Nitric oxide (NO) has earned the reputation of being a signaling mediator with many diverse and often opposing biological activities. The diversity in response to this simple diatomic molecule comes from the enormous variety of chemical reactions and biological properties associated with it. In the last few years, the importance of steady state NO concentrations have emerged as a key determinant of its biological function. Precise cellular responses are differentially regulated by specific NO concentration. We propose 5 basic distinct concentration levels of NO activity; cGMP mediated processes ([NO] <1-30 nM; Akt phosphorylation ([NO] = 30-100 nM); stabilization of HIF-1α ([NO] = 100-300 nM); phosphorylation of p53 ([NO] > 400 nM) and nitrosative stress (1 µM). In general, lower NO concentrations promote cell survival and proliferation, while higher levels favor cell cycle arrest, apoptosis, and senescence. Free radical interactions will also influence NO signaling. One of the consequences of reactive oxygen species (ROS) generation is to reduce NO concentrations. This antagonizes the signaling of nitric oxide and in some cases results in converting a cell cycle arrest profile to a cell survival one. The resulting reactive nitrogen species (RNS) that are generated from these reactions can also have biological effects and increase oxidative and nitrosative stress responses. A number of factors determine the formation of NO and its concentration, such as diffusion, consumption, and substrate availability which are referred to as Kinetic Determinants for Molecular Target Interactions. These are the chemical and biochemical parameters that shape cellular responses to NO. Herein we discuss signal transduction and the chemical biology of NO in terms of the direct and indirect reactions.

The redox siblings nitroxyl (HNO) and nitric oxide (NO) have often been assumed to undergo casual redox reactions in biological systems. However, several recent studies have demonstrated distinct pharmacological effects for donors of these two species. Here, infusion of the HNO donor Angeli's salt into normal dogs resulted in elevated plasma levels of calcitonin gene-related peptide, whereas neither the NO donor diethylamine͞NONOate nor the nitrovasodilator nitroglycerin had an appreciable effect on basal levels. Conversely, plasma cGMP was increased by infusion of diethylamine͞NONOate or nitroglycerin but was unaffected by Angeli's salt. These results suggest the existence of two mutually exclusive response pathways that involve stimulated release of discrete signaling agents from HNO and NO. In light of both the observed dichotomy of HNO and NO and the recent determination that, in contrast to the O2͞O 2 ؊ couple, HNO is a weak reductant, the relative reactivity of HNO with common biomolecules was determined. This analysis suggests that under biological conditions, the lifetime of HNO with respect to oxidation to NO, dimerization, or reaction with O2 is much longer than previously assumed. Rather, HNO is predicted to principally undergo addition reactions with thiols and ferric proteins. Calcitonin gene-related peptide release is suggested to occur via altered calcium channel function through binding of HNO to a ferric or thiol site. The orthogonality of HNO and NO may be due to differential reactivity toward metals and thiols and in the cardiovascular system, may ultimately be driven by respective alteration of cAMP and cGMP levels.Angeli's salt ͉ superoxide dismutase ͉ heme protein ͉ cGMP ͉ calcitonin gene-related peptide D uring the last two decades, discussion of the chemistry of nitric oxide (NO) in biological systems has primarily focused on the nitrosylation of heme proteins such as soluble guanylyl cyclase and the production of reactive nitrogen oxide species (RNOS) (1-3). The RNOS literature has largely been concerned with nitrogen dioxide (NO 2 ), dinitrogen trioxide (N 2 O 3 ), and peroxynitrite (ONOO Ϫ ), which are formed through reaction with molecular oxygen or superoxide (O 2 Ϫ ) (4-6). Recently, however, there has been increased interest in the one-electron reduction product of NO, nitroxyl (HNO͞NO Ϫ ; nitrosyl hydride͞nitroxyl anion). Of particular note are studies suggesting that oxidation of L-arginine by NO synthase (NOS) leads to production of nitroxyl rather than NO under certain conditions (7-10). In this light, elucidation of the chemical biology of nitroxyl has acquired new importance.Comparisons of the toxicological and pharmacological properties of nitrogen oxide donor compounds have revealed that NO and HNO in general elicit distinct responses under a variety of biological conditions. In vitro, HNO reacts with O 2 to generate potent oxidizing species capable of cleaving DNA, thereby augmenting oxidative damage (3, 11). The RNOS formed by NO autoxidation do not cause these cellular a...

We demonstrate herein dramatic acceleration of aqueous nitric oxide (NO) reaction with O 2 within the hydrophobic region of either phospholipid or biological membranes or detergent micelles and demonstrate that the presence of a distinct hydrophobic phase is required. Per unit volume, at low amounts of hydrophobic phase, the reaction of NO with O 2 within the membranes is approximately 300 times more rapid than in the surrounding aqueous medium. In tissue, even though the membrane represents only 3% of the total volume, we calculate that 90% of NO reaction with O 2 will occur there. We conclude that biological membranes and other tissue hydrophobic compartments are important sites for disappearance of NO and for formation of NO-derived reactive species and that attenuation of these potentially damaging reactions is an important protective action of lipid-soluble antioxidants such as vitamin E.Nitric oxide (NO) is an important mediator and messenger in mammalian systems and subserves an astonishing variety of roles in physiology and pathophysiology (1). One of its distinctive properties is its relatively short half-life (reported to be on the order of several seconds) in biological systems, which determines its spatial range and temporal extent of actions (2). One generally recognized mechanism for the disappearance of NO is reaction with O 2 , which is responsible for the formation of nitrite as a product of NO oxidation. Intermediates in this reaction are responsible for nitrosative reactions that result in the formation of biologically important species such as nitrosamines and nitrosothiols (2).The aqueous reaction of NO with dioxygen occurs with the following overall stoichiometry:and the rate of disappearance of NO is given bywith k ϭ 2 ϫ 10 6 M Ϫ2 ⅐s Ϫ1 at 25°C (3, 4). Because NO is approximately nine times more soluble in a hydrophobic solvent such as hexane than in water (5, 6), we [and others (7, 8)] have suspected that the presence of a hydrophobic phase (such as the interior of a lipid bilayer membrane) might accelerate the autooxidation of NO because of the concentration of reactants within the hydrophobic phase. Thus, biological membranes may act as a ''lens'' that can focus and magnify the autooxidation of NO. That is, even if the intrinsic rate constant of the reaction within the membrane hydrophobic phase is the same as in the aqueous cytosol, the reaction is accelerated overall because of the increased reactant concentrations within the membrane.To test this possibility, we used an electrochemical method to measure the rate of disappearance of NO in an aerobic buffered solution upon addition of various hydrophobic phases. METHODSHepatocyte Isolation and Cell Membrane Preparation. Rat hepatocytes were isolated as described (9). For membrane isolation, cells were suspended in 50 mM potassium phosphate (pH 7.4) containing 0.5 mM EDTA and sonicated (two 10-s bursts) while cooled on ice. The sonicated preparations were centrifuged at 5,000 ϫ g for 5 min at 4°C. The supernatant was subjected ...