Central role of cyclooxygenase in the response of canine peripheral airways to antigen

We studied the effects of antigen aerosol challenge on the airways of the canine peripheral lung and examined the roles of cyclooxygenase products, histamine, and cholinergic activity in the responses. One-minute deliveries of 1:10,000 or 1:100,000 concentrations of Ascaris suum antigen aerosol through a wedged bronchoscope resulted in mean maximal increases in collateral system resistance (Rcs) of 415 and 177%, respectively, after 4-8 min. Repeated antigen challenge (1:100,000) resulted in significantly decreased responsiveness to antigen after the initial exposure (P less than 0.005). Bronchoalveolar lavage fluid obtained from the isolated, challenged segment had a significant increase in mean (+/- SE) prostaglandin D2 (PGD2) concentration vs. control (222.0 +/- 65.3 vs. 72.7 +/- 19.5 pg/ml; P less than 0.05); histamine concentrations were variable and not significantly different (4.1 +/- 2.6 vs. 1.2 +/- 0.2 ng/ml; P greater than 0.05). In nine experiments, cyclooxygenase inhibition significantly attenuated the antigen-induced increase in Rcs by 53.4% (P less than 0.001), and the concentration of PGD2 in lavage fluid was reduced by 96.0% (P less than 0.01). Blockade of histamine H1-receptors (n = 8) or cholinergic receptors (n = 7) did not significantly affect the airway response (P greater than 0.05). These data indicate that the canine peripheral lung responds in a dose-dependent manner to antigen aerosol challenge and exhibits characteristics of antigen tachyphylaxis. Results also suggest that cyclooxygenase products play a central role in the acute bronchoconstrictive response of the lung periphery.     

Dry air-induced constriction: effects of pharmacological intervention and temperature

We studied airway wall temperature (Taw) during dry air challenge of the canine lung periphery. We measured collateral resistance (Rcs) before and after periods of elevated airflow using a wedged bronchoscope technique. As flow rate increased, Taw dropped and postchallenge Rcs rose. A significant negative correlation was found between Taw recorded during challenge and Rcs observed 5 min after challenge. Repetitive dry air challenge produced similar changes in Rcs and Taw. However, responses to warm moist air were significantly lower than consecutive responses to dry air. Taw was significantly lower during dry air challenge than during moist air challenge. Indomethacin (5 mg/kg) and atropine (1 mg/kg) reduced responses to dry airflow challenge. Indomethacin did not affect Taw during the challenge, whereas atropine reduced the fall in Taw. We conclude that temperature correlates negatively with peripheral lung tone 5 min after dry air challenge. This correlation holds under conditions where airflow is increased, air is humidified, or atropine is administered. The dissociation between Taw and physiological response after indomethacin likely reflects a decrease in mediators released during challenge.     

Dry air-induced constriction in lung periphery: a canine model of exercise-induced asthma

We studied the effects of the flow of dry air on collateral tone in the lung periphery. A bronchoscope was wedged in sublobar segments of anesthetized dogs, and measurements of collateral resistance (Rcs) were recorded before and after flow was increased from 200 to 2,000 ml/min for a 5-min period. Five minutes after exposure was completed, Rcs increased by an average of 117 +/- 25.2% (SE) over control. Maximum Rcs occurred 5 min after the challenge was concluded and required 48 +/- 10.5 min to return to base line. When flow rate was held constant and exposure period varied, Rcs increased with increased stimulus duration. With exposure times held constant, the response of the collateral system was positively associated with changes in stimulus strength (flow rate). No refractory period was observed with repetitive challenges. Finally, when dry air (delivered at 22 degrees C) and conditioned air (i.e., delivered at 28 degrees C; relative humidity = 80%) challenges were alternated in the same wedged segment, dry air produced a mean increase in Rcs of 93.2%, whereas challenge with warm moist air increased Rcs only 33.5%. Regardless of which challenge was presented first, dry air consistently produced a greater constrictor response. This response is similar to that observed in cold air- and exercise-induced asthma and indicates that the lung periphery in dogs, like larger airways in asthmatic subjects, has the potential to increase tone when exposed to dry air. Peripheral airways in dogs thus constitute a model that can be used for the investigation of exercise-induced asthma.     

