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The effects of two monocyclic monoterpenes, limonene and sobrerol, known as inhibitors of farnesyltransferase activity, were studied on monocrotaline (MCT)-induced lung injury, pulmonary hypertension and right ventricular hypertrophy in male Wistar rats. After 14 days, pulmonary arterial pressure values and the right ventricle to left ventricle plus septum weight ratios, RV/(LV + S), were markedly increased in rats subcutaneously injected with MCT (60 mg/kg). Limonene and sobrerol, administered daily at the oral dose of 400 mg/rat, markedly decreased the MCT-induced alterations. After treatment for 21 days, limonene still prevented pulmonary hypertension and the increase in RV/(LV + S). Both monoterpenes also reduced the increase in pulmonary arterial media thickness, the development of interstitial fibrosis and the increase in the number of macrophages in intra-alveolar spaces and of lymphocytes around the pulmonary veins. The present data indicate that treatment of rats with inhibitors of farnesyltransferase, like limonene and sobrerol, regulate the development of pulmonary hypertension.

The effect of the immunosuppressive compound, cyclosporin A, and the corticosteroid, betamethasone, was investigated on eosinophil accumulation in guinea-pig lung tissue induced by antigen, platelet-activating factor (PAF) and leukotriene B4 (LTB4). The accumulation of eosinophils in the peribronchial area was evaluated on histological preparations. The lung sections were stained with Luna's reagent specific for eosinophil granule content. Oral treatment of the guinea-pigs with cyclosporin, 10 mg.kg-1 three times a day for two days, and 10 mg.kg-1 1 h before antigen challenge, significantly reduced the accumulation of eosinophils observed at 4 and 24 h, in the peribronchial area of sensitized guinea-pig lung. Betamethasone (3 mg.kg-1), administered orally 24 h and 1 h before antigen challenge elicited a moderate but significant reduction of antigen-induced eosinophil accumulation. Pretreatment of the guinea-pigs with cyclosporin or betamethasone elicited a marked inhibition of the accumulation of eosinophils in the peribronchial area induced by aerosolized PAF (100 micrograms.ml-1) or LTB4 (5 micrograms.ml-1). Since cyclosporin and betamethasone significantly inhibit the antigen-induced eosinophil accumulation, these results suggest that antigen-induced lung eosinophilia is dependent of T-lymphocytes. However, cyclosporin and betamethasone may also reduce the chemotactic activity of PAF and LTB4 on guinea-pig eosinophils.

The effect of phosphoramidon on the increase in pulmonary inflation pressure (PIP) induced by endothelin-1 (ET-1) administered by aerosol in ovalbumin (OA)-sensitized and challenged guinea-pigs was investigated after pretreatment or not of the animals with the neutral endopeptidase inhibitor, phosphoramidon, the cyclooxygenase inhibitor, indomethacin or the platelet activating factor (PAF) antagonist, BN 50730. When guinea-pigs were pretreated by phosphoramidon (0.1 mM, aerosol for 15 min), a significant enhancement of PIP was observed after administration of ET-1 (1 or 3 micrograms ml-1, aerosol for 2 min), whereas these doses of the peptide were only slightly active in control animals. In sensitized and unchallenged guinea-pigs, ET-1 (1 or 3 micrograms.ml-1, aerosol for 2 min) induced, as in controls, a moderate increase in PIP. In contrast, aerosol exposure of OA in sensitized guinea-pigs developed an increased PIP following ET-1 (1 and 3 micrograms.ml-1, aerosol for 2 min) administration, that was non significantly affected by pretreatment of the animals with phosphoramidon after the dose of 3 micrograms ml-1 ET-1. Guinea-pigs exposed to phorphoramidon and treated with indomethacin (10 mg kg-1, i.v.) or BN 50730 (25 mg kg-1, per os) significantly reduced the increase in PIP upon administration of ET-1 (3 micrograms.ml-1, aerosol for 2 min). No inhibitory effect of indomethacin was noted when ET-1 (3 micrograms.ml-1, aerosol for 2 min) was administered to sensitized and OA-exposed guinea-pigs, pretreated or not with phosphoramidon. In contrast, BN 50730 significantly reduced the increase in PIP induced by ET-1 observed in sensitized and OA-exposed guinea-pigs. Moreover, this drug was moderately active in reducing the increase in PIP induced by ET-1, when the animals were pretreated by phosphoramidon. These results suggest that a phosphoramidon-sensitive endopeptidase-like enzyme, present in the airway tissue modulates the effect of ET-1. Furthermore, the increase in PIP to ET-1 observed in aerosol-sensitized and antigen-exposed guinea-pigs appears to be mediated by PAF rather than cyclooxygenase metabolites, even though the participation of other mediators in this process is open.

