The accumulation of diamines and polyamines into rat lung slices

The diamine cadaverine, and the polyamines spermidine and spermine have been shown to accumulate into rat lung slices by an uptake process which obeyed saturation kinetics. The apparent K_m values for the accumulation process of cadaverine, spermidine and spermine were 19, 11 and 15 μM respectively with V_max values of 937, 768 and 617 nmoles/g wet weight/hr respectively. The accumulation was KCN sensitive, indicative of an energy dependent process, although spermine did show some non-specific binding to lung tissue. Cadaverine, spermidine and spermine were not accumulated by slices of liver, kidney, heart and spleen to concentrations much greater than that in the medium. They were accumulated, however, by a KCN sensitive process into brain slices although the accumulation was much less than that which occurred in lung slices. The diamine, putrescine, exhibited a concentration-dependent inhibition of the ability of lung slices to accumulate cadaverine and the polyamines. These data have led us to conclude that the transport process in the lung, which has recently been shown to accumulate the diamine putrescine, is also capable of accumulating cadaverine, spermidine and spermine. Thus, by analogy with putrescine, there exists in specific lung cells a membrane receptor(s) which is selective in its acceptance and transport of diamines and polyamines.     

The accumulation of pentamidine into rat lung slices and its interaction with putrescine.

The aromatic diamidine, pentamidine, accumulated into rat lung slices by an uptake system that obeyed saturation kinetics, with an average Km value of 554 microM and a Vmax value of 4077 nmol/g lung wet wt/30 min, respectively. This system was not inhibited by metabolic inhibitors but was greatly diminished by lowering the temperature from 37 degrees to 4 degrees. Both compounds, pentamidine and putrescine, inhibited the uptake of the other and the inhibition of pentamidine accumulation by putrescine was demonstrated to be non-competitive. Uptake of putrescine was inhibited by increasing concentrations of pentamidine. As putrescine accumulates in epithelial type 1 and type 2 cells and in Clara cells, it is likely that pentamidine is also accumulated in these cell types but does not utilize the pulmonary uptake system for polyamine transport. Within the time period studied, toxic effects of the drug were not observed.     

The relationship between paraquat accumulation and covalent binding in rat lung slices

The accumulation and covalent binding of paraquat in rat lung slices were both linear for 6 hr in room air incubations. Binding continued to increase in slices transferred to paraquat-free buffer after 3 hr of incubation in paraquat although accumulated paraquat decreased. Binding in 100% O2 was decreased slightly. Active accumulation in 100% N2 did not occur, but binding proceeded at one-third the rate observed in room air. Ascorbate decreased accumulation in room air, although binding was unaffected. Reductants had no effect on binding in 100% nitrogen. Paraquat binding in slices of various organs was in the order of lung greater than liver greater than heart greater than kidney cortex. Mitochondrial proteins were found to have the highest concentration of bound paraquat in lung slices followed in order by microsomal protein greater than nuclear protein = cytosolic protein. The binding of paraquat is postulated to involve a reduced species, presumably the monovalent radical.     

The identification and characterization of an uptake system for taurine into rat lung slices.

The objective of this study was to determine whether taurine was accumulated by rat lung slices and if so, to establish the role of this uptake as a source of pulmonary taurine. We have shown that taurine is accumulated into rat lung by an active uptake process that was both ATP and Na(+)-dependent and obeyed saturation kinetics, exhibiting an apparent Km of 186 microM and Vmax of 970 nmol/g wet wt/hr. Substrate specificity of the system was high and only compounds possessing anionic and cationic groups separated by two methylene groups were able to competitively inhibit taurine uptake. Subsequent to its uptake, taurine was not significantly metabolized, and since the apparent Km for the uptake process is similar to the known plasma concentration of taurine, it can be inferred that this system will contribute to pulmonary taurine uptake in vivo. Taurine has been suggested to possess antioxidant and antiinflammatory properties, and we suggest that this uptake system may contribute to the defence of pulmonary tissue against oxidative stress.     

