Antagonism of thromboxane receptors by diclofenac and lumiracoxib.

BACKGROUND AND PURPOSE: Non-steroidal anti-inflammatory drugs (NSAIDs) are analgesic and anti-inflammatory by virtue of inhibition of the cyclooxygenase (COX) reaction that initiates biosynthesis of prostaglandins. Findings in a pulmonary pharmacology project gave rise to the hypothesis that certain members of the NSAID class might also be antagonists of the thromboxane (TP) receptor. EXPERIMENTAL APPROACH: Functional responses due to activation of the TP receptor were studied in isolated airway and vascular smooth muscle preparations from guinea pigs and rats as well as in human platelets. Receptor binding and activation of the TP receptor was studied in HEK293 cells. KEY RESULTS: Diclofenac concentration-dependently and selectively inhibited the contraction responses to TP receptor agonists such as prostaglandin D2 and U-46619 in the tested smooth muscle preparations and the aggregation of human platelets. The competitive antagonism of the TP receptor was confirmed by binding studies and at the level of signal transduction. The selective COX-2 inhibitor lumiracoxib shared this activity profile, whereas a number of standard NSAIDs and other selective COX-2 inhibitors did not. CONCLUSIONS AND IMPLICATIONS: Diclofenac and lumiracoxib, in addition to being COX unselective and highly COX-2 selective inhibitors, respectively, displayed a previously unknown pharmacological activity, namely TP receptor antagonism. Development of COX-2 selective inhibitors with dual activity as potent TP antagonists may lead to coxibs with improved cardiovascular safety, as the TP receptor mediates cardiovascular effects of thromboxane A2 and isoprostanes.

Increased airway responsiveness of a common fragrance component, 3-carene, after skin sensitisation--a study in isolated guinea pig lungs.

Lungs from skin-sensitised and non-sensitised guinea pigs were exposed via the airways to 3-carene (1900 mg/m3) and perfused with buffer containing either autologous plasma or lymphocytes. The experiments were performed in order to investigate the importance of blood components for the increased lung responsiveness seen in skin-sensitised animals. A reduction in lung function was noted in all lungs during 3-carene exposure. There was no difference in the 3-carene response between lungs from skin-sensitised animals versus lungs from non-sensitised animals when the perfusion buffer contained lymphocytes. However, when plasma diluted with buffer was used as perfusion medium, there was a significant enhancement in the response in lungs from sensitised versus lungs from non-sensitised animals. This implies that skin sensitisation increases lung responses to inhaled 3-carene and those components in plasma, and not the lymphocyte fraction, contributes to the observed increased lung responsiveness.

Human alveolar macrophage phagocytic function is impaired by aggregates of ultrafine carbon particles.

Alveolar macrophages (AM) were collected by bronchoalveolar lavage from healthy volunteers. The AM were loaded with small masses (0.03-3 microg/10(6) AM) of ultrafine carbon particle aggregates. The phagocytic activity of the cells was studied 20 h after the loading. Fluorescein-labeled silica particles (3 microm) were used as test particles and the attachment and ingestion processes were studied separately. In some experiments, AM were incubated with interferon-gamma (IFN-gamma) for 20 h before and during the test of phagocytic activity and during measurement of oxidative metabolism. The ingested carbon particles induced a dose-related impairment of both the attachment and the ingestion processes with a marked impairment down to a carbon particle dose around 0.2 microg/10(6) AM. Such levels should reasonably occur after inhalation of existing concentrations of urban air particles, which to a considerable extent consist of aggregates of ultrafine particles with a carbon skeleton. Incubation with IFN-gamma (12.5 U/ml) also induced significant impairments in both the attachment and the ingestion processes. Loading with carbon further aggravated the effect of IFN-gamma. In contrast to earlier studies in rat AM, IFN-gamma did not impair the oxidative metabolism at rest in these human AM; instead the oxidative metabolism was increased. This difference was due to a difference between rat and human AM and not between rat and human IFN-gamma. Our results suggest that ingested environmental particles in AM, e.g., after an episode of high particle concentration, may impair phagocytic capacity of the cells, especially after infections that induce an increased production of IFN-gamma. Consequently, there might be a risk for additional infections. Moreover, inhaled particles not phagocytized by AM might damage the lung tissue.

Increased airway responsiveness after skin sensitisation to 3-carene, studied in isolated guinea pig lungs.

