Precursor utilization of 5-hydroxytryptophan for 5-hydroxytryptamine biosynthesis in isolated and perfused rabbit and rat lungs

The present study was designed to investigate whether lungs can utilize 5-hydroxytryptophan (5-HTP), formed elsewhere and transported, for the synthesis of 5-hydroxytryptamine (5-HT). [14C]5-HTP uptake was 7.7 +/- 1.1 and 3.9 +/- 0.2% by rabbit and rat lungs, respectively, after 1 h of perfusion with 10 microM [14C]5-HTP. There was an increase in the lung uptake of [14C]5-HTP when the lungs were preperfused with 0.5 mM chlorphentermine (CP) and the uptake was low when the lungs were preperfused with 0.1 mM hydroxybenzylhydrazine dihydrochloride (HBH). The perfusate concentration of 5-hydroxyindole acetic acid (5-HIAA) increased significantly (3-4 micrograms/100 mL) during rabbit lung perfusion with 10 microM [14C]5-HTP and this did not change significantly when the lungs were preperfused with 0.5 mM CP. However, 5-HT increased with time in the perfusate. 5-HT, but not 5-HIAA, was detected in the perfusate and increased with time of perfusion when the rat lungs were perfused either with 10 microM 5-HTP or with 0.5 mM CP and 10 microM 5-HTP. However, no metabolites were detected in either the rabbit lung or rat lung perfusates when they were preperfused with 0.1 mM HBH. Lung contents of 5-HT and 5-HIAA were significantly higher in the rat lungs and only 5-HIAA increased in rabbit lungs after 1 h of perfusion with 10 microM 5-HTP. Preperfusion with 0.5 mM CP resulted in a greater increase in the 5-HT content of both rabbit and rat lungs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of halothane on metabolism of 5-hydroxytryptamine by rat lungs perfused in situ

The effect of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) on the uptake of 14C-labelled 5-hydroxytryptamine (5-HT) and its metabolism to 5-hydroxyindol-3-ylacetic acid (5-HIAA) was investigated in rat lungs perfused in situ. The rate of accumulation of 14C-labelled 5-HIAA in the tissue, monitored as an index of 5-HT metabolism, was linear with time, displayed saturation kinetics and remained stable for at least 180 min of perfusion. Exposure of the lungs to halothane (4%) for 60 min reversibly reduced production of 5-HIAA through an increase in the apparent Km for metabolism of the amine from 1.45 to 3.52 microM (P less than 0.001); the anaesthetic had no effect on the Vmax. of the process. The magnitude of the inhibition increased with time of exposure to the anaesthetic. Halothane exposure did not alter the distribution of [3H]sorbitol or [14C]5-HT, pulmonary vascular resistance, levels of ATP or the kinetics of amino acid transport in the tissue. Inhibition of protein synthesis by cycloheximide did not mimic the effect of the anaesthetic. These observations, together with those made in lungs exposed to inhibitors of 5-HT uptake and metabolism, were consistent with a halothane-mediated inhibition of 5-HT uptake, which did not appear to involve non-specific changes in membrane permeability.     

Sites of uptake of3H-5-hydroxytryptamine in rat isolated lung

Summary The localisation of radioactivity in rat lungs after perfusion of³H-5-hydroxytryptamine (5-HT) was studied by autoradiography. Rat isolated lungs, perfused with Krebs bicarbonate solution, via the pulmonary circulation, were rapidly frozen after the infusion of³H-5-HT. All subsequent handling was carried out at −30 to −40°C. Developed sections were examined by the light microscope and showed that radioactivity was concentrated around the alveoli with little or no label in other parts of the lung. Lungs perfused with³H-5-HT in the presence of mebanazine, a monoamine oxidase inhibitor, showed label in the endothelial cells of arteries and arterioles as well as the alveolar label. Lungs treated with amitriptyline were essentially devoid of label. These results indicate that the site of the avid uptake and metabolism of 5-HT in the lung is the endothelial cells of the vasculature.     

C-receptor ligand blocks pulmonary clearance of atrial natriuretic peptide in isolated rat lungs.

