Mark J. S. Miller

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.

PEROXYNITRITE-INDUCED APOPTOSIS IN T84 AND RAW 264.7 CELLS : ATTENUATION BY L-ASCORBIC ACID

The free radicals nitric oxide and superoxide react to form peroxynitrite (ONOO-), a potent cytotoxic oxidant. This study was designed to evaluate whether addition of L-Ascorbic acid (AsC) into the culture medium decreases peroxynitrite-induced apoptosis in human intestinal epithelial (T84) and murine macrophage (RAW 264.7) cell lines. In Experiment 1, T84 and RAW 264.7 cells were divided in two protocols: (1) treated with 100-300 microM ONOO- and incubated for 4 h, and (2) treated with 10-100 microM ONOO- and incubated overnight (14 h). In Experiment 2, T84 and RAW 264.7 cells were treated with 300 microM ONOO- and 500 microM AsC and incubated for 4 h. In Experiment 3, T84 and RAW 264.7 cells were preincubated for 2 h with 500 microM AsC then exposed to 300 microM ONOO- for 4 h. Cell viability (necrosis) was assessed by trypan blue dye exclusion. Apoptosis was quantified with a cell death detection ELISA assay. In the 4 h protocol, ONOO- induced apoptosis in T84 and RAW 264.7 cells, at levels of 100-300 microM. Concentrations of ONOO- greater than 300 microM caused necrosis. In contrast, extension of the protocol to 14 h indicated that ONOO- induced apoptosis at lower concentrations (50;-75 microM), with concentrations > 75 microM resulting in necrosis. AsC administered to the media or with preincubation plus washout, decreased peroxynitrite-induced apoptosis in T84 and RAW 264.7 cells. These results indicate that ONOO- may contribute to the pathophysiology of gut inflammation by promoting cell death and ascorbic acid may protect against peroxynitrie-induced damage.

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.

Nitric oxide as a mediator of inflammation?—You had better believe it

Nitric oxide has enigmatic qualities in inflammation. In order to appreciate the precise contributions of nitric oxide to a pathophysiological process, one must account for enzyme source, coproduction of oxidants and antioxidant defences, time, rate of nitric oxide production, cellular source, peroxynitrite formation and effects on DNA (mutagenesis/apoptosis). We contend that there is ample evidence to consider nitric oxide as a molecular aggressor in inflammation, particularly chronic inflammation. Therapeutic benefit can be achieved by inhibition of inducible nitric oxide synthase and not the donation of additional nitric oxide. Furthermore, there is growing appreciation that nitric oxide and products derived thereof, are critical components linking the increased incidence of cancer in states of chronic inflammation.

Anti-inflammatory properties of interleukin-10 administration in hapten-induced colitis.

Therapeutic efficacy of interleukin-10 administration in colonic inflammation was assessed in rats. Following intracolonic instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS), subcutaneous administration of 1-1000 micrograms/kg per day interleukin-10, or a placebo (0.9% NaCl) was commenced and continued for 5 days. Interleukin-10 administered at 1, 10 and 100 micrograms/kg per day significantly reduced myeloperoxidase activity by 34, 57, and 28%, respectively, compared to the placebo-treated group, which was paralleled by an attenuation of colonic tumor necrosis factor alpha (TNF-alpha) content. In contrast, the severity of mucosal necrosis was not affected by interleukin-10 administration at the dose range used. In addition, the 10-fold elevation in nitric oxide release, 5-fold rise in colonic nitrite production and enhanced expression of inducible nitric oxide synthase, associated with TNBS colitis, was not suppressed by interleukin-10. Interleukin-10 gene expression was elevated during the first 14 days of TNBS colitis. We conclude that 5 days administration of interleukin-10 in TNBS colitis displays mild anti-inflammatory properties which were not mediated via a nitric oxide-dependent pathway, but may involve TNF-alpha.

Fetal growth retardation in rats may result from apoptosis: Role of peroxynitrite

Administration of the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) results in fetal growth retardation. This study was designed to further examine the influence of NO on fetal growth, specifically, the potential role of inducible NOS and to evaluate the possibility that apoptosis contributed to uteroplacental dysfunction. L-NAME administration caused a paradoxical increase in NO synthesis determined by direct detection of NO by electrochemistry, nitrite accumulation, and cGMP levels, indicating that a lack of NO was not the cause of the fetal growth retardation. Additionally, supplemental L-arginine or NO donors failed to reverse the effects of L-NAME on fetal and placental size. Administration of low dose endotoxin (30 micrograms/kg IP daily for 6 d) also caused significant reductions in fetal and placental size and increased NO synthesis comparable to that seen with L-NAME. Inducible NOS was constitutively expressed in the pregnant uterus (smooth muscle and epithelia) and placenta (sinusoids and macrophages) but was absent in the nonpregnant state as determined by RT-PCR and immunohistochemistry. Neither L-NAME nor endotoxin modified the expression of iNOS. In situ evidence for apoptosis (DNA fragmentation) was minimal to absent in control pregnant rats, but markedly evident in the placenta (decidua) and uterus of rats treated with L-NAME or endotoxin. Immunohistochemical evidence for nitrotyrosine, a marker for peroxynitrite formation, was absent in control rats but colocalized with apoptosis in the L-NAME and LPS groups. We conclude that L-NAME-induced fetal growth retardation is not due to a lack of NO, but as for endotoxin, results from a net reduction in cellular proliferation due to the induction of apoptosis, possibly in response to peroxynitrite formation.