Thromboxane contributes to the immediate antigenic response of canine peripheral airways

The actions of specific humoral mediators in the immediate response of the canine peripheral airways to antigen challenge are not well understood. Using a method which allows localized exposure of the peripheral lung to antigen, we investigated the role of locally released thromboxane A2 (TxA2) in the immediate response of collateral airways to aerosolized antigen. In dogs with native sensitivity to Ascaris suum antigen, resistance to flow through the collateral system (Rcs) was measured using a wedged bronchoscope technique. Local administration of antigen aerosol (25 microliters, 1:10,000 dilution) produced a gradual increase in Rcs which reached a maximum of 365% of base line in 4-8 min. Analysis of bronchoalveolar lavage fluid obtained from the exposed segment at the peak of the response demonstrated significantly more TxB2 compared with control lavage samples (41.8 +/- 7.8 pg/ml vs. 27.9 +/- 8.3; P less than 0.025). After inhibition of thromboxane synthase with UK-37,248 (3 mg/kg iv) or OKY-046 (5 mg/kg iv), the increase in Rcs was significantly reduced at 40 s (P less than 0.001) and 2 min (P less than 0.01) after antigen delivery, and the maximal increase was attenuated by 41% (P less than 0.005). In contrast, the magnitude and time course of the airway response to aerosols of a stable thromboxane analog (U-46619) were not affected by blockade. Despite a similar attenuation (42%) of the maximal increase in Rcs by sodium meclofenamate (3 mg/kg iv), this cyclooxygenase inhibitor had no effect on the time course of the antigenic response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tonic beta-sympathetic activity in the lung periphery in anesthetized dogs

The present study was undertaken to determine whether beta-adrenoceptors could be physiologically detected in the lung periphery and whether they were under tonic stimulation in the resting state in anesthetized dogs. A fiberoptic bronchoscope was wedged in a sublobar segment of lung in anesthetized male mongrel dogs for measurement of resistance through the collateral system (Rcs). beta-Agents were delivered locally as aerosols through the bronchoscope, and the response was evaluated by changes in Rcs. Distilled water alone produced a mean increase of 8.5 +/- 2.43% (SE) in Rcs at 2 min in six dogs, whereas dl-isoproterenol produced a mean decrease of 8.9 +/- 2.10% (P less than 0.03), thus demonstrating the presence of submaximally stimulated beta-receptors. To test whether the beta-receptors were under tonic stimulation, we compared the effect of aerosolized d- and dl-propranolol in 5 dogs. d-Propranolol that lacks significant beta-blocking activity and dl-propranolol both produced large transient increases in Rcs. However, with d-propranolol, Rcs had returned to base line at 15 min, whereas with dl-propranolol Rcs remained elevated at a mean of 20% above base line for greater than 2 h (P less than 0.01). Local timolol aerosol also produced a sustained increase in Rcs. After pretreatment with reserpine or after bilateral adrenalectomy, both d- and dl-propranolol still produced large transient increases in Rcs, but dl-propranolol no longer produced a sustained increase. Neither isoproterenol nor atropine affected Rcs in the presence of dl-propranolol, nor did pretreatment with atropine affect the response of Rcs to dl-propranolol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pronounced eosinophilic lung inflammation and Th2 cytokine release in human lipocalin-type prostaglandin D synthase transgenic mice.

PGD(2) is a major lipid mediator released from mast cells, but little is known about its role in the development of allergic reactions. We used transgenic (TG) mice overexpressing human lipocalin-type PGD synthase to examine the effect of overproduction of PGD(2) in an OVA-induced murine asthma model. The sensitization of wild-type (WT) and TG mice was similar as judged by the content of OVA-specific IgE. After OVA challenge, PGD(2), but not PGE(2), substantially increased in the lungs of WT and TG mice with greater PGD(2) increment in TG mice compared with WT mice. The numbers of eosinophils and lymphocytes in the bronchoalveolar lavage (BAL) fluid were significantly greater in TG mice than in WT mice on days 1 and 3 post-OVA challenge, whereas the numbers of macrophages and neutrophils were the same in both WT and TG mice. The levels of IL-4, IL-5, and eotaxin in BAL fluid were also significantly higher in TG mice than in WT mice, although the level of IFN-gamma in the BAL fluid of TG mice was decreased compared with that in WT mice. Furthermore, lymphocytes isolated from the lungs of TG mice secreted less IFN-gamma than those from WT mice, whereas IL-4 production was unchanged between WT and TG mice. Thus, overproduction of PGD(2) caused an increase in the levels of Th2 cytokines and a chemokine, accompanied by the enhanced accumulation of eosinophils and lymphocytes in the lung. These results indicate that PGD(2) plays an important role in late phase allergic reactions in the pathophysiology of bronchial asthma.     

