Endothelin: a knockout in London.
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Amino Acid Sequence↗
Biomedical subjects
B Battistini
Publications and source records attributed to B Battistini.
Contractile activity of endothelin precursors in the isolated gallbladder of the guinea-pig: presence of an endothelin-converting enzyme.
A single addition of 3 x I0-7 M ET-1, ET-2 or ET-3 produced contractions that reached a steady state in 28.2 +/- 4.2, 21.1 +/- 1.3 and 24.0 +/- 3.8 min, respectively and took 2.7 +/- 0.4, 2.1 + 0.1 and 1.6 +/- 0.1 min to reach half of this steady-state response.4. Contractions induced by 3 x I0-7 M big ET-11-38 or big ET-11- 39 reached a plateau in 38.5 +/- 3.6 and 35.6 +/- 3.3 min, respectively, and half of these responses were attained in 12.0 +/- 2.5 and 7.1 +/- 1.1 min.Thus, these contractions developed more slowly than those induced by ET-1. Contractions induced by 3 x 10-7 M big ET-21-38 were also much slower to develop than those to ET-2, for these took 49 +/- 2 min to reach plateau and 19.4 +/- 2.1 min to attain half that response. Contractions induced by 3 x 10-7 M big ET-31-41 amide took 50.2 +/- 3.7 min to reach a plateau and 27.3 +/- 3.0 min to reach half of this response.5. Phosphoramidon (0.1, 1 and 3 x 10-4 M) inhibited contractions induced by big ET-11.39. For instance,the contractions induced by 3 x 10-7 M big ET-11-39 were inhibited by 10-4 M or 3 x 10-4 M of phosphoramidon by 62.8 +/- 6.7% or 74.5 +/- 4.6%, respectively. Similarly, contractions induced by ET-21-38 were inhibited by 91.3 +/- 5.4% and the small response induced by big ET-3l-4l amide was abolished by 3 x 10-4M phosphoramidon. Conversely, the neutral endopeptidase (EC 24.11) inhibitor DL-thiorphan(3 x 10-4 M) had no effect. Captopril (10-5 M), pepstatin A (10-5 M), phenylmethylsulphonylfluoride(PMSF, 10-3 M), aprotinin (10-5 M), E-64 (10-5 M), cystatin (10-6 M), leupeptin (10-4 M),chymostatin (10-4 M), or bestatin (10-5 M) did not inhibit but rather increased to a similar, but small degree the contractions induced by 3 to 30 x 10-9 M big ET-11-39. Only captopril (10-5 M) or leupeptin(10-4 M) increased the contraction induced by 3 x 10-7 M big ET-11-39. Phosphoramidon (10-4 M),pepstatin (10-5 M) or PMSF (10-3 M) did not affect contractions induced by ET-1.6. Removal of the epithelium increased by 70% the size of the contraction induced by 5 microM histamine(1.08 +/- 0.05 g; n = 160 to 1.84 +/- 0.14 g; n = 12) but did not affect, in absolute terms, the contraction induced by ET-1 (as a % of the response to histamine, these responses were, of course, apparently depressed). Epithelium removal did, however, increase the size of the contractions induced by 3 to 30 x 10-9 M big ET-1 -39 which was very similar to the effect of the protease inhibitors.7. In competition binding studies on membranes prepared from the guinea-pig gallbladder, 10-11 MET-1 inhibited by 76.9 +/- 3.1% the binding of [125]-ET-I while porcine big ET-11-39 caused no inhibition(0.7 +/- 3.0; n = 3). ET-1 (10-6 M) inhibited binding by 95.7 =/- 1.1% (n = 3) while at this much higher concentration, big ET-11-39 inhibited binding by only 16.8 +/- 4.2% (n = 3). This clearly suggests that big ET-11-39 does not bind directly to ET receptors.8. Thus, a phosphoramidon-sensitive endothelin-converting enzyme (ECE), different from neutral endopeptidase (NEP; EC 24.11) and not located on the epithelium, converts big ET-1 into ET-1 in the gallbladder of the guinea-pig. This ECE appears to act preferentially on big ET-1 or big ET-2 over bigET-3.
Animals↗
Characterization of endothelin receptors mediating mechanical responses to the endothelins in the isolated stomach strip of the rat.