Pulmonary clearance of atrial natriuretic peptide (ANP) was measured by indicator dilution technique in isolated perfused rat lungs with and without ANP clearance receptor (C-receptor) blockade. Approximately 50% of a bolus injection of 125I-ANP was removed during a single pass through the lungs compared with the intravascular marker 14C-dextran. Pulmonary clearance of 125I-ANP was suppressed in a dose-dependent fashion by unlabeled ANP. C-receptor blockade suppressed pulmonary clearance of 125I-ANP to the same degree as unlabeled ANP. High-performance liquid chromatography analysis of the pulmonary venous effluent from lungs treated with C-receptor ligand demonstrated intact 125I-ANP. We conclude that virtually all of the pulmonary vascular uptake of 125I-ANP during a single pass through isolated lungs is secondary to removal by ANP C-receptors.     

Determinants of platelet kinetics: effects of pulmonary microembolism

We examined the mechanisms of platelet uptake in the lungs after alpha-thrombin-induced pulmonary microembolism. Platelets labeled with 111In-oxine were reinfused into chronically prepared sheep. Pulmonary microembolism resulted in an increase in lung platelet radioactivity (95.5 +/- 15.3%; n = 4), which was followed by an exponential washout (half-life = 115 +/- 4 min). Platelet uptake in the lungs was more sustained after prior fibrinolytic inhibition with tranexamic acid (half-life = 178 +/- 11 min), although the initial increase was similar (90.7 +/- 9.6%; n = 7). Prior depletion of fibrinogen with ancrod (Arvin), blunted the initial increase in lung platelet uptake after alpha-thrombin challenge (31.7 +/- 11.3%, n = 5), indicating that the effect of thrombin was markedly dependent on fibrinogen. We examined the role of circulating granulocytes, since platelets may bind to subendothelial matrix exposed after granulocyte-mediated lung vascular injury. Maximal pulmonary platelet uptake after thrombin in granulocytopenic sheep was not different from control (71.7 +/- 14.4%; n = 4). The results indicate that pulmonary microembolism results in lung platelet sequestration. Platelet uptake is not dependent on granulocyte-mediated vascular injury but requires fibrin deposition and is sustained if fibrinolysis is inhibited.     

Inhibition of gut- and lung-derived serotonin attenuates pulmonary hypertension in mice

Decreasing the bioavailability of serotonin (5-HT) by inhibiting its biosynthesis may represent a useful adjunctive treatment of pulmonary hypertension (PH). We assessed this hypothesis using LP533401, which inhibits the rate-limiting enzyme tryptophan hydroxylase 1 (Tph1) expressed in the gut and lung, without inhibiting Tph2 expressed in neurons. Mice treated repeatedly with LP533401 (30-250 mg/kg per day) exhibited marked 5-HT content reductions in the gut, lungs, and blood, but not in the brain. After a single LP533401 dose (250 mg/kg), lung and gut 5-HT contents decreased by 50%, whereas blood 5-HT levels remained unchanged, suggesting gut and lung 5-HT synthesis. Treatment with the 5-HT transporter (5-HTT) inhibitor citalopram decreased 5-HT contents in the blood and lungs but not in the gut. In transgenic SM22-5-HTT+ mice, which overexpress 5-HTT in smooth muscle cells and spontaneously develop PH, 250 mg/kg per day LP533401 or 10 mg/kg per day citalopram for 21 days markedly reduced lung and blood 5-HT levels, right ventricular (RV) systolic pressure, RV hypertrophy, distal pulmonary artery muscularization, and vascular Ki67-positive cells (P < 0.001). Combined treatment with both drugs was more effective in improving PH-related hemodynamic parameters than either drug alone. LP533401 or citalopram treatment partially prevented PH development in wild-type mice exposed to chronic hypoxia. Lung and blood 5-HT levels were lower in hypoxic than in normoxic mice and decreased further after LP533401 or citalopram treatment. These results provide proof of concept that inhibiting Tph1 may represent a new therapeutic strategy for human PH.     

Inhibition of pulmonary prostaglandin metabolism by exposure of animals to oxygen or nitrogen dioxide.

