Isolated Rat Lung Perfusion Research Articles

Calcyclin gene expression is increased by mechanical strain in fibroblasts and lung.

Mechanical tension extending throughout the structural elements of the lung is a potential stimulus for cell proliferation and gene expression. Pulmonary fibroblasts located in the interstitial space of the capillary wall throughout the lung parenchyma and within the large vessels and airways are uniquely situated to sense changes in mechanical force. Therefore, we used the polymerase chain reaction-based method of differential display analysis to screen for altered gene expression in fetal human lung fibroblasts exposed to increased cyclic stretch. IMR-90 cells were seeded at 3 x 10(4) cells/cm(2) on laminin-coated plates. Cells were subsequently exposed to mechanical strain on a Flexercell apparatus, resulting in a maximal elongation of 20% at a rate of 60 cycles/min over a period of 48 h. A complementary DNA corresponding to the cell cycle-regulated gene calcyclin was identified in mechanically strained fibroblasts. Increased calcyclin messenger RNA levels were confirmed by Northern blot analysis. Further, calcyclin gene expression was upregulated in isolated-perfused rat lungs exposed to increased mechanical strain by ventilation at high states of lung inflation for 4 h. These data suggest that calcyclin gene expression plays a role in the response of pulmonary fibroblasts to increased mechanical tension and may alter the regulation of the fibroblast cell cycle.

Comparison of Euro-Collins Solution, Low-Potassium Dextran Solution Containing Glucose, and ET-Kyoto Solution for Lung Preservation in an Extracorporeal Rat Lung Perfusion Model

We have recently developed a modified ET-Kyoto solution by adding N-acetylcysteine, nitroglycerin, and dibutyryl cyclic adenosine monophosphate to the previously reported ET-Kyoto solution. The purpose of this study was to compare the new ET-Kyoto solution with the regular Euro-Collins solution and a low-potassium dextran solution containing glucose (Perfadex^®) in an isolated rat lung perfusion model. Rat lung blocks were preserved with ET-Kyoto solution, with Euro-Collins solution, or with Perfadex for 4 h. Arterial oxygen tension, shunt fraction, peak inspiratory pressure, mean pulmonary arterial pressure, and pulmonary vascular resistance were assessed up to 50 min after reperfusion. ET-Kyoto solution provided a significantly better lung function after reperfusion than Euro-Collins solution and Perfadex, while Perfadex was also superior to the Euro-Collins solution. We conclude that lung preservation with ET-Kyoto solution is significantly superior to Euro-Collins solution and to Perfadex in an isolated rat lung perfusion model.

Involvement of Fibrinogen in Protamine-Induced Pulmonary Hypertension

Protamine reversal of heparin anticoagulation occasionally results in pulmonary hypertension as well as systemic hypotension. To examine the contribution of blood components to this induction of pulmonary hypertension, we developed an isolated rat lung perfusion model and perfused heparinized plasma, heparinized serum, and Hepes (4% bovine serum albumin, 20 mMN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 5 mM glucose, in warm physiological saline) buffer solution with or without fibrinogen. Perfusion with heparinized plasma and Hepes buffer solution with fibrinogen caused pulmonary hypertension; perfusion with heparinized serum or Hepes buffer solution without fibrinogen did not, suggesting that fibrinogen is involved in the induction of pulmonary hypertension. We also labeled protamine with125I and compared the amounts of protamine accumulating in the lung with different concentrations of fibrinogen. The amount of protamine trapped in the lung increased according to the concentration of fibrinogen. Fibrinogen may accelerate the reaction between pulmonary endothelial cells and protamine or protamine–heparin complexes. In the mechanism of protamine-induced pulmonary hypertension, fibrinogen, as well as heparin and protamine, may be an essential component.

Peripheral hypertension and alterations in pulmonary vascular regulation.

