Reversal of the endocrine toxicity of commercially produced perfluorochemical emulsion

Perfluorochemicals provide a biologically inert system for oxygen transport to tissue. The purpose of the present study was to determine if a simple clean-up procedure could reverse the endocrine toxicity of a commercially produced perfluorochemical emulsion, Oxypherol-E.T. The clean-up procedure consisted of a combined resin and dialysis treatment. The endocrine toxicity of the untreated and treated perfluorochemical emulsions was tested by determining their effect on testosterone secretion by rat testes perfused in vitro. Rat testes perfused with untreated Oxypherol-E.T. secreted low amounts of testosterone. However, the treated Oxypherol-E.T. was an effective and nontoxic oxygen carrier for testes perfused in vitro. The results are significant because they suggest that the endocrine toxicity of Oxypherol-E.T. is caused by toxic contaminants and not the perfluorochemicals. Additional experiments revealed that the fluoride ion may be the primary toxic contaminant of Oxypherol-E.T. The data support the efficacy of perfluorochemicals as oxygen carriers for rat testes perfused in vitro.     

Induction of cytochrome P-450 in rat liver microsomes by perfluorodecalin

Perfluorodecalin was incorporated into phospholipid liposomes and injected intraperitoneally in various dozes. The maximal cytochrome P-450 induction is reached 48 hours after perfluorodecalin injection. Cytochrome P-450 content increases 4 times after perfluorodecalin injection in dose of 0.6 ml/kg in homogenate, and 6 times after perfluorodecalin injection in a dose of 0.4 ml/kg in microsomes. Phenobarbital and perfluorodecalin induce several cytochrome P-450 isozymes and cause the appearance of a new isozyme with mass 56 kD absent in microsomes of intact CBA mice. Perfluorodecalin induction strongly increased the rate of NADPH-dependent aminopyrine nN-demethylation (6-7 times per mg of microsomal protein and 1.5 times per nmol cytochrome P-450). The rate of NADPH-dependent hydroxylation of aniline was not affected by perfluorodecalin induction.     

Fluorocarbon/lecithin emulsions: identification of EYP-coated fluorocarbon droplets and fluorocarbon-empty vesicles by freeze-fracture electron microscopy

Freeze-fracture has been used to examine perfluorodecalin/egg yolk phospholipid emulsions (70:8, w/v%) destined to be used as injectable oxygen carriers. The fluorocarbon displays a specific granular aspect upon freeze-fracture which makes it readily recognizable and allows the distinction between two populations of objects on the micrographs: fluorocarbon droplets and water-filled lipid vesicles.     

Effect of the interfacial surfactant layer on oxygen transfer through the oil/water phase boundary in perfluorocarbon emulsions

The effect of interfacial surfactant molecules on oxygen transfer through oil/water phase boundary has been studied in FlurO(2) (TM) emulsions, i.e., perfluorocarbon (PFC) emulsions developed as oxygen carriers in cell culture. Measurements of oxygen permeability were made with a polarographic oxygen electrode in pure PFCs and in emulsions with various PFC volume fractions. Comparison of the experimental results with the theoretically derived values of relative oxygen permeability clearly indicates that the mass transfer resistance caused by the interfacial surfactant layer in PFC emulsions is insignificant. Therefore, oxygen dissolved in the enclosed PFC phase is readily available to cells growing in the aqueous media and FlurO(2) emulsions with very fine emulsion particles (< 0.2 mum) can be used to effectively enhance gas/liquid interfacial oxygen transfer in bioreactors. The inadequacy in describing mass transfer in heterogeneous systems, such as the PFC emulsions, by conventional concentration-based oxygen diffusion coefficients has also been discussed.     

Enhancing oxygen tension and cellular function in alginate cell encapsulation devices through the use of perfluorocarbons

