Paraquat disposition in rats, guinea pigs and monkeys

LD50 doses of ¹⁴C-labeled paraquat were administered to rats, guinea pigs and monkeys by gavage, and radioactivity was determined in excreta and tissues. Rat urine was analyzed for paraquat metabolites by thin-layer chromatography. [¹⁴C]Paraquat was absorbed from the gastrointestinal tract and reached highest serum values 0.5–1 hr after administration. Disappearance of [¹⁴C]paraquat from serum was characterized by a rapid initial decline followed by a prolonged slow decline. Tissue paraquat values were higher than serum values in rats and guinea pigs. Relative to other tissues, paraquat accumulated transiently in the lung and reached peak concentration 32 hr after administration. In rats a major portion of administered paraquat was not absorbed from the gastrointestinal tract. At 32 hr after paraquat, 52% of the administered dose remained in the gastrointestinal tract and 17 and 14% of the administered dose was excreted in the feces and urine, respectively. No radioactivity was recovered in expired air or flatus. Excretion of paraquat in urine and feces was prolonged in all species. In monkeys paraquat was measured in urine and feces 21 days after administration. Chromatography of urine from [¹⁴C]paraquat-treated rats revealed no metabolites. The primary pathologic changes induced by paraquat in the lung may be related to the transient uptake of the chemical by that organ.     

Chronic toxicity, teratologic, and reproduction studies with hair dyes.

Administration of paraquat to mice, 1.67 and 3.35 mg/kg ip or 20 mg/kg po, daily on Days 8–16 of gestation induced no significant teratogenic effects, although a slight increase in nonossification of sternabrae was observed. Radioactivity reaching the mouse embryo after ip or po administration of [¹⁴C]paraquat on Day 11 of gestation was low. The fetal toxicity of diquat in rats, as measured by the number of dead and resorbing fetuses, was greater than that caused by paraquat after 15 mg/kg iv doses on various days of gestation, which correlated with higher fetal concentrations of [14C]diquat compared to [¹⁴C]paraquat. In perinatal organ distribution studies, more radioactivity from [¹⁴C]paraquat was retained in lung tissue of postnatal mice and rats than that in liver and kidney tissue. In prenatal studies, however, [¹⁴C]paraquat was retained in lung tissue of fetal rats after maternal administration of paraquat on Day 21 of gestation but not in lung tissue of fetal mice after maternal paraquat on Day 16 of gestation. This may be indicative of prenatal development of binding sites or of an active transport process for the uptake of paraquat into the lung or that elevated oxygen tensions in postnatal lungs contributes to paraquat retention in lung tissue.     

Studies on the mechanism of the lung toxicity of paraquat: comparison of tissue distribution and some biochemical parameters in rats and rabbits

After iv injection of [¹⁴C]paraquat (20 mg/kg) tissue localization was preferential in lungs of rats as well as rabbits although the latter did not show any histopathologic or biochemical signs of lung damage. No preferential subcellular localization of [¹⁴C]paraquat was found in lungs of either species, but all subcellular levels decreased more rapidly in the rabbit than in the rat. [¹⁴C]Paraquat was not covalently bound to tissue macromolecules. In vitro measurements of lipid peroxidation, H₂O₂ formation and lung lysosomal stability failed to account adequately for the lung damage in the rat.     

A comparison of the effects of paraquat and diquat on lung compliance,lung volumes and single breath diffusing capacity in the rat

Paraquat intoxication in its initiaal stage in characterized histologically in the lungs by atelectasis, hyaline membrane formation, alveolar edema and vascular hemorrhage often into the interstitium or air spaces. Information on the functional modification of paraquat-damaged lungs has been lacking.We evaluated lung volumes, single breath diffusing capacity of the lungs for carbon monoxide (D_LCO) and static lung compliance (C_st(L))_in rats treated with paraquat or diquat. Measurements were made 12, 24, 48, and 72 h after treatment. Paraquat by intratracheal (i.t.) installation 0.5 mg/kg or by intraperitoneal injection (i.p.) 27 mg/kg significantly decreased (P<0.01) the body weight, total lung capacity (TLC), functional residual capacity (FRC), vital capacity (VC), residual volume (RV), D_LCO, apparent alveolar volume (V_A) and C_st(L). At a lower dose level (13.5 mg/kg), the effects of paraquat peaked at about 24 h following treatment, causing a significantly decreased (P<0.01) VC and TLC. Diquat i.t. or i.p. had effect on the lungs. However, diquat i.p. decreased body weight (P<0.01) and caused a slight increase (P<0.05) in VC. The data obtained are consistent with the known pathological changes seen in paraquat-damaged lungs in that, by both routes, paraquat caused severe lung damage associated with decreased elasticity of the lungs and thorax, destruction of gas exchanging alveolar surfaces, and edema. These changes were detected reliably by lung function measurements.     

