Interaction between locally released endothelin-1 (ET-1) and the endothelial ET(B)receptor could modulate pulmonary vascular tone. We evaluated pulmonary ET-1 clearance and ET-1-ET(B)receptor interaction in the modulation of pulmonary vascular tone. Controls and rats with Monocrotaline (MCT)-induced pulmonary hypertension (PH) were studied. Lungs were isolated and perfused under constant pressure. The effect of the selective ET(B)antagonist BQ-788 (10(-12)-10(-8)mole) on perfusion flow rate and(125)I-ET-1 extraction was determined. Baseline(125)I-ET-1 extraction was reduced from 62+/-5% in controls to 49+/-10% in PH (P=0.012). BQ-788 inhibited extraction with a higher half-inhibitory dose in the MCT group (-Log ID(50)= 8.9+/-0.4 vs. 9.5+/-0.1, P=0.03). BQ-788 induced a mild reduction in perfusion flow rate of 0.7+/-0.3 ml/min in controls. In the MCT group, this occurred at a lower dose and was more pronounced with a maximal reduction of 3.3+/-0.7 ml/min (P<0.01 vs. control). ET-1 was undetectable in the effluent at baseline but was present in similar concentrations in both groups after ET(B)blockade. Addition of 2 pg/ml ET-1 to lung perfusate did not modify pulmonary ET-1 clearance or the effect of BQ788 on perfusion flow rate in control lungs. In normal rat lungs, the ET(B)receptor plays a minor regulatory role on vascular tone. In MCT hypertension however, despite a reduction in ET(B)mediated extraction, luminal production of ET-1 attenuates the increase in pulmonary vascular tone.

Background-Nitric oxide (NO) and endothelin (ET) have been implicated in the pathogenesis of pulmonary hypertension (PH). Chronic ETA antagonist therapy reduces PH in monocrotaline (MCT)-treated rats. Interactions between the L-arginine-NO pathway and the ET system have been described. We therefore studied the effect of long-term treatment with an oral ETA antagonist (LU 135252) on NO-related vasodilation in isolated lungs from control rats and rats with MCT-induced PH.Methods and Results-Three weeks after MCT injection, PH was associated with an increase in right ventricular pressure (from 27.4+/-0.9 to 66.6+/-4.1 mm Hg) and a decrease in endothelium-independent vasodilation in response to sodium nitroprusside (10(-10) to 10(-5) mol/L; Delta E-max, from 11.1+/-0.9 to 2.7+/-0.3 mm Hg). Endothelium-dependent vasodilation in response to acetylcholine (10(-9) to 10(-4) mol/L) and the calcium ionophore A23187 (10(-9) to 10(-7) mol/L) remained unaffected. Treatment with LU 135252 did not significantly affect the endothelium-dependent and -independent vasodilations in control rats. However, in MCT-treated rats, LU 135252 therapy significantly reduced right ventricular pressure (39.7+/-2.1 mmHg), potentiated acetylcholine-induced vasodilatation (Delta E-max, from 1.6+/-0.2 to 3.7+/-0.4 mm Hg), and improved the responses to sodium nitroprusside (Delta E-max from 2.7+/-0.3 to 5.6+/-0.6 mm Hg). LU 135252 did not significantly alter the non-receptor-mediated endothelium-dependent vasodilation to A23187 or pulmonary constitutive NO synthase activity.Conclusions-MCT PH is associated with a reduced smooth muscle responsiveness to NO but a maintained endothelium-dependent vasodilatory potency. Long-term ETA antagonist therapy not only restores smooth muscle responsiveness to NO but also increases endothelium-dependent dilation in response to acetylcholine. This mechanism may contribute to the therapeutic benefit of ETA antagonists in PH.

The effects of leukotriene D4 (LTD4) on the concentration of intracellular cytosolic free calcium ([Ca++]i) and on phosphoinositide hydrolysis were studied in cultured guinea pig tracheal smooth muscle cells. In Fura-2-loaded cells, LTD4 (10(-9)-10(-6) M) induced concentration-dependent changes in [Ca++]i consisting of a slow, transient increase followed by a sustained phase. Preincubation of cells with LTD4 receptor antagonist MK-571 (10(-6) M) blocked the increase in [Ca++]i. Similarly, LTD4-induced inositol phosphate ([3H]InsP(s) synthesis was transient, concentration-dependent and inhibited by the LTD4 antagonist. In the absence of extracellular Ca++, LTD4 failed to induce [Ca++]i increases and [3H]InsP(s) formation. Accordingly, NiCl2 completely inhibited the LTD4-stimulated [3H]InsP(s) synthesis. Nifedipine (10(-5) M) had a slight inhibitory effect on [Ca++]i increase but significantly reduced (40-50%) the [3H]lnsP(s) accumulation. These findings indicate that LTD4-stimulated inositol phosphate synthesis and [Ca++]i increases in tracheal smooth muscle cells are receptor-mediated events and are dependent on the availability of extracellular Ca++. It is suggested that Ca++ influx plays a major role in the LTD4 signal transduction mechanism.