1. Intra-arterial injection of endothelin-1 (ET-1, 400 pmol; 1 microgram) in guinea-pig isolated perfused lungs, induced increases in pulmonary inflation pressure (PIP) and perfusion pressure (PPP), associated with oedema formation and thromboxane B2 (TxB2) release but not with the generation of sulphidopeptide leukotrienes or release of histamine. In contrast, aerosol administration of ET-1 (3, 6, 10 micrograms ml-1, for 2 min) evoked a dose-dependent increase in PIP, without significant changes in PPP, oedema formation or TxB2 release. 2. Addition of indomethacin (5 microM) or BW 755C (10 or 100 microM), but not nordihydroguaiaretic acid (NDGA, 50 microM) or FPL 55712 (10 microM), to the perfusion medium led to a significant inhibition of the increases in PIP and PPP, TxB2 release and oedema formation evoked by intra-arterial injection of 400 pmol ET-1. In contrast, indomethacin (5 microM), BW 755C (100 microM) or FPL 55712 (10 microM), added to the perfusion medium 10 min prior to challenge, did not affect the increase in PIP induced by a 2-min aerosol of a solution of ET-1 10 micrograms ml-1. 3. In vivo aerosol administration of indomethacin (100 mg ml-1, for 20 min) to non-anaesthetized guinea-pigs, 15 min before lung removal, did not modify the bronchopulmonary response evoked in isolated perfused lungs by an aerosol of ET-1 10 micrograms ml-1. However, under the same experimental conditions, indomethacin significantly inhibited TxB2 release evoked by aerosolized arachidonic acid (2 mg ml-1). 4. In conclusion, the present study shows that when injected by the intra-arterial route, ET-1 effects are mediated primarily via the generation of cyclo-oxygenase metabolites of arachidonic acid, whereas when the aerosol route is used, the peptide appears to act on airway smooth muscle cells, through an indomethacin-insensitive process which may involve some other, as yet unidentified, mediator(s).

In anesthetized and ventilated guinea pigs intravenous injection of ET-1, ET-2, or ET-3 (1 or 2 nmol/kg) induced similar dose-dependent increases in pulmonary inflation pressure (PIP) associated with increases in mean arterial blood pressure (MBP). Pretreatment of the guinea pigs with 1 mg/kg intravenous indomethacin significantly inhibited the increase in PIP evoked by 2 nmol/kg of ET-1, ET-2, or ET-3. In contrast, the increase in MBP following injection of the various ET isotypes was not significantly affected by indomethacin. Injection of ET-1, ET-2, or ET-3 (40, 120, and 400 pmol) via the pulmonary artery of isolated and perfused guinea pig lungs induced dose-dependent increases in PIP and pulmonary perfusion pressure (PPP), thromboxane B2 (TXB2) release, and formation of lung edema. In keeping with the in vivo results, no marked differences were observed between the activities of ET-1, ET-2, and ET-3 on isolated and perfused guinea pig lungs. Indomethacin (5 microM) added to the perfusion medium significantly inhibited the alterations of PIP and PPP, TXB2 release, and edema formation evoked by 400 pmol ET-1, ET-2, or ET-3. High-affinity binding sites for ET-1, ET-2, and ET-3 exhibiting similar characteristics were identified on guinea pig lung membrane. Therefore ET-1, ET-2, and ET-3 exert comparable bronchopulmonary and pressor activities in the guinea pig and probably act via interaction with the same binding site. In addition, the ET-induced increase in PIP and pulmonary vasoconstriction are primarily mediated via the production of cyclooxygenase metabolites.