Tissue and cellular disposition of paraquat in mice

Male C57B1/6J mice were injected intravenously with [methyl-¹⁴C]paraquat dichloride and frozen at 1, 3, 9, and 24 hr for whole-body autoradiography or with [methyl-³H]paraquat dichloride and the lungs were removed at 3, 24, and 48 hr for cellular autoradiography. The methods do not allow thawing or exposure of the tissues to solvents; this prevents translocation or removal of radioactivity. The whole-body autoradiographs revealed localization of radioactivity at all time intervals in melanin, lung, choroid plexus, muscle, and excretory pathways such as proximal tubules of kidney, urine, liver, gallbladder, and intestinal contents. The radioactivity in lung was much higher in certain discrete, unidentified areas at all time intervals except at 1 hr. The concentration was high in myocardium at 1 and 3 hr. Cellular resolution autoradiography revealed that the radioactivity within the lung was confined almost entirely to cells having the distribution of alveolar type II cells at the three time intervals studied. The radioactivity in these cells was easily washed away indicating that an active transport process was probably involved instead of binding to a cellular constituent. The localization suggested that choline might be antidotal to paraquat toxicity. However, there was not a significant increase in survival of mice given 100 mg/kg choline chloride simultaneously to or following treatment with 50 mg/kg paraquat chloride.     

Methcathinone is a Substrate for the Serotonin Uptake Transporter*

Abstract: We previously reported that the psychostimulant drug methcathinone inhibits serotonin accumulation via the plasma membrane serotonin uptake transporter. By analogy to known substrates for the serotonin transporter, we hypothesized that methcathinone is also a substrate for this transporter and that inhibition of serotonin uptake by methcathinone occurs in part through competition for substrate recognition sites within the transporter. To test the hypothesis we preloaded human platelets with [³H]5‐HT then superfused the platelets with either methcathinone or with the known serotonin uptake transporter substrate para‐methylthioamphetamine. Under superfusion conditions, transporter substrates will evoke an increase in released [³H]5‐HT through a carrier‐mediated exchange process. For direct assessment of methcathinone transport via the serotonin uptake transporter, we tested whether [³H]methcathinone would be accumulated by cells stably expressing the cloned human serotonin uptake transporter (293SERT cells). Supporting the hypothesis, superfusion of [³H]5‐HT‐containing platelets with methcathinone or with para‐methylthioamphetamine produced a large increase in tritium efflux. The efflux declined when the drugs were removed. When increasing concentrations of [³H]methcathinone were incubated with 293SERT cells under conditions used to assess serotonin transport, saturable, single‐site accumulation of radiolabel was observed. The uptake of [³H]methcathinone was temperature, inhibitor, and sodium‐sensitive, and was not observed in wild‐type HEK 293 cells. Non‐linear regression analysis of specific [³H]methcathinone uptake produced values for K_M and V_max of 244±51 nM and 202±25 fmol/min./mg protein, respectively. These data support the notion that the reported serotonergic neurotoxicity of methcathinone may arise through accumulation of the drug within serotonergic neurones.     

