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.     

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.     

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.     

Sodium-dependent accumulation of 5-hydroxytryptamine by rat blood platelets

1. The influence of sodium and potassium on the accumulation of 5-hydroxytryptamine (5-HT) by rat blood platelets was investigated.2. An absolute dependence of 5-HT uptake on the sodium concentration in the medium was found.3. Removal of potassium reduced the uptake by about 60%. High concentrations of potassium inhibited sodium-dependent accumulation.4. The observations have been discussed in terms of a carrier-mediated transport process for 5-HT operating in the platelet membrane.     

The relationship between 5-hydroxytryptamine and adenosine triphosphate in blood platelets

In normal platelets a proportionality was found between the amount of adenosine triphosphate (ATP) and amount of 5-hydroxytryptamine (5-HT) in platelets both before and after incubating them in plasma to which 5-HT had been added. In patients receiving reserpine and in others with myeloid leukaemia, the amount of 5-HT in the platelets and the uptake of 5-HT by them were depressed, while the amount of ATP was normal. The possibility that ATP is involved in the accumulation of 5-HT by platelets is discussed.     

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.     

Effects of fenfluramine on accumulation of 5-hydroxytryptamine and other neurotransmitters into synaptosomes of rat brain.

Earlier studies have demonstrated a marked long-lasting reduction in the brain level of 5-hydroxytryptamine after injection of fenfluramine. The decrease in the brain levels of dopamine and noradrenaline were less marked and disappeared one day after treatment. In this study, the effects of fenfluramine upon the uptake of 5-hydroxytryptamine and other neurotransmitters (dopamine, γ-aminobutyric acid) or their precursors (tryptophan, tyrosine and glutamic acid) into synaptosomes of rat brain were investigated. In vivo, fenfluramine is a competitive inhibitor of the high affinity synaptosomal uptake of 5-hydroxytryptamine. Even though fenfluramine produced a small reduction in the uptake of dopamine, γ-aminobutyric and glutamic acids 4 hr after injection, this effect disappeared 1 day later. In vitro, addition of fenfluramine into a synaptosomal fraction reduced uptake of 5-hydroxytryptamine, dopamine, γ-aminobutyric acid, glutamic acid, tryptophan and tjrosine. [¹⁴C] Fenfluramine itself was bound to synaptosomes. Ten days after 5–6 dihydroxytryptamine treatment, both the accumulation of [¹⁴C] fenfluramine in the brain in vivo, and the binding of [¹⁴C] fenfluramine to synaptosomes in vitro, were reduced. These results indicate that 5-hydroxytryptamine synaptosomal sites are involved in some actions of fenfluramine.     

Relationship between internal Na+/K+ and the accumulation of 14C-5-hydroxytryptamine by rat platelets

1. 5-Hydroxytryptamine (5-HT) transport has been investigated in rat blood platelets poisoned with dinitrophenol-sodium fluoride or ouabain.2. The inhibition of transport produced by different concentrations of the metabolic inhibitors has been correlated with changes in the internal Na(+) and K(+) concentrations of the platelets.3. Platelets poisoned in a high K(+) medium maintained a high internal K(+) concentration in the absence of cellular metabolism. When transferred to Krebs solutions containing different concentrations of Na(+) they accumulated 5-HT by a process that was related to the magnitudes of the internal and external Na(+) concentrations.4. The results are consistent with the hypothesis that the spontaneous movement of ions through the platelet membrane is capable of providing, at least in part, the energy requirements for 5-HT transport.     

The metabolism of 5-hydroxytryptamine and beta-phenylethylamine in perfused rat lung and in vitro.

1 Metabolism of 5-hydroxytryptamine (5-HT) and beta-phenylethylamine (PHE) by monoamine oxidase (MAO) was investigated in rat isolated lungs and in mitochondrial preparations from rat lung. 2. In perfused lungs 5-HT metabolism had an apparent Km of 2 microgram and PHE metaoblism a Km of 54 microgram, whereas in vitro the Km values were 330 microgram and 28 microgram respectively. 3 In vitro, MAO activity had substrate and inhibitor specificities compatible with the presence of A and B types of MAO. 4 In perfused lung, metabolism of 5-HT but not that of PHE was inhibited by desmethylimipramine. 5 These results show that PHE metabolism in perfused lung, unlike that of other metabolized amines, is not limited by transport and the transport process for PHE is unlike that of 5-HT or noradrenaline. 6 These results also show that the kinetic parameters obtained for MAO activity in vitro do not generally apply to the isolated lung where transport of substrate can be the deciding factor. This discrepancy emphasizes that the enzymic properties of the whole organ cannot relaibly be deduced from its enzymic content.     