Mucosal injury and eicosanoid kinetics during hyperventilation-induced bronchoconstriction.

Bronchoalveolar lavage (BAL) of canine peripheral airways was performed at various times after hyperventilation, and BAL fluid (BALF) cell and mediator data were used to evaluate two hypotheses: 1) hyperventilation-induced mucosal injury stimulates mediator production, and 2) mucosal damage is correlated with the magnitude of hyperventilation-induced bronchoconstriction. We found that epithelial cells increased in BALF immediately after a 2- and a 5-min dry air challenge (DAC). Prostaglandins D(2) and F(2alpha) and thromboxane B(2) were unchanged immediately after a 2-min DAC but were significantly increased after a 5-min DAC. Leukotriene C(4), D(4), and E(4) did not increase until 5 min after DAC. Hyperventilation with warm moist air did not alter BALF cells or mediators and caused less airway obstruction that occurred earlier than DAC. BALF epithelial cells were correlated with mediator release, and mediator release and epithelial cells were correlated with hyperventilation-induced bronchoconstriction. These observations are consistent with the hypothesis that hyperventilation-induced mucosal damage initiates peripheral airway constriction via the release of biochemical mediators.     

Clearance of different-sized proteins from the alveolar space in humans and rabbits.

Investigation of the clearance of proteins from the air spaces is important for an understanding of the resolution of pulmonary edema and also because of current interest in delivery of therapeutic peptides via the distal air spaces. Few experimental studies have examined the size dependence for alveolar clearance of large macromolecules; there have been no human studies. In anesthetized rabbits, we measured clearance of cyanocobalamin and different-sized human proteins instilled into the air spaces. After 8 h, the amounts of instilled tracer recovered in the lungs were [57Co]cyanocobalamin, 19.4 +/- 3.0% (Stokes radius 0.65 nm); 125I-labeled insulin, 64.6 +/- 3.9% (1.2 nm); 131I-labeled albumin, 87.0 +/- 4.0% (3.5 nm); and 125I-labeled immunoglobulin G, 91.8 +/- 3.3% (5.5 nm) (P < 0.05). Sieving of different-sized proteins occurred across the alveolar epithelial barrier because tracer concentrations in air space lavage fluid after 8 h were decreased more for the smaller tracers than the larger ones. Size selectivity for alveolar protein clearance in humans with resolving alveolar edema was investigated by measuring the changes in albumin and total protein concentration. The fraction of total protein concentration made up of albumin was greater in the edema fluid than in the plasma initially. The albumin fraction decreased with time in 9 of 10 patients with resolving edema, from 0.62 +/- 0.2 to 0.58 +/- 0.10 (P < 0.05) after 10 +/- 5 h. Thus both rabbit studies and human studies provide evidence for size-dependent clearance of protein from the air spaces of the lung.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium chelators induce bronchoconstriction in the canine lung periphery

We studied the mechanism by which Na2EDTA, a divalent cation chelator, induces bronchoconstriction in the lung periphery of mongrel dogs as a model of nonspecific small airway hyperresponsiveness. Using a wedged bronchoscope technique, we measured collateral system resistance (Rcs) before and after challenges with aerosolized Na2EDTA. An isotonic solution (4% Na2EDTA, 0.28 osmol/kg) increased Rcs 91 +/- 21%. Na2EDTA increased Rcs in a dose-dependent fashion after challenges of increasing concentration (0, 1, 3, and 6%) or duration (15, 30, 60, and 90 s) with 6% Na2EDTA. Atropine (1 mg/kg iv) significantly (P = 0.01) attenuated the response to an aerosol challenge with distilled H2O. Atropine did not significantly (P = 0.35) alter the response to a challenge with 4% Na2EDTA. Challenge with 6% Na2EDTA (0.42 osmol/kg) increased Rcs to a significantly greater (P less than 0.01) extent than did challenge with 6% CaNa2EDTA (0.37 osmol/kg, 250 +/- 55 vs. 29 +/- 11%, respectively). We conclude that Na2EDTA induces bronchoconstriction in the canine lung periphery in a dose-dependent fashion. As suggested by the Na2EDTA-CaNa2EDTA comparison, hyperosmolality of the solution alone cannot explain this phenomenon. The mechanism does not depend on muscarinic activity and appears to involve chelation of calcium.     

