Bruno Battistini

ENDOTHELIN-1 INDUCES PROSTACYCLIN RELEASE FROM BOVINE AORTIC ENDOTHELIAL CELLS

The effects of endothelin-1 (ET-1) on the release of prostacyclin from cultured bovine aortic endothelial cells were studied. ET-1 induced a time- and dose-dependent release of 6-keto PGF1 alpha, the stable metabolite of prostacyclin, with an apparent EC50 value of 3.0 +/- 0.9 nM (n = 6). ET-1 up to a concentration of 500 nM did not affect cellular integrity. Preincubation of the cells for 30 min with 10 microM indomethacin inhibited ET-1 (100 nM) - induced prostacyclin release by 90%. These findings indicate that ET-1 can directly stimulate prostacyclin release from endothelial cells probably through a receptor mediated mechanism.

Induction by endothelin-1 of epithelium-dependent relaxation of guinea-pig trachea in vitro: role for nitric oxide.

1 The purpose of the present experiments was to study the underlying mechanisms responsible for the relaxant action of endothelin‐1 (ET‐1) in the guinea‐pig trachea in vitro. 2 In tracheal strips precontracted (60–70% of the maximum) with carbachol, ET‐1 (1–100 nm) evoked slowly developing concentration‐dependent relaxations. Preincubation of the tissues with the thromboxane A2/prostaglandin H2 receptor antagonist, BM 13505 (5 μm) significantly potentiated the relaxant response to ET‐1. 3 Removal of the epithelium changed the response of precontracted tracheal preparations to ET‐1 from a relaxation to a sustained contraction. 4 ET‐1‐induced relaxations were abolished by methylene blue (10 μm) and were almost completely attenuated by oxyhaemoglobin (5 μm) and NG‐monomethyl‐l‐arginine (l‐NMMA, 100 μm), an inhibitor of nitric oxide synthesis, but were not altered by indomethacin (10 μm). 5 In tracheal strips under passive tension, ET‐1 (1–100 nm) elicited dose‐dependent contractions. The sensitivity of tissues to ET‐1 was significantly enhanced by removal of the epithelium (apparent EC50 values were 28.1 ± 4.1 and 12.5 ± 0.8 nm in intact and rubbed trachea, respectively, n = 7, P < 0.01). 6 Preincubation of intact tracheal strips with methylene blue, oxyhaemoglobin or l‐NMMA did not mimic the effect of epithelium removal on ET‐1‐induced contractions. 7 There was a concentration‐dependent increase in thromboxane A2 but not in PGE2 and prostacyclin release from intact tracheal strips following stimulation with ET‐1 (5–100 nm). 8 These results show that ET‐1 exerts a dual action on guinea‐pig isolated trachea: it evokes contractions at low resting tone, whereas it induces relaxations at higher resting tone. The relaxant action of ET‐1 may be mediated by nitric oxide released from epithelial cells and resultant activation of smooth muscle guanylate cyclase.

ENDOTHELIN INDUCES THROMBOXANE RELEASE AND CONTRACTION OF ISOLATED GUINEA-PIG AIRWAYS

The possible role of thromboxane (TX) A2 in mediating the bronchopulmonary effect of endothelin (ET) was studied in isolated guinea pig airways (trachea, upper bronchi and parenchyma). ET (1-100 nM) evoked a concentration-dependent contraction of all three tissues. The contractile response was significantly attenuated by pretreatment of the tissues with the specific thromboxane receptor blockers, BM 13177 (10-50 microM) and BM 13505 (1-5 microM). Furthermore, ET caused a dose-dependent TXB2 release from trachea and parenchyma. These findings suggest that the bronchopulmonary action of ET is mediated, in part, by the release of TXA2.