Chlorphentermine inhibits oxidative energy metabolism in rat lung slices

In an attempt to assess possible functional consequences of chlorphentermine (CP) to pulmonary tissue, we investigated the effects of this drug on energy metabolism in slices and isolated mitochondria of control rat lungs. Oxygen consumption in 0.5-mm thick slices was inhibited in a concentration-dependent manner by 0.5 to 2.0 mm CP (max. depression = 91%). In isolated mitochondria, the respiratory control ratio (RCR) was decreased by 1.0 to 5.0 mm CP. In addition, the ADP-mediated respiratory burst (State III) and the subsequent recovery (State IV) were decreased at 2.5 and 5.0 mm CP. When mitochondria were isolated from lung slices preincubated with CP, RCR was again decreased by 1.0 to 5.0 mm CP. At 2.5 mm CP, oxidative phosphorylation was uncoupled. In addition, aerobic glucose metabolism in lung slices was impaired by CP concentrations above 2.0 mm. The metabolism of [6-¹⁴C]- and [1-¹⁴C]glucose was inhibited 95 and 70%, respectively, suggesting a preferential inhibition of glycolysis in comparison to the hexose monophosphate shunt. These data show that CP inhibits oxidative functions in lung preparations and suggest that CP has the potential for disrupting energy metabolism in the intact organ.     

Putrescine and 5-hydroxytryptamine accumulation in rat lung slices: cellular localization and responses to cell-specific lung injury

The cellular localization of putrescine (1,4-diaminobutane) and 5-hydroxytryptamine (5HT) following the accumulation of tritium-labeled putrescine (2.5 microM) or 5HT (0.5 microM) into rat lung slices was determined by autoradiography at the light microscope level. Putrescine labeling was found to occur in type II alveolar epithelial cells and in branchiolar nonciliated (Clara) cells, and possibly also in type I alveolar epithelial cells. The pattern of 5HT labeling was clearly different from that with putrescine, since the parenchyma was diffusely labeled with no preferential location in type II cells, but with strong labeling of the endothelium of large vessels and also the pleural mesothelium. The apparent kinetic parameters for the tissue uptake of [3H]putrescine (2.5 to 80 microM) and [14C]5HT (0.5 to 16 microM; both being simultaneously present in a 5 to 1 molar ratio) were studied in lung slices from normal rats and rats pretreated with O,S,S-trimethyl phosphorodithioate (OSSMe, 11 to 95 mg/kg, po), with paraquat (20 mg/kg, ip), or with alpha-naphthylthiourea (ANTU, 5 or 10 mg/kg, ip). OSSMe and paraquat were used as models for pulmonary epithelium-damaging agents, and ANTU was taken as a model for a pulmonary endothelium-damaging agent. The Vmax for the uptake of 5HT was significantly increased (without change in Km) following treatment with OSSMe and paraquat. Following ANTU treatment the Vmax for the uptake of 5HT was unchanged (5 mg/kg) or increased (10 mg/kg, Km also increased). These results indicate that in lung slices the response to lung injury may be associated with an increased accumulation of 5HT. The Vmax for the uptake of putrescine was significantly decreased (without change in Km) following treatment with OSSMe and paraquat. Following ANTU treatment the Vmax for the uptake of putrescine was unchanged (5 mg/kg) or decreased (10 mg/kg, no change in Km). These results suggest that a decreased putrescine uptake is a sensitive index of pulmonary epithelial damage.     

Specific polyclonal and monoclonal antibody prevents paraquat accumulation into rat lung slices

