l-citrulline prevents asymmetric dimethylarginine-mediated reductions in nitric oxide and nitrosative stress in primary human airway epithelial cells.

BACKGROUND: Asthma is associated with reduced systemic levels of l-arginine and increased asymmetric dimethylarginine (ADMA). This imbalance leads to nitric oxide synthase (NOS) uncoupling with reduced nitric oxide (NO) formation and greater oxidative and nitrosative stress. Whether this imbalance also occurs in bronchial epitheliumof asthmatics is unknown. OBJECTIVES: We used primary human bronchial epithelial cells (HBECs) from asthmatics and healthy controls to evaluate: (i) ADMA-mediated NOS uncoupling reduces epithelial production of NO and increases oxygen and nitrogen reactive species, and (ii) l-citrulline can reverse this mechanism by recoupling NOS, restoring NO production and reducing oxidative and nitrosative stress. RESULTS: In HBECsIL-13 and INFγ stimulated NOS2 and increased NOx levels. The addition of ADMA reduced NOx and increased H2 O2 levels (p<0.001). Treatment with l-citrulline (800, 1600 µm) rescued NOx when the l-arginine media concentration was 25 µm but failed to do so with higher concentrations (100 µm). Under reduced l-arginine media conditions, HBECs treated with l-citrulline increased the levels of argininosuccinate, an enzyme that metabolizes l-citrulline to l-arginine. l-citrulline prevented the ADMA-mediated increase in nitrotyrosine in HBECs in cells from asthmatics and controls. CONCLUSIONS AND CLINICAL RELEVANCE: Increasing ADMA reduces NO formation and increases oxidative and nitrosative stress in airway epithelial cells. l-citrulline supplementation restores NO formation, while preventing nitrosative stress. These results, suggest that l-citrulline supplementation may indeed be a powerful approach to restore airway NO production and may have a therapeutic potential in diseases in which there is a defective production of NO.

Na,K-ATPase gene transfer mitigates an oxidant-induced decrease of active sodium transport in rat fetal ATII cells.

We investigated whether adenovirus-mediated transfer of genes encoding for subunits of the Na,K-ATPase increases transepithelial Na(+) transport in rat fetal distal lung epithelial (FDLE) monolayers and renders them more resistant to hydrogen peroxide injury. FDLE cells, isolated from rat fetuses at a gestational age of 19 to 20 d (22 d = term), were seeded on filters and infected with replication-incompetent human type 5 adenoviruses containing complementary DNAs encoding for rat Na,K-ATPase alpha(1) or beta(1) subunits (ad alpha(1) and ad beta(1), respectively). Once confluent monolayers were formed, the filters were mounted in Ussing chambers and short circuit currents (I(SC)) were measured. Increased levels of alpha(1) or beta(1) subunit proteins after infection with ad alpha(1) and ad beta(1), respectively, were confirmed by Western blot analysis. Baseline I(SC) increased after transfection with 2 plaque-forming units (pfu) of ad beta(1) from 5.1 +/- 0.3 to 6.1 +/- 0.3 microA/cm(2) (mean +/- SEM; P < 0.05). Permeabilization of the apical membrane with amphotericin B caused a large increase in I(SC); the ouabain-sensitive component of the amphotericin B-elicited I(SC) (ouab(max)) was increased from 4.0 +/- 0.2 (n = 69) in controls to 4.8 +/- 0.2 (n = 15), 5.9 +/- 0.3 (n = 53), 6.9 +/- 0.4 (n = 25), 7.7 +/- 0.9 (n = 16) in monolayers infected with 1, 2, 11, and 22 pfu of ad beta(1), respectively; transfection with ad alpha(1) had no effect on any measured variables. Further, transfection with ad beta(1) in comparison to noninfected monolayers resulted in higher baseline and ouab(max) I(SC) after injury with 500 microM H(2)O(2). We conclude that overexpression of the beta(1) subunit of the Na,K-ATPase may help maintain normal levels of vectorial Na(+) transport across ATII cell monolayers in pathologic conditions.

The p23 molecular chaperones act at a late step in intracellular receptor action to differentially affect ligand efficacies.