The effects of exposure of animals to 100% O2 and NO2 on the rate of prostaglandin metabolism by lung and kidney were studied in vitro. Exposure of guinea pigs to 100% O2 for 48 h inhibited the metabolism of prostaglandin F2 alpha by both NAD+- and NADP+-dependent prostaglandin dehydrogenase in lung, but had no effect on the metabolism in kidney. Succinate dehydrogenase, but not glucose 6-phosphate dehydrogenase, in guinea-pig lung was inhibited by exposure to 100% O2. Exposure to 46 p.p.m. but not 16 or 29 p.p.m. NO2 for 6 h inhibited guinea-pig lung prostaglandin dehydrogenase in vitro. The inhibition of pulmonary prostaglandin dehydrogenase by exposure to 100% O2 or to 49 p.p.m. NO2 was dependent on the duration of exposure, but returned to control values within 7 days after cessation of the exposure. The pulmonary transport system responsible for removing circulating prostaglandins from the blood was not affected by exposure to 100% O2 as measured by using the isolated perfused lung. Kinetic analysis of the inhibition of pulmonary prostaglandin dehydrogenase activity in guinea pig exposed to 100% O2 showed non-competitive inhibition with respect to both prostaglandin F2 alpha and NAD+, which suggests destruction or inactivation of the enzyme. Pulmonary prostaglandin dehydrogenase appears to be inhibited by exposure to oxidant gases, which may lead to elevated prostaglandin concentrations in the lungs or in the systemic circulation.     

Serotonin uptake and configurational change of bovine pulmonary artery smooth muscle cells in culture

Although it is well known that endothelial cells transport serotonin (5-HT) from extracellular to intracellular locations, it has been generally assumed that smooth muscle cells do not accumulate 5-HT but, rather, respond to 5-HT through a receptor activity unrelated to uptake of this amine or via stimulation of endothelial-derived relaxing factor. In the present study smooth muscle cells (PASMC), isolated and cultured from bovine pulmonary artery, were evaluated for 5-HT uptake under a variety of conditions. 5-HT uptake was linear up to 15 min and the rate was seven- to eightfold higher than that by bovine pulmonary artery endothelial cells. There was intracellular metabolism of 5-HT to 5-hydroxyindoleacetic acid (5-HIAA). The uptake was inhibited by exposure to 4 degrees C, absence of Na+ from the medium, and agents such as imipramine, verapamil, ketanserin, and methiothepin. Like that of endothelial cells, 5-HT uptake by PASMC was stimulated by exposure of cells to anoxia for 24 hr. Unlike endothelial cells that showed no morphological changes, PASMC at early passage showed dendritic formation after 30-60 min exposure to 5-HT at a concentration as low as 10(-8) M. Although this configurational change in response to 5-HT was lost with passage of cells, transport of 5-HT by these cells was retained. The configurational change was blocked by agents that inhibited 5-HT uptake, such as imipramine, verapamil, ketanserin, and methiothepin; it was unaffected by inhibitors of protein kinase C, phospholipase C, and calmodulin or absence of Ca2+ from the medium. We conclude that PASMC, as well as endothelial cells, accumulate 5-HT; there appears to be a close relationship between 5-HT uptake and configurational change of early passaged PASMC in culture. The factor(s) required for the configurational change are absent in endothelial cells and lost during passage of PASMC.     

Review of the significance of pulmonary intravascular macrophages with respect to animal species and age

Pulmonary intravascular macrophages (PIMs) have been recognized as the site of substantial uptake of blood-borne particles in the lungs of a number of domestic animal species. Concomitantly, there is a pronounced lung susceptibility to endotoxin in calves, goats, sheep, pigs, and cats. Hemodynamic changes and initial lung injury after endotoxin administration are mediated by arachidonic acid metabolites from a pulmonary source. A significant role of PIMs in regulating pulmonary hemodynamics is implicated.     

