Stop-flow studies of solute uptake in rat lungs

Stop-flow studies were used to characterize solute uptake inisolated rat lungs. These lungs were perfused at 8 or 34 ml/min for10-28 s with solutions containing¹²⁵I-albumin and two or more ofthe following diffusible indicators: [³H]mannitol,[¹⁴C]urea,³HOH,²⁰¹Tl⁺,or⁸⁶Rb⁺.After this loading period, flow was stopped for 10-300 s and thenresumed to flush out the perfusate that remained in the pulmonary vasculature during the stop interval. Concentrations of²⁰¹Tl⁺and⁸⁶Rb⁺in the venous outflow decreased after the stop interval, indicating uptake from exchange vessels during the stop interval. The amount ofthese K⁺ analogs lost from thecirculation during the stop interval was greater when the intervalswere longer. However, losses of²⁰¹Tl⁺at 90 s approached those at 300 s. Because extraction continued afterthe vasculature had been flushed, vascular levels had presumably fallento negligible levels during the stop interval. By 90 s of stop flow thevascular volume that was cleared of²⁰¹Tl⁺averaged 0.657 ± 0.034 (SE) ml in the experiments perfused at 8 ml/min and 0.629 ± 0.108 ml in those perfused at 34 ml/min. Increases in perfusate K⁺decreased the cleared volumes of²⁰¹Tl⁺and⁸⁶Rb⁺.Uptake of[³H]mannitol,[¹⁴C]urea, and³HOH during the stop intervals wasobserved only when the lungs were loaded at high flow for shortintervals. Decreases in²⁰¹Tl⁺and⁸⁶Rb⁺concentrations in the pulmonary outflow can be used to identify thefraction of the collected samples that were within exchange vessels ofthe lung during the stop interval and may help determine thedistribution of solute and water exchange along the pulmonary vasculature.

     

Enzyme histochemical studies on rat lungs 2. Normal enzyme distribution in the alveolar tissue of the mature lung

Synopsis The activity and distribution of the following eighteen oxidative and hydrolytic enzyme systems have been investigated in the lung of the adult rat: reduced NAD dehydrogenase, reduced NADP dehydrogenase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase, glutamate dehydrogenase, -hydroxybutyrate dehydrogenase, acid phosphatase, alkaline phosphatase, glucose-6-phosphatase, adenosine triphosphatase, 5-nucleotidase, non-specific esterase, cytochrome oxidase and -glucuronidase.The low concentration of cells in sections of inflated lung may have made histochemical demonstration of some enzymes impossible because the enzyme concentration was below that detectable by the method employed.The carboxylic acid cycle and the hexose monophosphate shunt were potentially active but fatty acid metabolism was not indicated.The granular reaction sometimes encountered in alveolar cell cytoplasm may be useful for differentiating alveolar cell types, but further cytochemical studies are required to resolve the possible metabolic differences of alveolar cells.     

Derecruitment of filtration surface area in paraquat-injured isolated dog lungs

The capillary filtration coefficient (Kf,c) is a sensitive and specific index of vascular permeability if surface area remains constant, but derecruitment might affect Kf,c in severely damaged lungs with high vascular resistance. We studied the effect of high and low blood flow rates on Kf,c in papaverine-pretreated blood-perfused isolated dog lungs perfused under zone 3 conditions with and without paraquat (PQ, 10(-2) M). Three Kf,cs were measured successively at hourly intervals for 5 h. These progressed sequentially from isogravimetric blood flow with low vascular pressure (I/L) to high flow with low vascular pressure (H/L) to high flow with high vascular pressure (H/H). The blood flows of H/L and H/H were greater than or equal to 1.5 times that of I/L. There were no significant changes in Kf,c in lungs without paraquat over a 50-fold range of blood flow rates. At 3 h after PQ, I/L-Kf,c was significantly increased and both isogravimetric capillary pressure and total protein reflection coefficient were decreased from base line. At 4 and 5 h, H/L-Kf,c was significantly greater than the corresponding I/L-Kf,c (1.01 +/- 0.22 vs. 0.69 +/- 0.09 and 1.26 +/- 0.19 vs. 0.79 +/- 0.10 ml.min-1.cmH2O-1.100 g-1, respectively) and isogravimetric blood flow decreased to 32.0 and 12.0% of base line, respectively. Pulmonary vascular resistance increased to 12 times base line at 5 h after PQ. We conclude that Kf,c is independent of blood flow in uninjured lungs. However, Kf,c measured at isogravimetric blood flow underestimated the degree of increase in Kf,c in severely damaged and edematous lungs because of a high vascular resistance and derecruitment of filtering surface area.     