The interaction between noradrenaline and 5-hydroxytryptamine in the isolated perfused rat hindlimb

The vascular response to bolus injections of 5-hydroxytryptamine (5HT) over the range 0.01-10.0 microgram was measured in isolated perfused rat hindlimbs. The hindlimb vasculature displayed a vasoconstrictor response to 5HT, which was potentiated by the addition of 0.01 microgram/ml noradrenaline (NA) to the perfusate at all doses tested. The response to 5HT was also enhanced by the monoamine oxidase (MAO) inhibitor, tranylcypromine (0.05 mg/ml), suggesting that MAO located in the vascular wall is involved in the termination of the response to 5HT in the peripheral circulation. MAO inhibition abolishes the potentiation of 5HT by NA, indicating that this potentiation results from competition for MAO by both amines, leaving more amine intact to activate membrane receptors. Corticosterone (0.01 mg/ml), catecholamine extraneuronal uptake inhibitor, did not alter the response to 5HT, nor the increase of this response by NA, it thus appears that 5HT and NA have different membrane transport systems in the peripheral vasculature.     

Effect of paraquat treatment of rats on disposition of 5-hydroxytryptamine and angiotensin I by perfused lung.

Rats were injected with the herbicide, paraquat dichloride (25 mg/kg, i.p.), and their lungs were perfused 2–28 days later. Isolated lungs from rats treated with paraquat (PQ) 3 or 4 days before perfusion removed significantly less perfused 5-hydroxytryptamine (5-HT) than did saline-injected controls. This effect was not caused by PQ directly, since perfusion of lungs from untreated animals with PQ did not alter removal of co-perfused 5-HT. Monoamine oxidase activity of600 g supernatan fractions of homogenates of lungs from PQ-treated rats was also reduced compared to controls. Although removal of perfused angiotensin I (1 ng/ml) by isolated lungs was not altered by PQpretreatment, antgiotensin-converting enzyme activity in 600 g supernatant fractions of lung homogenates was reducedd significantly. These results suggest that PQ damages pulmonary endothelium and impairs the metabolic function of lung.     

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.     

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.     

Trichlorethylene and halothane inhibit uptake of 5-hydroxytryptamine in the isolated perfused rat lung

Lung uptake of 5-hydroxytryptamine (5-HT) was determined in isolated perfused and ventilated rat lung, and was found to decrease with time according to a two-compartmental model. When the lungs were exposed to either trichlorethylene (TRI) or halothane, the uptake of 5-HT was drastically reduced. Both TRI and halothane gave log dose inhibition curves, which were superimposed, i.e. they were equally potent to inhibit lung uptake of 5-HT. At a concentration TRI of 18,000 ppm, the extraction of 5-HT was inhibited by 80 ± 2 (x? ± S.E.M.) per cent, at 8500 ppm the inhibition was 65 ± 6 per cent and 25 ± 1 per cent at 3000 ppm. When the lungs were exposed to halothane, the inhibition was 85 ± 6 per cent at 40,000 ppm, 48 ± 1 per cent at 6000 ppm, and 15 ± 0.3 per cent at 2000 ppm. When exposure to the solvent was discontinued, extraction of 5-HT was rapidly normalized. There was no detectable displacement of [³H]-5-HT from lungs saturated with the atnine when they subsequently were exposed to solvent-containing atmosphere. This inhibition of lung uptake of 5-HT from the circulation is therefore postulated as to be an effect dependent on concentration solvent in the tissue, and is probably due to a reversible membrane stabilization of the endothelium.     

Effect of selective proteolysis on the accumulation of 5-hydroxytryptamine by intact rat blood platelets

1 The effect of perturbation of intact blood platelets with proteolytic enzymes was studied with respect to 5-hydroxytryptamine (5-HT) transport, adenine transport and intracellular Na(+) and K(+) levels.2 Leucine aminopeptidase and thrombin reduced 5-HT transport, released 5-HT from pre-labelled platelets and disturbed the gradient to monovalent cations. Leucine amino-peptidase, but not thrombin, inhibited adenine transport.3 alpha-Chymotrypsin and carboxypeptidases A and B were without effect on the parameters studied.4 Trypsin selectively reduced 5-HT transport. It did not release 5-HT from blood platelets or inhibit adenine transport.5 The action of proteolytic enzymes is discussed in terms of proteins localized in the external membrane that may function in part as membrane carriers.