Encapsulation devices are often hindered by the inability to achieve sufficient oxygen levels for sustaining long-term cell survival both in vivo and in vitro. We have investigated the use of synthetic oxygen carriers in alginate gels to improve metabolic activity and viability of HepG2 cells over time. Perfluorocarbons (PFCs), specifically perfluorotributylamine (PFTBA) and perfluorooctylbromide (PFOB), were emulsified with alginate and used to encapsulate HepG2 cells in a spherical geometry. Cellular state was assessed using the MTT assay and Live/Dead stain as well as through analysis of both lactate and lactate dehydrogenase (LDH) levels which are indirect indicators of oxygen availability. Addition of 1% surfactant resulted in stable emulsions with evenly dispersed PFC droplets of the order of 1-2 microm in diameter, with no influence on cell viability. Both PFCs evaluated were effective in increasing cellular metabolic activity over alginate-only gels. The presence of 10% PFOB significantly increased cellular growth rate by 10% and reduced both intracellular LDH and extracellular lactate levels by 20-40%, improving glucose utilization efficiency. The characteristic drop in cellular metabolic activity upon encapsulation was eliminated with addition of 10% PFC and viability was better maintained throughout the bead, with a significant decrease in necrotic core size. Results were consistent under a physiologically relevant 5% oxygen environment. The incorporation of PFC synthetic oxygen carriers into encapsulation matrices has been successfully applied to improve cell function and viability with implication for a variety of tissue engineering applications.     

Effects of perfluorodecalin and perfluorooctylbromide emulsions on peak contractile force and ATP levels in the guinea pig left atrium.

The isolated, electrically-driven, guinea pig left atrium was used to study the ability of two perfluorocarbon emulsions to prevent anaerobic hypofunction in the myocardium. A 20% perfluorodecalin (PFD) emulsion maintained peak tension at 80% of aerated levels for more than 20 minutes. Emulsions of perfluorooctylbromide (PFOB) ranging from 25 to 100% produced a similar result, except that the 25% emulsion could not maintain contractions for the entire time period. Peak tension of atria bathed in K-H solution decreased to less than 50% over the same time period. Both 20% PFD and 100% PFOB maintained myocardial ATP levels at pre-hypoxic levels for at least twenty minutes after aeration was terminated. Unaerated atria, bathed in Krebs-Henseleit solution only, exhibited a significant decline in tissue ATP levels at this time. It appears that perfluorocarbon emulsions may delay oxygen desaturation and thereby protect cardiac tissue from ATP depletion and impaired cardiac function associated with hypoxia. This tissue preparation was found to be very useful for determining the efficacy of potential oxygen carriers.     

Pulmonary vascular effects of red blood cells containing S-nitrosated hemoglobin

The role of S-nitrosated hemoglobin (SNO-Hb) in the regulation of blood flow is a central and controversial question in cardiopulmonary physiology. In the present study, we investigate whether intact human red blood cells (RBCs) synthesized to contain high SNO-Hb levels are able to export nitric oxide bioactivity and vasodilate the pulmonary circulation, and whether SNO-Hb dependent vasodilation occurs secondary to an intrinsic oxygen-linked, allosteric function of Hb. RBCs containing supraphysiological concentrations (100-1,000x normal) of SNO-Hb (SNO-RBCs) were synthesized and added to isolated, perfused rat lungs during anoxic or normoxic ventilation, and during normoxic ventilation with pulmonary hypertension induced by the thromboxane mimetic U-46619. SNO-RBCs produced dose-dependent pulmonary vasodilation compared with control RBCs during conditions of both normoxic (U-46619) and hypoxic pulmonary vasoconstriction. These effects were associated with a simultaneous, rapid, and temperature-dependent loss of SNO from Hb. Both vasodilatory effects and the rate of SNO-Hb degradation were independent of oxygen tension and Hb oxygen saturation. Furthermore, these effects were not affected by inhibition of the RBC membrane band 3 protein (anion exchanger-1), a putative membrane facilitator of NO export from RBCs. Whereas these data support observations by multiple groups that synthesized SNO-Hb can vasodilate, this effect is not under intrinsic oxygen-dependent allosteric control, nor likely to be relevant in the pulmonary circulation at normal physiological concentrations.     

Synergistic enhancement of protoplast growth by oxygenated perfluorocarbon and Pluronic F-68

Summary Cell suspension-derived protoplasts of albino Petunia hybrida were grown for 10 d at the interface between aqueous culture medium (KM8P) and an oxygenated (10 mbar for 15 min) perfluorocarbon liquid, perfluorodecalin. Protoplasts synthesised new cell walls and divided normally at the perfluorodecalin/culture medium interface, with a mean viability after 10 d of > 92.0%. The mean plating efficiency of protoplasts was elevated by 37% (P<0.05) following culture at the perfluorodecalin/medium interface, but was unaltered by perfluorodecalin or oxygen separately. The mean plating efficiency of protoplasts cultured at the interface was further increased to a maximium of 52% above control, in the presence of oxygenated perfluorodecalin and KM8P medium supplemented with the non-ionic, co-polymer surfactant, Pluronic F-68 at 0.01% (w/v). These findings demonstrate the effectiveness of oxygenated perfluorodecalin for promoting protoplast growth, by facilitating oxygen delivery. The finding that Pluronic F-68 further increased the plating efficiency of protoplasts cultured at the perfluorocarbon/aqueous interface suggests that these agents improve growth through separate, but cumulative, mechanisms.Abbreviations ATP adenosine triphosphate - PFCs perfluorochemicals - STP standard temperature and pressure     

