cysteines were nitrosylated without modifying TGase activity. In the presence of Ca(2+), up to 15 cysteines were found to be nitrosylated and this modification resulted in an inhibition of TGase activity. The addition of Ca(2+) to nitrosylated tTG was able to trigger the release of NO groups (i.e. denitrosylation). tTG nitrosylated in the absence of Ca(2+) was 6-fold more susceptible to inhibition by Mg-GTP. When endothelial cells in culture were incubated with tTG and stimulated to produce NO, the exogenous tTG was S-nitrosylated. Furthermore, S-nitrosylated tTG inhibited platelet aggregation induced by ADP. In conclusion, we provide evidence that Ca(2+) regulates the S-nitrosylation and denitrosylation of tTG and thereby TGase activity. These data suggest a novel allosteric role for Ca(2+) in regulating the inhibition of tTG by NO and a novel function for tTG in dispensing NO bioactivity.


Subject(s)
Calcium/physiology , GTP-Binding Proteins/metabolism , Mercaptoethanol , Nitric Oxide/metabolism , Phosphorylcholine/analogs & derivatives , S-Nitrosothiols , Sphingosine/analogs & derivatives , Transglutaminases/metabolism , Adenosine Diphosphate/physiology , Adenosine Triphosphate/pharmacology , Animals , Cations, Divalent/pharmacology , Cattle , Cells, Cultured , Cysteine/analogs & derivatives , Cysteine/pharmacology , Endothelium, Vascular/enzymology , Endothelium, Vascular/metabolism , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , GTP-Binding Proteins/antagonists & inhibitors , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/genetics , Guanosine Triphosphate/pharmacology , Guinea Pigs , Humans , Kinetics , Nitroso Compounds/metabolism , Nitroso Compounds/pharmacology , Phosphorylcholine/metabolism , Platelet Aggregation , Protein Conformation , Protein Glutamine gamma Glutamyltransferase 2 , Recombinant Proteins/metabolism , Sphingosine/metabolism , Transglutaminases/antagonists & inhibitors , Transglutaminases/chemistry , Transglutaminases/genetics
11.
Nature ; 410(6827): 490-4, 2001 Mar 22.
Article in English | MEDLINE | ID: mdl-11260719

ABSTRACT

Considerable evidence indicates that NO biology involves a family of NO-related molecules and that S-nitrosothiols (SNOs) are central to signal transduction and host defence. It is unknown, however, how cells switch off the signals or protect themselves from the SNOs produced for defence purposes. Here we have purified a single activity from Escherichia coli, Saccharomyces cerevisiae and mouse macrophages that metabolizes S-nitrosoglutathione (GSNO), and show that it is the glutathione-dependent formaldehyde dehydrogenase. Although the enzyme is highly specific for GSNO, it controls intracellular levels of both GSNO and S-nitrosylated proteins. Such 'GSNO reductase' activity is widely distributed in mammals. Deleting the reductase gene in yeast and mice abolishes the GSNO-consuming activity, and increases the cellular quantity of both GSNO and protein SNO. Furthermore, mutant yeast cells show increased susceptibility to a nitrosative challenge, whereas their resistance to oxidative stress is unimpaired. We conclude that GSNO reductase is evolutionarily conserved from bacteria to humans, is critical for SNO homeostasis, and protects against nitrosative stress.


Subject(s)
Aldehyde Oxidoreductases/metabolism , Glutathione/analogs & derivatives , Mercaptoethanol , Nitroso Compounds/metabolism , S-Nitrosothiols , Aldehyde Oxidoreductases/genetics , Amino Acid Sequence , Animals , Cell Line , Conserved Sequence , Escherichia coli/enzymology , Escherichia coli/genetics , Evolution, Molecular , Humans , Macrophages , Mice , Molecular Sequence Data , Nitric Oxide , Nitro Compounds , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Substrate Specificity
12.
J Biol Chem ; 276(19): 15625-30, 2001 May 11.
Article in English | MEDLINE | ID: mdl-11278999