Newborn response to cationic amphiphilic drugs

Administration of various cationic amphiphilic drugs in utero results in induction of a phospholipid storage disorder in many tissues, particularly in lungs. In addition to the phospholipidosis in utero, drug exposure results in toxicity to the offspring; newborn rats die within 48 h of birth. Although drug-induced pulmonary pathological changes appear to be involved in the observed mortality, this relationship remains unclear. In contrast to mammals, administration of cationic amphiphilic drugs to the chick embryo seems not to induce phospholipid storage in the tissues examined. Treatment of newborn rats directly with these drugs also induces phospholipidosis in several tissues including lung and kidney; however, mortality does not occur. Concurrent administration of phenobarbital and chlorphentermine reduces or prevents amphiphilic drug-induced phospholipid storage in newborn rat lung and kidney. Modification of chlorphentermine actions by phenobarbital may be caused by alterations in amphiphilic drug excretion, metabolism, and catabolic phospholipase activity. Evidence thus indicates that regardless of age, animals appear susceptible to the effects of cationic amphiphilic drugs; however, species and tissues examined, as well as specific drug administration, play an important role in the observed qualitative and quantitative responses.     

Selective effect of phorbol ester on serotonin removal and ACE activity in rabbit lungs

The effect of phorbol myristate acetate (PMA) on pulmonary removal of [14C]serotonin (5-[14C]HT) and metabolism of [3H]benzoyl-phenylalanyl-alanyl-proline (BPAP), a synthetic substrate for angiotensin-converting enzyme (ACE), was evaluated in isolated rabbit lungs perfused in situ with Krebs-albumin. Metabolic functions were assessed before, during, and after perfusion with 80 nM PMA (n = 11), or PMA plus 133 microM papaverine (n = 10) or PMA diluent (dimethyl sulfoxide, n = 11). Organ kinetic parameters (apparent Vmax, Km) were calculated by use of indicator-dilution techniques and by a mathematical model of whole-organ metabolism. PMA treatment resulted in a significant decline in Vmax for BPAP metabolism (from 52 +/- 4 to 30 +/- 4 nmol/s) and 5-HT removal (from 2.1 +/- 0.2 to 1.1 +/- 0.1 nmol/s). Km for BPAP was not significantly altered, whereas Km for 5-HT removal was higher after treatment (before treatment, 1.1 +/- 0.1 microM; after treatment, 2.3 +/- 0.6 microM). Coperfusion with papaverine, which attenuated the pressor response to PMA, abolished PMA-induced changes in Vmax for BPAP metabolism and in Km for 5-HT removal but left PMA-induced changes in Vmax for 5-HT removal intact. We conclude that PMA alters endothelial metabolic function by both hemodynamic and biochemical mechanisms that are independent of circulating blood cells. Pulmonary capacity for BPAP metabolism may largely reflect perfused surface area, and capacity for 5-HT removal may be more sensitive to frank endothelial cell dysfunction in this model.     

N-oxidation of imipramine by isolated perfused rat and rabbit lung

Pulmonary uptake and metabolism of imipramine (IMP) was investigated in isolated perfused rat (IPrL) and rabbit (IPRL) lung preparations. Perfusate containing ¹⁴C-IMP (1.2 μmole/g lung) was recirculated through the pulmonary artery in artificially ventilated lungs. The radioactivity in the perfusate declined rapidly and about 80% of the dose was taken up by the lungs within 10 minutes in both IPrL and IPRL preparations. A steady-state was apparently reached thereafter in the IPRL, while a portion of the radiolabel effluxed into the perfusate of the IPrLs, thus reducing the net lung content to 54% of added IMP by 60 minutes. After 60 minutes perfusion, metabolites of IMP accounted for the major radioactivity (80%) in the perfusate, while the lung contained mainly (83%) the unchanged parent compound. The principal metabolite was identified as IMP-N-oxide (IMP-NO) which was found in the perfusate after 5 minutes of perfusion. Only 3% of the added IMP was metabolized by IPRL in 60 minutes. SKF-525A, an inhibitor of cytochrome P-450-mediated monooxygenase system, did not inhibit but enhanced the metabolism of IMP by IPrL to IMP-NO. IMP was principally metabolized to IMP-NO by incubations of 9,000 g supernatant fractions of rat lungs to a significantly higher extent than similar rabbit lung preparations. Including SKF-525A significantly accelerated the metabolism of IMP to IMP-NO in accordance with the perfusion experiments. These results suggest that in contradiction to publishedd reports, IMP is appreciably metabolized by the rat lung $\text{via}$ N-oxidation by non-cytochrome P-450 pathway and the metabolite formed in the lung is released into the circulation indicating its low affinity for the lung tissue.     