Multiple molecular chaperones, including Hsp90 and p23, interact with members of the intracellular receptor (IR) family. To investigate p23 function, we compared the effects of three p23 proteins on IR activities, yeast p23 (sba1p) and the two human p23 homologs, p23 and tsp23. We found that Sba1p was indistinguishable from human p23 in assays of seven IR activities in both animal cells and in yeast; in contrast, certain effects of tsp23 were specific to that homolog. Transcriptional activation by two IRs was increased by expression of any of the p23 species, whereas activation by five other IRs was decreased by Sba1p or p23, and unaffected by tsp23. p23 was expressed in all tissues examined except striated and cardiac muscle, whereas tsp23 accumulated in a complementary pattern; hence, p23 proteins might contribute to tissue-specific differences in IR activities. Unlike Hsp90, which acts on IR aporeceptors to stimulate ligand potency (i.e., hormone-binding affinity), p23 proteins acted on IR holoreceptors to alter ligand efficiencies (i.e., transcriptional activation activity). Moreover, the p23 effects developed slowly, requiring prolonged exposure to hormone. In vitro, p23 interacted preferentially with hormone-receptor-response element ternary complexes, and stimulated receptor-DNA dissociation. The dissociation was reversed by addition of a fragment of the GRIP1 coactivator, suggesting that the two reactions may be in competition in vivo. Our findings suggest that p23 functions at one or more late steps in IR-mediated signal transduction, perhaps including receptor recycling and/or reversal of the response.

Endothelial injury from a circulating mediator following rat liver ischemia.

Ischemia-reperfusion to the liver results in increased microvascular permeability in a nonischemic lung. We hypothesized that a circulatory mediator released from ischemic liver contributed to endothelial cell (EC) damage. Isolated rat livers, made ischemic for 2 h, were reperfused for 10 min. Bovine ECs were incubated for 5 h with pooled liver effluent collected before ischemia (Baseline) or after 10 min of reperfusion (Reperfusion). In the Reperfusion group, there was increased endothelial cell injury, as determined by release of 8-[14C]adenine, (39 +/- 2%) compared to the Baseline group (22 +/- 2%). Permeability of ECs to rhodamine B-labeled dextran (70,000 Mr) was also increased in the Reperfusion group by 54 +/- 9%. There was no significant attenuation in EC injury following incubation with reperfusion effluent stored for 24 h, supplementation with antioxidants (superoxide dismutase + catalase), or inhibition of xanthine oxidase with allopurinol or tungstate. We conclude that the reperfused liver releases a long-lived circulatory mediator of EC injury, which may produce the clinical microvascular injury observed following hepatic ischemia. The mechanism of injury in our model is independent of oxidants or oxidants generated from the circulating xanthine oxidase released from reperfused ischemic liver.

Clinical surfactant preparations mediate SOD and catalase uptake by type II cells and lung tissue.

Pulmonary surfactant mixtures are rapidly taken up by alveolar type II cells and thus may serve as vectors for the pulmonary delivery of antioxidant enzymes to the alveolar epithelium. We prepared emulsions of Survanta with superoxide dismutase (CuZn-SOD) and catalase and quantified their cellular uptake both in vitro and in vivo. Incubations of fetal lung epithelial cells with an emulsion of Survanta plus SOD and catalase mixtures resulted in significant augmentation of SOD and catalase activities (12.8 +/- 4.6 U SOD/microgram DNA; 7.49 +/- 2.21 U catalase/microgram DNA). These numbers were significantly greater than those obtained in controls (1.8 U SOD/microgram DNA; 0.55 +/- 0.52 U catalase/microgram DNA, Survanta alone (0.43 U SOD/microgram DNA; 0.16 U catalase/microgram DNA), and SOD and catalase alone (3.47 +/- 5.2 U SOD/microgram DNA; 4.24 +/- 3.0 U catalase/microgram DNA). Intratracheal instillation of the Survanta plus SOD and catalase mixture resulted in significant augmentation of enzymes by the rat lung homogenates. Confocal microscopic analysis revealed the presence of antioxidant enzymes in the cytoplasm of epithelial cells. We concluded that Survanta supplementation, in addition to replenishing surfactant stores, can also enhance the delivery of antioxidant enzymes to alveolar epithelium both in vitro and in vivo.

Synchronous proctocolectomy and ileoanal pouch formation and the risk of Crohn's disease.

There is controversy on the advisability of one-stage proctocolectomy and the formation of an ileoanal pouch. Accurate preoperative diagnosis is essential to avoid the error of constructing a pouch in a patient with Crohn's disease. Twenty-four consecutive patients undergoing subtotal colectomy for inflammatory bowel disease were reviewed. All patients had been treated with systemic steroids, 23 were on 5-aminosalicylates and 11 on azathioprine. The preoperative diagnoses, based on a combination of clinical features, colonoscopy, barium enema and biopsy histology, were ulcerative colitis (19), Crohn's disease (four) and inflammatory bowel disease (unclassified) (one). The final diagnosis was made on histological examination of the resected specimen. A discrepancy between initial and final diagnosis occurred in eight patients. In three, the diagnosis was changed from ulcerative colitis to Crohn's disease. Three preoperative diagnoses of Crohn's disease were changed to ulcerative colitis (one), Behçet's disease (one) and diverticulitis (one) on final histology. These data suggest that caution should be exercised in performing synchronous proctocolectomy with the formation of an ileoanal pouch.