Inhalation of 3-carene has been shown to induce bronchoconstriction in concentrations not far from the threshold limit value. In this study, one group of guinea-pigs were sensitised by dermal exposure to 3-carene according to the modified Cumulative Contact Enhancement Test protocol and another group of animals was used as controls. Lungs from the skin-sensitised and control guinea-pigs were perfused with diluted autologous blood (13 ml blood/87 ml buffer) and exposed to 3-carene at an air concentration of 3000 mg/m(3). In both groups there was a reduction in compliance and conductance but this reduction was significantly (P<0.05) more pronounced (2.5-3 times) in lungs obtained from sensitised animals than from control animals. In a previous study with similar design, but with plain buffer instead of diluted autologous blood as perfusate, we found no statistically significant difference in lung bronchoconstriction. Thus, it is concluded that skin sensitisation can increase lung reactivity to 3-carene and that important mediators of this effect seem to be present in the blood.

Ingested aggregates of ultrafine carbon particles and interferon-gamma impair rat alveolar macrophage function.

Alveolar macrophages (AM), obtained by lavage from the rat lung, were allowed to ingest aggregated ultrafine carbon particles, about 1 microgram/10(6) AM, which is a realistic result of long-term exposure to ambient air. The effects of the ingested carbon on the phagocytosis of test particles and oxidative metabolism of the AM were studied. In addition, the effects of short-term (40 min or 2 h) and long-term (28 or 44 h) incubation with interferon gamma (IFN-gamma) on AM loaded and unloaded with carbon were investigated. Phagocytic activity was studied using fluorescein-labeled 3.2-microgram silica particles. The attachment and ingestion processes were evaluated separately. The ingested carbon markedly impaired the phagocytosis of silica particles; the accumulated attachment (sum of attached and ingested particles per AM) decreased from 5.0 to 4.2 particles/AM and the ingested fraction (number of ingested particles per AM divided with accumulated attachment) from 0.42 to 0.27. The short-term incubation with IFN-gamma tended to increase the accumulated attachment (from 5.0 to 5.7 particles/AM) and decreased the ingested fraction (from 0.42 to 0.34) in unloaded AM. Long-term incubation with IFN-gamma markedly impaired both the accumulated attachment (to 3.8 particles/AM) and the ingested fraction (to 0.24) in unloaded AM and the carbon load further decreased the accumulated attachment to 2.8 particles/AM, and the ingested fraction to 0.21. The oxidative metabolism was not effected by the ingested carbon or the short-term incubation with IFN-gamma, but the long-term incubation with IFN-gamma increased it with a factor of almost 3. Our results suggest that ingested environmental particles in AM may markedly impair their phagocytic capacity, especially during long-term exposure to IFN-gamma as after infections, and there might be an increased risk for additional infections. Moreover, during an episode of high ambient particle concentration the inhaled particles will not be efficiently phagocytized and may thereby damage the Lung tissue.

Amitriptyline-induced release of endothelin-1 in isolated perfused and ventilated rat lungs.

We have previously shown that tricyclic antidepressants can induce vaso- and bronchoconstriction as well as oedema formation in isolated perfused lungs. This is an effect similar to that seen clinically in adult respiratory distress syndrome. In order to investigate whether endothelin can be a mediator of this reaction, isolated perfused rat lungs were exposed to 0.1 mM amitriptyline via the pulmonary circulation, perfusate was collected and endothelin-1 present in the perfusate and lavage fluids was determined by radioimmunoassay. A significant increase in perfusate concentration of endothelin-1 was noted, with the highest release seen within the first 10 min. of exposure. Histamine and thromboxane have also been proposed as mediators in induction of adult respiratory distress syndrome. However, no increased amounts of these mediators were detected in the perfusate. Experiments where lungs were exposed to exogenous endothelin-1(0.1-1 nmol), both via the perfusate and via intratracheal instillation were conducted. Similar effects as observed with amitriptyline (0.1 mM) on lung function and perfusion flow were detected. In conclusion, the detection of endothelin-1 release in our lung model proposes a role for endothelin-1 in amitriptyline-induced vaso- and bronchoconstriction and possibly in adult respiratory distress syndrome type reaction. Further studies with this model are interesting in order to elucidate mechanisms behind the complex issue of adult respiratory distress syndrome-induction.

Does airway responsiveness increase after skin sensitisation to 3-carene: a study in isolated guinea pig lungs.

Guinea pigs were sensitised by dermal exposure to 3-carene according to the modified cumulative contact enhancement test (CCET) protocol. Lungs from sensitised and non-sensitised animals were then perfused with buffer and exposed for a period of 10 min to two different air concentrations of 3-carene, 600 and 3000 mg/m3. 3-Carene caused a statistically significant bronchoconstriction even at the relatively low concentration of 600 mg/m3 and the constriction was dose dependent. 600 mg/m3 of 3-carene caused a reduction of 19% in conductance capacity and 16% in compliance capacity. 3000 mg/m3 of 3-carene decreased lung compliance and conductance by 43 and 31%, respectively. The lungs from sensitised animals tended to show a greater response than lungs obtained from control animals. The lower concentration of 3-carene is close to and may even be below, occupational limit values in Sweden, Germany and USA.