We have recently reported in normal isolated-perfused rat lungs that low basal tone appears to be regulated by nitric oxide (NO)-dependent and -independent mechanisms of soluble guanylate cyclase activation. In this study, we examined the role of NO in the regulation of pulmonary artery (PA) tone from rats with renin-dependent hypertension. Rats were made hypertensive by ligating the abdominal aorta above the left and below the right renal artery (aortic coarctation, AC). Mean arterial pressure significantly increased from 119 +/- 8.4 mmHg in control animals to 156 +/- 15 mmHg 7-14 days after AC surgery. PA pressures, however, remained unchanged (8.5 +/- 3.4 mmHg in control animals vs. 11 +/- 3.3 mmHg in AC animals). Hypoxic contractions in U-46619 precontracted isolated small PA (160-260 microns diameter) were significantly increased from 51 +/- 13 mg in the control group to 142 +/- 38 mg (P < or = 0.05) in AC animals. Nitro-L-arginine (NLA; 100 microM) contractions were also enhanced in the AC animal. The enhanced NLA response may correlate with an increase in endothelial cell NO synthase (NOS) as detected by Western blotting (132 +/- 28% of control; P < 0.05). These data suggest that, in this renin-dependent model of systemic hypertension, there is increased endothelial cell NOS activity that maintains low PA tone, preventing the lung from developing increased pressures.

Endothelial cells internalize monoclonal antibody to angiotensin-converting enzyme.

We investigated the fate of MAb 9B9, a monoclonal antibody to angiotensin-converting enzyme (ACE), which binds to endothelium both in vitro and in vivo. Using cultured human umbilical vein endothelial cells (HUVEC) and isolated perfused rat lungs (IPL), we demonstrated specific and saturable binding of 125I-labeled MAb 9B9 at 4 degrees C [affinity constant (Kd) = 20-50 nM, maximal number of binding sites (Bmax) = 1.5-3.0 x 10(5) sites/cell]. When 125I-MAb 9B9 was bound to HUVEC at 37 degrees C, only 40% of cell-associated radioactivity was acid elutable, suggesting antibody internalization. This was confirmed by finding that 1) the amount of MAb 9B9 uptake at 37 degrees C was higher than at 4 degrees C both in HUVEC and IPL; 2) binding of 125I-labeled streptavidin with HUVEC and IPL pretreated with biotinylated MAb 9B9 (b-MAb 9B9) was diminished in a temperature- and time-dependent fashion at 37 degrees C; and 3) b-MAb 9B9 bound to HUVEC at 37 degrees C was found intracellularly by ultrastructural analysis using streptavidin gold. Intracellular 125I-MAb 9B9 was found in microsomal fractions of lung homogenate from IPL and after intravenous (iv) injections in rats. Degradation of internalized MAb 9B9 was minimal, since > 90% of cell-associated 125I label remained precipitable by trichloracetic acid in HUVEC, IPL, and in vivo. Autoradiography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of lung homogenates made as late as several days after iv injections of 125I-MAb 9B9 in rats demonstrated a predominant band above 140 kDa. These data indicate that endothelial cells either in vitro or in vivo internalize the ACE ligand MAb 9B9 without significant intracellular degradation. Therefore MAb 9B9 may be useful for selective intracellular delivery of drugs to the pulmonary vascular endothelium after systemic administration.

Effect of polycations on barrier and transport properties of alveolar epithelium in situ.

We examined the effect of polycations, classes of which are released by activated leukocytes, on the transport properties of the alveolar epithelium in isolated-perfused rat lungs. Protamine, polylysines, and ruthenium red produced rapid, dose-dependent increases in mannitol permeability (PAmann) when instilled into airspaces. The coupling between active transepithelial Na+ transport and alveolar fluid absorption was not altered, despite > 10-fold increases in PAmann. The increase in albumin permeability compared with that in mannitol suggested preservation of alveolar barrier-size selectivity. Tracheal instillation of protamine produced no cellular abnormality, whereas its addition to the perfusate resulted in damage to endothelial and type I cells. Protamine produced an even larger (P < 0.05) increase in PAmann in the presence of isoproterenol or dibutyryl adenosine 3',5'-cyclic monophosphate + 3-isobutyl-1-methyl-xanthine. The stimulation of Na+ and fluid transport by these agents was unaffected by protamine. Mastoparan, a peptide that activates G proteins, produced effects comparable to those of the polycations. The protamine- and mastoparan-induced increase in PAmann was abolished by barium, a K+ channel blocker, but not by zinc, a membrane-protective cation. Other K+ channel blockers, tetraethylammonium and quinine, had no effect. Thus short-term apical application of polycations and mastoparan alter alveolar epithelium paracellular permeability by a noncytotoxic mechanism that is inhibited by barium. The resulting increase in paracellular permeability does not alter fluid absorption driven by active Na+ transport. Polycations have very different effects, depending on whether they are present on one side or the other side of the alveolar capillary barrier.

Effects of low-dose hydrogen peroxide in the isolated perfused rat lung.