Artificial carrier for oxygen supply in biological systems

Several poly (dimethylsiloxanes) (PDMS) copolymers of dimethylsiloxane (DMS) with ethylene or propylene oxide were tested as artificial carriers for the delivery of oxygen to biological systems. Copolymers with a DMS content of 33% or lower enhanced glucose oxidation by 200% in contrast to the 25% increase produced by the same concentration of perfluorodecalin. When 0.05% of the copolymer with 18% DMS was included in the growth media of Bacillus thuriginensis, the biomass (growth rate) increased 1.5-fold. With 0.1% of this copolymer, actinorhodin production by Streptomyces coelicolor A3 (2) occurred in half the normal time and with an increased yield. In conclusion, these PDMS copolymers are a good alternative to perfluorodecalin as oxygen carriers in biotechnological processes.     

Mechanism of inhibiting the cytochrome P-450-dependent monooxygenase system in liver with fluorocarbons]

The inducer of the liver monooxygenase system perfluorodecalin added to microsomes as a submicron emulsion forms an enzyme-substrate complex with cytochrome P-450. The K(app) values for the perfluorodecalin binding to cytochrome P-450 in microsomes isolated from the livers of control and phenobarbital-treated rats are 5 x 10(-5) M and 2.3 x 10(-6) M, respectively. Perfluorodecalin competitively inhibits the binding of substrates to cytochrome P-450 and decreases the rates of monooxygenase reactions. Perfluorodecalin extrusion from the active center of cytochrome P-450 occurs when an excess of perfluorocarbons non-interacting with cytochrome P-450 is added to microsomes. There is a significant vagueness in the rates of various monooxygenase reactions because of simultaneous induction and inhibition of monooxygenase enzymes after perfluorodecalin administration to rats. The data obtained are consistent with the hypothesis that constitutive forms of cytochrome P-450 are primary receptors for xenobiotic-inducers of phenobarbital-type cytochrome P-450 isoforms.     

Two carbon fluxes to reserve starch in potato (Solanum tuberosum L.) tuber cells are closely interconnected but differently modulated by temperature

Parenchyma cells from tubers of Solanum tuberosum L. convert several externally supplied sugars to starch but the rates vary largely. Conversion of glucose 1-phosphate to starch is exceptionally efficient. In this communication, tuber slices were incubated with either of four solutions containing equimolar [U-1?C]glucose 1-phosphate, [U-1?C]sucrose, [U-1?C]glucose 1-phosphate plus unlabelled equimolar sucrose or [U-1?C]sucrose plus unlabelled equimolar glucose 1-phosphate. C1?-incorporation into starch was monitored. In slices from freshly harvested tubers each unlabelled compound strongly enhanced 1?C incorporation into starch indicating closely interacting paths of starch biosynthesis. However, enhancement disappeared when the tubers were stored. The two paths (and, consequently, the mutual enhancement effect) differ in temperature dependence. At lower temperatures, the glucose 1-phosphate-dependent path is functional, reaching maximal activity at approximately 20 °C but the flux of the sucrose-dependent route strongly increases above 20 °C. Results are confirmed by in vitro experiments using [U-1?C]glucose 1-phosphate or adenosine-[U-1?C]glucose and by quantitative zymograms of starch synthase or phosphorylase activity. In mutants almost completely lacking the plastidial phosphorylase isozyme(s), the glucose 1-phosphate-dependent path is largely impeded. Irrespective of the size of the granules, glucose 1-phosphate-dependent incorporation per granule surface area is essentially equal. Furthermore, within the granules no preference of distinct glucosyl acceptor sites was detectable. Thus, the path is integrated into the entire granule biosynthesis. In vitro C1?C-incorporation into starch granules mediated by the recombinant plastidial phosphorylase isozyme clearly differed from the in situ results. Taken together, the data clearly demonstrate that two closely but flexibly interacting general paths of starch biosynthesis are functional in potato tuber cells.     