Delivery of superoxide dismutase to pulmonary epithelium via pH-sensitive liposomes.

Respiratory insufficiency, when treated with oxygen supplementation, or exposure to diverse pulmonary toxins can cause lung damage as a result of increased oxygen radical production. Enzymes such as superoxide dismutase (SOD) may attenuate this pathological process, but the intracellular delivery and antioxidant action of SOD is impeded by its inability to cross cellular membranes. One approach for facilitating intracellular delivery of macromolecules is to entrap SOD into liposomes. The delivery of SOD to lung cells was accomplished using pH-sensitive liposomes, made with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1-oleoyl-2-oleoyl-sn-glycero-3-succinate (DOSG), added to cultured fetal rat lung distal epithelial (FRLE) cells. FRLE cells, obtained from fetuses at day 19 gestation, expressed a high-affinity receptor for surfactant protein A (SP-A) with an apparent dissociation constant (Kd) = 3.6 +/- 0.2 micrograms/ml (5.5 x 10(-9) M) and a capacity of 130 +/- 3 ng/10(6) cells (125,000 +/- 3,000 binding sites/cell). This receptor was utilized for targeting liposomes to cells, after incorporating SP-A during liposome membrane formation. Liposomes were uniformly small (180 +/- 77 nm; mean +/- SD) and stable at 4 degrees C for 1 wk, entrapping 10 +/- 4% of initially added SOD. After incubation of pH-sensitive liposomes containing entrapped SOD with cultured FRLE cells, cell-associated SOD activity was increased 5.1-fold from 7.8 +/- 2.5 to 40.1 +/- 3.3 U SOD/mg cell protein. Incorporation of SP-A into liposomes increased by 6.2-fold the delivery of liposomal SOD to cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Superoxide and peroxynitrite in atherosclerosis.

The role of reactive oxygen species in the vascular pathology associated with atherosclerosis was examined by testing the hypothesis that impaired vascular reactivity results from the reaction of nitric oxide (.NO) with superoxide (O2-), yielding the oxidant peroxynitrite (ONOO-). Contractility studies were performed on femoral arteries from rabbits fed a cholesterol-supplemented diet. Cholesterol feeding shifted the EC50 for acetylcholine (ACh)-induced relaxation and impaired the maximal response to ACh. We used pH-sensitive liposomes to deliver CuZn superoxide dismutase (SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) to critical sites of .NO reaction with O2-. Intravenously injected liposomes (3000 units of SOD per ml) augmented ACh-induced relaxation in the cholesterol-fed group to a greater extent than in controls. Quantitative immunocytochemistry demonstrated enhanced distribution of SOD in both endothelial and vascular smooth muscle cells as well as in the extracellular matrix. SOD activity in vessel homogenates of liposome-treated rabbits was also increased. Incubation of beta very low density lipoprotein with ONOO- resulted in the rapid formation of conjugated dienes and thiobarbituric acid-reactive substances. Our results suggest that the reaction of O2- with .NO is involved in the development of atherosclerotic disease by yielding a potent mediator of lipoprotein oxidation, as well as by limiting .NO stimulation of vascular smooth muscle guanylate cyclase activity.

The role of cytochrome c and mitochondrial catalase in hydroperoxide-induced heart mitochondrial lipid peroxidation.

The role of cytochrome c and catalase in hydroperoxide-induced lipid peroxidation of rat heart mitochondria was investigated. Mitoplasts were prepared from hearts of aminotriazole-treated rats which displayed both an 80-90% reduction in matrix catalase activity and rate of H2O2 consumption. Catalase-depleted mitochondria were more susceptible to H2O2-dependent lipid peroxidation and had similar extents of tert-butyl hydroperoxide (t-BuOOH)-induced lipid peroxidation compared with control mitochondria. The magnitude of lipid peroxidation induced by H2O2 was greater than that for t-BuOOH in catalase-depleted mitochondria, while t-BuOOH induced soybean phosphatidylcholine (PC) liposome lipid peroxidation to a greater extent than H2O2. The t-BuOOH- and H2O2-dependent mitochondrial lipid peroxidation was inhibited 50 and 7%, respectively, by cytochrome c3+ depletion of mitochondria. Similar relative sensitivities to t-BuOOH- and H2O2-dependent peroxidation occurred for cytochrome c(3+)-supplemented soybean PC liposomes. These data show a critical role for cytochrome c3+ in hydroperoxide-induced mitochondrial lipid peroxidation and demonstrate the importance of matrix catalase in protecting heart mitochondria from the toxicity of H2O2.

