Halina Sadowska-Krowicka

DIFFUSION-LIMITED REACTION OF FREE NITRIC OXIDE WITH ERYTHROCYTES

Concentration changes of nitric oxide (NO) were monitored using an NO-sensitive electrode in phosphate-buffered saline (PBS) with either free oxyhemoglobin (oxyHb) or red blood cells (RBCs). In aerated PBS, the half-life of 0.9 μm NO is greater than 4 min. NO is undetectable (<50 nm) when added to a solution of oxyHb because the reaction of NO with oxyHb is rapid. The disappearance rate of NO in PBS containing RBCs is rapid, compared with PBS, but it is much slower (by a factor of approximately 650) than with an equivalent solution of free oxyHb. The half-life of NO is inversely proportional to the concentration of RBCs, independent of oxyHb concentration inside RBCs, and the disappearance rate of NO is first order in NO concentration and first order in the concentration of RBCs. After all the oxyHb reacts with NO to form methemoglobin, the disappearance rate of NO slows greatly. These data indicate that the reaction of NO with oxyhemoglobin within RBCs is limited by the diffusion of NO into the cell, which has also been shown previously for the reaction of O2 with deoxyhemoglobin. Experimental data show that the half-life of NO in the presence of 2.1 × 106 RBCs/ml is 4.2 s. From this value, we estimate that the half-life of NO in whole blood (5 × 109RBCs/ml) will be 1.8 ms. A simple analytical expression for the half-life of NO in PBS with RBCs was derived in this study based on a spherical diffusion model. The calculated half-life of NO from the expression is in good agreement with the experimental values.

Genistein and gut inflammation: role of nitric oxide.

Abstract 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 antiinflammatory properties which may, in part, involve an attenuation of nitric oxide release via inducible nitric oxide synthase, and the formation of peroxynitrite.

Inhibition of angiogenic initiation and disruption of newly established human vascular networks by juice from Morinda citrifolia (noni).

Abstractnoni, 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.

Nitric oxide: the Jekyll and Hyde of gut inflammation.

We studied the effects of seven day treatment with the nitric oxide synthase (NOS) inhibitorNG-nitro-l-arginine (l-NAME), administered in the drinking water (100 μg/mlad lib) of female guinea pigs. The effects of NOS inhibition were evaluated in naive animals and in guinea pigs with ileitis induced by intraluminal trinitrobenzenesulfonic acid (TNBS). After 7 days, animals were anesthetized, a sterile saline lavage injected into an ileal loop and removed after 30 min for analysis. In naive guinea pigs,l-NAME caused a marked increase in ileal myeloperoxidase activity and conversion of the mucosa from an absorptive to a secretory state. TNBS-treated guinea pigs had a similar, marked increase in granulocyte infiltration and a mucosal secretory response. However, in contrast to naive animals,l-NAME treatment was anti-inflammatory, reverting all responses to the basal state. We conclude that intestinal nitric oxide serves an antiinflammatory role under basal conditions, whereas in the TNBS model of chronic ileitis, nitric oxide is a critical mediator of gut injury.

Inhibition of calcium-dependent nitric oxide synthase causes ileitis and leukocytosis in guinea pigs

As nitric oxide reduces gut epithelial permeability, we designed a study to determine if chronic nitric oxide synthase inhibition predisposes the gut to inflammation. Nitric oxide synthase (NOS) inhibitors were administered in the drinking waterad libitum, for seven days: aminoguanidine (10 µg/ml), a selective inhibitor of the inducible form of nitric oxide synthase; andNG-nitro-l-arginine methyl ester (l-NAME, 1, 10, and 100 µg/ml), which inhibits both the constitutive and inducible forms. Control animals drank tap water only or water withd-NAME, the inactive enantiomer. After one week, circulating leukocyte count and tissue myeloperoxidase activity were measured.l-NAME (100 µg/ml), but notd-NAME or aminoguanidine, caused a twofold increase in a circulating leukocyte numbers. This increase in leukocyte numbers was time- and dose-dependent, but the differential count was unaltered. Tissue myeloperoxidase (MPO) activity as an index of granulocyte infiltration was comparable in all groups in the stomach, jejunum, colon, liver, lung, kidney, heart, and skeletal muscle. However, ileal MPO activity was elevated threefold in thel-NAME- (100 µg/ml) treated group (P<0.05). Results in thed-NAME and aminoguanidine groups were similar to controls.l-NAME administration resulted in a reduction in NOS activity ([14C]citrulline formation) in the ileum but not jejunum, whereas cGMP levels were elevated in both ileum and jejunum. We conclude that chronic inhibition of the constitutive form of nitric oxide synthase predisposes the ileum to inflammation and leads to a progressive leukocytosis.

