Jonathan S. Stamler

Protein S-nitrosylation: purview and parameters.

S-nitrosylation, the covalent attachment of a nitrogen monoxide group to the thiol side chain of cysteine, has emerged as an important mechanism for dynamic, post-translational regulation of most or all main classes of protein. S-nitrosylation thereby conveys a large part of the ubiquitous influence of nitric oxide (NO) on cellular signal transduction, and provides a mechanism for redox-based physiological regulation.

Adverse vascular effects of homocysteine are modulated by endothelium-derived relaxing factor and related oxides of nitrogen.

Elevated levels of homocysteine are associated with an increased risk of atherosclerosis and thrombosis. The reactivity of the sulfhydryl group of homocysteine has been implicated in molecular mechanisms underlying this increased risk. There is also increasingly compelling evidence that thiols react in the presence of nitric oxide (NO) and endothelium-derived relaxing factor (EDRF) to form S-nitrosothiols, compounds with potent vasodilatory and antiplatelet effects. We, therefore, hypothesized that S-nitrosation of homocysteine would confer these beneficial bioactivities to the thiol, and at the same time attenuate its pathogenicity. We found that prolonged (> 3 h) exposure of endothelial cells to homocysteine results in impaired EDRF responses. By contrast, brief (15 min) exposure of endothelial cells, stimulated to secrete EDRF, to homocysteine results in the formation of S-NO-homocysteine, a potent antiplatelet agent and vasodilator. In contrast to homocysteine, S-NO-homocysteine does not support H2O2 generation and does not undergo conversion to homocysteine thiolactone, reaction products believed to contribute to endothelial toxicity. These results suggest that the normal endothelium modulates the potential, adverse effects of homocysteine by releasing EDRF and forming the adduct S-NO-homocysteine. The adverse vascular properties of homocysteine may result from an inability to sustain S-NO formation owing to a progressive imbalance between the production of NO by progressively dysfunctional endothelial cells and the levels of homocysteine.

A metabolic enzyme for S -nitrosothiol conserved from bacteria to humans

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.

(S)NO Signals: Translocation, Regulation, and a Consensus Motif

(Table 1; Stamler et al., 1992b). This is well exemplified Nitric oxide (NO) is a signaling molecule that has capin the immune system in work by DeGroote and Fang tured our imagination. According to the common view, (DeGroote et al., 1996). These researchers found that NO diffuses over a large sphere of influence, moving bacterial virulence is conferred by a gene that protects freely through membranes of target cells to raise levels against the lethal effects of SNOs produced by the (muof cGMP. In the brain, NO influences synaptic plasticity, rine) host, whereas NO is harmless against the same apoptosis, neuronal development, and even complex bacteria. Molecular recognition of the SNO onslaught behavioral responses. This image has been reinforced and the activation of bacterial resistance is achieved by by observations in the cardiovascular and immune sysS-nitrosylation of proteins involved in defense (Hauslatems, for example, the relaxation of blood vessels by den et al., 1996). Thus, in this system, S-nitrosylation is cGMP, and the killing of tumor cells and bacteria by the the signal and the regulator of the response. macrophage NO synthase (NOS). A similar NO signal is used by mammalian cells. Images can also be misleading. First, a wide sphere For example, the endothelium-derived relaxation factor of NO diffusion implies that it travelsdown concentration (EDRF)/NO-mediated relaxation of blood vessels occurs gradients that are established by extracellular sinks partly by direct activation of a potassium channel (Lancaster, 1994; Stamler, 1996). The problem with this through reactions of EDRF with critical thiols (Bolotina picture is that NO cannot achieve local action: it would et al., 1994). Likewise in the heart, SNO and peroxynitrite be leaving cells more rapidly than it reacts within. Sec(OONO) directly activate calcium channels by a redox ond, we have come to appreciate that many NO signals mechanism that opposes the effects of cGMP (Campbell are cGMP independent. These pathways, typically et al., 1996). Ion channel activation may also account grouped under the broad heading of “redox”, have not for NO/SNO-mediated relaxations of third to fourth order been incorporated into the theory of NO action in the human airways (Gaston et al., 1994) and canine (Koh et nervous system. However, redox-related NO signals can al., 1995) or rat proximal colon (Takeuchi et al., 1996);

REDOX-BASED REGULATION OF SIGNAL TRANSDUCTION: PRINCIPLES, PITFALLS, AND PROMISES

Oxidants are produced as a by-product of aerobic metabolism, and organisms ranging from prokaryotes to mammals have evolved with an elaborate and redundant complement of antioxidant defenses to confer protection against oxidative insults. Compelling data now exist demonstrating that oxidants are used in physiological settings as signaling molecules with important regulatory functions controlling cell division, migration, contraction, and mediator production. These physiological functions are carried out in an exquisitely regulated and compartmentalized manner by mild oxidants, through subtle oxidative events that involve targeted amino acids in proteins. The precise understanding of the physiological relevance of redox signal transduction has been hampered by the lack of specificity of reagents and the need for chemical derivatization to visualize reversible oxidations. In addition, it is difficult to measure these subtle oxidation events in vivo. This article reviews some of the recent findings that illuminate the significance of redox signaling and exciting future perspectives. We also attempt to highlight some of the current pitfalls and the approaches needed to advance this important area of biochemical and biomedical research.