Ethanol induces the generation of reactive free radicals by neural crest cells in vitro.

Developmental craniofacial anomalies related to the neural crest derived ectomesenchymal cell population are associated with fetal alcohol syndrome (FAS). Information regarding any potential relationship between ethanol, free radicals, and the viability, proliferation, etc., of isolated neural crest cells was sought. The hypersensitivity of neural crest cells to ethanol was observed. This drug severely depressed cell viability while simultaneously inducing the generation of such reactive oxygen intermediates (ROI) as superoxide, hydrogen peroxide, and hydroxyl anions. Addition of the free radical scavenging enzyme superoxide dismutase to the culture medium significantly reversed these effects of ethanol. The cytotoxicity of ethanol was further confirmed by the release of radiolabeled chromium (51Cr) from cells prelabeled prior to ethanol treatment. This effect was also depressed by the addition of superoxide dismutase. Interestingly, an assay for superoxide dismutase activity showed that neural crest cells may be devoid of this enzyme. The latter may help to explain the overt sensitivity of these cells to such a broad spectrum of teratogens, many of which can either dissociate directly into ROI, or cause the radicalization of biological structures and molecules. Plasmalemmal lipids (via lipid peroxidation) and DNA are at an especially high risk from uncontrolled ROI. Changes in neural crest cell surface morphology, i.e., loss of microvilli, formation of xeiotic blebs, as well as the "leakage" of radiolabeled Cr from prelabeled cells, would seem to show that ethanol, as a result of induced free radical formation, alters the physiology and biochemistry of the cell membrane. These findings however, should not exclude other potential sites for ETOH-induced cell injury related to free radicals, especially the nuclei (DNA), mitochondria, organelle membranes, and the cytoskeleton.

Hyperoxia increases H2O2 production by brain in vivo.

Hyperoxia and hyperbaric hyperoxia increased the rate of cerebral hydrogen peroxide (H2O2) production in unanesthetized rats in vivo, as measured by the H2O2-mediated inactivation of endogenous catalase activity following injection of 3-amino-1,2,4-triazole. Brain catalase activity in rats breathing air (0.2 ATA O2) decreased to 75, 61, and 40% of controls due to endogenous H2O2 production at 30, 60, and 120 min, respectively, after intraperitoneal injection of 3-amino-1,2,4-triazole. The rate of catalase inactivation increased linearly in rats exposed to 0.6 ATA O2 (3 ATA air), 1.0 ATA O2 (normobaric 100% O2) and 3.0 ATA O2 (3 ATA 100% O2) compared with 0.2 ATA O2 (room air). Catalase inactivation was prevented by pretreatment of rats with ethanol (4 g/kg), a competitive substrate for the reactive catalase-H2O2 intermediate, compound I. This confirmed that catalase inactivation by 3-amino-1,2,4-triazole was due to formation of the catalase-H2O2 intermediate, compound I. The linear rate of catalase inactivation allows estimates of the average steady-state H2O2 concentration within brain peroxisomes to be calculated from the formula: [H2O2] = 6.6 pM + 5.6 ATA-1 X pM X [O2], where [O2] is the concentration of oxygen in ATA that the rats are breathing. Thus the H2O2 concentration in brains of rats exposed to room air is calculated to be about 7.7 pM, rises 60% when O2 tension is increased to 100% O2, and increases 300% at 3 ATA 100% O2, where symptoms of central nervous system toxicity first become apparent. These studies support the concept that H2O2 is an important mediator of O2-induced injury to the central nervous system.

Liposome-mediated augmentation of brain SOD and catalase inhibits CNS O2 toxicity.

Enzymes specific for O-2 and H2O2 metabolism [superoxide dismutase (SOD) and catalase] can be delivered to the rat brain following entrapment in liposomes and intravenous injection and will protect against hyperbaric O2-induced convulsions in rats. Liposome-mediated superoxide dismutase and catalase augmentation of brain enzyme activity was 2.7-fold and 1.9-fold, respectively, 15 min after intravenous injection of superoxide dismutase plus catalase-entrapped liposomes. Rats treated with liposomes containing superoxide dismutase plus catalase 2 h before 6 ATA 100% O2 exposure had the time to convulsion extended three times that of controls. This protective effect was dose-dependent and was primarily due to augmentation of catalase activity. These findings show O-2 and H2O2 are important mediators of hyperbaric O2-induced central nervous system toxicity and that liposome-mediated augmentation of brain antioxidant enzymes has a biological effect.