We have characterized the receptors mediating the mechanical responses of the isolated stomach strip to endothelin-1 (ET-1), endothelin-3 (ET-3) and the ETB-selective receptor agonists sarafotoxin 6c (SX6c) and IRL 1620. As antagonists we used BQ-123 (ETA receptor selective), BQ-788 (ETB receptor selective) and PD 145065 (ETA/ETB receptor nonselective). We have also compared the responses of the mature peptides to their precursors human big ET-1(1-38), porcine big ET-1(1-39) and big ET-3(1-41) amide. ET-1, ET-3, SX6c and IRL 1620 produced equipotent concentration-dependent contractions of the rat stomach strips that were antagonized by PD 145065 (10(-5) M) or BQ-788 (10(-5) M) but not by BQ-123 (10-5 M). This indicates that the ETB receptor mediates contractions to the endothelins in this preparation. In preparations precontracted with PGE2 (3 x 10(-8) M), ET-1, but not SX6c (both 3 x 10(-9) M), caused a transient (< 2 min) relaxation (approx. 40% of the induced tone). This relaxation was antagonized by BQ-123 (10(-5) M) but prolonged by BQ-788, and therefore mediated by ETA receptors. A single administration of 3 x 10(-7) M ET-1, ET-3, SX6c or IRL 1620 produced contractions that reached a maximal response after 1 to 3 min. The contractions were not maintained, although responses to ET-1 or ET-3 lost their tone less rapidly than those to SX6c or IRL 1620.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Structure-activity studies of the C-terminal segment of structurally reduced analogues of ET-1.
Structurally reduced analogues of endothelin-1 (ET-1) were synthesized by linking via aminocaproic acid (Aca) the segment 3-11 of ET-1 to C-terminal fragments of various lengths [16-21, 17-21,...,21]. Analogues were studied in their linear or cyclic form in the absence or presence of a formyl group on the Trp21 side-chain, and their biologic activities were tested using guinea pig lung parenchymal strips. The absence of the first disulfide bridge and the presence of the Aca spacer caused a slight decrease in activity. The presence of His16 is essential for the contractile activity of the monocyclic peptides. Formylation of these monocyclic analogues did not modify this behavior. Similarly, linear analogues carrying S-acetamidomethyl (Acm) functions and an Aca linker were still active. However, most formylated linear derivatives were partial agonists, whereas the addition of His16 caused a decrease in contractile activity. In these latter analogues, molecular modeling studies suggested that formylation produces different conformations.
Animals↗
Endothelin-1 mediates the increase in ex vivo coronary vascular resistance induced by hemorrhagic shock in the anesthetized rat.
Cytokines such as tumor necrosis factor-alpha (TNF-alpha), given exogenously or liberated endogenously, cause the release of endothelin (ET) into the circulation. In the rat, increase in ET causes marked ex vivo coronary vasoconstriction. Hemorrhage also increases the circulating levels of cytokines such as TNF-alpha. Here we show that in rats subjected to hemorrhagic shock there is a marked increase in ex vivo coronary vascular resistance, which is mediated by ET. Hemorrhagic shock was induced in anesthetized rats by withdrawing sufficient blood to reduce mean arterial blood pressure to 40 mm Hg for a period of 30 min, after which all the withdrawn blood was retransfused over a period of 15 min. Hearts were perfused ex vivo at a constant flow of 10 ml/min according to the Langendorff technique. After 90 min in vitro, the coronary perfusion pressure in hearts removed from control rats was 76 +/- 1 mm Hg (n = 5), whereas in hearts taken from rats after hemorrhage it was 114 +/- 6 mm Hg (n = 5; p < 0.05 from control). After the same time in vitro, the coronary perfusion pressure of hearts from rats treated with TNF-alpha (4 micrograms/kg, i.v.) was 122 +/- 4 mm Hg (n = 4; p < 0.05 from control). The increases in coronary perfusion pressure caused by hemorrhagic shock or TNF-alpha were abolished by pretreating rats with the nonselective ET receptor (ETA/ETB) antagonist SB209670 (3 mg/kg, i.v.) (coronary perfusion pressure at 90 min 80 +/- 1 mm Hg after hemorrhage; 73 +/- 4 mm Hg after TNF-alpha, p < 0.05 compared to hemorrhage or TNF-alpha controls, respectively; n = 3-5). Interestingly, pretreatment with polyclonal antibodies to TNF-alpha (3 mg/kg, i.v.) did not significantly attenuate the rise in coronary perfusion pressure caused by hemorrhage. Therefore, hemorrhage followed by retransfusion causes marked coronary vasoconstriction assessed ex vivo owing to the release of ET by factors including TNF-alpha.