ABSTRACT

The skeletal muscle Ca(2+) release channel/ryanodine receptor (RyR1) is a prototypic redox-responsive ion channel. Nearly half of the 101 cysteines per RyR1 subunit are kept in a reduced (free thiol) state under conditions comparable with resting muscle. Here we assessed the effects of physiological determinants of cellular redox state (oxygen tension, reduced (GSH) or oxidized (GSSG) glutathione, and NO/O(2) (released by 3-morpholinosydnonimine)) on RyR1 redox state and activity. Oxidation of approximately 10 RyR1 thiols (from approximately 48 to approximately 38 thiols/RyR1 subunit) had little effect on channel activity. Channel activity increased reversibly as the number of thiols was further reduced to approximately 23/subunit, whereas more extensive oxidation (to approximately 13 thiols/subunit) inactivated the channel irreversibly. Neither S-nitrosylation nor tyrosine nitration contributed to these effects. The results identify at least three functional classes of RyR1 thiols and suggest that 1) the channel may be protected from oxidation by a large reservoir of functionally inert thiols, 2) the channel may be designed to respond to moderate oxidative stress by a change in activation setpoint, and 3) the channel is susceptible to oxidative injury under more extensive conditions.


Subject(s)
Glutathione/metabolism , Molsidomine/pharmacology , Muscle, Skeletal/physiology , Nitric Oxide Donors/pharmacology , Ryanodine Receptor Calcium Release Channel/physiology , Sarcoplasmic Reticulum/physiology , Sulfhydryl Compounds/metabolism , Animals , Glutathione Disulfide/metabolism , Kinetics , Molsidomine/analogs & derivatives , Nitric Oxide/metabolism , Oxidation-Reduction , Protein Subunits , Rabbits , Ryanodine/pharmacokinetics , Ryanodine Receptor Calcium Release Channel/drug effects , Superoxides/metabolism
14.
Nature ; 409(6820): 622-6, 2001 Feb 01.
Article in English | MEDLINE | ID: mdl-11214321

ABSTRACT

Previous studies support a model in which the physiological O2 gradient is transduced by haemoglobin into the coordinate release from red blood cells of O2 and nitric oxide (NO)-derived vasoactivity to optimize oxygen delivery in the arterial periphery. But whereas both O2 and NO diffuse into red blood cells, only O2 can diffuse out. Thus, for the dilation of blood vessels by red blood cells, there must be a mechanism to export NO-related vasoactivity, and current models of NO-mediated intercellular communication should be revised. Here we show that in human erythrocytes haemoglobin-derived S-nitrosothiol (SNO), generated from imported NO, is associated predominantly with the red blood cell membrane, and principally with cysteine residues in the haemoglobin-binding cytoplasmic domain of the anion exchanger AE1. Interaction with AE1 promotes the deoxygenated structure in SNO-haemoglobin, which subserves NO group transfer to the membrane. Furthermore, we show that vasodilatory activity is released from this membrane precinct by deoxygenation. Thus, the oxygen-regulated cellular mechanism that couples the synthesis and export of haemoglobin-derived NO bioactivity operates, at least in part, through formation of AE1-SNO at the membrane-cytosol interface.


Subject(s)
Erythrocytes/metabolism , Mercaptoethanol , Nitric Oxide/metabolism , S-Nitrosothiols , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Animals , Antiporters/metabolism , Aorta , Biological Transport , Chloride-Bicarbonate Antiporters , Erythrocyte Membrane/drug effects , Erythrocyte Membrane/metabolism , Hemoglobins/metabolism , Humans , In Vitro Techniques , Nitroso Compounds/metabolism , Rabbits , Vasoconstriction
15.
Physiol Rev ; 81(1): 209-237, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11152758