Pulmonary antioxidant enzyme maturation in the fetal and neonatal rat. II. The influence of maternal iron supplements upon fetal lung catalase activity.

It was observed that the lung catalase activity of premature (day 21 of gestation; term = 22 days) rat pups is affected by maternal iron intake. Pups from control dams receiving Purina Lab Chow and water ad libitum have only 50% of the lung catalase activity of pups from dams who received 1 mg/kg parenteral iron dextran daily from day 7 to day 20 of gestation. Other oxygen-protective enzymes, copper-zinc and manganese superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase, were unaffected by maternal iron supplements.

Toxicities with intravenous and subcutaneous administration of a petroleum distillate.

Although ingestion of petroleum distillates is common, injection of these products has not been widely reported. The effects of subcutaneous administration have not been fully documented. Presented is a 27-year-old male who developed a sterile abscess and pneumonitis following concurrent intravenous and subcutaneous administration of a petroleum distillate.

Fatty acid secretion and metabolism in 'activated' rabbit alveolar macrophages.

The lipid content of bronchoalveolar lavages collected from both control rabbits and rabbits undergoing a pulmonary inflammatory response induced by intravenous injection of complete Freund's adjuvant was examined. A maximum of 4 x 10(8) alveolar macrophages could be recovered from the lavage of injected rabbits, a 10-fold greater number of cells than could be obtained from control rabbits. Increased amounts of unsaturated free fatty acids were present in the lavage lipids of injected rabbits, and no change in the amount and degree of saturation of lavaged phosphatidylcholine occurred. Alveolar macrophages recovered from injected rabbits contained 25 to 40% less lipid per cell, as measured by total fatty acid composition. Free fatty acids are released from phospholipids of adjuvant-induced alveolar macrophages incubated in vitro following lavage. Concentrations of N-formylmethionyl-phenylalanine similar to those which stimulate macrophage chemotaxis and bactericidal activity enhance this fatty acid release. Alveolar macrophages incorporate both saturated and unsaturated fatty acids with similar efficiency, primarily into phospholipids and triacylglycerols. Thus, activation of alveolar macrophages which results in a relative increase in internal phospholipase activity with concomitant large losses in cellular phospholipid results not only in liberation of chemokinetic fatty acids but also in considerable loss of membrane components.

Intelligibility of time-expanded speech with normally hearing and elderly subjects.

The effects of time-expanded monosyllabic words (NU-6) on the auditory discrimination performance of 15 young adults with normal hearing and 20 elderly subjects were studied. Three conditions of time expansion, 30, 60 and 100%, plus a 0% control condition, were presented at four sensation levels (8, 16, 24 and 32 dB). For the normally hearing subjects, auditory discrimination performance at all ratios of time expansion was equal to the 0% condition. Results for the elderly subjects indicated intelligibility was inversely related to time expansion at the 30 and 60% conditions. However, at the 100% condition, speech intelligibility improved over the 60% condition at 8 and 16 dB sensation level. At 24 and 32 dB sensation level, performance at 100% was equal to the 60% condition. With the normal and elderly subjects, ear and list effects were minimal. The results are discussed in terms of the clinical value of this procedure and in light of literature that reviews the performance of subjects on tests employing various temporally altered stimuli.

Dietary aureomycin and the response of the fowl to stressors.

1. Conventional or gnotobiotic chicks, when injected from 1 d to 3 weeks of age with adrenocorticotrophic hormone (120IU/kg, three times weekly), showed a depressed growth rate, adrenal hypertrophy and depletion of cholesterol form the adrenal glands. 2. Feeding a diet supplemented with aureomycin (10 mg/kg) did not have any consistent ameliorating effect on the response of the stressed bird as judged by the above parameters. 3. It was found that treating germfree chicks with five daily injections of sterile milk on days 3 to 7 did not depress growth rate at any time, nor could differences in adrenal size or cholesterol stores be detected at the end of the 21-d experimental period. The responses were not modified by feeding an aureomycin-supplemented diet.

Effects of differential rearing condition on heart rate conditioning and response suppression.

Rats were reared in either an enriched or isolated environment for 60 days following weaning. Neither rearing environment nor sex affected heart rate conditioning. However, when the CS used during conditioning was subsequently presented in a drinking situation, males reared in isolation showed a greater degree of response suppression than did similarly reared females. Males and females reared in the enriched environment did not differ in degree of response suppression.