Pulmonary hypertension is associated with endothelial dysfunction that may mediate or contribute to the disease process; among those abnormalities is an increase in circulating endothelin-1 levels. We investigated the effect of the orally active endothelin A receptor antagonist LU 135252 (LU) on the development of monocrotaline (MCT)-induced pulmonary hypertension and endothelial metabolic dysfunction. Rats were assigned to four groups by receiving a single dose of MCT or saline, followed by once-daily gavage with LU (50 mg/kg) or saline for 3 weeks. Plasma immunoreactive endothelin-1 levels doubled after MCT and were unaffected by LU therapy. The MCT-induced increase in right ventricular systolic pressure (72.5 +/- 15.9 mmHg) and hypertrophy (right ventricle/[left ventricle plus septum weight]; 0.58 +/- 0.08) were reduced by LU to 42.7 +/- 8.5 mmHg (P < .01) and 0.42 +/- 0.05 (P < .01), respectively. LU, however, did not modify MCT-induced pulmonary artery medial hypertrophy. Pulmonary vascular endothelial metabolic activity was evaluated in isolated lungs by measuring endothelium-bound angiotensin-converting enzyme activity using a synthetic angiotensin-converting enzyme substrate, 3H-benzoyl-phenylalanly-glycyl-proline. MCT reduced fractional 3H-benzoyl-phenylalanly-glycyl-proline hydrolysis (0.488 +/- 0.051, P < .01) which was normalized by LU therapy (0.563 +/- 0.050). LU treatment alone had no significant effect on any of these parameters. We conclude that the endothelin A antagonist LU reduces MCT-induced pulmonary hypertension and right ventricular hypertrophy and restores endothelial metabolic function. These results support the development of endothelin antagonists for the treatment of pulmonary hypertension and associated endothelial metabolic abnormalities.

The influence of inflammatory cells on airway reactivity was investigated on arachidonic acid-induced relaxations of guinea-pig trachea and on arachidonic acid metabolism in guinea-pig tracheal epithelial cells. The presence of either eosinophils or neutrophils (1.0 x 10(7) cells/ml), from bronchoalveolar lavage, decreased the tracheal relaxations induced by arachidonic acid (1.0-30 mu M). The basal synthesis of prostaglandin E(2) was increased in epithelial cells (from 176 +/- 36 to 7920 +/- 898 pg/ml), eosinophils (from 360 +/- 56 to 2693 +/- 686 pg/ml) and neutrophils (from 352 +/- 81 to 4400 +/- 272 pg/ml) following incubation with arachidonic acid (10 mu M) The co-incubation of either eosinophils or neutrophils with epithelial cells, in the presence of arachidonic acid, decreased the synthesis of prostaglandin E(3) (2600 +/- 686 and 4400 +/- 272 pg/ml respectively) but increased the synthesis of thromboxane B-2 (from 60 +/- 6 to 11634 +/- 840 and 9282 +/- 485 pg/ml respectively). Similarly, when major basic protein-treated (100 mu g/ml) epithelial cells were incubated with arachidonic acid, the prostaglandin E(2) synthesis decreased (75%) but thromboxane B-2 synthesis was unaffected. The results suggest that eosinophils and neutrophils may impair arachidonic acid metabolism in guinea-pig epithelium in favor of production of bronchoconstrictor prostanoids.

Leukotriene (LT) C4 receptors have been characterized on freshly isolated guinea pig tracheal epithelial cells (tracheocytes). The [3H]LTC4 receptor affinity was enhanced by increasing the sodium (60-160 mM) and the magnesium (0-10 mM) concentrations. Low concentrations of calcium (0-3 mM) increased [3H]LTC4 binding, but high concentrations (3-10 mM) decreased it. The pH (6.5-8.0) had no effect on [3H]LTC4 binding to tracheocytes. Under our experimental conditions, binding equilibrium was reached after 20 min. The association and the dissociation rate constants were estimated to be 2.75 +/- 0.25 x 10(6) M-1.min-1 and 0.093 +/- 0.008 min-1, respectively. The Kd (35.4 +/- 8.6 nM) and the Bmax values (2.4 +/- 0.6 x 10(5) receptors/cell) were determined by Scatchard analysis. LTB4, LTD4 and LTE4 did not inhibit [3H]LTC4 binding to the receptors. However, the compound FPL 55712 inhibited the binding of [3H]LTC4 with an IC50 value of 9.0 +/- 1.0 microM. [3H]LTC4 was not metabolized during the binding assays, as confirmed by reverse-phase high-performance liquid chromatography. The lack of [3H]LTC4 binding to glutathione-S-transferase was demonstrated in the presence of an excess of reduced glutathione. LTC4 produced a concentration-dependent increase of free Ca++ in tracheocytes. Our results suggest that guinea pig tracheocytes possess a specific LTC4 receptor coupled to a Ca++ signaling pathway. This LTC4 receptor may play a key role in the epithelium-dependent responses of airway smooth muscle.