Inducible Nitric Oxide Synthase and the Regulation of Central Vessel Caliber in the Fetal Rat

BACKGROUND The purpose of this study was to evaluate the possibility that inducible nitric oxide synthase (iNOS) regulates the fetal circulation. METHODS AND RESULTS Positive evidence for iNOS gene expression was noted in heart central vessels and placenta of untreated rat fetuses. Rats in the last week of pregnancy were treated for 5 days with L-NG-(1-Iminoethyl)lysine (L-NIL), a selective inhibitor of iNOS, at 1, 10, and 100 micrograms/mL in the drinking water. To raise NO levels, lipopolysaccharide (LPS) 30 micrograms/kg was given by intraperitoneal injection, and sodium nitroprusside (SNP) was placed in mini-osmotic pumps to deliver 10 micrograms/kg per minute. Control animals were undisturbed. On day 21 of gestation, dams were anesthetized and fetuses were delivered by cesarean section and rapidly frozen in isopentane chilled in liquid nitrogen. Frozen sections (10 microns) were used to reconstruct a computer-generated three-dimensional image of the great vessels and ductus arteriosus. Significant constriction of the great vessels and ductus arteriosus was observed with L-NIL, whereas both LPS and SNP dilated these vessels. The vasorelaxant effect of LPS was blocked by L-NIL. NO release from placental explants was 633 +/- 41 nmol/L under basal conditions, increasing to 4.0 +/- 0.4 mumol/L with LPS administration, although placental iNOS message and protein levels were unchanged. CONCLUSIONS We suggest that nitric oxide, generated by iNOS, plays a significant role in control of major vessel and ductus arteriosus caliber in the rat fetus. In regard to the nitrergic regulation of the circulation, the fetus is clearly different from the adult.

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.

Perinatal nitric oxide synthase inhibition retards neonatal growth by inducing hypertrophic pyloric stenosis in rats

ABSTRACT: Administration of the nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) during pregnancy has been shown to compromise fetal growth. This study was designed to determine whether aminoguanidine, a predominate inhibitor of inducible NOS, affects fetal outcome. In addition, we extended the prenatal administration of L-NAME into the postnatal period (14 d) to determine whether neonatal growth and maturation were also affected. L-NAME, but not aminoguanidine, compromises fetal and placental growth. When compared with control 14-d-old pups, postnatal L-NAME compromised neonatal growth, whether it was given directly (intraperitoneally) (39.7 ± 1.1 versus 24.1 ± 1.0 g) or indirectly (38.6 ± 0.5 versus 22.2 ± 1.2 g) via maternal breast milk. Neonatal growth retardation was asymmetric, with brain sparing, suggesting a nutritional origin. L-NAME administration resulted in growth retardation that extended into adulthood, without evidence of catch-up growth. Treated neonates displayed the hallmarks of hypertrophic pyloric stenosis. Significant increases in stomach weight/pup weight (9.9 ± 0.3 versus 8.2 ± 0.4 X 103) and stomach volume/pup weight (12.0 ± 0.6 versus 9.4 ± 0.6 mL/100 g) with a concomitant decrease in small intestine weight/length (2.10 ± 0.08 versus 3.18 ± 0.13 g/100 cm) was noted in treated versus control pups (p < 0.05). Muscularis hypertrophy at the pyloric sphincter in the L-NAME-treated pups was noted by histology. Blood pressure was elevated in the L-NAME-treated pups (93 ± 6 versus 60 ± 5 mm Hg in control pups, p < 0.05). These findings are consistent with inhibition of neuronal and endothelial NOS activity. We conclude that NO, formed via the constitutive isoforms of NOS, is a critical determinant of fetal and neonatal growth and maturation.

Limb Reduction Defects after Prenatal Inhibition of Nitric Oxide Synthase in Rats

ABSTRACT: To determine the influence of nitric oxide (NO) on vascular tone during fetal development, timed pregnant rats received the NO synthase inhibitor NG-nitro-L-arginine methyl ester for consecutive 4, 7, or 14 d before parturition (postorganogenesis). Offspring demonstrated limb reduction defects (incidence, 53%) involving either or both hindlimbs, whereas forelimbs were uniformly spared. Defects were dose-dependent but independent of the duration of administration occurring with equal frequency in 4-, 7-, and 14-d treatment groups. Histologic analysis revealed features characteristic of vascular disruption with hemorrhagic necrosis and loss of structure. The defects were prevented by concurrent maternal administration of L-arginine or the NO donors S-nitroso-N-acetyl-penicillamine and sodium nitroprusside. Defects were not seen after prenatal treatment with aminoguanidine. To study basal and agonist-mediated NO release, newborn femoral and brachial arteries were cannulated with a glass micropipette under constant pressure, and changes in intraluminal diameter (micrometers) were measured in response to acetylcholine and the NO synthase inhibitor Nω-nitro-L-arginine. Newborn femoral and brachial vessels demonstrated a dramatic (59%) decrease in resting diameter compared with adult vessels (16%). These findings suggest that basal NO release is upregulated during fetal development concurrent with the processes that increase maternal NO release. The data also suggest that up-regulation of NO release occurs throughout the fetal systemic circulation and is not restricted to hindlimbs. This is the first study to demonstrate inhibition of NO release in the pathogenesis of limb reduction defects.