Acute lung failure induced by tricyclic antidepressants.

Overdosing of several drugs, such as tricyclic antidepressants, salicylates, and opiates, is known to induce effects like those seen in patients with adult respiratory distress syndrome. By exposing isolated perfused and ventilated rat lungs via the perfusate to six different tricyclic antidepressants (amitriptyline, nortriptyline, imipramine, desipramine, mianserine, and maprotiline), we investigated possible effects on ventilation (conductance and dynamic compliance), lung perfusion flow, and edema formation. The effects of these substances were pronounced and appeared within 15 min after exposure. Amitriptyline was studied in greater detail and caused a dose-related (0.01-1.0 mM) reduction in ventilation and perfusion flow. At the highest drug concentration pronounced lung edema was observed. Morphological studies were conducted with a transmission electron microscope. The microscopic preparations showed dose-related edema (amitriptyline 0.1 and 1.0 mM). The effects noted in our experimental studies are similar to those described in patients who have taken an overdose of tricyclic antidepressants. This emphasizes the possibility of a noncardiogenic edema component in these patients.

Hexamethylene diisocyanate (HDI)-induced lung impairment: studies in isolated perfused and ventilated guinea pig lungs.

Isolated, perfused and ventilated guinea pig lungs were exposed to hexamethylene diisocyanate via the air passages. Two air concentrations of hexamethylene diisocyanate were studied (3.5 and 11 mg/m3). There was a statistically significant (P < 0.05-0.001) dose-related reduction in both conductance and compliance but no effects were noted on the pulmonary circulation. With 3.5 mg/m3 hexamethylene diisocyanate the conductance capacity was reduced with 38% and compliance with 30% after 60 min. exposure. Eleven mg/m3 hexamethylene diisocyanate reduced the conductance and compliance capacity with 86 and 69%, respectively, on an average. The reduction in lung function (with 11 mg/m3) was abolished when 100 microM diclofenac, a cyclooxygenase inhibitor, was added to the perfusate (P < 0.01). The thromboxane A2 antagonist L-670, 596 (20 microM) exerted a partial protective effect. The capacity of conductance and compliance decreased with 46 and 32%, respectively, on an average, after preperfusion with L-670, 596 and a following exposure of 11 mg/m3 hexamethylene diisocyanate for 60 min. Statistically significant protection (P < 0.05) was obtained on compliance and the P-value was < 0.1 for conductance. Thus, these data indicate that hexamethylene diisocyanate-induced bronchoconstriction is mediated via arachidonic acid release and thromboxane formation, in isolated, perfused and ventilated guinea pig lungs.

Analysis of the reactivity of [14C]toluene diisocyanate (TDI) in an isolated, perfused lung model.

An isolated, perfused, guinea pig lung model was used to investigate the molecular events which occur when a 14C-labeled TDI vapor reaches the airways. Exposure concentrations of 0.2 and 0.7 ppm were tested. Perfusate composition included: Krebs Ringer buffer only, as well as buffer containing either guinea pig serum albumin, human serum albumin, or diluted guinea pig plasma. Radioactivity was detected in the perfusate within minutes of exposure, and following a delay, increased linearly. The rate of uptake was dependent on TDI concentration and the composition of the perfusate. Biochemical characterization of the state of the 14C-labeled material in the perfusate was performed. The distribution between low and high molecular weight reaction products was determined by molecular sieve fractionation and varied as a function of perfusate composition but no variability was observed as a function of time during the 45 min of exposure. An increase in nucleophile concentration in the perfusate was associated with both a higher percentage of conjugated products (from 15% with buffer only to 45% with diluted guinea pig plasma) and an increase in the rate of TDX uptake (from 0.5 microns Eq/min with buffer alone to 0.1 micrograms Eq/min with diluted GPSA as perfusate at 0.7 ppm). GC-MS analysis of the samples for free TDA, before and after acid hydrolysis, showed that the low molecular weight product(s), which represented from 55-85% of the circulating radioactivity, was composed of hydrolyzable and non-hydrolyzable conjugates and metabolites with approximately 4% of the label associated with free TDA. Although the distribution between high and low molecular weight species varies, this result is analogous to the findings from in vivo studies and suggests that the isolated, perfused lung (IVPL) system may be a useful tool in investigating the molecular mechanisms of isocyanate-induced disease and metabolic activity of the lung.