Chloroquine attenuates paraquat-induced lung injury in mice by altering inflammation,oxidative stress and fibrosis

Paraquat is one of the most extensively used herbicides and has high toxicity for humans and animals. However, there is no effective treatment for paraquat poisoning. The aim of the present study was to evaluate the effects of chloroquine on paraquat-induced lung injury in mice. Mice received a single intraperitoneal injection of paraquat and a daily intraperitoneal injection of the indicated dosages of chloroquine or dexamethasone. The histological changes, inflammation and oxidative stress in the lungs were examined at day 3, and the degree of pulmonary fibrosis was examined at day 28. H&E staining showed that chloroquine markedly attenuated lung injury induced by paraquat. In addition, the inflammatory responses induced by paraquat were inhibited after treatment with chloroquine, as indicated by the decreased number of leukocytes, the reduced levels of TNF-α, IL-1β and IL-6 in the bronchoalveolar lavage fluid, the reduced NO content, and downregulation of iNOS expression in lung tissues. No different effect was found between high-dose chloroquine and dexamethasone. Additionally, the treatment with chloroquine increased the activity of SOD and decreased the level of MDA in the lung tissues. The expressions of the anti-oxidative proteins, Nrf2, HO-1 and NQO1, were also upregulated by chloroquine treatment. The high-dose chloroquine was more effective than dexamethasone in its anti-oxidation ability. Finally, the results of Masson's staining illustrated that chloroquine markedly attenuated fibrosis in the paraquat-exposed lungs. Immunohistochemistry staining showed that the expressions of the pro-fibrotic proteins TGF-β and α-SMA were downregulated after treatment with chloroquine. In conclusion, chloroquine effectively attenuated paraquat-induced lung injury in mice.     

Binding of [methyl-3H]paraquat to rat,rabbit, hamster,mouse and guinea pig lung proteins,in vitro

When incubated with the rat lung slices [methyl-³H] paraquat was found to bind covalently to acid-insoluble proteins. The evidence in the present study indicated that the binding of radioactivity was not due to the formation of a reactive metabolite secondary to mixed-function oxidase (MFO)-mediated bioactivation of paraquat but was probably due to formation of paraquat free radicals. Preincubation of lung slices with CN?, 2,4-dinitrophenol (DNP), p-hydroxymercuribenzoate and cysteine inhibited binding, while anaerobic conditions has a marked stimulatory effect. There were no significant differences in the extent of covalent binding of [methyl-³H]paraquat to lung protein of rat, rabbit, mouse, guinea pig, and hamster.     

Uptake and displacement of [3H]dihydroalprenolol, [3H]epinephrine and [3H]clonidine in isolated perfused rabbit lung

Uptake and displacement of three adrenergic receptor ligands, [³H]dihydroalprenolol ([³H]DHA), [³h]epinephrine ([³H]EPI) and [³H]clonidine ([³H]CLON), were examined in isolated rabbit lungs by recirculating perfusion. Removal of [³h]DHA was the most extensive (85% uptake; 6.6 $\text{ml}\text{min}$ clearance), [³H]CLON removal was intermediate (50%; 3.8 $\text{ml}\text{min}$), and [³H]EPI removal was the lowest (33%; 1.2 $\text{ml}\text{min}$). Specific displacement of each radioligand from lung was attempted using several competing agents. Both (?)- and (+)-propranolol equally displaced [³H]DHA from lung. Phentolamine, (?)-phenylephrine and (?)-epinephrine were unable to displace 10 nM [³H]EPI from lung, although the latter two agents did produce concentration-dependent increases in perfusion pressure. High concentrations of (?)-epinephrine, which produced near maximal physiological responses, inconsistently displaced 30–40 nM [³H]EPI from lung. [³H]Clonidine was displaced by unlabeled clonidine at concentrations that caused increases in perfusion pressure. Pretreatment of lungs with either 10 μM phentolamine or phenoxybenzamine did not alter the total amount of [³H]CLON displaced by clonidine, suggesting that [³H]CLON was displaced predominantly from non-specific sites, perhaps preventing detection of [³H]CLON displacement from specific (receptor) sites. Alternatively, these results may be interpreted as inhibition of uptake of each radioligand. Thus, both (?)- and (+)-propranolol interfered with [³H]DHA removal, suggesting a common mechanism for uptake and/or retention for these two β-adrenergic receptor antagonists. Inhibition of ³H]EPI removal was observed only at high concentrations of (?)-epinephrine which indicates that pulmonary removal of epinephrine occurs through a low affinity uptake system. [³H] Clonidine removal was effectively inhibited by the same (μM) concentrations of unlabeled clonidine that produced physiological responses. Neither phentolamine nor phenoxybenzamine was able to interfere with pulmonary removal of [³H]CLON. Therefore, uptake and displacement of these adrenergic receptor radioligands showed no correlation with pharmacological effects produced by these agents in isolated perfused rabbit lung. The results are more closely associated with inhibition of removal and/or non-specific retention of the radioligands examined.     