Time course of bronchoconstriction induced by dry gas hyperpnea in guinea pigs

We examined the effects of hyperpnea duration and abrupt changes in inspired gas heat and water content on the magnitude and time course of hyperpnea-induced bronchoconstriction (HIB) in anesthetized mechanically ventilated male Hartley guinea pigs. In 12 animals subjected to 5, 10, and 15 min (random order) of dry gas isocapnic hyperpnea [tidal volume (VT) 4-6 ml, 150 breaths/min) followed by quiet breathing of humidified air (VT 2-3 ml, 60 breaths/min), severe bronchoconstriction developed only after the cessation of hyperpnea; the magnitude of respiratory system resistance (Rrs) increased with the duration of dry gas hyperpnea [peak Rrs 1.0 +/- 0.2, 1.8 +/- 0.3, and 2.3 +/- 0.3 (SE) cmH2O.ml-1.s, respectively]. Seven other guinea pigs received, in random order, 10 min of warm humidified gas hyperpnea, 10 min of room temperature dry gas hyperpnea, and 5 min of dry gas hyperpnea immediately followed by 5 min of warm humidified gas hyperpnea. After each hyperpnea period, the animal was returned to quiet breathing of humidified gas. Rrs rose appreciably after the 10 min of dry and 5 min of dry-5 min of humidified hyperpnea challenges (peak Rrs 1.3 +/- 0.2 and 0.7 +/- 0.2 cmH2O.ml-1.s, respectively) but not after 10 min of humidified hyperpnea (0.2 +/- 0.04 cmH2O.ml-1.s). An additional five animals received 10 min of room temperature dry gas hyperpnea followed by quiet breathing of warm humidified air and 10 min of room temperature dry gas hyperpnea followed by 30 min of warm humidified gas hyperpnea in random order.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of magnesium sulfate on bronchoconstriction in the lung periphery

Magnesium sulfate has been shown to be effective clinically as a bronchodilator, but its mechanism of action is unknown. We used a wedged bronchoscope technique to study the ability of MgSO4 at clinically relevant concentrations to attenuate hypocapnia-, acetylcholine- (ACh), and dry air-induced bronchoconstriction in the canine lung periphery. Control experiments demonstrated that consecutive challenges of either hypocapnia or ACh resulted in greater collateral system resistance (Rcs) after the second challenge compared with the first. Intravenous infusion of MgSO4 diminished the maximum response to a second hypocapnic challenge (Rcs = 1.59 +/- 0.29 cmH2O.ml-1.s prechallenge vs. 1.12 +/- 0.20 postchallenge) but had no effect on either ACh- or dry air-induced bronchoconstriction. Serum magnesium levels before MgSO4 administration were 1.59 +/- 0.04 meq/l and rose to 6.20 +/- 0.13 during the infusion. Previous studies demonstrated that nifedipine, like MgSO4 in this study, attenuates hypocapnia-induced bronchoconstriction in the canine lung periphery but has no effect on ACh- or dry air-induced bronchoconstriction. We conclude that these results are consistent with the idea that, like nifedipine, magnesium acts in the airway as a voltage-sensitive calcium channel blocker.     

O3-induced change in bronchial reactivity to methacholine and airway inflammation in humans

The increase in airway responsiveness induced by O3 exposure in dogs is associated with airway epithelial inflammation, as evidenced by an increase in the number of neutrophils (polymorphonuclear leukocytes) found in epithelial biopsies and in bronchoalveolar lavage fluid. We investigated in 10 healthy, human subjects whether O3-induced hyperresponsiveness was similarly associated with airway inflammation by examining changes in the types of cells recovered in bronchoalveolar lavage fluid obtained after exposure to air or to O3 (0.4 or 0.6 ppm). We also measured the concentrations of cyclooxygenase and lipoxygenase metabolites of arachidonic acid in lavage fluid. We measured airway responsiveness to inhaled methacholine aerosol before and after each exposure and performed bronchoalveolar lavage 3 h later. We found more neutrophils in the lavage fluid from O3-exposed subjects, especially in those in whom O3 exposure produced an increase in airway responsiveness. We also found significant increases in the concentrations of prostaglandins E2, F2 alpha, and thromboxane B2 in lavage fluid from O3-exposed subjects. These results show that in human subjects O3-induced hyperresponsiveness to methacholine is associated with an influx of neutrophils into the airways and with changes in the levels of some cyclooxygenase metabolites of arachidonic acid.     