Exogenous Tryptophan Increases Synthesis, Storage, and Intraneuronal Metabolism of 5-Hydroxytryptamine in the Rat Hypothalamus

The effects of tryptophan administration on neurochemical estimates of synthesis [5-hydroxytryptophan (5-HTP) accumulation following administration of a decarboxylase inhibitor], storage [5-hydroxytryptamine (5-HT) concentrations], and metabolism [5-hydroxyindoleacetic acid (5-HIAA) concentrations] of 5-HT in selected regions of the hypothalamus were determined using HPLC coupled to an electrochemical detector. Tryptophan methyl ester HCl (30-300 mg/kg i.p.) produced a dose-dependent increase in the rate of 5-HTP accumulation throughout the hypothalamus but had no effect on the rate of accumulation of 3,4-dihydroxyphenylalanine. Peak 5-HTP levels were attained by 30 min following administration of tryptophan (100 mg/kg i.p.) and were maintained for an additional 60 min. Tryptophan also produced concomitant dose-dependent increases in 5-HT and 5-HIAA concentrations in these same regions without changes in the 5-HIAA/5-HT ratio. These results indicate that exogenous tryptophan administration selectively increases the synthesis, storage, and metabolism of 5-HT in the hypothalamus without altering the synthesis of catecholamines. Inhibition of 5-HT uptake with chlorimipramine or fluoxetine produced modest (10-40%) reductions in 5-HIAA concentrations throughout the hypothalamus, revealing that only a minor portion of 5-HIAA is derived from released and recaptured 5-HT, whereas the major portion of this metabolite reflects intraneuronal metabolism of unreleased 5-HT. In both chlorimipramine- and fluoxetine-treated rats, 5-HIAA concentrations were significantly increased by tryptophan administration, indicating that the increase in synthesis of 5-HT following precursor loading is accompanied by an increase in the intraneuronal metabolism of 5-HT.     

Effects of 4-methyl-α-ethyl-tyramine, 4, α-dimethyl-tyramine and tetrabenazine on the release and uptake of 5-hydroxytryptamine by human blood platelets invitro

4-Methyl-α-ethyl-tyramine and its 4, α-dimethyl analogue release 5-HT from human blood platelets $\text{in}$$\text{vitro}$. At lower concentrations they inhibit the uptake of 5-HT into platelets. Tricyclic antidepressant drugs do not block 5-HT release by these compounds. On removal of the depletor, platelets recover their ability to take up 5-HT; platelets preloaded with exogenous 5-HT lose the same proportion of amine as those containing only endogenous 5-HT. Tetrabenazine behaves similarly, but its actions are partial, whereas those of the tyramines are more complete. The temperature dependence of spontaneous and drug-induced 5-HT release has been measured. The results are discussed in terms of the action of these drugs and with special reference to the use of human blood platelet as a model of a 5-HT-containing nerve ending.     

Pulmonary neutrophil kinetics in sheep: effects of altered hemodynamics

We investigated the effect of elevated left atrial pressure and reduced cardiac output on pulmonary neutrophil kinetics in the sheep. Sheep neutrophils were isolated, labeled with 111In-oxine, and reinfused. Erythrocytes were labeled with [99mTc]pertechnetate. A gamma camera measured the lung activities of the labeled neutrophils and erythrocytes. The results indicated that 38.5% of the total injected neutrophils marginated in the lung. Pulmonary hemodynamics were altered by inflating a left atrial balloon three times in each sheep for 15-30 min to achieve 5- to 25-mmHg increments in pulmonary arterial wedge pressure. At least a 30-min recovery period was allowed between inflations. After each left atrial balloon inflation, neutrophil uptake remained unchanged from base line, despite decreased mean cardiac output to 0.67 +/- 0.24 (+/- SD) 1/min and increased pulmonary blood volume. The absence of pulmonary neutrophil uptake was confirmed by arterial-venous measurements. Increased pulmonary blood volume had little effect on lung neutrophil uptake, suggesting that most of the pulmonary neutrophils are marginated. We conclude that the lungs have a large marginated neutrophil pool compared with the circulating pool and that reduced cardiac output and elevated left atrial pressure have no effect on pulmonary neutrophil kinetics in the sheep.     

