Nitric oxide (NO) 1 is one of the 10 smallest, stable molecules of the hundreds of millions in nature (1). According to Stokes' Law, the diffusibility of a molecule in the condensed phase is inversely proportional to its molecular radius, which thus makes NO one of the most rapidly diffusible molecules known. Its diffusion constant (D) is approximately 3300 -3800 m 2 /s, whether measured in aqueous solution (2) or in intact tissue (e.g. brain (3)). Membranes and other hydrophobic structures in tissue are no barrier to diffusion of NO because of its solubility in hydrophobic phases (4).The reaction of free NO with oxyhemoglobin is rapid (bimolecular rate constant k ϭ 3.4 ϫ 10 7 M Ϫ1 s Ϫ1 (5)), and from this rate constant it can be calculated that the half-life of NO in the presence of a concentration of hemoglobin equivalent to that in the bloodstream (15 g/dl) would be very short, approximately 2 ϫ 10 Ϫ6 s. As we have pointed out previously (6, 7), the extremely rapid diffusibility of NO coupled with its rapid reaction with oxyhemoglobin apparently poses a difficulty in the postulate that free NO is the endothelium-derived relaxing factor.Using an electrochemical method, we describe here the results of measurements of the disappearance of NO upon reaction with either oxyhemoglobin in solution or oxyhemoglobin when contained within intact erythrocytes. We find that, as reported in 1927 for the reaction of O 2 with deoxyhemoglobin (8), the NO reaction with intact RBCs is considerably slower than with an equivalent concentration of free oxyhemoglobin. We present a mathematical analysis of this phenomenon, which demonstrates that the rate of the reaction of NO with intraerythrocytic hemoglobin is limited by the rate of diffusion of NO into the cell. From our data, we estimate that in whole blood the half-life of NO will be less than 2 ms, which, although quite rapid, is considerably longer than in the presence of free hemoglobin. EXPERIMENTAL PROCEDURESPreparation of NO Solution-6 ml of phosphate-buffered saline (PBS: 15 mM phosphate (potassium) plus 0.09% NaCl pH 7.4) in a plastic vial was used in preparing saturated NO solution. The solution was bubbled with argon gas (Aldrich) for 30 min and then changed to NO gas (Aldrich) for 20 min. The NO gas was passed first through a gaswashing bottle containing 1 M deaerated KOH solution.RBC and Free Hemoglobin Preparation-Blood was withdrawn from rats and centrifuged at 2300 ϫ g for 10 min. The plasma and buffy coat were discarded, and the RBC pellet was washed 3 times with PBS (pH 7.4). The packed RBCs then were added to PBS and the solution was stirred gently. Cells were counted with a hemocytometer and were stored on ice for use. To prepare free oxyHb, 2 ml of counted RBCs was centrifuged at 2300 g for 10 min (4°C). The packed RBCs were then added to 40 ml of 5 mM phosphate solution (pH 8), stirred and allowed to incubate for 30 min for hemolysis.Electrochemical Measurements-All electrochemical measurements were carried out at 25 Ϯ 2°C by a BAS 100B electrochemical 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 ...

Recent studies reveal a novel role for hemoglobin as an allosterically regulated nitrite reductase that may mediate nitric oxide (NO)-dependent signaling along the physiological oxygen gradient. Nitrite reacts with deoxyhemoglobin in an allosteric reaction that generates NO and oxidizes deoxyhemoglobin to methemoglobin. NO then reacts at a nearly diffusion-limited rate with deoxyhemoglobin to form iron-nitrosyl-hemoglobin, which to date has been considered a highly stable adduct and, thus, not a source of bioavailable NO. However, under physiological conditions of partial oxygen saturation, nitrite will also react with oxyhemoglobin, and although this complex autocatalytic reaction has been studied for a century, the interaction of the oxy-and deoxy-reactions and the effects on NO disposition have never been explored. We have now characterized the kinetics of hemoglobin oxidation and NO generation at a range of oxygen partial pressures and found that the deoxy-reaction runs in parallel with and partially inhibits the oxy-reaction. In fact, intermediates in the oxy-reaction oxidize the heme iron of ironnitrosyl-hemoglobin, a product of the deoxy-reaction, which releases NO from the iron-nitrosyl. This oxidative denitrosylation is particularly striking during cycles of hemoglobin deoxygenation and oxygenation in the presence of nitrite. These chemistries may contribute to the oxygen-dependent disposition of nitrite in red cells by limiting oxidative inactivation of nitrite by oxyhemoglobin, promoting nitrite reduction to NO by deoxyhemoglobin, and releasing free NO from iron-nitrosyl-hemoglobin.