Tracheal vascular and smooth muscle responses to air temperature and humidity in dogs

Twenty-five dogs were anesthetized, paralyzed, and artificially ventilated. Their cranial tracheal arteries were perfused bilaterally with blood at constant flow, and the perfusion pressures (Patr) were measured. Tracheal smooth muscle function was assessed by recording changes in external diameter (delta Dtr). The perfused segment of the trachea was exposed to air at a constant unidirectional airflow of 25 l/min. Group 1 (n = 6) was exposed to cold dry air, ambient room air, and hot dry and hot humid air, each for 10 min with exposures starting from zero flow. The tracheal vascular responses to all four conditions were small vasodilations (delta Patr from -2 to -6%) followed by recovery or small vasoconstrictions. In group 2 (n = 19), exposures to cold dry and hot humid air were preceded and followed by body-temperature fully humidified air. Cold dry air caused a sustained vasodilation (delta Patr -9.0 +/- 1.1%), and hot humid air usually caused a biphasic response: a vasoconstriction (delta Patr 4.4 +/- 1.0%) followed by a vasodilation (delta Patr -5.7 +/- 1.9%). The warm humid air after cold dry air or hot humid air caused a further vasodilation, which lasted a short time after cold dry air (delta Patr -3.7 +/- 0.4%) but greater than 10 min after hot humid air (delta Patr -13.8 +/- 1.4%). In both groups, all exposures that cooled the trachea (cold dry air, ambient room air, and hot dry air) caused smooth muscle contraction, and hot humid air that warmed the trachea caused relaxation.     

Effects of neutrophils on collateral ventilation and peripheral lung reactivity in dogs

We studied the effects of neutrophil activation on collateral ventilation and peripheral lung reactivity in anesthetized dogs. A fiberoptic bronchoscope was wedged into a segmental airway under direct vision. Ventilation beyond the obstruction thus occurred only through collateral channels. Through one lumen of a double-lumen catheter threaded through the suction port of a bronchoscope, 5% CO2 in air was infused at a known constant rate (V coll). Through the other lumen, pressure at the tip of the bronchoscope was monitored (Pb). For measurements of resistance to flow through the collateral system (Rcs), the ventilation was stopped at functional residual capacity (FRC). Histamine was delivered through the bronchoscope to the obstructed lung segment in the form of an aerosol mist generated by an ultrasonic nebulizer. Measurements of Rcs were used as a parameter of the peripheral lung reactivity to histamine challenge. Within one hour after intravenous infusion of phorbol myristate acetate (PMA), a neutrophil activator, the reactivity to histamine significantly increased. After this, Rcs increased even without histamine challenge. This increase may have been due to an edematous injury of lung caused by PMA. The nature of the injury was confirmed by wet to dry weight ratios. In the other group, the white cell count dropped below 1000 per cu. mm. after intravenous infusion of nitrogen mustard. The same experimental protocols were followed. The Rcs did not increase even with histamine challenge. Our results suggested that substances such as oxygen radicals and arachidonic acid metabolites, which can be released by activated neutrophils, may not not only increase peripheral lung reactivity, but may also induce pulmonary edema.     

Characterization of primate bronchoalveolar mast cells. I. IgE-dependent release of histamine, leukotrienes, and prostaglandins