Mechanisms of 3-carene-induced bronchoconstriction in the isolated guinea pig lung.

Inhaled 3-carene at a concentration of 5,000 mg/m3 caused bronchoconstriction in isolated, ventilated and perfused guinea pig lungs. This effect was inhibited by the cyclooxygenase inhibitor diclofenac (100 microM) and the thromboxane/prostaglandin endoperoxide-receptor antagonist L-670,596 (1 microM). 3-Carene exposure also increased the amount of thromboxane in the perfusate from the lungs. In cultured calf pulmonary arterial endothelial cells 3-carene caused a dose-related release of arachidonic acid. Thus, the results obtained in this experimental model may have implications in the understanding of the pathophysiology of 3-carene-induced obstructive pulmonary disease in humans.

Menadione-mediated membrane fluidity alterations and oxidative damage in rat hepatocytes.

Menadione toxicity in isolated rat hepatocytes was mitigated by the antioxidant 4b,5,9b,10-tetrahydroindeno[1,2-b]indole at low concentrations (less than 100 microM), but not at high concentrations (greater than 200 microM) of menadione. When hepatocytes were incubated with menadione, there was a time-dependent and concentration-dependent inhibition of lipid peroxidation in intact cells, as well as an increase in the antioxidative potency of acetone extracts, suggesting that metabolites of menadione could inhibit oxidative stress, and that at high menadione concentrations a different mechanism was involved in cytotoxicity. A possible mechanism was suggested by the ability of acetone extracts from hepatocytes that had been incubated with menadione to increase osmotic fragility in red blood cells. This increase correlated with an increase in membrane fluidity in red blood cells, determined by flourescence polarization using the membrane probe 1,6-diphenyl-1,3,5-hexatriene. At 200 microM menadione, an increase in membrane fluidity was also observed in hepatocytes. The thiol dithiothreitol protected hepatocytes from 50 microM menadione toxicity, but not from greater than or equal to 100 microM menadione. The results suggest that while oxidative stress and arylation may be the critical mechanisms of toxicity at low menadione concentrations, at higher concentrations another mechanism such as enhanced membrane fluidity is operative.

On the mechanisms of glutathione depletion in hepatocytes exposed to morphine and ethylmorphine.

Incubation of isolated rat hepatocytes with either morphine or ethylmorphine resulted in glutathione (GSH) depletion followed by loss of cell viability. Pretreatment of cells with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to inactivate glutathione reductase did not markedly affect the rates of GSH depletion seen in untreated cells. In contrast, hexobarbital stimulated H2O2 production in isolated liver microsomes, incubated aerobically with NADPH, whereas the effects of morphine and ethylmorphine on microsomal H2O2 production were minimal. Finally, incubation of hepatocytes with radioactively labeled morphine resulted in formation of 2 glutathione conjugates, one of which was tentatively identified as formyl glutathione. We conclude that GSH consumption during the metabolism of morphine or ethylmorphine by hepatocytes is due mainly to formation of glutathione conjugates.

Effects of oxidative stress caused by hyperoxia and diquat. A study in isolated hepatocytes.

The effects of oxidative stress caused by hyperoxia or administration of the redox active compound diquat were studied in isolated hepatocytes, and the relative contribution of lipid peroxidation, glutathione (GSH) depletion, and NADPH oxidation to the cytotoxicity of active oxygen species was investigated. The redox cycling of diquat occurred primarily in the microsomal fraction since diquat was found not to penetrate into the mitochondria. Depletion of intracellular GSH by pretreatment of the animals with diethyl maleate promoted lipid peroxidation and sensitized the cells to oxidative stress. Diquat toxicity was also greatly enhanced when glutathione reductase was inhibited by pretreatment of the cells with 1,3-bis(2-chloroethyl)-1-nitrosourea. Despite extensive lipid peroxidation, loss of cell viability was not observed, with either hyperoxia or diquat, until the GSH level had fallen below approximately 6 nmol/10(6) cells. The iron chelator desferrioxamine provided complete protection against both diquat-induced lipid peroxidation and loss of cell viability. In contrast, the antioxidant alpha-tocopherol inhibited lipid peroxidation but provided only partial protection from toxicity. The hydroxyl radical scavenger alpha-keto-gamma-methiol butyric acid, finally, also provided partial protection against diquat toxicity but had no effect on lipid peroxidation. The results indicate that there is a critical GSH level above which cell death due to oxidative stress is not observed. As long as the glutathione peroxidase - glutathione reductase system is unaffected, even relatively low amounts of GSH can protect the cells by supporting glutathione peroxidase-mediated metabolism of H2O2 and lipid hydroperoxides.