The role of sodium cromolyn in treatment of paraquat-induced pulmonary fibrosis in rat.

Paraquat is a non-selective herbicide which is widely used in agriculture. It has potential of producing pulmonary fibrosis. In this study the therapeutic effect of different doses of sodium cromolyn on the development of paraquat-induced pulmonary fibrosis was investigated. In order to produce lung fibrosis, rats were given single oral dose of paraquat (40 mg kg(-1)). Two groups of animals were nebulised with 6 or 8 mg day(-1) cromolyn (divided into two doses per day) 5 days prior and 2 weeks after paraquat treatment. Control animals were given equivalent volume of normal saline and treated with cromolyn similar to the test groups. Rats were killed at the end of treatment course and lung tissues were tested histologically and biochemically. Histological examination of paraquat-treated animals showed marked infiltration of inflammatory cells in the alveolar spaces, septal thickening and fibrosis. Lesions were evident in many places of sections. Sodium cromolyn could markedly reduce such damages in lung tissue. Lung weight, hydroxyproline and collagen content of lung tissues were elevated significantly (P < 0.05) in paraquat group compared with control group. In cromolyn-treated groups such factors were near to control value and were significantly lower than paraquat group (P < 0.05). Results of this study indicate that sodium cromolyn can decrease the fibrogenic effect of paraquat on lung. Such effect may be due to the stabilising of mast cells and inhibition of inflammatory mediators by sodium cromolyn.     

Effects of mazindol on rat brain synaptosomal monoamine uptake

The effects of the anorectic drug mazindol on the uptake of [³H]NA and (³H]5HT by rat hypothalamic synaptosomes and the uptake of [³H]DA by rat striatal synaptosomes were investigated. In in vitro studies drugs were added to the incubation medium. In ex vivo experiments drugs were injected i.p. at various times prior to death and synaptosomal [³H] monoamine uptake subsequently determined. Two other anorectics (d-amphetamine and dl-fenfluramine) and two inhibitors of monoamine uptake (chlorimipramine and desipramine) were included for comparative purposes. Mazindol was a potent inhibitor of [³H]NA and [³H]DA uptake in vitro being approx. 0.5 times as potent as desipramine and d-amphetamine respectively. The abilities of mazindol, fenfluramine and desipramine to block the in vitro uptake of [³H]5HT were comparable and all three drugs were appreciably less potent than chlorimipramine. Following 1hr pretreatment, d-amphetamine was the most potent of the five drugs at inhibiting synaptosomal [³H]NA and [³H]DA uptake. Mazindol was approx. 2.5 times more potent than desipramine at blocking [³H]NA uptake. In contrast to the other drugs, pretreatment with large doses of mazindol had essentially no effect on hypothalamic synaptosomal [³H]5HT uptake. Results of ex vivo studies thus confirm in vivo findings that mazindol is a selective inhibitor of rat brain catecholamine uptake.     

Effects of lysine aspirin on lung AQP5 expression and lymphocyte apoptosis in paraquat-poisoned rats

Objective: This study explored the effects of lysine aspirin on lung aquaporin 5 (AQP5) expression and lymphocyte apoptosis in paraquat-poisoned rats. Methods: Thirty healthy male Wales rats were randomly divided into three groups (n?=?10): the control group received 0.9% sodium chloride (0.4?mL, intragastric administration; 0.8?mL, intraperitoneal injection); the paraquat group received 40?mg/kg paraquat (intragastric administration) and 0.9% sodium chloride (intraperitoneal injection); and the paraquat + lysine aspirin group received 40?mg/kg paraquat (intragastric administration) and 20?mg/kg lysine aspirin (intraperitoneal injection). Rats were killed at 24 and 48?h. RT-PCR and immunohistochemical staining were performed in lung tissue to determine the AQP5 mRNA and protein expression. Blood from the arterial vein was used to evaluate lymphocyte apoptosis. Results: The lung tissue of paraquat-treated rats displayed pulmonary hemorrhage, interstitial edema and inflammatory cell infiltration. AQP5 mRNA and protein expression in the paraquat-treated group decreased after 24 and 48?h, whereas the peripheral blood lymphocyte apoptosis ratio significantly increased. In contrast, paraquat + lysine aspirin treatment ameliorated these changes. Conclusion: Paraquat decreases AQP5 expression in rat lungs and increases peripheral blood lymphocyte apoptosis. Lysine aspirin can reduce these alterations.     