Effect of complement activation with cobra venom factor on pulmonary vascular permeability

We examined the effect of acute complement activation on lung vascular permeability to proteins in awake sheep prepared with lung lymph fistulas. Complement was activated by cobra venom factor (CVF) infusion (400 U/kg for 1 h iv). Studies were made in two groups of sheep: 1) infusion of CVF containing the endogenous phospholipase A2 (PLA2) (n = 6); and 2) infusion of CVF pretreated with bromophenacyl bromide to inhibit PLA2 activity (n = 5). Intravascular complement activation transiently increased mean pulmonary arterial pressure (Ppa) and pulmonary vascular resistance (PVR) in both groups. Pulmonary lymph flow (Qlym) and lymph protein clearance (Qlym X lymph-to-plasma protein concentration ratio) were also transiently increased in both groups. Pulmonary vascular permeability to proteins was assessed by raising left atrial pressure and determining the lymph-to-plasma protein concentration ratio (L/P) at maximal Qlym. In both groups the L/P at maximal Qlym was not different from normal. In a separate group (n = 4), CVF-induced complement activation was associated with 111In-oxine granulocyte sequestration in the lungs. In vitro plasma from CVF-treated animals aggregated neutrophils but did not stimulate neutrophils to produce superoxide anion generation. Therefore, CVF-induced complement activation results in pulmonary neutrophil sequestration and in increases in PVR and lymph protein clearance. The increase in lymph protein clearance is due to increased pulmonary microvascular pressure and not increased vascular permeability to proteins.     

Enhanced clearance of surfactant protein D during LPS-induced acute inflammation in rat lung

Pulmonary surfactant participates in the regulation of alveolar compliance and lung host defense. Surfactant homeostasis is regulated through a combination of synthesis, secretion, clearance, recycling, and degradation of surfactant components. The extracellular pool size of surfactant protein (SP) D fluctuates significantly during acute inflammation. We hypothesized that changes in SP-D levels are due, in part, to altered clearance of SP-D. Clearance pathways in rats were assessed with fluorescently labeled SP-D that was instilled into control lungs or lungs that had been treated with lipopolysaccharide (LPS) 16 h earlier. SP-D clearance from lavage into lung tissue was time dependent from 5 min to 1 h and 1.7-fold greater in LPS-treated lungs than in control lungs. Analysis of cells isolated by enzymatic digestion of lung tissue revealed differences in the SP-D-positive cell population between groups. LPS-treated lungs had 28.1-fold more SP-D-positive tissue-associated neutrophils and 193.6-fold greater SP-D association with those neutrophils compared with control lungs. These data suggest that clearance of SP-D into lung tissue is increased during inflammation and that tissue-associated neutrophils significantly contribute to this process.     

Local delivery of GM-CSF protects mice from lethal pneumococcal pneumonia

The growth factor GM-CSF has an important role in pulmonary surfactant metabolism and the regulation of antibacterial activities of lung sentinel cells. However, the potential of intra-alveolar GM-CSF to augment lung protective immunity against inhaled bacterial pathogens has not been defined in preclinical infection models. We hypothesized that transient overexpression of GM-CSF in the lungs of mice by adenoviral gene transfer (Ad-GM-CSF) would protect mice from subsequent lethal pneumococcal pneumonia. Our data show that intra-alveolar delivery of Ad-GM-CSF led to sustained increased pSTAT5 expression and PU.1 protein expression in alveolar macrophages during a 28-d observation period. Pulmonary Ad-GM-CSF delivery 2-4 wk prior to infection of mice with Streptococcus pneumoniae significantly reduced mortality rates relative to control vector-treated mice. This increased survival was accompanied by increased inducible NO synthase expression, antibacterial activity, and a significant reduction in caspase-3-dependent apoptosis and secondary necrosis of lung sentinel cells. Importantly, therapeutic treatment of mice with rGM-CSF improved lung protective immunity and accelerated bacterial clearance after pneumococcal challenge. We conclude that prophylactic delivery of GM-CSF triggers long-lasting immunostimulatory effects in the lung in vivo and rescues mice from lethal pneumococcal pneumonia by improving antibacterial immunity. These data support use of novel antibiotic-independent immunostimulatory therapies to protect patients against bacterial pneumonias.     

