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Biomedical subjects

A J Gow

Publications and source records attributed to A J Gow.

15 recordsLinked to original sources
PubMed2001

S-nitrosothiol repletion by an inhaled gas regulates pulmonary function.

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.

Administration, Inhalation↗
PubMed2001

Nitric oxide chemistry and cellular signaling.

Nitric oxide (NO) has been shown to mediate a number of different physiological functions within every major organ system. This wide variety of functional roles is made all the more remarkable when one considers that NO is a simple diatomic molecule. However, despite the simplicity of the molecule, NO possesses a wide range of chemical reactivity and multiple potential reactive targets. It is the variability of NO reactivity, which leads to its capability to control such a vast range of biological functions. In essence the functionality of NO is controlled by its chemical reactivity. In order to understand this possibility further it is necessary to consider the biologically relevant reactions of nitric oxide.

Animals↗
PubMed2001

Methamphetamine neurotoxicity: necrotic and apoptotic mechanisms and relevance to human abuse and treatment.

Research into methamphetamine-induced neurotoxicity has experienced a resurgence in recent years. This is due to (1) greater understanding of the mechanisms underlying methamphetamine neurotoxicity, (2) its usefulness as a model for Parkinson's disease and (3) an increased abuse of the substance, especially in the American Mid-West and Japan. It is suggested that the commonly used experimental one-day methamphetamine dosing regimen better models the acute overdose pathologies seen in humans, whereas chronic models are needed to accurately model human long-term abuse. Further, we suggest that these two dosing regimens will result in quite different neurochemical, neuropathological and behavioral outcomes. The relative importance of the dopamine transporter and vesicular monoamine transporter knockout is discussed and insights into oxidative mechanisms are described from observations of nNOS knockout and SOD overexpression. This review not only describes the neuropathologies associated with methamphetamine in rodents, non-human primates and human abusers, but also focuses on the more recent literature associated with reactive oxygen and nitrogen species and their contribution to neuronal death via necrosis and/or apoptosis. The effect of methamphetamine on the mitochondrial membrane potential and electron transport chain and subsequent apoptotic cascades are also emphasized. Finally, we describe potential treatments for methamphetamine abusers with reference to the time after withdrawal. We suggest that potential treatments can be divided into three categories; (1) the prevention of neurotoxicity if recidivism occurs, (2) amelioration of apoptotic cascades that may occur even in the withdrawal period and (3) treatment of the atypical depression associated with withdrawal.

Amphetamine-Related Disorders↗
PubMed2000

Two distinct mechanisms of nitric oxide-mediated neuronal cell death show thiol dependency.

To better understand the mechanism(s) underlying nitric oxide (. NO)-mediated toxicity, in the presence and absence of concomitant oxidant exposure, postmitotic terminally differentiated NT2N cells, which are incapable of producing. NO, were exposed to PAPA-NONOate (PAPA/NO) and 3-morpholinosydnonimine (SIN-1). Exposure to SIN-1, which generated peroxynitrite in the range of 25-750 nM/min, produced a concentration- and time-dependent delayed cell death. In contrast, a critical threshold concentration (>440 nM/min) was required for. NO to produce significant cell injury. Examination of cells by electron microscopy shows a largely necrotic injury after peroxynitrite exposure but mainly apoptotic-like morphology after. NO exposure. Cellular levels of reduced thiols correlated with cell death, and pretreatment with N-acetylcysteine (NAC) fully protected from cell death in either PAPA/NO or SIN-1 exposure. NAC given within the first 3 h posttreatment further delayed cell death and increased the intracellular thiol level in SIN-1 but not. NO-exposed cells. Cell injury from. NO was independent of cGMP, caspases, and superoxide or peroxynitrite formation. Overall, exposure of non-. NO-producing cells to. NO or peroxynitrite results in delayed cell death, which, although occurring by different mechanisms, appears to be mediated by the loss of intracellular redox balance.

Acetylcysteine↗
PubMed1999

Ascaris haemoglobin is a nitric oxide-activated 'deoxygenase'.

The parasitic nematode Ascaris lumbricoides infects one billion people worldwide. Its perienteric fluid contains an octameric haemoglobin that binds oxygen nearly 25,000 times more tightly than does human haemoglobin. Despite numerous investigations, the biological function of this molecule has remained elusive. The distal haem pocket contains a metal, oxygen and thiol, all of which are known to be reactive with nitric oxide. Here we show that Ascaris haemoglobin enzymatically consumes oxygen in a reaction driven by nitric oxide, thus keeping the perienteric fluid hypoxic. The mechanism of this reaction involves unprecedented chemistry of a haem group, a thiol and nitric oxide. We propose that Ascaris haemoglobin functions as a 'deoxygenase', using nitric oxide to detoxify oxygen. The structural and functional adaptations of Ascaris haemoglobin suggest that the molecular evolution of haemoglobin can be rationalized by its nitric oxide related functions.

Anaerobiosis↗
PubMed1999

The oxyhemoglobin reaction of nitric oxide.