Histochemical studies of rat lungs after the short-term influence of soil dust

There was examined the biological activity of soil dusts using histochemical methods. Intratracheal administration of dusts used in this study is a common method for testing activity of industrial dusts. The used soil dusts were characterized by high content of free silicon dioxide, 3 times higher than its content in dusts from power stations released in the process of burning coal and approximately to the amount in graphite dust. Both coal dusts and graphite dusts absorbed by the lungs cause silicoanthracosis. The investigations have been shown that soil dusts caused stimulation of the mitochondrial metabolism giving an increase of the activity of succinic dehydrogenase, lactic dehydrogenase, and ATPase induced by Mg++ ions. This was additionally confirmed by an increase of NADP activity which is an enzyme binding a chain or reactions regulating the hydrocarbonic metabolism. There was also observed an increased activity of the hydrolytic enzyme acid phosphatase. High activity occurred in the epithelium of bronchi and bronchioli and focally in pulmonary parenchyma.     

Perfusion heterogeneity in rat lungs assessed from the distribution of 4-microm-diameter latex particles.

Pulmonary vascular perfusion has been shown to follow a fractal distribution down to a resolution of 0.5 cm(3) (5E11 microm(3)). We wanted to know whether this distribution continued down to tissue volumes equivalent to that of an alveolus (2E5 microm(3)). To investigate this, we used confocal microscopy to analyze the spatial distribution of 4-microm-diameter fluorescent latex particles trapped within rat lung microvessels. Particle distributions were analyzed in tissue volumes that ranged from 1.7E2 to 2.8E8 microm(3). The analysis resulted in fractal plots that consisted of two slopes. The left slope, encompassing tissue volumes less than 7E5 microm(3), had a fractal dimension of 1.50 +/- 0.03 (random distribution). The right slope, encompassing tissue volumes greater than 7E5 microm(3), had a fractal dimension of 1.29 +/- 0.04 (nonrandom distribution). The break point at 7E5 microm(3) corresponds closely to a tissue volume equivalent to that of one alveolus. We conclude that perfusion distribution is random at tissue volumes less than that of an alveolus and nonrandom at tissue volumes greater than that of an alveolus.     

Effect of alveolar hypoxia on pulmonary fluid filtration in in situ dog lungs

We have studied the effect of alveolar hypoxia on fluid filtration characteristics of the pulmonary microcirculation in an in situ left upper lobe preparation with near static flow conditions (20 ml/min). In six dogs (group 1), rate of edema formation (delta W/delta t, where W is weight and t is time) was assessed over a wide range of vascular pressures under two inspired O2 fraction (FIO2) conditions (0.95 and 0.0 with 5% CO2-balance N2 in both cases). delta W/delta t was plotted against vascular pressure, and the best-fit linear regression was obtained. There was no significant difference (paired t test) in either threshold pressure for edema formation [18.3 +/- 1.8 and 17.1 +/- 1.2 (SE) mmHg, respectively] or the slopes (0.067 +/- 0.008 and 0.073 +/- 0.017 g.min-1. mmHg-1.100g-1, respectively). In another seven dogs (group 2), delta W/delta t was obtained at a constant vascular pressure of 40 mmHg under four FIO2 conditions (0.95, 0.21, 0.05, and 0.0, with 5% CO2-balance N2). Delta W/delta t for the four conditions averaged 0.60 +/- 0.11, 0.61 +/- 0.11, 0.61 +/- 0.10, and 0.61 +/- 0.10 (SE) g.min-1.mmHg-1.100g-1, respectively. No significant differences (ANOVA for repeated measures) were noted. We conclude that alveolar hypoxia does not alter the threshold for edema formation or delta W/delta t at a given microvascular pressure.     