Ectodomain shedding of cell surface membrane-anchoring proteins is an important process in a wide variety of physiological events(1, 2). Tumor necrosis factor alpha (TNF-alpha) converting enzyme (TACE) is the first discovered mammalian sheddase responsible for cleavage of several important surface proteins, including TNF-alpha, TNF p75 receptor, L-selectin, and transforming growth factor-a. Phorbol myristate acetate (PMA) has long been known as a potent agent to enhance ectodomain shedding. However, it is not fully understood how PMA activates TACE and induces ectodomain shedding. Here, we demonstrate that PMA induces both reactive oxygen species (ROS) generation and TNF p75 receptor shedding in Mono Mac 6 cells, a human monocytic cell line, and l-selectin shedding in Jurkat T-cells. ROS scavengers significantly attenuated PMA-induced TNF p75 receptor shedding. Exogenous H2O2 mimicked PMA-induced enhancement of ectodomain shedding, and H2O2-induced shedding was blocked by TAPI, a TACE inhibitor. Furthermore, both PMA and H2O2 failed to cause ectodomain shedding in a cell line that lacks TACE activity. By use of an in vitro TACE cleavage assay, H2O2 activated TACE that had been rendered inactive by the addition of the TACE inhibitory pro-domain sequence. We presume that the mechanism of TACE activation by H2O2 is due to an oxidative attack of the pro-domain thiol group and disruption of its inhibitory coordination with the Zn++ in the catalytic domain of TACE. These results demonstrate that ROS production is involved in PMA-induced ectodomain shedding and implicate a role for ROS in other shedding processes.

Although irreversible reaction of NO with the oxyheme of hemoglobin (producing nitrate and methemoglobin) is extremely rapid, it has been proposed that, under normoxic conditions, NO binds preferentially to the minority deoxyheme to subsequently form S-nitrosohemoglobin (SNOHb). Thus, the primary reaction would be conservation, rather than consumption, of nitrogen oxide. Data supporting this conclusion were generated by using addition of a small volume of a concentrated aqueous solution of NO to a normoxic hemoglobin solution. Under these conditions, however, extremely rapid reactions can occur before mixing. We have thus compared bolus NO addition to NO generated homogeneously throughout solution by using NO donors, a more physiologically relevant condition. With bolus addition, multiple hemoglobin species are formed (as judged by visible spectroscopy) as well as both nitrite and nitrate. With donor, only nitrate and methemoglobin are formed, stoichiometric with the amount of NO liberated from the donor. Studies with increasing hemoglobin concentrations reveal that the nitrite-forming reaction (which may be NO autoxidation under these conditions) competes with reaction with hemoglobin. SNOHb formation is detectable with either bolus or donor; however, the amounts formed are much smaller than the amount of NO added (less than 1%). We conclude that the reaction of NO with hemoglobin under normoxic conditions results in consumption, rather than conservation, of NO. O ne of the most important experimental findings that led to the postulate that the endothelium-derived relaxing factor (EDRF) is identical to nitric oxide (nitrogen monoxide, NO) was the demonstration that endothelium-dependent relaxation is exquisitely sensitive to inhibition by hemoglobin, which rapidly reacts with NO (1). Curiously, however, even though very small concentrations of hemoglobin are quite potent in preventing EDRF-dependent relaxation, little attention was initially paid to the problem this raises with the NO͞EDRF hypothesis, namely, that in vivo NO is produced immediately adjacent to a pool of very high (mM) concentrations of hemoglobin, which will act as a potent sink for the NO, thus decreasing its concentration at all locations and preventing it from reaching a physiologically functional level. Mathematical modeling has illustrated the validity of this concept (2-4).There have emerged two hypotheses to explain how NO might still function as EDRF. According to one (5-10), rather than irreversibly consuming NO, hemoglobin actually conserves it, in the form of a nitrosothiol moiety, and the linkage of the accessibility͞ reactivity of the thiol group involved (␤cys93) to the oxygenationdependent allosteric transitions in hemoglobin establishes a respiratory cycle whereby NO and O 2 are simultaneously taken up in the lung and then delivered to the tissue during the arterial͞venous transit. Thus, hemoglobin would deliver to oxygen-deficient vascular beds not only the oxygen required for sustained metabolism but also a vasodilator (NO).Th...