Anesthesia↗
Endothelin receptor antagonists: actions and rationale for their development.
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Animals↗
Comparison of PD 145065 and Ro 46-2005 as antagonists of contractions of guinea-pig airways induced by endothelin-1 or IRL 1620.
Cumulative concentrations of endothelin-1 or IRL 1620 (Suc-[Glu9,Ala11,15]endothelin-1-(8-21)), and endothelin ETB receptor selective agonist were similarly potent in contracting guinea-pig upper bronchi, whereas endothelin-1 was more potent than IRL 1620 in contracting lung parenchymal strips. PD 145065 (10(-5) M and 10(-4) M), a non-selective endothelin ETA/ETB receptor antagonist (Ac-D-Bhg-L-Leu-L-Asp-L-Ile-L-Ile-L-Trp), significantly attenuated the contractions induced by endothelin-1 in either preparation whereas Ro 46-2005 (3 x 10(-5) M and 10(-4) M), a non-peptide non-selective receptor antagonist (4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-3-methoxy-phenoxy)-4-pyrimidinyl (benzenesulfonamide)), was without effect. PD 145065 (10(-6) M and 10(-5) M) also strongly inhibited contractions induced by IRL 1620, whereas Ro 46-2005 caused much less antagonism. Thus, PD 145065 is a more potent antagonist than Ro 46-2005 of contractions induced by endothelin-1 or IRL 1620 and mediated by endothelin ETB receptors in these guinea-pig airway preparations.
Animals↗
Dual cardiovascular effects of endothelin-1 dissociated by BQ-153, a novel ETA receptor antagonist.
Endothelin-1 (ET-1) is a potent endothelium-derived vasoconstrictor peptide that elicited both vasodilator and vasoconstrictor responses in anesthetized pigs. Within 1.0 min after the first injection of ET-1 (0.4 nmol/kg, intravenously, i.v.) there was a transient decrease in mean arterial pressure (MAP 82 +/- 4 to 64 +/- 5 mm Hg; p < or = 0.01). The vasodepressor response was accompanied by reductions in left ventricular (LV) + dp/dtmax (1,834 +/- 104 to 1,493 +/- 87 mm Hg/s, p < or = 0.001), LV - dp/dt (2,600 +/- 149 to 1,865 +/- 136 mm Hg/s; p < or = 0.001) and cardiac output (CO 2.6 +/- 0.1 to 2.0 +/- 0.1 L/min, p < or = 0.001). The short (< 3.0 min) vasodilatory phase was followed by a prolonged (> 15 min) vasopressor response in which MAP (82 +/- 4 to 103 +/- 5 mm Hg; p < or = 0.001) and pulmonary arterial pressure (PAP 11 +/- 1 to 15 +/- 1 mm Hg; p < or = 0.01) increased. With each subsequent dose (0.4 nmol/kg i.v.) of ET-1, the initial vasodilatory component diminished progressively, only a monophasic vasoconstrictor response was observed after the fourth dose. The reductions in CO progressively decreased from 2.6 to 0.1 to 1.7 +/- 0.1 L/min (p < or = 0.001) by the end of the experiment. In contrast to the systemic circulation effects, ET-1 produced consistent and reproducible reductions in renal blood flow (RBF 105 +/- 16 to 21 +/- 6 mm Hg; p < or = 0.004) that lasted approximately 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Characterization of endothelin (ET) receptors in the isolated gall bladder of the guinea-pig: evidence for an additional ET receptor subtype.