Sheep polyclonal and mouse monoclonal antibodies have been produced that bind to the bipyridyl herbicide, paraquat. The binding capacities and affinities of the various antibody solutions (serum, ascites, purified tissue culture supernatant) to paraquat were determined using a radioimmunoassay. All antibody solutions bound paraquat with high affinity (Ka = 10(9)-10(10) l/mol). The sheep polyclonal antisera, the mouse ascites fluid, and the purified culture supernatant had mean binding capacities of 8, 1 and 22 micrograms paraquat/ml respectively. All the antibody preparations were able to prevent the in vitro accumulation of paraquat into rat lung tissue. The amount of antibody to achieve this was dependent upon the binding capacity of the antibody solution, i.e. when the binding capacity of the antibody was equal to the amount of paraquat present in the incubation medium a total blockade of uptake was achieved. When antibody was added to lung tissue that had been accumulating paraquat for 1 hr, the inhibition of uptake was immediate and was complete for at least 2 hr. Both the radioimmunoassay and lung slice experiments indicate that an equivalent of 1 mg of IgG is required to bind 2.5 micrograms of paraquat ion. Preincubation of lung tissue with antibody did not affect the subsequent accumulation of paraquat, nor did it result in a detectable degree of intracellular neutralisation of paraquat as measured by paraquat's ability to stimulate the pentose phosphate pathway. The rate of efflux of paraquat from lung slices prepared from rats dosed intravenously with paraquat was not increased by the presence of antibody in the incubation medium. In conclusion, neutralising antibodies to paraquat have been produced. They bind to paraquat in solution with high affinity and render the paraquat unavailable for its in vitro accumulation into lung cells.     

Trichloroethylene and halothane inhibit uptake and metabolism of 5-hydroxytryptamine in rat lung slices

The effect of exposure to organic solvents on uptake and metabolism of 5-HT was studied in rat lung slices. It was found that under control conditions 5-HT was both taken up and metabolized to 5-HIAA. When halothane (35,000 ppm) or trichloroethylene (18,000 ppm) were equilibrated with the incubation medium the uptake of 5-HT decreased by approximately 50% after 30 min of incubation, and the production of 5-HIAA was inhibited by approximately 70% and 80%, respectively. The results are consistent with earlier studies using a much more elaborate technique, in which halothane and trichloroethylene were found to depress 5-HT uptake in isolated perfused rat lungs. Our results demonstrate that the simpler technique employing lung slices can also be used, to investigate factors affecting pulmonary uptake of endogenous amines, and, potentially, the uptake of other compounds as well.     

Pulmonary elimination and metabolism of 5-fluoro-2'-deoxyuridine in isolated perfused rat lung and lung slices

Elimination kinetics and metabolism of the cytostatic drug 5-fluoro-2'-deoxyuridine (FUDR) were studied in isolated perfused rat lung and in incubated lung slices. The intact organ exhibited a low clearance of 0.2 to 0.8 ml/min and a calculated first-pass extraction of 2 to 7% of the drug inflow. Thus, the pulmonary uptake of the fluorinated nucleoside from the circulation is low. Within 120 min of perfusion, however, 30 to 45% of the initial FUDR dose was metabolized by the isolated rat lung. The nucleobase metabolite 5-fluorouracil (FU) represented almost all FUDR metabolites in the medium, indicating that this metabolic pathway, mediated by thymidine phosphorylase, is active in lung while the enzymic activity for further pyrimidine degradation is low. This was demonstrated in incubated lung slices, which have a high capacity to transform FUDR into FU, comprising 83 to 95% of the metabolites in the medium. The final catabolic metabolite, alpha-fluoro-beta-alanine, was present in trace amounts only. It is concluded that the pulmonary tissue contains a marked "intrinsic" capacity to transform FUDR into FU, while the metabolic activity for catabolism of the nucleobase metabolites FU and 5,6-dihydrouracil is virtually lacking.     