A novel protective effect of erythropoietin in the infarcted heart

Erythropoietin (EPO) has been shown to protect neurons from ischemic stroke, but can also increase thrombotic events and mortality rates in patients with ischemic heart disease. We reasoned that benefits of EPO might be offset by increases in hematocrit and evaluated the direct effects of EPO in the ischemic heart. We show that preconditioning with EPO protects H9c2 myoblasts in vitro and cardiomyocytes in vivo against ischemic injury. EPO treatment leads to significantly improved cardiac function following myocardial infarction. This protection is associated with mitigation of myocyte apoptosis, translating into more viable myocardium and less ventricular dysfunction. EPO-mediated myocyte survival appears to involve Akt activation. Importantly, cardioprotective effects of EPO were seen without an increase in hematocrit (eliminating oxygen delivery as an etiologic factor in myocyte survival and function), demonstrating that EPO can directly protect the ischemic and infarcted heart.

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 ≈ 107 M−1 s−1) (refs 1, 2) and once bound, the NO activity is largely irretrievable (Kd ≈ 10−5 s−1) (refs 3–10); 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 re-examination of NO interactions with other haem proteins containing allosteric-site thiols may be warranted.

S-nitrosylation in health and disease

S-nitrosylation is a ubiquitous redox-related modification of cysteine thiol by nitric oxide (NO), which transduces NO bioactivity. Accumulating evidence suggests that the products of S-nitrosylation, S-nitrosothiols (SNOs), play key roles in human health and disease. In this review, we focus on the reaction mechanisms underlying the biological responses mediated by SNOs. We emphasize reactions that can be identified with complex (patho)physiological responses, and that best rationalize the observed increase or decrease in specific classes of SNOs across a spectrum of disease states. Thus, changes in the levels of various SNOs depend on specific defects in both enzymatic and non-enzymatic mechanisms of nitrosothiol formation, processing and degradation. An understanding of these mechanisms is crucial for the development of an integrated model of NO biology, and for effective treatment of diseases associated with dysregulation of NO homeostasis.

Nitric oxide regulates basal systemic and pulmonary vascular resistance in healthy humans.

BACKGROUND The endothelium synthesizes and releases a relaxing factor with the physiochemical properties of nitric oxide (NO). However, the role of endothelium-derived NO in the basal regulation of systemic and pulmonary vascular resistance in humans is not known. Our primary objectives were to determine the effects of inhibiting NO synthesis on blood pressure and systemic vascular resistance and to establish the role of endothelium-derived NO in the regulation of normoxic pulmonary vascular tone. METHODS AND RESULTS We studied the systemic and pulmonary hemodynamic effects of NG-monomethyl-L-arginine (L-NMMA, 0.03 to 1.0 mg.kg-1.min-1 IV), an NO synthase inhibitor, in 11 healthy volunteers, aged 33 +/- 2 years. An arterial cannula and a pulmonary artery catheter were placed in each subject to measure blood pressure, pulmonary artery pressure, and pulmonary capillary wedge pressure. Cardiac output was determined by the Fick technique, and systemic and pulmonary vascular resistances were calculated. Serum NO levels (free and protein bound) were measured by chemiluminescence in 5 subjects. Six of the subjects also received phenylephrine (25 to 100 micrograms/min IV) to compare the cardiac hemodynamic effects of L-NMMA with those of a direct-acting vasoconstrictor. L-NMMA caused dose-dependent increases in both blood pressure and systemic vascular resistance. At the highest dose of L-NMMA, there was a 15.5 +/- 1.3% increase in mean blood pressure and a 63.4 +/- 8.2% increase in systemic vascular resistance (each P < .01). Pulmonary vascular resistance increased 39.8 +/- 9.4% (P < .01), whereas mean pulmonary artery pressure did not change. Administration of L-NMMA also reduced cardiac output by 27.8 +/- 2.9% and stroke volume by 15.4 +/- 3.5% (each P < .01). Serum NO levels decreased 65 +/- 10% from basal values (P < .05), confirming inhibition of endogenous NO production. Phenylephrine increased blood pressure to a level comparable to that observed with L-NMMA. The decline in stroke volume was greater with L-NMMA than with phenylephrine (P < .01). CONCLUSIONS This study demonstrates that basal release of endothelium-derived NO is directly involved in the determination of systemic vascular resistance and, therefore, blood pressure in healthy humans. In addition, NO regulates basal normoxic pulmonary vascular tone. The complex hemodynamic effects of NO are composite properties of its actions on systemic and pulmonary vascular resistance and cardiac function.

Nitric Oxide Produced by Human B Lymphocytes Inhibits Apoptosis and Epstein-Barr Virus Reactivation

Nitric oxide (NO) produced by murine macrophages is important in murine resistance to ectromelia virus, herpes simplex virus, and vaccinia virus infection. In contrast, NO production by human mononuclear cells has been difficult to demonstrate, and a role for NO in human responses to infection is uncertain. We report constitutive, low level, macrophage-type NO synthase (iNOS) expression in Epstein-Barr virus (EBV)-transformed human B lymphocytes and Burkitts lymphoma cell lines. Immune NOS activity is involved in maintaining EBV latency through down-regulation of the expression of the immediate-early EBV transactivator Zta. NO also inhibits apoptosis in B lymphocyte cell lines. The effects of NO are largely independent of cGMP and influential on signaling pathways regulated by (sulfhydryl) redox status. These results suggest that NO plays a physiological role in human B cell biology by inhibiting programmed cell death and maintaining viral latency.