1. We have characterized the receptors mediating contractions induced by endothelin-1 (ET-1), ET-2, ET-3 and the ETB-selective receptor agonists, sarafotoxin 6c (SX6c), IRL 1620, BQ-3020, [Ala1,3,11,15]ET-1 and ET (16-21) in strips of the isolated gall bladder of the guinea-pig (GPGB). We used as antagonists BQ-123 (ETA receptor selective) and PD 145065 (ETA/ETB receptor non-selective). 2. ET-1, ET-2 and ET-3 (10(-10) M to 3 x 10(-7) M) caused similar slowly-developing concentration-dependent contractions of the GPGB. Contractile effects induced by ET-1, ET-2 or ET-3 (at 3 x 10(-7) M) were also similar (230 +/- 25, 241 +/- 7 and 287 +/- 37% of that to histamine at 5 x 10(-6) M, n = 7, 6, 12, respectively). However, the threshold concentration for ET-1 or ET-2 was 10(-10) M whereas it was 3 x 10(-9) M for ET-3. 3. SX6c (10(-10) M to 3 x 10(-7) M) also caused slowly-developing concentration-dependent contractions at a threshold concentration of 10(-10) M (n = 16). However, the contraction caused by SX6c at 3 x 10(-7) M was 116 +/- 9% of that to histamine at 5 x 10(-6) M, which was half of that induced by the same concentration of the ET isopeptides. The contraction induced by IRL 1620 at 3 x 10-7 M (n = 9) was 43 +/- 9% of that to histamine at 5 x 10-6 M, which was one fifth of that produced by the same concentration of ET-1. Contractions induced by BQ-3020 or [Ala1,3,11,15]ET-I at 3 x 10-7 M were even less than those produced by IRL 1620. ET (16-21) was inactive up to 10-5 M. Addition of a concentration of 3 x 10-7 M of ET-1 to tissues with developed contractions induced by the bolus addition of 3 x 10-7 M SX6c caused a further contraction of the GPGB to the level observed with ET-1 alone at 3 x 10-7M (n = 8).4. BQ-123 (10-5 M) did not affect the concentration-response curve to ET-1 and the contraction induced by 3 x 10- M was also not affected (n = 5; 239 +/- 19% of histamine at 5 x 10-6 M). PD 145065(10-5 M) shifted the ET-1 concentration-response curve to the right and the contraction induced by ET-1at 3 x 10-7 M was inhibited by 15% (n = 6; NS). A higher concentration of BQ-123 (10-4 M) caused a significant shift to the right of the ET-1 concentration-response curve similar to that caused by PD 145065 (10-s M) and caused a 24% (n = 6; NS) inhibition of the contractions induced by ET-1 at 3 x 10-7 M. PD 145065 (10-4 M) abolished contractions induced by ET-1 (up to 10- M) and inhibited the response to ET-1 at 3 x 10-7 M by 52% (n = 4; P< 0.05).5. Contractions induced by ET-3 were more sensitive to inhibition by the antagonists. BQ-123 (10-6,10-5 or 10-4 M) inhibited responses to 3 x 10-7 M ET-3 by 66, 71 and 83%, respectively (n = 5, 5, 3;P< 0.05). PD 145065 (10-6, 10-5 or 10-4 M) attenuated more strongly than did BQ-123 the contractions induced by ET-3. For instance, the contractions caused by ET-3 at 3 x 10-7 M were decreased by 73 and 80% (n = 5, 5; P<0.05) in the presence of PD 145065 (10-6 or 10-5 M, respectively). PD 145065(10-4 M) completely abolished contractions to ET-3 (n = 4; up to 3 x 10-7M).6. Contractions induced by SX6c, especially those observed at concentrations lower than 10-8 M, were attenuated by BQ-123 (up to 10-4 M). PD 145065 (10-5M) shifted to the right the concentration response curve to SX6c and inhibited by 38% (P<0.05) the contractions induced by 3 x 10-7M.However, the contractions induced by a bolus addition of a high concentration of SX6c (3 x 10-7 M)and the subsequent addition of an identical concentration of ET-1 on top of SX6c were not affected byBQ-123 (10-6 or 10-5 M).7. These results suggest that ETB receptors are involved in the contractions induced by endothelins in the GPGB. However, SX6c and other selective ETB agonists produced only half or less than half of the contractile response induced by non-selective agonists. In addition, the responses to ET-1 but not to ET-3, were insensitive to the antagonist action of BQ-123 at 10-5 M whereas BQ-123 or PD 145065 at 10-5 M strongly antagonized contractions induced by ET-3. Finally, BQ-123 at 10-4 M inhibited contractions to ET-1 and SX6c. Thus, within the GPGB there may well be additional ET receptor(s) not conforming to the established ETA/ETB receptor subtype classification, as well as ETB receptors.
Amino Acid Sequence↗
Characterization of ETB receptors mediating contractions induced by endothelin-1 or IRL 1620 in guinea-pig isolated airways: effects of BQ-123, FR139317 or PD 145065.