Phosphatidylglycerol of rat lung. Intracellular sites of formation de novo and acyl species pattern in mitochondria, microsomes and surfactant.

The subcellular site of phosphatidylglycerol (PG) formation for lung surfactant has not been convincingly clarified. To approach this problem we analysed the acyl species pattern of lung PG in mitochondria, microsomes and surfactant by h.p.l.c. separation of its 1,2-diacyl-3-naphthylurethane derivatives. Both mitochondrial and microsomal PG proved identical with surfactant PG, containing the major species 1-palmitoyl-2-oleoyl-PG and 1,2-dipalmitoyl-PG. The fatty acid composition of mitochondrial PG differs markedly from that of diphosphatidylglycerol. This may be taken as an indication that mitochondrial PG is synthesized on purpose to form surfactant, rather than being only the precursor of diphosphatidylglycerol. In vitro, sn-[U-14C]glycerol 3-phosphate incorporation into PG of mitochondria or microsomes occurs in the presence of CTP, ATP and CoA but independently of the supply of exogenous lipoidic precursors. Although the rate in vitro of autonomous PG synthesis, and the endogenous PG content, are higher in mitochondria than in microsomes, it is assumed that both subcellular fractions are involved in PG formation for surfactant.     

Effects of emulsified perfluorochemicals on liver cytochromes P-450 in rats

1. The effects of intravenous (i.v.) injection of various perfluorochemical (PFC) emulsions or different fractions of the non-ionic poloxamer surfactant, Pluronic F-68, have been studied separately in male and female rats. 2. Injection of 10 ml/kg body wt of either Fluosol-DA 20% (F-DA) or a novel perfluorodecalin emulsion containing a C-16 oil additive in male rats increased liver weight up to 7 days later; no corresponding effect occurred in response to injection of Oxypherol (FC-43). 3. Liver weight was also increased in female rats at 72 hr after injection of the novel emulsion but this was less pronounced than in males; liver weight in female rats was unchanged in response to injection of either F-DA or FC-43. 4. Mean liver microsomal cytochromes P-450 concentrations in male rats were increased 2-3 fold at 72 hr after injection of either F-DA or the novel emulsion with a less pronounced increase also seen at 7 days in animals receiving the novel emulsion. No significant alterations in cytochrome concentration occurred in response to injection of FC-43 or either commercial grade or purified pluronic solution. 5. Liver cytochromes P-450 concentrations in female rats were unaffected by any of the experimental treatments. 6. These results show that injection of a single low dose of emulsified PFCs into male rats can increase hepatic microsomal cytochromes P-450 concentration but the response is highly variable, depending on composition of emulsion injected.     

Influence of emulsion type and atmospheric oxygen on the fading of latent image in electron microscopic radioautography

Summary The intensity of radioautographic reactions in Ilford L4, Sakura NR-H2 and Kodak NTE emulsions was compared after exposure in either dry air or dry helium gas at 4°C to test the stability of latent images in the presence or absence of oxygen. A light proof container is described in which slides bearing radioactive sections coated with the three emulsions were exposed in dry helium at a constant pressure of approximately 0.5 atm. The comparison of air and helium atmospheres during exposure of radioautographs was estimated qualitatively for ¹²⁵I-labeled thyroid sections stored for several years and, in addition, quantitative data was derived from ³H-labeled methacrylate sections stored from 21 days to 1 year.With the three emulsions under study, the background fog remains low under both exposure conditions at 4°C for as long as several years duration. Using L4 emulsion, similar high grain densities are obtained in air and helium and, therefore, the latent images in L4 emulsion remain stable in the presence of oxygen. In the case of NTE and NR-H2 emulsions, as the exposure time increases, substantially lower reaction intensities are observed in air than in helium. This difference in reaction intensity is evident by 3 weeks with NTE and after 4 weeks with NR-H2. Hence, there is fading of the latent images in the latter emulsions in the presence of oxygen.It is concluded that reliable results may be obtained with the L4 emulsion by exposure of radioautographs in dry air, whereas with the NR-H2 and NTE emulsions, exposure should be in an oxygen-free medium, such as is provided by a dry helium atmosphere.     