Large numbers of functional mast cells were obtained by bronchoalveolar lavage (BAL) of Macaca arctoides monkeys that had been infected with the nematode Ascaris suum. These lavage cells, of which 21% were mast cells, released histamine, LTC4, and PGD2 in a concentration-dependent fashion when challenged with ascaris antigen or antibody to human IgE. However, there was no release of histamine when these cells were challenged with compound 48/80. The amount of mediator released was highly dependent on the sensitivity of the cells to immunologic challenge, but was generally in the range of 2 to 5 micrograms histamine (30 to 70% of total), 20 to 80 ng LTC4, and 100 to 300 ng PGD2 per 10(6) mast cells when maximally challenged. Other eicosanoids measured were released only in much smaller quantities. Maximal values were 4 ng LTB4, 2 ng PGE2, and approximately 10 to 20 ng PGF2 alpha per 10(6) mast cells. The amount of LTC4 and PGD2 released correlated with the release of histamine, the calculated regression line indicating that 18 ng LTC4 and 50 ng PGD2 were released per microgram of histamine released. This correlation suggests that the majority of the LTC4 and PGD2 released was probably mast cell-derived. Further support for this conclusion was given by the observation that when lavage cells were fractioned on continuous Percoll gradients, the ability to release LTC4 and PGD2 on immunologic challenge coincided with the peak of mast cells.     

Tracheobronchial and upper airway blood flow in dogs during thermally induced panting

Tracheobronchial blood flow increases two- to fivefold in response to isocapnic hyperventilation with warm dry or cold dry air in anesthetized, tracheostomized dogs. To determine whether this response is governed by central nervous system thermoregulatory control or is a local response to the drying and/or cooling of the airway mucosa, we studied eight anesthetized spontaneously breathing dogs in a thermally controlled chamber designed so that inspired air temperature, humidity, and body temperature could be separately regulated. Four dogs breathed through the nose and mouth (group 1), and four breathed through a short tracheostomy tube (group 2). Dogs were studied under the following conditions: 1) a normothermic control period and 2) two periods of hyperthermia in which the dogs panted with either warm 100% humidified air or warm dry (approximately 10% humidified) air. Radiolabeled microspheres (15 +/- 3 micron diam) were injected into the left ventricle as a marker of nasal, lingual, and tracheobronchial blood flow. After the final measurements, the dogs were killed and tissues of interest excised. Results showed that lingual and nasal blood flow (ml.min-1.g-1) increased during panting (P less than 0.01) in both groups and were not affected by the inspired air conditions. In group 1, tracheal mucosal blood flow barely doubled (P less than 0.01) and bronchial blood flow did not change during humid and dry air panting. In group 2, there was a sevenfold increase in tracheal mucosal and about a threefold increase in bronchial blood flow (P less than 0.01), which was only observed during dry air panting.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution

Bronchoalveolar lavage is a powerful technique for sampling the epithelial lining fluid (ELF) of the lower respiratory tract but also results in a significant dilution of that fluid. To quantify the apparent volume of ELF obtained by bronchoalveolar lavage, urea was used as an endogenous marker of ELF dilution. Since urea diffuses readily through the body, plasma and in situ ELF urea concentrations are identical; thus ELF volume can be calculated using simple dilution principles. Using this approach, we determined that with a standard lavage procedure, the volume of ELF recovered from a normal human is 1.0 +/- 0.1 ml/100 ml of recovered lavage fluid. Time course experiments in which the saline used for lavage was permitted to remain in the lower respiratory tract for various "dwell times" suggested that diffusion of urea from sources other than recovered ELF can contribute to the total urea recovered resulting in an overestimate of the volume of ELF recovered. Thus, while reasonably accurate, the volume of ELF determined by urea must be considered an overestimate, or "apparent" volume. The ELF albumin concentration based on the apparent ELF volume was 3.7 +/- 0.3 mg/ml, a value that is in good agreement with direct measurements made by other techniques in experimental animals. The density of all inflammatory and immune effector cells on the epithelial surface of the lower respiratory tract, based on the apparent ELF volume, was 21,000 +/- 3,000 cells/microliter, a value that is twofold greater than that in blood.(ABSTRACT TRUNCATED AT 250 WORDS)     

An improved LC-MS/MS method for the quantification of prostaglandins E(2) and D(2) production in biological fluids