Epithelial cells from the guinea pig trachea (tracheocytes) have been separated from the lamina propria by incubation with EDTA. Contaminating eosinophils were removed by plating on guinea pig immunoglobulin G (IgG)-coated plates. Purity of the cell suspension was assessed by electron microscopy. Enzyme immunoassay analyses of supernatants of guinea pig tracheocytes incubated with 10 microns arachidonic acid (AA) in the presence or absence of 2 microns ionophore A23187 showed that the cells released up to eight times more thromboxane, prostaglandin E2, and 6-ketoprostaglandin F1 alpha than control cells. Ionophore A23187 by itself stimulated to a smaller extent the release of these eicosanoids and did not potentiate the effect of AA. The release of cyclooxygenase products by these cells was highly modified by cell densities in the incubation media. The release of leukotrienes (LT) by stimulated epithelial cells was also investigated using reverse-phase high-performance liquid chromatography. Our results showed that the cells could not release LT on incubation with ionophore A23187 and/or AA. However, the cells released LTB4 in the medium on incubation with 2 microns LTA4. Peptido-leukotrienes were not detected. These results indicated that guinea pig tracheocytes have the cyclooxygenase and the LTA4 hydrolase but not the 5-lipoxygenase. It is postulated that tracheocytes may participate in transcellular metabolism of LTA4 generated by other cell types.

Mechanical removal of guinea pig bronchial and tracheal epithelium by rubbing the luminal surface resulted in a significant dose-dependent increase of the contractile responses to leukotrienes (LT) A4, C4, D4, E4 and histamine. Histological examinations were made to confirm either the presence or the absence of the epithelium and the integrity of underlying tissues. The contractions elicited by the high doses of LTA4, C4, D4 and E4 were potentiated by 2.7, 3.2, 1.9 and 4.5 fold respectively on trachea and by 2.1, 1.9, 2.3 and 1.9 fold respectively on upper bronchi in the absence of the epithelial layer. Histamine and LTD4 induced the release of prostaglandins (PG) E2 and I2 by intact or epithelium-denuded tracheal and bronchial rings. Diphenhydramine (4.0 x 10(-6) M) inhibited significantly the release of relaxant prostanoids induced by histamine, whereas the compound MK-571 (1.0 x 10(-6) M) did not inhibit leukotriene D4-stimulated release. These antagonists inhibited tracheal and bronchial contractions in either the absence or presence of epithelium. Indomethacin (2.8 x 10(-6) M) enhanced the myotropic activities of histamine and LTD4 on the trachea and bronchi deprived of their epithelial layer. These results suggest that PGE2 and I2 are major components of the epithelium-derived relaxing factor. Our results suggest however that underlying tissues are also responsible for the release of these relaxant prostanoids.

The pharmacological activity of leukotrienes (LT) A4, C4, D4, E4, and histamine was investigated on guinea pig upper and lower bronchi. The contractions of the upper bronchi to histamine, LTA4, C4 and D4 were enhanced by cyclooxygenase inhibitors aspirin (1.67 × 10−5 and 1.67 × 10−6 M) and indomethacin (2.8 × 10−6 and 2.8 × 10−5 M) whereas the responses to LTE4 were not affected. The myotropic activity of the lower bronchi to all agonists were either very slightly or not at all modified by the presence of cyclooxygenase inhibitors. The thromboxane synthetase inhibitor OKY-046 (1.77 × 10−5 and 1.77 × 10−6 M) did not change the responses of higher bronchi to the agonists which suggested that the response of the upper bronchi may be mediated by prostaglandins but not by thromboxanes. The responses of the lower bronchi to leukotrienes A4, C4, D4 and E4 were inhibited by compound OKY-046. Blockade of thromboxane receptors together with inhibition of lipoxygenases by compound L-655,240 (2.53 × 10−8 to 2.53 × 10−5 M) had a slight effect on the stimulation of upper and lower bronchi by leukotrienes and histamine. The compound FPL-55712 (1.92 × 10−6 and 1.92 × 10−5 M) strongly reduced the contractions of the upper and lower bronchi to leukotrienes but did not affect the responses to histamine. These results suggest that the contractile effects of leukotrienes on upper bronchi is modulated by bronchorelaxant prostaglandins whereas the responses of the lower bronchi are mediated by thromboxanes. The responses of both upper and lower bronchi to histamine do no appear to be mediated by cyclooxygenase products.

Strips, of guinea pig bronchus dissected from the upper, middle, and lower lobe and divided in two segments referred to as external and internal bronchi, were analyzed for their reactivity to several icosanoids. The external bronchi produced much more contractile force than the internal bronchi, and the reactivity to the agonists was different. The order of potency of prostanoids and histamine on the external bronchus was U44069≃histamine>PGF2α and on the internal bronchus U44069>histamine> PGF2α. The internal bronchus did not react to PGE2, whereas this agonist produced a dose-dependent relaxation on the external bronchus. The order of potency of leukotrienes and histamine on the external bronchus was LTD4 >LTC4>LTE4>LTA4> histamine and on the internal bronchus LTC4> LTD4>LTA4=LTE4> histamine. LTB4 has a significant myotropic activity on guinea pig bronchus. Because of its sensitivity and characteristic responses to icosanoids, it is suggested that the guinea pig bronchus may be as suitable (and possibly more) a pharmacological preparation as the trachea or the parenchyma to study the bronchoreactivity.