The uptake and metabolism of 3H-catecholamines in rat cerebral cortex slices

Summary The accumulation and metabolism of ³H-catecholamines were studied in cerebral cortex slices obtained from rats pretreated with reserpine, during 30 min of incubation with 50 nmol/l of the ³H-amines. In some experiments neuronal uptake (uptake,) was inhibited by the presence of 0.3 mol/l desipramine, in others COMT was inhibited by 30 mol/l U-0521. When both MAO and COMT were intact, most of the metabolism of ³H-noradrenaline was neuronal (i. e., desipramine-sensitive). For ³H-adrenaline rates of neuronal metabolism were much lower than for ³H-noradrenaline, non-neuronal O-methylation accounting for about 50% of total metabolism. Rates of metabolism of ³H-dopamine were similar to those of ³H-noradrenaline, but with a predominance of non-neuronal metabolism, which involved O-methylation and deamination. Under these conditions, very little ³H-catecholamine was recovered from the tissues; moreover, desipramine tended to increase tissue levels. Hence, tissue content then appears to partly reflect extracellularly distributed ³H-amines. After block of MAO rates of metabolism of ³H-noradrenaline and ³H-dopamine were greatly reduced, and tissue levels were increased. Desipramine now antagonized the accumulation of ³H-amines in the tissue, while U-0521 increased it. Rates of O-methylation (in the presence of desipramine) increased in the order ³H-noradrenaline < ³H-dopamine. It is concluded that neuronal uptake is associated with MAO only, and rates of neuronal deamination increased in the order: ³H-adrenaline < ³H-dopamine « ³H-noradrenaline. Non-neuronal uptake is associated with both, COMT and MAO, and rates of non-neuronal metabolism increased in the order: ³H-adrenaline < ³H-noradrenaline « ³H-dopamine.Abbreviations COMT catechol-O-methyl transferase - DOMA dihydroxymandelic acid - DOPAC dihydroxyphenylacetic acid - DOPEG dihydroxyphenylglycol - DOPET dihydroxyphenylethanol - HVA homovanillic acid - MAO monoamine oxidase - MN metanephrine - MOPEG methoxyhydroxyphenylglycol - MOPET methoxyhydroxyphenylethanol - NMN normetanephrine - 3-OMT 3-O-methyl-tyramine - VMA vanillylmandelic acid Supported by the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to U. Trendelenburg at the above addresswith the technical assistance of M. Babl     

Inhibition of paraquat accumulation in rat lung slices by a component of rat plasma and a variety of drugs and endogenous amines

The accumulation of paraquat by slices of rat lung has been shown to be inhibited in vitro by the addition of rat plasma. For a given concentration of plasma, inhibition was constant with time and the amount of inhibition increased with increasing concentration of plasma. This suggests that there are components of rat plasma which inhibit paraquat accumulation by rat lung slices in a concentration-dependent manner. An ultrafiltrate of plasma also inhibited paraquat uptake, indicating that the inhibitor is a small molecular weight compound. A number of endogenous amines including noradrenaline, 5-hydroxytryptamine and histamine have been shown to reduce the concentration of paraquat accumulated into lung slices, as have several other drugs including imipramine, propranalol, burimamide and betazole. The relevance of these findings to the prevention of paraquat accumulation by lung is discussed.     

The effect of taurine on motor behaviour,body temperature and monoamine metabolism in rat brain

Summary Taurine, a putative inhibitory neurotransmitter, was injected in various doses intracerebroventricularly to conscious rats via pre-implanted polythene cannulas. The formation of DOPA and 5-hydroxytryptophan (5-HTP) in various brain regions was investigated by measuring the accumulation of these monoamine precursors induced within 30 min by the intraperitoneal injection of 3-hydroxybenzyl hydrazine HCl (NSD 1015, 100 mg/kg), an inhibitor of the aromatic L-amino acid decarboxylase readily penetrating into the brain. DIPA formation, but not 5-HTP formation was significantly enhanced by taurine in dose-related manner in all brain regions studied, indicating an increased synthesis of both dopamine and noradrenaline. Dopamine depletion induced by -methyltyrosine was significantly retarded by taurine, whereas noradrenaline depletion tended to be enhanced. Endogenous levels of dopamine were increased, whereas the following brain constituents were unchanged: tyrosine, tryptophan, noradrenaline, 5-HT and 5-hydroxyindoleacetic acid. In the exoeriments with NSD 1015, a dose-related decrease in rectal temperature and in motility was observed after taurine treatment, as compared to treatment with the decarboxylase inhibotor alone. Systemic parenteral administration of taurine caused no significant changes in brain monoamines, body temperature or behaviour but decreased the heart noradrenaline levels. The data indicate that taurine, which apparently has to be given intracerebroventricularly in order to reach the brain in sufficient amounts, causes inhibition of firing in central dopamine neurons but has the opposite effect on noradrenaline neurons, perhaps also peripherally, whereas 5-HT neurons appear to be unaffected. In addition, taurine appears to interfere with motor behaviour and temperature regulation, possibly via effects on catecholaminergic systems.     