ABSTRACT

In the past five years, skeletal muscle has emerged as a paradigm of "nitric oxide" (NO) function and redox-related signaling in biology. All major nitric oxide synthase (NOS) isoforms, including a muscle-specific splice variant of neuronal-type (n) NOS, are expressed in skeletal muscles of all mammals. Expression and localization of NOS isoforms are dependent on age and developmental stage, innervation and activity, history of exposure to cytokines and growth factors, and muscle fiber type and species. nNOS in particular may show a fast-twitch muscle predominance. Muscle NOS localization and activity are regulated by a number of protein-protein interactions and co- and/or posttranslational modifications. Subcellular compartmentalization of the NOSs enables distinct functions that are mediated by increases in cGMP and by S-nitrosylation of proteins such as the ryanodine receptor-calcium release channel. Skeletal muscle functions regulated by NO or related molecules include force production (excitation-contraction coupling), autoregulation of blood flow, myocyte differentiation, respiration, and glucose homeostasis. These studies provide new insights into fundamental aspects of muscle physiology, cell biology, ion channel physiology, calcium homeostasis, signal transduction, and the biochemistry of redox-related systems.


Subject(s)
Drosophila Proteins , Muscle, Skeletal/metabolism , Nitric Oxide/metabolism , Animals , Carrier Proteins/metabolism , Caveolin 3 , Caveolins/metabolism , Cell Differentiation/drug effects , Cell Respiration/physiology , Dyneins , Glucose/metabolism , Humans , Isoenzymes/classification , Isoenzymes/metabolism , Mammals , Membrane Potentials/physiology , Muscle Contraction/drug effects , Muscle Contraction/physiology , Muscle, Skeletal/blood supply , Muscle, Skeletal/drug effects , Muscular Dystrophy, Duchenne/metabolism , Nitric Oxide/pharmacology , Nitric Oxide Synthase/classification , Nitric Oxide Synthase/metabolism , Physical Exertion/physiology , Regional Blood Flow/drug effects , Regional Blood Flow/physiology , Ryanodine Receptor Calcium Release Channel/metabolism
16.
FASEB J ; 14(13): 1889-900, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11023973

ABSTRACT

A growing body of evidence suggests that the cellular response to oxidative and nitrosative stress is primarily regulated at the level of transcription. Posttranslational modification of transcription factors may provide a mechanism by which cells sense these redox changes. In bacteria, for example, OxyR senses redox-related changes via oxidation or nitrosylation of a free thiol in the DNA binding region. This mode of regulation may serve as a paradigm for redox-sensing by eukaryotic transcription factors as most-including NF-kappaB, AP-1, and p53-contain reactive thiols in their DNA binding regions, the modification of which alters binding in vitro. Several of these transcription factors have been found to be sensitive to both reactive oxygen species and nitric oxide-related species in vivo. It remains entirely unclear, however, if oxidation or nitrosylation of eukaryotic transcription factors is an important mode of regulation, or whether transcriptional activating pathways are principally controlled at other redox-sensitive levels.-Marshall, H. E., Merchant, K., Stamler, J. S. Nitrosation and oxidation in the regulation of gene expression.


Subject(s)
Gene Expression Regulation , Nitric Oxide/metabolism , Oxidative Stress/genetics , Reactive Oxygen Species/metabolism , Transcription Factors/metabolism , Models, Genetic , NF-kappa B/metabolism , Nitrosation , Oxidation-Reduction
17.
Cell ; 102(4): 499-509, 2000 Aug 18.
Article in English | MEDLINE | ID: mdl-10966111

ABSTRACT

Ion channels have been studied extensively in ambient O2 tension (pO2), whereas tissue PO2 is much lower. The skeletal muscle calcium release channel/ryanodine receptor (RyR1) is one prominent example. Here we report that PO2 dynamically controls the redox state of 6-8 out of 50 thiols in each RyR1 subunit and thereby tunes the response to NO. At physiological pO2, nanomolar NO activates the channel by S-nitrosylating a single cysteine residue. Among sarcoplasmic reticulum proteins, S-nitrosylation is specific to RyR1 and its effect on the channel is calmodulin dependent. Neither activation nor S-nitrosylation of the channel occurs at ambient PO2. The demonstration that channel cysteine residues subserve coupled O2 sensor and NO regulatory functions and that these operate through the prototypic allosteric effector calmodulin may have general implications for the regulation of redox-related systems.