Stimulation of glycogenolysis and vasoconstriction in the perfused rat liver by the thromboxane A2 analogue U-46619

Infusion of the thromboxane A2 analogue U-46619 into isolated perfused rat livers resulted in dose-dependent increases in glucose output and portal vein pressure, indicative of constriction of the hepatic vasculature. At low concentrations, e.g. less than or equal to 42 ng/ml, glucose output occurred only during agonist infusion; whereas at concentrations greater than or equal to 63 ng/ml, a peak of glucose output also was observed upon termination of agonist infusion coincident with relief of hepatic vasoconstriction. Effluent perfusate lactate/pyruvate and beta-hydroxybutyrate/acetoacetate ratios increased significantly in response to U-46619 infusion. Hepatic oxygen consumption increased at low U-46619 concentrations (less than or equal to 20 ng/ml) and became biphasic with a transient spike of increased consumption followed by a prolonged decrease in consumption at higher concentrations. Increased glucose output in response to 42 ng/ml U-46619 was associated with a rapid activation of glycogen phosphorylase, slight increases in tissue ADP levels, and no increase in cAMP. At 1000 ng/ml, U-46619 activation of glycogen phosphorylase was accompanied by significant increases in tissue levels of AMP and ADP, decreases in ATP, and slight increases in cAMP. In isolated hepatocytes, U-46619 did not stimulate glucose output or activate glycogen phosphorylase. Reducing the perfusate calcium concentration from 1.25 to 0.05 mM resulted in a marked reduction of the glycogenolytic response to U-46619 (42 ng/ml) with no efflux of calcium from the liver. U-46619-induced glucose output and vasoconstriction displayed a similar dose dependence upon the perfusate calcium concentration. Thus, U-46619 exerts a potent agonist effect on glycogenolysis and vasoconstriction in the perfused rat liver. The present findings support the concept that U-46619 stimulates hepatic glycogenolysis indirectly via vasoconstriction-induced hypoxia within the liver.     

Optimization of oxygen mass transfer in a multiphase bioreactor with perfluorodecalin as a second liquid phase

Oxygenation is an important parameter involved in the design and operation of mixing-sparging bioreactors and it can be analyzed by means of the oxygen mass transfer coefficient (k(L)a). The operational conditions of a stirred, submerged aerated 2-L bioreactor have been optimized by studying the influence of a second liquid phase with higher oxygen affinity (perfluorodecalin or olive oil) in the k(L)a. Using k(L)a measurements, the influence of the following parameters on the oxygen transfer rate was evaluated: the volume of working medium, the type of impellers and their position, the organic phase concentration, the aqueous phase composition, and the concentration of inactive biomass. This study shows that the best experimental conditions were achieved with a perfluorodecalin volume fraction of 0.20, mixing using two Rushton turbines with six vertical blades and in the presence of YPD medium as the aqueous phase, with a k(L)a value of 64.6 h(-1). The addition of 20% of perfluorodecalin in these conditions provided a k(L)a enhancement of 25% when pure water was the aqueous phase and a 230% enhancement when YPD medium was used in comparison to their respective controls (no perfluorodecalin). Furthermore it is shown that the presence of olive oil as a second liquid phase is not beneficial to the oxygen transfer rate enhancement, leading to a decrease in the k(L)a values for all the concentrations studied. It was also observed that the magnitude of the enhancement of the k(L)a values by perfluorodecalin depends on the biomass concentration present.     

Diminution of phagocytosed perfluorocarbon emulsions using perfluoroalkylated polyethylene glycol surfactant

Perfluorocarbon emulsions have been considered as potential blood substitutes for years due to their high capacity of dissolving respiratory oxygen and carbon dioxide. However, they have been reported to associate with side effects (e.g., flu-like syndrome) after being injected into animal's bloodstream. The cause of these side effects is related to the phagocytosis of perfluorocarbon emulsions by cells (e.g., macrophages). Inspired by the approach of using polyethylene glycol (PEG) to camouflage liposomes, we synthesized a perfluoroalkylated PEG (R(F)-PEG) surfactant to provide steric hindrance for decreasing phagocytosis of perfluorocarbon emulsions. The R(F)-PEG surfactant along with Pluronic F-68 and egg yolk phospholipid mediated perfluorocarbon emulsions were incubated individually with J774A.1 macrophages to examine the degree of phagocytosis. 19F NMR studies were used to quantitatively determine the amount of perfluorocarbon emulsions phagocytosed by macrophages. Results showed that the degree of phagocytosis was diminished to a large extent for perfluorocarbon microparticles emulsified by the R(F)-PEG surfactant.     

