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first through a gaswashing bottle containing 1 M deaerated KOH solution.RBC and Free Hemoglobin Preparation-Blood was withdrawn from rats and centrifuged at 2300 ϫ g for 10 min.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...

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Background: Uncaria tomentosa is a vine commonly known as cat’s claw or ‘uña de gato’ (UG) and is used in traditional Peruvian medicine for the treatment of a wide range of health problems, particularly digestive complaints and arthritis. Purpose: The aim of this study was to determine the proposed anti‐inflammatory properties of cat’s claw. Specifically: (i) does a bark extract of cat’s claw protect against oxidant‐induced stress in vitro, and (ii) to determine if UG modifies transcriptionally regulated events. Methods: Cell death was determined in two cell lines, RAW 264.7 and HT29 in response to peroxynitrite (PN, 300 μM). Gene expression of inducible nitric oxide synthase (iNOS) in HT29 cells, direct effects on nitric oxide and peroxynitrite levels, and activation of NF‐κB in RAW 264.7 cells as influenced by UG were assessed. Chronic intestinal inflammation was induced in rats with indomethacin (7.5 mg/kg), with UG administered orally in the drinking water (5 mg/mL). Results: The administration of UG (100 μg/mL) attenuated (P < 0.05) peroxynitrite‐induced apoptosis in HT29 (epithelial) and RAW 264.7 cells (macrophage). Cat’s claw inhibited lipopolysaccharide‐induced iNOS gene expression, nitrite formation, cell death and inhibited the activation of NF‐κB. Cat’s claw markedly attenuated indomethacin‐enteritis as evident by reduced myeloperoxidase activity, morphometric damage and liver metallothionein expression. Conclusions: Cat’s claw protects cells against oxidative stress and negated the activation of NF‐κB. These studies provide a mechanistic evidence for the widely held belief that cat’s claw is an effective anti‐inflammatory agent.

noni, the juice of the fruit from the Morinda citrifolia plant, has been used for centuries as a medicinal agent. We tested the effects of noni juice in a three-dimensional fibrin clot matrix model using human placental vein and human breast tumor explants as sources for angiogenic vessel development. Noni in concentrations of 5% (vol/vol) or greater was highly effective in inhibiting the initiation of new vessel sprouts from placental vein explants, compared with initiation in control explants in media supplemented with an equivalent amount of saline. These concentrations of noni were also effective in reducing the growth rate and proliferation of newly developing capillary sprouts. When used at a concentration of 10% in growth media, noni was able to induce vessel degeneration and apoptosis in wells with established capillary networks within a few days of its application. We also found that 10% noni juice in media was an effective inhibitor of capillary initiation in explants from human breast tumors. In tumor explants which did show capillary sprouting, the vessels rapidly degenerated (2-3 days) in those exposed to media supplemented with 10% noni.

Genistein, a principal soy isoflavone, has been identified as a protein kinase inhibitor that possesses immunosuppressive and anti-inflammatory properties. The aim of the study was to determine if genistein modified chronic ileitis in guinea pigs induced by the hapten trinitrobenzene sulfonic acid (TNBS), and the activity index of cultured macrophages (RAW 264.7 cells) stimulated with lipopolysaccharide (LPS). Genistein at low doses (0.1 mg/kg, s.c.) had mild anti-inflammatory effects in TNBS ileitis. Therapeutic benefit included a reduction in nitric oxide production, granulocyte infiltration and improved mucosal architecture. Genistein, at low doses, also appeared to attenuate immunohistochemical staining for inducible nitric oxide synthase (iNOS) and nitrotyrosine. The beneficial effects of genistein were not apparent at doses above 0.1 mg/kg. We found that genistein also inhibited LPS-induced nitrite production by cultured macrophages and protected against LPS-induced necrosis despite its ability to cause apoptosis. These results indicate that genistein displayed mild anti-inflammatory properties which may, in part, involve an attenuation of nitric oxide release via inducible nitric oxide synthase, and the formation of peroxynitrite.