Subject(s)
Muscle, Skeletal/physiology , Nitric Oxide/metabolism , Oxygen/metabolism , Ryanodine Receptor Calcium Release Channel/physiology , Signal Transduction , Animals , Calcium/metabolism , Calcium-Transporting ATPases/metabolism , Calmodulin/metabolism , Oxidation-Reduction , Rabbits , Ryanodine/metabolism , Sarcoplasmic Reticulum/metabolism
18.
Proc Natl Acad Sci U S A ; 97(9): 4672-6, 2000 Apr 25.
Article in English | MEDLINE | ID: mdl-10758168

ABSTRACT

Yeast hemoglobin was discovered close to half a century ago, but its function has remained unknown. Herein, we report that this flavohemoglobin protects Saccharomyces cerevisiae from nitrosative stress. Deletion of the flavohemoglobin gene (YHB1) abolished the nitric oxide (NO)-consuming activity of yeast cells. Levels of protein nitrosylation were more than 10-fold higher in yhb1 mutant yeast than in isogenic wild-type cells after incubation with NO donors. Growth of mutant cells was inhibited by a nitrosative challenge that had little effect on wild-type cells, whereas the resistance of mutant cells to oxidative stress was unimpaired. Protection conferred by yeast flavohemoglobin against NO and S-nitrosothiols was seen under both anaerobic and aerobic conditions, consistent with a primary function in NO detoxification. A phylogenetic analysis indicated that protection from nitrosative stress is likely to be a conserved function among microorganismal flavohemoglobins. Flavohemoglobin is therefore a potential target for antimicrobial therapy.


Subject(s)
Hemeproteins/metabolism , Nitric Oxide/metabolism , Oxidative Stress/physiology , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/physiology , Aerobiosis , Animals , Dioxygenases , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Deletion , Hemeproteins/genetics , Kinetics , Phylogeny , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism
19.
J Biol Chem ; 275(22): 16738-45, 2000 06 02.
Article in English | MEDLINE | ID: mdl-10747928

ABSTRACT

S-Nitrosohemoglobin (SNO-Hb) is a vasodilator whose activity is allosterically modulated by oxygen ("thermodyamic linkage"). Blood vessel contractions are favored in the oxygenated structure, and vasorelaxant activity is "linked" to deoxygenation, as illustrated herein. We further show that transnitrosation reactions between SNO-Hb and ambient thiols transduce the NO-related bioactivity, whereas NO itself is inactive. One remaining problem is that the amounts of SNO-Hb present in vivo are so large as to be incompatible with life were all the S-nitrosothiols transformed into bioactive equivalents during each arterial-venous cycle. Experiments were therefore undertaken to address how SNO-Hb conserves its NO-related activity. Our studies show that 1) increased O(2) affinity of SNO-Hb (which otherwise retains allosteric responsivity) restricts the hypoxia-induced allosteric transition that exchanges NO groups with ambient thiols for vasorelaxation; 2) some NO groups released from Cys(beta93) upon transition to T structure are autocaptured by the hemes, even in the presence of glutathione; and 3) an O(2)-dependent equilibrium between SNO-Hb and iron nitrosylhemoglobin acts to conserve NO. Thus, by sequestering a significant fraction of NO liberated upon transition to T structure, Hb can conserve NO groups that would otherwise be released in an untimely or deleterious manner.


Subject(s)
Hemoglobins/metabolism , Oxygen/metabolism , Allosteric Regulation , Animals , Aorta, Thoracic/metabolism , Aorta, Thoracic/physiology , Glutathione/metabolism , Humans , In Vitro Techniques , Phosphates/metabolism , Protein Binding , Rabbits
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