Isolated perfused rat lungs (IPRL) were used to determine if treatment with hydrogen peroxide would result in measurable changes in exhaled ethane during the early stages of capillary leak. Pulmonary capillary filtration coefficient, pulmonary vascular resistance, and dynamic pulmonary compliance were measured at two time points in an IPRL. Additionally, exhaled ethane was determined before and after the addition of 0.25 mM H2O2 to the perfusate in a second group of lungs. Lung wet/dry weight ratios were measured at the termination of the experiments. The ethane in the exhaled alveolar gas from IPRLs ventilated with 5%CO2/20%O2/balance N2 was quantitated using gas chromatography before and after the addition of 0.25 mM H2O2 to Krebs Ringer's 5% albumin perfusate. H2O2 (0.25 mM) caused a small but significant increase in capillary filtration coefficient from 0.0122 (+/- 0.0008) to 0.0173 (+/- 0.0013) mL/min/cm H2O/g dry lung weight (p < .05). Wet/dry lung weight ratios were increased in the H2O2-treated lungs (6.0654 +/- 0.1024 versus 5.4149 +/- 0.1143; p < .05). Exhaled ethane did not increase over the period of time hydrogen peroxide was present in the perfusate. In other experiments in closed-chested rats, 0.25 mM peroxide did not cause increased exhaled ethane, whereas 1 mM H2O2 did. This latter increase in ethane was not noted in similarly perfused open-chested rats. These data indicate that small amounts of H2O2 may increase pulmonary capillary permeability without affecting exhaled ethane measurements.

An improved setup for the isolated perfused rat lung

This article will describe a method for perfusing and ventilating rat lungs which allows assessment of lung mechanics, segmental vascular resistance, gas exchange, and vascular permeability. Isolated perfused rat lungs (IPL) were ventilated by negative pressure ventilation and were perfused at constant pressure with recirculating, albumin-containing Krebs-Henseleit buffer. A newly constructed respirator pump facilitated well-defined ventilation of the lungs and permitted switching between negative and positive pressure ventilation. Using this setup lungs were ventilated with positive pressure during both surgery and measurement of lung weight. Once the lungs were prepared, electronic control circuits ensured stability of the perfusate pH and perfusate pressure. Such preparations were stable for at least 3 hr. Breathing mechanics, for example, tidal volume, pulmonary compliance, and pulmonary resistance, perfusate characteristics, such as pulmonary vascular resistance, pulmonary pre- and postcapillary resistance, perfusate pH, Po 2, and Pco 2, and the capillary filtration coefficient were determined. All measured parameters were within normal physiological range, and the lungs were biochemically active, and responded to various stimuli.

Interaction of atrial natriuretic peptide with DA1 receptors in preconstricted isolated perfused rat lungs.

Using an in situ isolated salt-perfused rat lung preparation, we investigated the pulmonary vascular response to atrial natriuretic peptide (ANP). ANP was infused in graded doses ranging from 0.01 to 100.0 micrograms/kg during prostaglandin F2 alpha-induced pulmonary vasoconstriction. These experiments were repeated after selective dopamine1 (DA1) receptor blockade with SCH 23390 and after catecholamine depletion by reserpine. ANP at doses of 0.01, 0.1, 1.0, 10.0, or 100.0 micrograms/kg was injected into the pulmonary artery (n = 5-7/dose). In the unblocked group ANP infusion resulted in a dose-dependent decrease in the mean pulmonary arterial pressure (PAP) with a maximum effect at 10 micrograms/kg (delta PAP = -3.4 +/- 0.2 mm Hg). In the DA1 blockade groups the ANP dose-response curve was shifted to the right, in a parallel fashion. After catecholamine depletion with reserpine, the ANP dose-response curve was identical to that of the unblocked groups. With the parallel, rightward shift of the ANP dose-response curves by SCH 23390 and no attenuation of ANP effect after catecholamine depletion, it appears that ANP vasoactive properties in the pulmonary vasculature involve an interaction with the vascular DA1 receptors. This observation differs from the ANP-dopaminergic interactions seen in the kidney in which ANP action depends on endogenous dopamine transmission.

Lung Vascular Permeability Enhanced by Sympathetic Nerve Stimulation in Rats.