The oxidation of nitric oxide (NO) to nitrate by oxyhemoglobin is a fundamental reaction that shapes our understanding of NO biology. This reaction is considered to be the major pathway for NO elimination from the body; it is the basis for a prevalent NO assay; it is a critical feature in the modeling of NO diffusion in the circulatory system; and it informs a variety of therapeutic applications, including NO-inhalation therapy and blood substitute design. Here we show that, under physiological conditions, this reaction is of little significance. Instead, NO preferentially binds to the minor population of the hemoglobin's vacant hemes in a cooperative manner, nitrosylates hemoglobin thiols, or reacts with liberated superoxide in solution. In the red blood cell, superoxide dismutase eliminates superoxide, increasing the yield of S-nitrosohemoglobin and nitrosylated hemes. Hemoglobin thus serves to regulate the chemistry of NO and maintain it in a bioactive state. These results represent a reversal of the conventional view of hemoglobin in NO biology and motivate a reconsideration of fundamental issues in NO biochemistry and therapy.

Electron Spin Resonance Spectroscopy↗
PubMed1999

Fas-induced caspase denitrosylation.

Only a few intracellular S-nitrosylated proteins have been identified, and it is unknown if protein S-nitrosylation/denitrosylation is a component of signal transduction cascades. Caspase-3 zymogens were found to be S-nitrosylated on their catalytic-site cysteine in unstimulated human cell lines and denitrosylated upon activation of the Fas apoptotic pathway. Decreased caspase-3 S-nitrosylation was associated with an increase in intracellular caspase activity. Fas therefore activates caspase-3 not only by inducing the cleavage of the caspase zymogen to its active subunits, but also by stimulating the denitrosylation of its active-site thiol. Protein S-nitrosylation/denitrosylation can thus serve as a regulatory process in signal transduction pathways.

Animals↗
PubMed1999

Immunotargeting of glucose oxidase: intracellular production of H(2)O(2) and endothelial oxidative stress.

Extracellular and intracellular reactive oxygen species attack different targets and may, therefore, result in different forms of oxidative stress. To specifically study an oxidative stress induced by a regulated intracellular flux of a defined reactive oxygen species in endothelium, we used immunotargeting of the H(2)O(2)-generating enzyme glucose oxidase (GOX) conjugated with an antibody to platelet-endothelial cell adhesion molecule (PECAM)-1, an endothelial surface antigen. Anti-PECAM-(125)I-GOX conjugates specifically bind to both endothelial and PECAM-transfected cells. Approximately 70% of cell-bound anti-PECAM-(125)I-GOX was internalized. The cell-bound conjugate was enzymatically active and generated H(2)O(2) from glucose. Use of the fluorescent dye dihydrorhodamine 123 revealed that 70% of H(2)O(2) was generated intracellularly, whereas 30% of H(2)O(2) was detected in the cell medium. Catalase added to the cells eliminated H(2)O(2) in the medium but had little effect on the intracellular generation of H(2)O(2) by anti-PECAM-GOX. Both H(2)O(2) added exogenously to the cell medium (extracellular H(2)O(2)) and that generated by anti-PECAM-GOX caused oxidative stress manifested by time- and dose-dependent irreversible plasma membrane damage. Inactivation of cellular catalase by aminotriazole treatment augmented damage caused by either extracellular H(2)O(2) or anti-PECAM-GOX. Catalase added to the medium protected either normal or aminotriazole-treated cells against extracellular H(2)O(2), yet failed to protect cells against injury induced by anti-PECAM-GOX. Therefore, treatment of PECAM-positive cells with anti-PECAM-GOX leads to conjugate internalization, predominantly intracellular H(2)O(2) generation and intracellular oxidative stress. These results indicate that anti-PECAM-GOX 1) provides cell-specific intracellular delivery of an active enzyme and 2) causes intracellular oxidative stress in PECAM-positive cells.

Antibodies, Monoclonal↗
PubMed1998

Nitrosative stress: metabolic pathway involving the flavohemoglobin.

Nitric oxide (NO) biology has focused on the tightly regulated enzymatic mechanism that transforms L-arginine into a family of molecules, which serve both signaling and defense functions. However, very little is known of the pathways that metabolize these molecules or turn off the signals. The paradigm is well exemplified in bacteria where S-nitrosothiols (SNO)-compounds identified with antimicrobial activities of NO synthase-elicit responses that mediate bacterial resistance by unknown mechanisms. Here we show that Escherichia coli possess both constitutive and inducible elements for SNO metabolism. Constitutive enzyme(s) cleave SNO to NO whereas bacterial hemoglobin, a widely distributed flavohemoglobin of poorly understood function, is central to the inducible response. Remarkably, the protein has evolved a novel heme-detoxification mechanism for NO. Specifically, the heme serves a dioxygenase function that produces mainly nitrate. These studies thus provide new insights into SNO and NO metabolism and identify enzymes with reactions that were thought to occur only by chemical means. Our results also emphasize that the reactions of SNO and NO with hemoglobins are evolutionary conserved, but have been adapted for cell-specific function.

Biological Evolution↗
PubMed1998

Reactions between nitric oxide and haemoglobin under physiological conditions.