Antifreeze Emulsions Produced from Linoleic Acid and Water Using Enzymatically Modified Gelatin

An enzymatically modified gelatin with covalently attached leucine dodecyl ester, referred to as EMG-12, was used as a surfactant to prepare emulsions with different properties by changing the surfactant concentration, oil volume fraction, and pH in the water phase. The emulsions generally resisted the freezing of their constituent bulk water at approximately ?10°C, but similar emulsions produced with soy protein isolate, casein, or Tween-80 as control agents were less resistant. The freezing (or unfreezing) of the bulk water in these emulsions depended on the kind of agent used, not on the emulsion properties such as average area of the oil/water interface, stability against coalescence, and stability against creaming. The emulsion produced with EMG-12, like that produced with polyglycerol stearate, tended to maintain its unfrozen state even in the presence of silver iodide crystals added as heterogeneous ice-nuclei. The significance of producing such an antifreeze emulsion is discussed from the standpoint of cryopreservation of cold-sensitive food and biological systems.     

Effect of stobadine, U-74389G, trolox and melatonin on resistance of rat hippocampal slices to oxidative stress

Reactive oxygen species have been suggested to participate in the impairment of nervous tissue by oxidative stress, induced by hypoxia (HYP) followed by reoxygenation (ROX). Although the mechanisms of such injury are rather complex, antioxidants might exert some protective action under such circumstances. This study tested the effect of a series of compounds interfering with the generation and action of reactive oxygen species on impairment of synaptic transmission in the CA1 region of rat hippocampal slices exposed to HYP followed by ROX in vitro. Shortlasting HYP (typically 4.5-7.5 min under the conditions used) resulted in fast decay of the amplitude of population spikes evoked in the CA1 neurons by stimulation of Sch?ffer collaterals. The impairment was mostly irreversible. However, in the presence of the antioxidants stobadine, 21-aminosteroid U-74389G, melatonin and trolox (with optimal concentrations of 10-30 micromol/l, 10 micromol/l, 30-100 micromol/l and 200 micromol/l, respectively), the irreversible damage of the transmission was significantly diminished. The decay of the synaptic transmission failure during HYP was also delayed by stobadine, U-74389G and melatonin. The results demonstrated that compounds with antioxidant activity may effectively protect nervous tissue during HYP and ROX.     

Intratracheal adenoviral-mediated delivery of iNOS decreases pulmonary vasoconstrictor responses in rats.

We hypothesized that adenovirus-mediated inducible nitric oxide synthase (iNOS) gene transduction of the lung would result in time-dependent iNOS overexpression and attenuate the vascular constrictor responses to a thromboxane mimetic, U-46619. Rats were treated via the trachea with surfactant alone (sham), surfactant containing an adenoviral construct with a cytomegalovirus promoter-regulated human iNOS gene (Adeno-iNOS), or an adenoviral construct without a gene insert (Adeno-Control). Adeno-iNOS-transduced rats demonstrated human iNOS mRNA and increased iNOS protein levels only in the lungs. Immunohistochemistry of lungs from Adeno-iNOS-treated animals demonstrated transgene expression in alveolar wall cells. In the lungs from Adeno-iNOS-transduced rats, the expression of iNOS protein and exhaled nitric oxide concentrations were increased on days 1-4 and 7 but returned to baseline values by day 14. The administration of the selective iNOS inhibitor L-N6-(1-iminoethyl)lysine dihydrochloride (L-NIL) decreased exhaled nitric oxide concentrations to levels found in Adeno-Control-transduced lungs. In a second group of rats, the segmental vasoconstrictor responses to U-46619 were determined in isolated, perfused lungs 3 days after transduction. Lungs from rats transduced with Adeno-iNOS had reduced total, arterial, and venous vasoconstrictor responses to U-46619 compared with sham, Adeno-Control, and control groups. In a third set of experiments, the response to 400 nM U-46619 in the presence of 10 microM L-NIL was not different in the isolated lungs from Adeno-Control- and Adeno-iNOS-transduced rats. We conclude that adenovirus-mediated iNOS gene transduction of the lung results in time-dependent iNOS overexpression, which attenuates the vascular constrictor responses to the thromboxane mimetic U-46619.