1. We have characterized the receptors mediating contractions to endothelin-1 (ET-1) or IRL 1620, an ETB receptor selective agonist, in isolated strips of tissue prepared from different parts of the guinea-pig airways. We used as antagonists BQ-123 and FR139317 (ETA receptor-selective) and PD 145065 (ETA/ETB receptor non-selective). 2. ET-1 and IRL 1620 (10(-10) M to 10(-6) M) caused similar concentration-dependent contractions of strips of guinea-pig trachea and upper bronchus. In the guinea-pig trachea without epithelium or lung parenchyma, IRL 1620 was less potent than ET-1. 3. In the trachea, contraction to ET-1 (< 10(-8) M) was preceded by a transient relaxation which was inhibited by BQ-123 (10(-5) M) or FR 139317 (10(-5) M) or by the removal of the epithelium. The concentration-response curve to ET-1 in the trachea was shifted to the right by PD 145065 (10(-5) M to 10(-4) M). PD 145065 (10(-4) M) also inhibited the response to ET-1 (3 x 10(-7) M) by 55%. Contractions induced by IRL 1620 were not affected by BQ-123 (10(-6) M) or FR139317 (10(-6) M) but were significantly attenuated by 10(-5) M of either antagonist. PD 145065 at 10(-6) M strongly attenuated and at 10(-5) M abolished contractions induced by IRL 1620. 4. In the trachea, removal of the epithelium potentiated the effects of both agonists. BQ-123 (10-5 M)had no effect on contractions of the trachea without epithelium induced by ET-1, but FR139317 (10-5 M)caused a significant inhibition. PD 145065 (10-5 M to 10-4 M) caused a shift to the right of the ET-1 concentration-response curve without affecting the contractile effect at 3 x 10-7 M. All three antagonists inhibited contractions induced by IRL 1620.5. In the upper bronchus, BQ-123 (10-5 M) did not affect contractions induced by ET-1, whileFR139317 (10-5 M) attenuated (20-26%) only contractions induced by 1-3 x 10-7 M ET-1. PD 145065(10-5 M to 10-4 M) caused a shift to the right of the ET-1 concentration-response curve. The contractions induced by IRL 1620 were inhibited by BQ-123 or FR139317 (10-5M to 10-4 M). PD 145065(10-6 M) strongly inhibited contractions induced by IRL 1620 and PD 145065 (10-5 M) totally abolished them.6. The contractile action of ET-1 in the lung parenchyma was significantly and similarly attenuated by BQ-123 (10-5 M) or indomethacin (10-5 M), while FRI39317 (10-5 M) was less effective. PD 145065(10-6 to 10-5 M) inhibited contractions to ET-1. IRL 1620, which is less potent than ET-1 in this preparation, was antagonized by PD 145065 (10-5 to 10-6 M) but unaffected by BQ-123 (10-6 M to10-5M) or FR139317 (10-6 M).7. Thus, ETB receptors mediate contractions to ET-1 in all four guinea-pig airway preparations. In addition, contractions to ET-1 in the trachea and lung parenchyma are mediated in part by ETA receptors. In the latter tissue, these ETA receptors mediate contraction through the release of cyclooxygenase metabolites. Similarly, ETA receptors located on the epithelial cells also mediate the release of prostanoids in the trachea with epithelium but they are responsible for transient relaxations. Interestingly,contractions induced by IRL 1620 were more susceptible to inhibition by the different antagonists,most probably because it binds to the endothelin receptors in a reversible manner. High concentrations(10-5 M) of ETA-selective antagonists also inhibit responses to IRL 1620, most probably by an effect at ETB receptors in both the trachea and the upper bronchus.
Animals↗
Endothelin ETA and ETB receptors mediate vasoconstriction and prostanoid release in the isolated kidney of the rat.
Endothelin-1 or sarafotoxin 6c (10(-12) to 10(-9) M) induced concentration-dependent increases in perfusion pressure in the isolated perfused kidney of the rat, and increased the release of 6-keto-prostaglandin F1 alpha, prostaglandin E2 and prostaglandin F2 alpha. The pressor effects of endothelin-1 were partially antagonised by BQ-123 (10(-6) M) and more strongly so by the endothelin ETA/B receptor non-selective antagonist PD 145065 (10(-6) M). PD 145065, but not BQ-123, also completely blocked the pressor effects of sarafotoxin 6c. The releases of prostanoids induced by endothelin-1 were greatly reduced by infusion of either BQ-123 or PD 145065, whereas PD 145065, but not BQ-123, strongly antagonised the releases induced by sarafotoxin 6c. These results indicate that the vasoconstrictions and the prostanoid releases induced by the endothelin/sarafotoxin peptides are mediated by the activation of both endothelin ETA and endothelin ETB receptors in the isolated perfused kidney of the rat.