The tenet of high-affinity nitric oxide (NO) binding to a haemoglobin (Hb) has shaped our view of haem proteins and of small diffusible signaling molecules. Specifically, NO binds rapidly to haem iron in Hb (k approximately 10[7] M[-1] s[-1]) and once bound, the NO activity is largely irretrievable (Kd approximately 10[-5] s[-1]); the binding is purportedly so tight as to be unaffected by O2 or CO. However, these general principles do not consider the allosteric state of Hb or the nature of the allosteric effector, and they mostly derive from the functional behaviour of fully nitrosylated Hb, whereas Hb is only partially nitrosylated in vivo. Here we show that oxygen drives the conversion of nitrosylhaemoglobin in the 'tense' T (or partially nitrosylated, deoxy) structure to S-nitrosohaemoglobin in the 'relaxed' R (or ligand-bound, oxy) structure. In the absence of oxygen, nitroxyl anion (NO-) is liberated in a reaction producing methaemoglobin. The yields of both S-nitrosohaemoglobin and methaemoglobin are dependent on the NO/Hb ratio. These newly discovered reactions elucidate mechanisms underlying NO function in the respiratory cycle, and provide insight into the aetiology of S-nitrosothiols, methaemoglobin and its related valency hybrids. Mechanistic reexamination of NO interactions with other haem proteins containing allosteric-site thiols may be warranted.

Allosteric Regulation↗
PubMed1998

Nitric oxide and peroxynitrite-mediated pulmonary cell death.

Nitric oxide (.NO) can be produced within the lung, and recently inhaled nitric oxide has been used as a therapeutic agent. Peroxynitrite1 (ONOO-), the product of the nearly diffusion-limited reaction between .NO and superoxide, may represent the proximal reactive species mediating .NO injury to pulmonary cells. To investigate the physiological and pathological reactivities of .NO and ONOO- at the molecular and cellular levels, bovine pulmonary artery endothelial cells (BPAEC) and rat type II epithelial cells were exposed to .NO (0.01-2.5 microM/min for 2 h) generated by spermine-NONOate and papa-NONOate and to the same fluxes of ONOO- generated by 1,3-morpholinosydnonimine (SIN-1). Exposure to SIN-1 resulted in cellular injury and death in both cell types. Epithelial cells displayed a concentration-dependent loss of cellular viability within 8 h of exposure. In contrast, BPAEC loss of cellular viability was evident after 18 h postexposure. Events preceding cell death in BPAEC include depolarization of the mitochondrial membrane, evident as early as 6 h postexposure, loss of cellular redox activity at 16 h, and DNA fragmentation detected by in situ staining at 18 h after exposure. Exposure of BPAEC to .NO did not affect the cellular viability, but type II cells were injured in a manner similar to ONOO- exposure. .NO-mediated cellular injury within type II cells was reduced by preincubation with N-acetylcysteine. The data imply that the pathological and physiological effects of .NO may be regulated by its reactions with superoxide and reduced thiols.

Acetylcysteine↗
PubMed1997

A novel reaction mechanism for the formation of S-nitrosothiol in vivo.

The objective of this study was to investigate the mechanism of S-nitrosothiol formation under physiological conditions. A mechanism is proposed by which nitric oxide (.NO) reacts directly with reduced thiol to produce a radical intermediate, R-S-N.-O-H. This intermediate reduces an electron acceptor to produce S-nitrosothiol. Under aerobic conditions O2 acts as the electron acceptor and is reduced to produce superoxide (O-2). The following experimental evidence is provided in support of this mechanism. Cysteine accelerates the consumption of .NO by 2.5-fold under physiological conditions. The consumption of O2 in the presence of .NO and cysteine is increased by 2.4-fold. The reaction orders of .NO and cysteine are second and first order, respectively. The second order of reaction for .NO may result from interaction between .NO and O-2 to form peroxynitrite. In the presence of Cu,Zn-superoxide dismutase, the reaction of .NO with cysteine generates hydrogen peroxide, indicating that the reaction generates O-2. Finally, the formation of S-nitrosothiol is demonstrated in an anaerobic environment and, as predicted by the mechanism, is dependent on the presence of an electron acceptor. These results demonstrate that under physiological conditions .NO reacts directly with thiols to form S-nitrosothiol in the presence of an electron acceptor.

Aerobiosis↗
PubMed1996

Effects of peroxynitrite-induced protein modifications on tyrosine phosphorylation and degradation.

The ability of protein tyrosine kinases to phosphorylate a synthetic peptide was inhibited 51% by peroxynitrite-mediated nitration of tyrosine. Exposure of endothelial cells to peroxynitrite decreased the intensity of tyrosine phosphorylated proteins and increased the intensity of nitrotyrosine-containing proteins. Peroxynitrite-modified BSA was degraded by human red blood cell lysates. However, human plasma in a concentration-, time-, and temperature-dependent manner, removed the protein nitrotyrosine epitope. These results suggest that tyrosine nitration interferes with phosphorylation and targets proteins for degradation. Specific enzymatic process(es) for removing nitrotyrosine may be present in vivo.

Animals↗