We report an improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that accurately measures prostaglandins D(2) (PGD(2)) and E(2) (PGE(2)) in cell culture supernatants and other biological fluids. The limit of detection for each prostaglandin was 20 pg/ml (0.20 pg, 0.55 fmol on-column), and the interday and intraday coefficients of variation were less than 5%. Both d(4)-PGE(2) and d(4)-PGD(2) were used as surrogate standards to control for differential loss and degradation of the analytes. Stability studies indicated that sample preparation time should be less than 8h to measure PGD(2) accurately, whereas preparation time did not affect PGE(2) measurement due to its greater stability in biological samples. As an application of the method, PGD(2) and PGE(2) were measured in culture supernatants from A549 cells and RAW 264.7 cells. The human lung alveolar cell line A549 was found to produce PGE(2) but no PGD(2), whereas the murine macrophage cell line RAW 264.7 produced PGD(2) and only trace amounts of PGE(2). This direct comparison showed that COX-2 gene expression can lead to differential production of PGD(2) and PGE(2) by epithelial cells and macrophages. Because PGE(2) is antiasthmatic and PGD(2) is proasthmatic, we speculate that the balance of production of these eicosanoids by epithelial cells and macrophages in the lung contributes to the pathogenesis of chronic obstructive pulmonary disease (COPD), bronchiectasis, asthma, and lung cancer.     

Air embolism-induced lung injury in isolated rat lungs.

Pulmonary air embolism causes physical obstruction of microvasculature and leads to permeability changes, release of mediators, and injury to lung tissue. In this study we employed an isolated perfused rat lung model to investigate the primary and secondary effects produced by infusion of air into the pulmonary artery. Infusion of various doses of air (0.10-0.25 ml) over a 1-min period produced a dose-dependent increase in pulmonary arterial pressure and lung weight gain. In contrast, when a constant air dose was administered over various periods of time (0.25 ml over 0.5-8.0 min), the pulmonary arterial pressure rose to the same extent regardless of the infusion rate, whereas the lung weight gain increased proportionately with the rate of infusion. Total vascular resistance rose from 1.41 +/- 0.04 to 5.04 +/- 0.09 mmHg.ml-1.min in rats given 0.25 ml air over 1 min (n = 14, P less than 0.001), with greater than or equal to 90% of this increase occurring in the arterial segments. Both thromboxane B2 and endothelin concentrations also increased in the perfusate, suggesting their involvement in this increased resistance. Furthermore the pulmonary filtration coefficient increased from 0.21 +/- 0.05 to 1.28 +/- 0.26 g.min-1.cmH2O-1.100 g (n = 8, P less than 0.001), and the protein concentration in lung lavage fluid also rose, indicating lung injury. Leukocyte counts in the perfusate were unaffected by embolization, but chemiluminescent activity was increased, indicating a possible role for activated leukocytes in lung injury induced by air emboli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Does CO2 enhance short-term storage success of Chinook salmon (Oncorhynchus tshawytscha) milt?

Successful short-term storage of salmonid milt depends on numerous factors, including temperature, fluid volume, and gaseous environment, with storage at low temperatures under an atmosphere of 100% O2 being the most common method. Salmonid sperm maintained in a storage environment with elevated carbon dioxide (CO2) levels, such as the approximately 4% CO2 in exhaled air, are not motile when activated. While these modest levels of CO2 inhibit sperm motility, the effect is reversible within hours after exposure to a CO2-free oxygenated environment. Therefore, the effect of CO2 (as a component gas in the storage environment) on chinook salmon (Oncorhynchus tshawytscha) sperm motility and viability was examined. The hypothesis of the current investigation was that CO2-exposure with subsequent CO2 removal would be beneficial during short-term chinook salmon milt storage. Milt samples were collected from mature (adult) and precocious (jack) male chinook salmon and stored under various CO2 and O2 levels at 3 to 4 degrees C for up to 14 days. Milt samples were then removed from the incubation environments and maintained under CO2-free humidified air with continuous mixing for 4 h at 10 degrees C before analysis of motility. The resultant motility of samples incubated under 3.5% or less CO2 was not different than controls during the 14 d incubation period; motility of samples stored under higher CO2 tensions were significantly lower. The motility of samples incubated under 3.5% CO2 reached the maximum recovered motility after 2 h exposure to CO2-free humidified air, while the motility of sperm incubated under 13.4% CO2 levels recovered no motility even after 6 h exposure to CO2-free humidified air. The motility of samples incubated under normoxia was significantly greater than that of samples incubated under hyperoxia (approximately 90% O2) at both 7 and 14 d, regardless of the CO2 level. Sperm viability was relatively unaltered by any of the incubation conditions examined. The results of this investigation suggest that there is no apparent advantage to storage of chinook salmon sperm in the presence of CO2 and that storage under hyperoxia negatively affects sperm function compared to storage under normoxia.