The effect of antiparkinsonian drugs on oxidative stress induced pathological [3H]dopamine efflux after in vitro rotenone exposure in rat striatal slices

An in vitro model of mitochondrial dysfunction with subsequent oxidative stress was elaborated and utilized to study the effect of drugs, currently used for the treatment of Parkinson's disease, on pathological H₂O₂-evoked [³H]dopamine efflux and the formation of toxic dopamine metabolites in rat striatal slices. 60 min rotenone (0.1–10 μM) pretreatment decreased dopamine content and [³H]dopamine uptake, as well as ATP level and energy charge of the slices. In addition, a robust potentiation of H₂O₂-evoked [³H]dopamine efflux and the formation of dopamine quinone in the effluent was detected. l-DOPA (200 μM) markedly elevated resting but not 100 μM H₂O₂-evoked and electrically-induced [³H]dopamine efflux. Furthermore, l-DOPA promoted the formation of dopamine quinone. Ropinirole (100 nM) did not affect resting and H₂O₂-evoked [³H]dopamine efflux and inhibited the electrically evoked release only in untreated slices. l-deprenyl, at concentration of 0.01 μM potentiated, whilst between 1 and 50 μM diminished H₂O₂-evoked [³H]dopamine efflux. Rasagiline (0.01–50 μM) slightly inhibited H₂O₂-evoked [³H]dopamine efflux, and it was able to prevent the generation of dopamine quinone. Neither of the drugs was able to suppress both the pathological H₂O₂-evoked [³H]dopamine efflux and the formation of dopamine quinone with simultaneous augmentation of electrically evoked [³H]dopamine release what should be a future concept of antiparkinsonian drug-design.     

Chymase mediates paraquat-induced collagen production in human lung fibroblasts

Survivors of paraquat poisoning may be left with pulmonary fibrosis and a restrictive type of pulmonary dysfunction. Chymase converts angiotensin (Ang) I to Ang II, which is closely involved with lung fibrosis. The role played by chymase in paraquat-induced lung fibrosis is unclear. We examined the effects of paraquat on chymase, renin–angiotensin system components, and collagen expression in murine and human lung fibroblasts (MRC-5). Lung chymase and collagen type I mRNA and protein expression were significantly increased and angiotensin-converting enzyme (ACE) mRNA and protein expression were comparable between the control and paraquat-treated mice 1 and 3 weeks after administration. Paraquat significantly upregulated angiotensinogen mRNA expression in a dose-dependent manner while ACE activity and protein expression were similar in MRC-5 cells. Furthermore, paraquat enhanced Ang II and collagen type I mRNA and protein expression, α-smooth muscle actin, and chymase protein and chymase small interfering RNA inhibited these effects. The cDNA sequence of chymase in MRC-5 cells is identical to that in human mast cells. This study found increased chymase expression in paraquat-treated human lung fibroblasts and confirmed in vitro and in an in vivo paraquat model of lung fibrosis that chymase generates Ang II and enhances collagen expression. These data suggest a role for chymase in the pathogenesis of paraquat-induced lung fibrosis.     

High-affinity [3H]desipramine binding in the peripheral and central nervous system: A specific site associated with the neuronal uptake of noradrenaline

The specific binding of [³H]desipramine to various brain regions and peripheral tissues of the rat was of high affinity, rapid and reversible. It was inhibited with high affinity only by tricyclic antidepressants and noradrenaline uptake blockers. There was a highly significant correlation between the potencies of a series of drugs for the inhibition of [³H]desipramine binding and for the inhibition of noradrenaline uptake. Substrates for the noradrenaline uptake system however inhibited the binding of [³H]desipramine only at very high concentrations. Postganglionic sympathetic denervation of the submaxillary gland and the heart both resulted in a pronounced decrease in [³H]desipramine binding sites, which paralleled the reduction in endogenous noradrenaline levels. High-affinity [³H]desipramine binding sites thus appear to be localised on noradrenergic nerve endings and are probably closely associated with the neuronal uptake system for noradrenaline.     