6-Ketoprostaglandin F1 alpha↗
Relaxation by dexamethasone of isolated guinea-pig airways precontracted with endothelin-1.
Dexamethasone (1-100 microM) produced concentration-dependent relaxations of isolated guinea-pig tracheal and bronchial strips precontracted with endothelin-1 (100 nM), which were not affected by the removal of the epithelium. Dexamethasone neither induced relaxation of tissues precontracted with carbachol (0.1 microM) or KCl (25 mM) nor inhibition of endothelin-1 (1-100 nM)-induced peak contractions after 10-min preincubation of the tissues with dexamethasone. These findings indicate that acute corticosteroid treatment can reverse the bronchoconstrictor action of endothelin-1.
Animals↗
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.
Anaphylaxis↗
Structure-activity relationships of ET-1 and selected analogues in the isolated guinea pig trachea: evidence for the existence of different ETB receptor subtypes.
We tested endothelin-1 (ET-1) derivatives on the guinea pig trachea (GPT) and compared their biologic activities to ET-1 in this tissue and to published reports using similar analogues in different assay systems, such as vascular smooth-muscle preparations. Six ET-1 analogues with no intramolecular disulfide bridges and/or with the substitution of one or more amino acids at position 7 and/or 21 were synthesized. Oxidation of the Met7 or formylation of the indole moeity of the Trp21 did not affect the biologic activity of ET-1 on the trachea. Acetamidomethylation of the Cys1-15 decreased the potency and intrinsic activity, but not as much as had been reported on vascular preparations. However, modifications involving one or both disulfide bridges, combined with formylation of the Trp21, yielded analogues with very weak contractile activity. These findings, when compared with the biologic activity of ET-1 analogues in other assay systems, suggest that a different population of receptors, probably non-ETA (i.e., an ETB subtype different from the one reported to be involved in ET-1-induced binding or activities in rabbit pulmonary artery or in rat cerebellum or vasculature) is present and responsible for ET-1-induced bronchoconstriction on the GPT.
Amino Acid Sequence↗
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 microM) 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 microM) and were almost completely attenuated by oxyhaemoglobin (5 microM) and NG-monomethyl-L-arginine (L-NMMA, 100 microM), an inhibitor of nitric oxide synthesis, but were not altered by indomethacin (10 microM). 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.
Animals↗
Endothelins: circulating plasma levels and presence in other biologic fluids.
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Animals↗
Characterization of ATP-diphosphohydrolase activities in the intima and media of the bovine aorta: evidence for a regulatory role in platelet activation in vitro.
The inner layer of the aorta contains the enzyme ATP diphosphohydrolase (ATPDase: EC 3.6.1.5) which catalyzes the sequential phosphorolysis of ATP----ADP----AMP. Two zones of the inner layer, the intima and media, were separated and both were shown to contain ATPDase activity of similar specific activity (0.08 and 0.10 U/mg protein, respectively). However, the media exhibited about 100-times more enzyme activity than the intima. Both preparations were virtually identical with respect to pH optima (7.5), migration patterns after electrophoresis under non-denaturing conditions, relative rates of ATP and ADP hydrolysis and potency to inhibit ADP-induced platelet aggregation in both human platelet-rich plasma and whole blood. The IC50 values for ADP (2 microM)-induced aggregation were 6.8 and 12.9 mU/ml in platelet-rich plasma and whole blood, respectively. Addition of ATPDase to platelets pre-aggregated with ADP resulted in a dose-dependent disaggregation in platelet-rich plasma (IC50 4.9 mU/ml), but not in whole blood. When both ATPDase (5.6-58.7 mU/ml) and ATP (0.5-10 microM) were added to platelet-rich plasma, there was an immediate dose-dependent aggregation of platelets followed by a slowly developing disaggregation. These data show that ATPDase is present in both the intima and media layers of bovine aorta and suggest a dual role for this enzyme in platelet activation. By converting ATP released from damaged cells into ADP, the enzyme could facilitate platelet aggregation at the site of vascular injury, whereas the subsequent conversion of ADP to AMP could inhibit or reverse platelet aggregation. The consequence of these activities would be to control the growth of a platelet thrombus.
Adenosine Triphosphate↗
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.
6-Ketoprostaglandin F1 alpha↗