Pharmacological modulation of endothelin‐induced contraction of guinea‐pig isolated airways and thromboxane release

1 The aim of the present experiments was to study the possible involvement of known bronchoconstrictor substances in mediating the myotropic action of endothelin‐1 (ET‐1, human‐porcine endothelin) in guinea‐pig isolated airways. 2 ET‐1 (1–100 nm) caused a dose‐dependent contraction of guinea‐pig trachea, upper bronchus and parenchyma. The contractions developed slowly, reaching maximal values 4–6 min after addition of the peptide. 3 The contractile action of ET‐1 was significantly attenuated by indomethacin (10 μm), a cyclo‐oxygenase blocker, BM 13505 (5 μm), a thromboxane receptor antagonist, FPL 55712 (19 μm) and YM 16638 (1 μm), antagonists of the sulphidopeptide leukotrienes, BN 52021 (10 μm) and WEB 2086 (1 μm), platelet‐activating factor receptor antagonists in all three tissue preparations studied. 4 Pretreatment of the airway tissues with compound U 75302 (3 μm), a selective leukotriene B4 receptor antagonist, or with a mixture of antagonists containing methysergide (0.75 μm), phentolamine (0.4 μm), propranolol (13 μm), atropine (0.4 μm) and diphenhydramine (0.45 μm) did not modify the myotropic action of ET‐1. 5 ET‐1, 10 and 100 nm induced three, and nine fold increases in thromboxane A2 release from lung parenchymal strips. 6 ET‐1‐induced thromboxane A2 release was completely abolished by indomethacin, and was significantly attenuated by BN 52021, WEB 2086 and FPL 55712. Neither BM 13505 nor YM 16638 exerted a significant effect on thromboxane release. 7 The present findings show that contraction of guinea‐pig airway smooth muscle by ET‐1 is mediated, in part, by the release of thromboxane A2, sulphidopeptide leukotrienes and platelet‐activating factor, and suggest that the increased thromboxane A2 release following ET‐1 is partly a consequence of enhanced synthesis of sulphidopeptide leukotrienes and platelet‐activating factor.

Potent thromboxane-mediated in vitro bronchoconstrictor effect of endothelin in the guinea-pig

The present data both confirm that endothelin is a potent constrictor of the guinea pig airways and suggest that this action of ET is mediated at least in part by the release of thromboxane A 2 , a well known bronchoconstrictor

Endothelin-induced constriction of guinea pig airways: role of platelet-activating factor.

Endothelin-1 (ET-1, 1-100 nM) evoked a concentration-dependent contraction of guinea pig trachea and upper bronchi in vitro. The response was significantly attenuated by BN 52021 (ginkgolide B, 10 microM), a specific receptor antagonist to platelet-activating factor. Precontraction of the tissues with endothelin (50 nM) desensitized tissues to platelet-activating factor (200 nM), while preincubation of the tissues with platelet-activating factor slightly inhibited the response to endothelin. These findings suggest that platelet-activating factor may mediate endothelin-induced airway contraction in the guinea pig.

Effects of Tranexamic Acid and Aprotinin, Two Antifibrinolytic Drugs, on PAF-induced Plasma Extravasation in Unanesthetized Rats

Two antifibrinolytic drugs, tranexamic acid (TXA), and aprotinin (APR), are currently used to improve the recovery of patients following major surgery while reducing blood loss. Their mechanisms of action have yet to be fully understood. Here, we examined (1) the effects of TXA or APR on basal vascular permeability (VP) and (2) the effects of TXA or APR on platelet-activating factor (PAF)-induced increase of VP in normal unanesthetized rats. Evans blue dye (EB) bound to albumin was used as the marker of extravasation in selected tissues. In normal rats, PAF (1 μg/kg i.v.) increased VP in most selected tissues including bronchi, aorta, duodenum and pancreas without affecting blood pressure. TXA (up to 300 mg/kg i.v.) had no significant effect on basal VP in any tissues, while APR (30 000 KIU/kg i.v.) decreased basal VP in 5 out of 8 tissues. Pre-treatment with TXA decreased PAF-induced increases of VP in the microcirculation of the thoracic and abdominal aorta, the duodenum and the pancreas, from 35% to 41%. TXA was mostly effective at an i.v. dose of 100 mg/kg with a 2 h of pre-treatment period. Pre-treatment with APR also reduced PAF-induced increases of VP in selected tissues by 35 to 61%. The i.v. dose of 30 000 KIU/mg was optimal when injected at least 30 min before the administration of PAF + Evans blue. These results suggest that the beneficial effect of APR and TXA, following cardiopulmonary bypass (CPB) and other type of surgeries, may be attributed to the inhibition of plasma exudation mediated, at least in part, by PAF. Thus, TXA and APR may improve patients recovery by reducting the capillary leakage of albumin, associated with interstitial edema formation, and maintaining intravascular fluid volume.