Role of sympathetic nerves in increasing the lung vascular permeability was investigated in the presence of papaverine using an isolated rat lung perfusion preparation. Sympathetic nerve stimulation was performed at different time points before and after pressure-induced weight gain. When stimulated at 3 or 7 min after the pressure associated increment, stimulation evoked a further increase in lung weight, particularly in the former case. In contrast, stimulation performed before or 15 min after the pressure increment elicited no such response. The histological finding of carbon particles in the perivascular wall after sympathetic nerve stimulation also provided direct evidence of increased lung vascular permeability.

Hydroxylamine is a vasorelaxant and a possible intermediate in the oxidative conversion of L-arginine to nitric oxide

Our objective was to determine whether hydroxylamine is a possible intermediate in the oxidative conversion of L-arginine to nitric oxide. Vasorelaxation by hydroxylamine is known to be mediated by nitric oxide. The vasorelaxant properties of hydroxylamine were examined using rat aortic rings and an isolated rat lung perfusion model. Hydroxylamine and acetylcholine were equally effective in relaxing norepinephrine-contracted intact aortic rings, whereas only hydroxylamine relaxed aortic rings with endothelium removed. This endothelium-independent vasorelaxation by hydroxylamine indicated that the hydroxylamine-converting enzyme is not localized solely within endothelial cells. Catalase, an enzyme known to oxidize hydroxylamine to nitric oxide, was present in homogenates of intact and endothelium-denuded rings. Cyanamide, another catalase substrate and a known precursor of nitroxyl (HNO), was not a vasorelaxant of aortic rings or of isolated, hypoxia-constricted lungs. These results suggest that free nitroxyl is not an intermediate in the oxidation of hydroxylamine to nitric oxide. An overall pathway for the oxidative conversion of L-arginine through an hydroxylamine intermediate to nitric oxide is proposed.

Accumulation mechanism of basic drugs in the isolated perfused rat lung.

The mechanism of the accumulation of basic drugs was investigated by isolated rat lung perfusion. Treatment with various metabolic inhibitors or non-basic drugs did not affect the accumulation of a basic drug in the lung, but a second basic drug inhibited the accumulation of the first basic drug depending on its lipid solubility. The basic drug already accumulated was rapidly displaced by the second drug except for poorly lipid-soluble basic drugs and non-basic drugs. The ability of a second basic drug to displace the first basic drug was well correlated with its ability to inhibit accumulation. From the Scatchard plot, at least two independent sets of binding sites for basic drugs were found to be present in the isolated perfused lung. The maximum binding capacity for each basic drug was similar in both sites. These results indicate that specific common binding sites for basic drugs, which do not contribute to the active transport system, exist in the lung tissues and the affinity to the sites depends on the lipid solubility of the basic drugs.

An isolated rat lung perfusion system for use in tobacco smoke studies

An isolated rat lung perfusion system has been developed for use in tobacco smoke studies. The lungs are ventilated by means of subatmospheric (negative) pressure produced through operation of an artificial thorax. The system enables standard respiratory conditions to be employed and so eliminates variations in animal breathing characteristics. The various tests of viability which have been carried out have shown that the preparations are viable for periods of at least 1 hr. It was also demonstrated that transfer of 14C-nicotine from smoke to perfusate was rapid and linear over the period of smoke exposure and that first-pass metabolism of nicotine was of little significance.

Effects of various in vitro--inhibitors of benzo(a)pyrene metabolism in isolated rat lung perfusion.

Various in vitro-inhibitors were added with 3H-benzo(a)pyrene (BP) into the perfusion fluids in isolated rat lung perfusions to see whether their effects are dependent on the integrity of tissue. 3H-BP and its metabolites were measured by thin-layer chromatography and radiometry from both samples of perfusion medium and homogenates of lung tissue. The total covalent binding to lung tissue was used as a measure of the formation of reactive metabolites. In methylcholanthrene-induced rat lung, the metabolism of BP was inhibited by alpha-naphthoflavone, an inhibitor of monooxygenase, and less with diethylmaleate, a depletor of glutathione, with salicylamide, an inhibitor of conjugases, and, astonishingly, with D-saccharo-1,4-lactone, an inhibitor of beta-glucuronidase. With trichloropropene oxide, which inhibits epoxide hydratase, the metabolism was either decreased or unchanged. Nicotine had no effect on BP-metabolism. Nicotine and diethylmaleate increased statistically significantly and alpha-naphthoflavone and salicylamide decreased the covalent binding of radioactivity to lung tissue. In most cases, the changes in BP metabolism observed during perfusion can be explained on the basis of effects of modifiers on the enzyme systems.