We have studied the development of airway hyperresponsiveness and the pulmonary cell infiltration in a guinea-pig model in which both initial sensitization and subsequent exposure to the antigen were performed by aerosol. Enhanced bronchopulmonary response to aerosol administration of acetylcholine (ACh) and 5-hydroxytryptamine (5-HT) was observed 3-4 hr and 18-24 hr after antigen exposure of sensitized animals. In contrast, when ACh and 5-HT were administered intravenously 3-4 hr after the challenge, no significant alteration of the dose-response curves was observed. However, 18-24 hr after antigen challenge, a marked leftward shift of the dose-response curve was observed on intravenous injection of ACh or 5-HT. The increased bronchial reactivity to aerosolized ACh in sensitized and challenged guinea-pigs reached a maximum by days 2-4, was still significantly increased at day 5 and returned to the basal value by day 8. No further alteration of the dose-related bronchopulmonary response to aerosol or intravenous administration of ACh was recorded 24 hr after a second antigen challenge, performed 8 days after the initial one. The analysis of bronchoalveolar lavage fluids showed a significant increase in the number of polymorphonuclear neutrophils 3-4 hr after the exposure of sensitized animals to the antigen, which was also associated with a significant eosinophilia at 18-24 hr. Histological examination of lung specimens obtained from animals 3-4 hr following challenge demonstrated eosinophil infiltration in the peribronchial regions and bronchial walls, as well as within the epithelium. Furthermore, as compared to time 3-4 hr, less eosinophils in the peribronchial area and submucosa were counted 24 hr after antigen challenge. However, a role of eosinophil-derived products in the development of bronchial hyperresponsivenss in this experimental model remains to be established.

The effect of chronic administration of platelet-activating factor (PAF) on airway reactivity, cell recruitment and lung morphology in the guinea-pig has been investigated. Alzet osmotic minipumps delivering either PAF (7.2 mg/kg/14 days) in 0.25% (w/v) bovine serum albumin in saline (saline-BSA), acetylcholine or saline-BSA alone were implanted s.c. in the neck region of guinea-pigs and connected to the jugular vein. In some experiments, implanted and non-implanted animals were treated daily with the PAF antagonist, BN 52021 (15 mg/kg, twice a day, p.o.). On day 15 after minipump implantation, the animals were anesthetized with urethane (1.2 g/kg, i.p.) and tracheal cannula was inserted for mechanical ventilation. Pulmonary inflation pressure (PIP) was monitored and airway responsiveness was assessed by administration of increasing doses of histamine (0.2-100 micrograms/kg, i.v.). As compared to saline-BSA-treated or non-implanted guinea-pigs, chronic treatment of the animals with PAF induced a significant (p less than 0.01) increase in airway response. No significant change in airway responsiveness was observed following chronic acetylcholine administration. In contrast, regardless of the treatment of the animals, no change in the threshold dose of histamine inducing alteration in PIP was noted, suggesting that PAF induces bronchopulmonary hyperreactivity rather than hyperresponsiveness. In addition, no significant difference was observed in the in vitro responsiveness to histamine of lung parenchymal strips from animals having received PAF or saline-BSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelin binding in human isolated lung membrane fractions revealed a single class of high affinity recognition sites with a Kd of 1.33 +/- 0.15 nM and a Bmax of 9.61 +/- 1.44 pmol/mg protein. Endothelin inhibited [125I]endothelin binding with a Ki of 1.90 +/- 0.15 nM whereas structurally unrelated compounds had no effect. Endothelin was a potent contractile agonist on human isolated pulmonary arterial (HPA) and venous (HPV) muscle preparations (pD2 values: 9.64 and 10.36, respectively). Neither indomethacin (1 microM), nicardipine (0.01, 0.10, 1.0 microM) nor diltiazem (1, 10, 100 microM) altered the sensitivity of HPA to endothelin. Human isolated bronchial muscle preparations were less sensitive to endothelin than vascular tissues. These data suggest that pulmonary veins may be a major target for this constrictory peptide in the human lung.