Fluid filtration coefficient of isolated goat lungs was unchanged by endotoxin

The Starling fluid filtration coefficient (Kf) of blood-perfused excised goat lungs was examined before and after infusion of Escherichia coli endotoxin. Kf was calculated from rate of weight gain as described by Drake et al. [Am. J. Physiol. 234 (Heart Circ. Physiol. 3): H266-H274, 1978]. These calculations were made twice during base line and then at hourly intervals for 5 h after infusion of 5 mg (approximately 250 micrograms/kg) of E. coli endotoxin or after injection of oleic acid (47 microliter/kg). All lungs were perfused at constant arterial and venous pressure under zone 3 conditions. Base-line Kf averaged 27 +/- 10 and 20 +/- 4 (SD) microliter.min-1.cmH2O-1.g dry wt-1 for endotoxin and oleic acid groups, respectively. It was unchanged in the endotoxin group throughout the experiment but approximately doubled in the oleic acid lungs. Pulmonary arterial and venous pressures were not changed significantly during the course of these experiments in either group. Lung wet-to-dry weight ratios of these lungs were 5.6 +/- 0.6 and 6.1 +/- 0.5 ml/g for the endotoxin and oleic acid groups, respectively. This compares with 4.6 +/- 0.5 ml/g for normal, freshly excised but not perfused goat lungs. The small change in lung water and unchanged pulmonary pressures after both endotoxin and oleic acid suggest that lung injury was minimal. We conclude that 1) endotoxin does not cause a direct injury to the endothelium of isolated lungs during the first 5 h of perfusion, and 2) neutrophils are not sufficient to cause increased Kf after endotoxin infusion in this preparation.     

Liquid chromatographic determination of minocycline in brain-to-plasma distribution studies in the rat

An isocratic reversed-phase high-performance liquid chromatographic procedure was developed for the determination of minocycline in rat plasma and brain and applied to brain-to-blood (plasma) distribution studies. The procedure is based on isolation of the compound and the internal standard (either demeclocycline or tetracycline may be used) from plasma and brain constituents using the Oasis HLB cartridge, with satisfactory recovery and specificity, and separation on a Symmetry Shield RP8 (15 cm x 4.6 mm, 3.5 microm) column coupled with a UV detector set at 350 nm. The assay was linear over a wide range, with a lower limit of quantification of 50 ng ml(-1) or g(-1), using 0.2 ml of plasma and about 200 mg of brain tissue. Precision and accuracy were acceptable. In the rat minocycline crossed the blood-brain barrier slowly, achieving mean brain concentrations between 30 and 40% of the equivalent systemic exposure, regardless of the dose and route of administration.     

Immunocytochemical studies on the distribution pattern of daunomycin in rat gastrointestinal tract

The cancer drug daunomycin is used in treatment of leukemia but possesses severe side effects that involve the gastrointestinal tract. We therefore used a newly developed immunocytochemical procedure to determine the distribution of DM in the gastrointestinal tracts of rats after i.v. injection. Two hours after injection, DM was diffusely distributed in nuclei and most parts of the cytoplasm of intestinal epithelial cells. The cytoplasmic immunoreactivity for DM was most pronounced in small granules of the apical cytoplasm. Sixteen hours after injection, DM immunostaining was by and large absent in the villous epithelium but persisted in the intestinal crypts. In addition, staining was also detected in endothelial cells, scattered cells of the lamina propria and in smooth muscle cells. After 5 days, only little staining for DM remained. Similar findings were made in the colon. In the gastric mucosa, DM accumulation persisted at 16 h in some glandular cells but was lost from the surface epithelium. No staining was detected in saline-injected control rats. The distribution of DM accumulation correlated partially with the distribution of apoptotic cells as detected by the TUNEL procedure. Our results pinpoint that DM may exert prolonged effects on glandular and regenerative cells of the gastrointestinal tract—an observation that may explain the gastrointestinal toxicity of the drug. It seems possible that DM accumulation in surface epithelial cells is rapidly cleared through drug transporters.     

Multiple-breath washout experiments in rat lungs.

Multiple-breath washouts were performed on 30 Wistar rats postmortem in a study in which breaths of 90% O2-5% He-5% SF6 were given. Preliminary comparison of alveolar plateau slopes obtained from anesthetized rats in vivo and postmortem showed that ventilation distribution remains the same within 1 h after the animals were killed. For maneuvers with different preinspiratory lung volumes and end-inspiratory breathholding, we computed the normalized N2 slope (Sn) and Fowler and Bohr dead spaces [VDF(n) and VDB(n), respectively] as a function of breath number (n). For all maneuvers analyzed, Sn of all gases increased in the first two or three breaths and reached a horizontal asymptote thereafter. The value of Sn decreased, both with increasing preinspiratory lung volume and breath hold of 4 s. The fact that the horizontal Sn asymptote is reached after only two or three breaths suggests the absence of convection-dependent inhomogeneities (CDI) in rat lungs. This contrasts with multiple-breath washout experiments in humans, where interregional (gravity-dependent CDI) and intraregional CDI generate a marked increase in Sn throughout the entire washout. Also, in contrast with results in humans, VDF and VDB were independent of n. The present work suggests that rats may be used to study diffusion- and convection-dependent inhomogeneities without the influence of CDI or gas exchange.