Increased plasma levels of endothelin during anaphylactic shock in the guinea-pig

Intravenous injection of ovalbumin into actively and passively sensitized guinea-pig resulted in acute circulatory collapse. The plasma level of immunoreactive endothelin rose from 22 +/- 2 to 40 +/- 7 fmol/ml (n = 12, P < 0.01) and 29 +/- 5 fmol/ml (n = 12, P < 0.01) in actively and passively sensitized animals, respectively, within 5 min of antigen challenge, and it remained significantly higher in actively sensitized animals that survived for 15 min. The plasma immunoreactive endothelin level was inversely correlated with arterial blood pO2, but not with pH or pCO2, both in actively (rs = -0.585, n = 20, P < 0.05) and passively sensitized animals (rs = -0.558, n = 20, P < 0.05). When non-sensitized animals were bled (5 and 20 ml/kg body weight), the plasma immunoreactive endothelin level remained unchanged. These results suggest that the elevated plasma level of immunoreactive endothelin during anaphylactic shock is independent of hypotension, hypovolemia and respiratory insufficiency.

Prostaglandin E2 increases cyclic AMP and inhibits endothelin-1 production/secretion by guinea-pig tracheal epithelial cells through EP4 receptors

Prostaglandin E2 (PGE2) increased adenosine 3′ : 5′‐cyclic monophosphate (cyclic AMP) formation in tracheal epithelial cells and concomitantly decreased the production/secretion of immunoreactive endothelin (irET). Naturally occurring prostanoids and selective and non‐selective EP receptor agonists showed the following rank order of potency in stimulating cyclic AMP generation by epithelial cells: PGE2 (EP‐selective)>16,16‐dimethyl PGE2 (EP‐selective)>11‐deoxy PGE2 (EP‐selective)>>>iloprost (IP/EP1/EP3‐selective), butaprost (EP2‐selective), PGD2 (DP‐selective), PGF2α (FP‐selective). The lack of responsiveness of the latter prostanoids indicated that the prostanoid receptor present in these cells is not of the DP, FP, IP, EP1, EP2 or EP3 subtype. Pre‐incubating the cells with the selective TP/EP4‐receptor antagonists AH23848B and AH22921X antagonized the PGE2‐evoked cyclic AMP generation. This suggested that EP4 receptors mediate PGE2 effects. However, in addition to any antagonistic effects at EP4‐receptors, both compounds, to a different extent, modified cyclic AMP metabolism. The selective EP1, DP and EP2 receptor antagonist (AH6809) failed to inhibit PGE2‐evoked cyclic AMP generation which confirmed that the EP2 receptor subtype did not contribute to the change in cyclic AMP formation in these cells. The PGE2‐induced inhibition of irET production by guinea‐pig tracheal epithelial cells was due to cyclic AMP generation and activation of the cyclic AMP‐dependent protein kinase since this effect was reverted by the cyclic AMP antagonist Rp‐cAMPS. These results provide the first evidence supporting the existence of a functional prostaglandin E2 receptor that shares the pharmacological features of the EP4‐receptor subtype in guinea‐pig tracheal epithelial cells. These receptors modulate cyclic AMP formation as well as ET‐1 production/secretion in these cells.

A role for Na+/H+ exchange in contraction of guinea pig airways by endothelin-1 in vitro

Endothelin-1-induced contractions of guinea pig tracheal and bronchial strips were dose-dependently attenuated by the amiloride analogues 5-(N-ethyl-N-isopropyl)amiloride (EIPA, 1-10 microM) and 5-(N,N-hexamethylene)amiloride (HMA, 1-10 microM). The calculated Ki values for EIPA and HMA were 0.11 +/- 0.02 microM and 0.06 +/- 0.02 microM in the trachea, and 0.28 +/- 0.11 microM and 0.70 +/- 0.25 microM in the bronchus, respectively. These values are in the same order of magnitude as those reported for inhibition of the Na+/H+ exchange in cells. Amiloride (1-10 microM) was ineffective. These data suggest that activation of the Na+/H+ exchange by ET-1 may be involved in mediating its myotropic action in guinea pig airway smooth muscle.