Phencyclidine-induced release of [3H]dopamine from chopped striatal tissue

Phencyclidine was examined for its ability to release [³H]dopamine ([³H]DA) from prelabelled chopped rat striatal tissue. A dynamic perfusion system was used in order to minimize the effects of drugs on uptake mechanisms. Cocaine and S-(+)-amphetamine were used to distinguish uptake inhibition from a neurotransmitter releasing action. Phencyclidine, starting at 3 μM, caused a dose-dependent increase in efflux of [³H]DA from chopped striatal tissue. In this same preparation, cocaine, a known neuronal uptake inhibitor of dopamine, was unable to release [³H]DA except in the largest dose of 100 μM. S-(+)Amphetamine, a known releaser of neuronal dopamine, was found to be about ten times more potent than phencyclidine in causing a dose-dependent release of [³H]DA. The results of the above experiments are discussed in relation to the ability of phencyclidine to decrease the synaptosomal accumulation of [³H]DA. It is concluded that some of the psychoactive effects of phencyclidine may be due to the ability of phencyclidine to elicit a release of dopamine from dopaminergic     

Pharmacokinetics of Substrate Uptake and Distribution in Murine Brain After Nasal Instillation

No HeadingPurpose. This study was conducted to develop a physiologically relevant mathematical model for describing brain uptake and disposition of nasally administered substrates.Methods. [¹⁴C]-antipyrine, [¹⁴C]-diazepam, [³H]-sucrose, or [³H]-verapamil was administered nasally to CF-1 mice. P-glycoprotein (P-gp)-deficient mice also received [³H]-verapamil to probe the influence of P-gp on uptake/distribution. Mice were sacrificed at selected intervals, and 20 serial 300-m coronal brain sections were obtained to determine radioactivity. A series of compartmental pharmacokinetic models was developed and fit to concentration vs. time/distance data.Results. After nasal instillation, substrate concentration was highest in the olfactory bulb and decreased with distance. In the absence of transport-mediated flux, peak brain exposure occurred at 6 h. A catenary pharmacokinetic model with slice-specific brain-to-blood efflux rate constants and slice-to-slice diffusivity factors was capable of fitting the data. P-gp limited fractional absorption of [³H]-verapamil via efflux from the nasal cavity and olfactory epithelium. P-gp also increased the rate constants associated with [³H]-verapamil efflux 1.5- to 190-fold, depending on brain region. P-gp limited [³H]-verapamil uptake from the nasal cavity into brain and facilitated removal of [³H]-verapamil from brain during rostral-to-caudal distribution.Conclusions. Taken together, the data and associated modeling provide a comprehensive assessment of the influence of P-gp on brain uptake and disposition of nasally administered substrates.     

Transforming growth factor-beta1 upregulation is independent of angiotensin in paraquat-induced lung fibrosis

Transforming growth factor-beta1 (TGF-beta1) contributes to the fibrosis of injured organs. Angiotensin II (Ang II) is an inducer of TGF-beta1 in cells of the heart and kidneys, and the regulation of TGF-beta1 by Ang II has not yet been confirmed in lung tissue. We evaluated the role of TGF-beta1 and its relationship with Ang II in paraquat-induced lung fibrosis. Adult male Sprague-Dawley rats were treated intraperitoneally with paraquat (20mg/kg) or saline in the control group. On days 1, 3, 7, and 21 after paraquat treatment, TGF-beta1 and collagen gene expressions, TGF-beta1 protein, angiotensin-converting enzyme (ACE) activity, Ang II, and hydroxyproline contents were measured in lung tissue. Lung TGF-beta1 mRNA expression progressively increased and reached a peak on day 7 after paraquat treatment. Increases in TGF-beta1 mRNA expression and TGF-beta1 levels preceded the onset of increased collagen I mRNA expression and hydroxyproline contents. c-myc mRNA expressions were inversely correlated with TGF-beta1 protein levels in paraquat-treated lungs. Lung ACE activity decreased after paraquat administration and the decrement was maximal on day 7. Lung Ang II concentrations immediately decreased after paraquat administration and the values were not related to TGF-beta1 levels. We conclude that TGF-beta1 is upregulated and contribute to the paraquat-induced lung fibrosis and this effect is independent of the renin-angiotensin system.     