Protective efficacy of recombinant urease B and aluminum hydroxide against Helicobacter pylori infection in a mouse model.

Efforts are underway for the development of an effective vaccine against Helicobacter pylori infection. We prepared recombinant full-length (568 aa) H. pylori recombinant urease B (rUreB) protein and tested it for immunogenicity and protection. BALB/c mice received either rUreB (40 μg) plus CpG (10 μg) intranasally, rUreB (50 μg) plus 3% aluminum hydroxide (50 μL) intramuscularly or rUreB (25 μg) plus Freunds adjuvant (25 μL) subcutaneously, three times (weeks 0, 2 and 6). Intranasal rUreB plus CpG was neither immunogenic nor protective; intramuscular rUreB plus aluminum hydroxide was immunogenic and modestly protective, and subcutaneous rUreB plus Freunds adjuvant was immunogenic and highly protective. The fact that protection was improved with Freunds adjuvant indicates that rUreB is a good antigen for a vaccine but that it needs a stronger adjuvant than aluminum hydroxide.

Rabbit gut permeability in response to histamine chloramines and chemotactic peptide

Granulocyte-derived chlorinated amines and bacterial formyl peptides are thought to enhance epithelial permeability. In the current study, gut permeability to [51Cr]ethylenediaminetetraacetic acid (EDTA) was monitored in response to luminal formyl-methionyl-leucyl-phenylalanine (fMLP) and histamine monochloramine and dichloramine. Responses were determined in rabbits during states of basal and elevated permeability. Luminal fMLP had minimal effects of gut permeability in control and injured states. Histamine monochloramine or dichloramine enhanced epithelial permeability under basal conditions; this effect was exaggerated by a pre-existing injury. Both histamine monochloramine and dichloramine retained full histamine agonist properties, and a combination of antioxidant and antihistamine therapy was required to block this increase in gut permeability. Whereas histamine chloramines caused a dose-dependent cytotoxicity in rat-cultured enterocytes, marked histological changes to the mucosa were not evident, nor were mucosal glutathione levels depleted. As histamine chloramines retain the histaminergic and oxidizing potential of their precursors, they represent a unique form of inflammatory mediator, although their highly reactive nature precludes in vivo confirmation of their formation.

Chronic Administration of the Nitric Oxide Synthase Inhibitor, L-NAME, Increases Circulating Endothelin Levels in Guinea Pigs

Endothelin and nitric oxide are both endothelium-derived vasoactive factors which appear to play a role in a variety of circulatory disorders (Leppaluoto and Ruskoaho, 1992; Moncada et al., 1991; Nathan, 1992; Rubanyi and Parker Botelho, 1991). Boulanger and Luscher (1990) noted in isolated blood vessels that the release of endothelin and nitric oxide were inversely related. The intracellular levels of cGMP appear to be the determinant of this response, i.e., nitric oxide elevated cGMP and decreased endothelin release, while decreased cGMP promotes endothelin release. In contrast to the inhibition of endothelin release by nitric oxide, Hirata et al. (1993) noted that endothelin promotes nitric oxide release in cultured bovine endothelial cells.

Histamine is a transient marker of small intestinal injury induced by luminal acetic acid and casein

We compared the time course of histamine release with other markers of intestinal injury in a rabbit model of necrotizing enterocolitis. Injury was induced by luminal acetic acid (200 mM) and casein (10 mg/ml) and experiments terminated after 45 min or 3 hr. Compared to saline controls there was a significant elevation of epithelial permeability (51Cr-EDTA clearance) and luminal protein levels at both time points. Luminal fluid histamine levels were approximately 120-fold greater than saline controls at 45 min but were indistinguishable from control values at 3 hr. We conclude that although mast cell activation is a characteristic of this model, elevations in histamine levels are transient.

Misoprostol attenuates acetic acid-induced damage in rabbit distal small intestine

The influence of misoprostol pretreatment (100 μg/loop intraluminally) on small intestinal damage induced by acetic acid was evaluated in anesthetized rabbits. In this model injury was induced by intraluminal administration into loops of distal small intestine, of a solution of calcium gluconate (50 mg/ml) and acetic (200 mM). After 3 hr damage was associated with increase in loop fluid volume, loop fluid protein levels and epithelial permeability to51Cr-EDTA, all of which were attenuated by misoprostol pretreatment. Similar protective effects were noted 45 min after the insult, suggesting that misoprostol may be therapeutically useful in conditions where the epithelial barrier is compromised.