Uptake, Release, and Subcellular Localization of l-Methyl-4-Phenylpyridinium in Blood Platelets

The neurotoxic compound 1-[methyl-3H]-4-phenylpyridinium ([3H]MPP+) was actively taken up by human, rabbit, and guinea pig platelets incubated in plasma. In human platelets, the apparent Km of this uptake (22.6 microM) was 50 times higher than that for serotonin [5-hydroxytryptamine (5-HT]). The uptake of [3H]MPP+ by human platelets was inhibited by selective 5-HT uptake blockers [cianopramine, (-)-paroxetine, and clomipramine], by metabolic inhibitors (KCN and ouabain), and by drugs that interfere with amine storage in the 5-HT organelles (reserpine, mepacrine, and Ro 4-1284). Impairment of the transmembrane proton gradient by ionophores (monensin and nigericin) induced a marked release of radioactivity from platelets preincubated with [3H]MPP+. Fractionation of homogenates of rabbit platelets preincubated with [3H]MPP+ showed that the drug was concentrated to a great extent in the 5-HT organelle fraction. MPP+ competitively inhibited [14C]5-HT uptake by human platelets and reduced the endogenous 5-HT content of human, rabbit, and guinea pig platelets. These investigations show that MPP+ is transported into the platelets via the 5-HT carrier and is accumulated predominantly in the subcellular organelles that store 5-HT and other monoamines. It is suggested that an accumulation of MPP+ in amine storage vesicles of neurons may be involved in the effects of the drug in the CNS, e.g., by protecting other subcellular compartments from exposure to high concentrations of MPP+, by sustaining a gradual release of the toxin, or both.     

Generation of 5-lipoxygenase metabolites following pulmonary reperfusion in isolated rabbit lungs.

We characterized the release of arachidonic acid (AA) metabolites in lung effluent following lung ischemia-reperfusion since they may contribute to the pathophysiology of reperfusion lung injury. The left pulmonary artery of rabbits (N = 5) was occluded for 24 hrs with a surgically implanted vascular clip. At 24 hrs, the heart and lungs were removed en bloc and perfused with Ringers-albumin (0.5 gm%) at 60 ml/min while statically inflated with 95% O2-5% CO2. The lipid fraction of the lung effluent was concentrated using the Bligh-Dyer extraction and analyzed by gradient RP-HPLC. Samples obtained in the first minute of reperfusion showed significant increases in LTB4 (+180%), LTC4 (+3600%), 15-HETE (+370%), 5-HPETE (+270%), PGE2 (+140%), 6-keto-PGF1 alpha (+110%) and 12-HHT (+160%) compared to the effluent from the right control lung. The reperfusion-induced increases in LTB4, LTC4, LTD4 and 15-HETE were inhibited greater than or equal to 70% by pretreatment with the 5-LO inhibitors L663,536 or L651,392. The increases in lipid concentrations corresponded to significantly increased pulmonary arterial pressure from a baseline value of 9.5 +/- 0.3 to 29.3 +/- 2.9 (cmH2O) during the first min of reperfusion. The pulmonary arterial pressure remained elevated for at least 20 min of reperfusion. Reperfusion also resulted in PMN uptake (assessed by lung tissue myeloperoxidase content) in the reperfused lung versus control lung (25.0 +/- 2.4 vs. 10.5 +/- 2.5 units). The generation of lipoxygenase metabolites during the initial phase of reperfusion may contribute to post-reperfusion PMN uptake and pulmonary vasoconstriction.