Studies on the distinction between uptake inhibition and release of [3H]dopamine in rat brain tissue slices

There exists some confusion over the classification of drugs as either uptake inhibitors or releasing agents for biogenic amines. We have evaluated this problem with rat brain tissue slices (neostriatum and cortex), using [³H]dopamine and the following compounds: potassium chloride, tyramine, d-and l-amphetamine, cocaine, amantacline, desipramine. amitriptyline, nortriptyline and protriptyline. Additional experiments were performed with l-dopa and [³H]serotonin in slices from whole rat brain. Potassium chloride and tyramine. both strong releasing agents, diminished the accumulation of [³H]dopamine during uptake studies. l-Dopa also caused release of [³H]serotonin and an inhibition of [³H]serotonin accumulation. Whenever a releasing action was observed, there was always a diminution in the amount of [³H]amine accumulation and this action (expressed as a per cent effect) was at least equal in magnitude to the per cent released. On the other hand, cocaine was an example of a pure uptake inhibitor; it did not evoke a releasing action at concentrations where a powerful uptake inhibition was seen. From these data, it was possible to conclude that, in the tissue slice system, an experimentally observed release was real (that is. not materially affected by blockade of reuptake), whereas a releasing action evoked an apparent inhibition of uptake equal in magnitude to the releasing action. Before a drug can be designated as an uptake inhibitor, the dose-response curve for inhibition of [³H]amine accumulation should be distinctly to the left of the dose-response curve for release. Our data indicate that, in the neostriatum, all of the drugs studied except cocaine were pure releasing agents and that drugs previously designated as uptake inhibitors were releasing agents. In the cortex, strong inhibition of uptake without significant releasing action was evident for many of the drugs.     

Binding of some non-steroidal anti-inflammatory drugs to glucocorticoid receptors in vitro

The mechanism of action of non-steroidal anti-inflammatory drugs (NSAID) is felt to be via prostaglandin synthetase inhibition. Yet several clinical findings suggest these drugs may possess glucocorticoid agonist activity as well. To evaluate this possibility, a variety of non-steroidal antiinflammatory drugs was examined to determine whether they compete for rat kidney glucocorticoid receptor sites in vitro. Meclofenamic acid, MK 410 (an indomethacin analogue) and indomethacin were the most potent competitors requiring 7 × 10³-fold, 2 × 10⁴-fold and 3 × 10⁴-fold molar excess, respectively, to inhibit 50 per cent of the binding of 10 nM [³H]dexamethasone to kidney cytosol binding sites. The same drugs also inhibited the binding of [³H]dexamethasone in kidney slices requiring about 3-fold higher concentrations to achieve 50 per cent inhibition. At a 10⁵-fold molar ratio, sulfinpyrazone reduced cytosol binding to 60 per cent, phenylbutazone to 85 per cent and ibuprofen to 89 per cent of control levels; aspirin and naproxen did not compete even at this concentration. Similarly, prostaglandins failed to compete with [³H]dexamethasone for kidney receptors. Indomethacin inhibition of [³H]dexamethasone binding appeared competitive when analyzed by a double reciprocal plot. As expected, inhibition of [³H]dexamethasone cytosol binding by indomethacin prevented the appearance of the nuclear complex. Of the NSAID examined, none competed for plasma binding of [³H]corticosterone except indomethacin which reduced the binding to 50 per cent of control at a 10⁵-fold molar excess. In conclusion, these studies support the possibility that some NSAID may possess intrinsic glucocorticoid agonist activity. Furthermore, they indicate the glucocorticoid receptor site is not as specific as once thought and suggest that non-steroidal compounds may potentially serve as glucocorticoid agonists or antagonists.