The possibility that PAF is implicated in the alterations of the beta-adrenoceptor function observed during endotoxemia was investigated. Lung parenchymal strips (LPS) from endotoxin-treated guinea-pig demonstrated specific desensitization to low doses of PAF whereas the contractions induced by histamine and leukotriene D4 were slightly affected. In addition, histamine-contracted LPS from endotoxin-injected animals exhibited decreased responsiveness to isoproterenol, a phenomenon not observed with guinea-pigs also treated with the specific PAF antagonist, BN 52021. No alteration of the sensitivity to isoproterenol of LPS preincubated with PAF was noted, suggesting an indirect effect of the autacoid on beta-adrenoceptor function.

Intravenous injection of BN 52021 in anesthetized guinea-pigs, 5 min before challenge, inhibited in a dose-dependent fashion with an IC50 of 0.90 mg/kg the bronchoconstriction induced by PAF (60 ng/kg i.v.). However, BN 52021 did not prevent the leukopenia following PAF injection but significantly inhibited the thrombocytopenia induced by PAF. The dioxolan compound, BN 52111, dose-dependently reduced the bronchoconstriction (IC50 = 0.27 mg/kg) and at doses higher than 1 mg/kg partially antagonized the decrease in the number of circulating platelets and leukocytes induced by PAF. BN 52115 also markedly inhibited the bronchoconstriction (IC50 = 0.36 mg/kg) as well as the thrombocytopenia induced by PAF, but was without significant effect on the leukopenia. These results demonstrate that the two dioxolan compounds, BN 52111 and BN 52115, are more potent than BN 52021 in inhibiting the in vivo bronchopulmonary alterations induced by PAF. Since these alterations are related to the activation of platelets by the autacoid, these blood elements are probably the targets of BN 52111 and BN 52115. Injection of PAF (10 and 100 ng) via the pulmonary artery of ventilated and perfused guinea-pig lungs induced dose-dependent increases in pulmonary inflation pressure (PIP) and pulmonary perfusion pressure (PPP), associated with a dose-dependent release of thromboxane B2 (TxB2). Addition of BN 52021, BN 52111 or BN 52115 (0.1, 1 or 10 microM) to the perfusion medium, 15 min before challenge, dose-dependently inhibited the bronchopulmonary effects of PAF. Although BN 52111 was the more potent in inhibiting the PAF-induced increase in PIP, BN 52021 was the more active with respect to the PAF-evoked generation of TxB2, suggesting that the two phenomena are not directly related.

The possible role of PAF in the development of bronchial hyperreactivity was investigated using Alzet osmotic minipumps loaded with PAF or solvent alone and implanted in male Hartley guinea-pigs. Compared to animals treated with solvent alone, those receiving PAF (20 micrograms/kg/h for 15 days) exhibited a hyperreactivity to histamine. The lungs from PAF-treated were congestive in appearance, bronchi and the bronchioli were contracted, Reisseissen muscles were markedly hypertrophied and a muciparous metaplasia of the epithelium was observed. In addition, the number of eosinophils was significantly increased in the parenchyma, as was the number of mast cells in the peribronchial regions. These data indicate that long term-treatment with PAF induces profound alterations in the respiratory system resembling those observed in asthmatic patients. They also strengthen the also strengthen the role of PAF in the development of bronchial hyperreactivity.

To further substantiate the possible role of platelet-activating factor (PAF) in the development of bronchial hyperreactivity, Alzet osmotic minipumps loaded with the autacoid or solvent alone were placed under the back skin of male Hartley guinea pigs and connected to the jugular vein for 15 days. As compared to the animals treated with solvent alone, guinea pigs receiving PAF (20 micrograms/kg/h, 15 days) exhibited a hyperresponsiveness to histamine that was not observed in those simultaneously treated with PAF and the PAF antagonist BN 52021. Lungs from PAF-treated animals were congestive in appearance, bronchi and bronchioli were contracted, Reisseisen muscles were markedly hypertrophied, and a muciparous metaplasia of the endothelium was observed. The number of eosinophils was significantly increased in the parenchyma, as was the number of mast cells in the peribronchial zones in the lungs from guinea pigs chronically treated with PAF. Lungs from control animals did not show morphologic alterations or eosinophil and mast cell infiltration. These data indicate that long-term treatment with PAF induces profound alterations in the respiratory system, resembling those observed in asthmatic patients. This result is in keeping with the possible role of this autacoid in the development of bronchial hyperreactivity.