Metabolic and structural viability of precision-cut rat lung slices in culture

1. The principal objective was to evaluate the functional and structural integrity of precision-cut rat lung slices in culture over 72 h. 2. Lung slices metabolized 7-ethoxycoumarin in a time-dependent fashion, the major metabolites being the sulphate and glucuronide of 7-hydroxycoumarin with very low levels of the free compound. Prior treatment of rats with beta-naphthoflavone elevated markedly the rate of metabolism. The optimum slice thickness, as exemplified by the metabolism of 7-ethoxycoumarin, was about 600 microm. 3. Lung slices retained metabolic viability towards 7-ethoxycoumarin for 8 h, but after this point a marked decline in metabolic activity was noted. However, very low levels of activity were still evident following a 72 h incubation. 4. Morphological examination of lung slices revealed nuclear degeneration and loss of tissue architecture following 24h incubation. When cellular integrity was assessed using lactate dehydrogenase, a time-dependent leakage was evident with maximum loss occurring within 24h; longer incubations did not result in further leakage. 5. It is concluded that precision-cut rat lung slices, of 600 microm thickness, can be maintained metabolically viable in culture for some 8 h.     

Metabolic and structural viability of precision-cut rat lung slices in culture

1. The principal objective was to evaluate the functional and structural integrity of precision-cut rat lung slices in culture over 72?h.

2. Lung slices metabolized 7-ethoxycoumarin in a time-dependent fashion, the major metabolites being the sulphate and glucuronide of 7-hydroxycoumarin with very low levels of the free compound. Prior treatment of rats with β-naphthoflavone elevated markedly the rate of metabolism. The optimum slice thickness, as exemplified by the metabolism of 7-ethoxycoumarin, was about 600?µm.

3. Lung slices retained metabolic viability towards 7-ethoxycoumarin for 8?h, but after this point a marked decline in metabolic activity was noted. However, very low levels of activity were still evident following a 72?h incubation.

4. Morphological examination of lung slices revealed nuclear degeneration and loss of tissue architecture following 24?h incubation. When cellular integrity was assessed using lactate dehydrogenase, a time-dependent leakage was evident with maximum loss occurring within 24?h; longer incubations did not result in further leakage.

5. It is concluded that precision-cut rat lung slices, of 600?µm thickness, can be maintained metabolically viable in culture for some 8?h.

     

Adenosine receptor agonists inhibit inositol phosphate accumulation in rat striatal slices

The accumulation of inositol-1-phosphate in rat striatal slices was inhibited by the adenosine analogues 5'-N-ethylcarboxamide-adenosine and N6-phenylisopropyladenosine. Maximal inhibition (approximately 20%) was achieved by micromolar concentrations of either compound. Both basal and stimulated values could be inhibited, and the inhibition was reversible by the adenosine receptor antagonist 8-phenyltheophylline (10 microM). The results suggest that adenosine may exert a tonic inhibitory influence on inositol phospholipid-derived second messenger production in the striatum in vivo.     

The effect of acute ethanol administration on phosphorylcholine uptake and metabolism in rat liver slices

Liver slices obtained from normal and acutely ethanol-intoxicated rats were incubated with labelled choline plus unlabelled orthophosphate or labelled phosphorylcholine (PC). After variable times of incubation hydrosoluble compounds and choline phosphoglycerides (CPG) were extracted from the tissue and analyzed. When compared to controls, the slices obtained from intoxicated livers accumulated more PC and synthesized more CPG when incubated with PC; on the other hand, when incubated with choline, they accumulated less PC. From these results it can be concluded that PC is a better lipid precursor in intoxicated livers, than in normal ones. In any case CPG becomes better labelled after incubation with choline than with PC; base-exchange could be liable for this result.