Arginine contains the guanidinium group and thus has structural similarity to ligands of imidazoline and ␣-2 adrenoceptors (␣-2 AR). Therefore, we investigated the possibility that exogenous arginine may act as a ligand for these receptors in human umbilical vein endothelial cells and activate intracellular nitric oxide (NO) synthesis. Idazoxan, a mixed antagonist of imidazoline and ␣-2 adrenoceptors, partly inhibited L-arginine-initiated NO formation as measured by a Griess reaction. Rauwolscine, a highly specific antagonist of ␣-2 AR, at very low concentrations completely inhibited NO formation. Like L-arginine, agmatine (decarboxylated arginine) also activated NO synthesis, however, at much lower concentrations. We found that dexmedetomidine, a specific agonist of ␣-2 AR was very potent in activating cellular NO, thus indicating a possible role for ␣-2 AR in L-arginine-mediated NO synthesis. D-arginine also activated NO production and could be inhibited by imidazoline and ␣-2 AR antagonists, thus indicating nonsubstrate actions of arginine. Pertussis toxin, an inhibitor of G proteins, attenuated L-arginine-mediated NO synthesis, thus indicating mediation via G proteins. L-type Ca 2؉ channel blocker nifedipine and phospholipase C inhibitor U73122 inhibited NO formation and thus implicated participation of a second messenger pathway. Finally, in isolated rat gracilis vessels, rauwolscine completely inhibited the L-arginine-initiated vessel relaxation. Taken together, these data provide evidence for binding of arginine to membrane receptor(s), leading to the activation of endothelial NO synthase (eNOS) NO production through a second messenger pathway. These findings provide a previously unrecognized mechanistic explanation for the beneficial effects of L-arginine in the cardiovascular system and thus provide new potential avenues for therapeutic development. agmatine ͉ rauwolscine ͉ calcium A rginine is critical to normal growth and multiple physiological processes. It serves as a precursor for the synthesis not only of proteins but also of NO, urea, polyamines, and agmatine. The unequivocal demonstration that NO is the product of NO synthase (NOS)-catalyzed oxidation of L-arginine led to widespread interest in the actions of L-arginine. The K m of L-arginine for endothelial NOS (eNOS) is determined to be 2.9 M (1), and the intracellular L-arginine concentrations are in the range of 0.8-2.0 mM. In other words, cells maintain saturating levels of L-arginine as a substrate for NO synthases. However, an external supply of L-arginine is still required for the cellular production of NO (2). This requirement of exogenous arginine for the cellular NO production is termed ''arginine paradox.'' A number of mechanisms have been proposed to address this phenomenon, including endogenous NOS inhibitors and compartmentalization of intracellular L-arginine. Some have proposed that endogenous NOS inhibitors [e.g., asymmetric dimethylarginine (ADMA)] modulate NO levels by antagonizing intracellular L-arginine (3). An alternative...

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Ectodomain shedding of cell surface proteins is an important process in a wide variety of physiological and developmental events. Recently, tumor necrosis factor-␣-converting enzyme (TACE) has been found to play an essential role in the shedding of several critical surface proteins, which is evidenced by multiple developmental defects exhibited by TACE knockout mice. However, little is known about the physiological activation of TACE. Here, we show that nitric oxide (NO) activates TACEmediated ectodomain shedding. Using an in vitro model of TACE activation, we show that NO activates TACE by nitrosation of the inhibitory motif of the TACE prodomain. Thus, NO production activates the release of cytokines, cytokine receptors, and adhesion molecules, and NO may be involved in other ectodomain shedding processes.Ectodomain shedding is an essential phenomenon involved in the cleavage and release of cell membrane-bound molecules ranging from Alzheimer's amyloid precursor protein to angiotensin-converting enzyme (1). Shedding of ligand/receptor families is involved in diverse processes such as inflammation, hematopoiesis, and normal development (2, 3). Ectodomain shedding can be stimulated by protein kinase C activation and endotoxin (4,5). TACE 1 is a member of a disintegrin and metalloproteinase (ADAM) family, a group of unique zinc-binding transmembrane metalloproteinases (6 -8). TACE has been shown to mediate cleavage of TNF␣ as well as a variety of ectodomains including the TNF p75 receptor, L-selectin, and transforming growth factor-␣ (3). Despite its importance, the physiological regulation of TACE activity remains undefined. Endotoxin does not alter TACE transcription, steady-state mRNA levels, or the level of processed TACE at the cell surface (3). However, endotoxin can induce both nitric oxide (NO) and free radical production (9 -11). It has been shown that both hydrogen peroxide and NO can enhance macrophage production and the secretion of TNF␣ (12-16). However, the mechanism of this effect remains unclear. Many metalloproteinases can be activated by oxidation and dissociation of the cysteine thiol-zinc linkage from a latent enzymatic site, with this complex referred to as a "cysteine zinc switch" mechanism (17). TACE contains a consensus cysteine switch motif in the prodomain, and it has been shown previously that the cysteine in this portion of the molecule is required for the inhibition of TACE activity (18). In the present study, we tested the hypothesis that NO, a molecule produced in a variety of inflammatory conditions, regulates TACE activity and ectodomain shedding. EXPERIMENTAL PROCEDURES Chemicals-(Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)-amino]-diazen-, and 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one were obtained from Alexis Biochemicals (San Diego, CA). Actinomycin D and oxyhemoglobin were from Sigma. Murine interferon-␥ was provided by Genentech (South San Francisco, CA). Escherichia coli LPS 026:B6 was from Difco. All other chemicals were purchased from Sigma.Cell Culture-Mono Mac 6 and Jurkat...