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Endothelin-1 and its Effects on Isolated Respiratory Tissue.

Peachey, Julie A. (1992) Endothelin-1 and its Effects on Isolated Respiratory Tissue. Doctoral thesis, University of Surrey (United Kingdom)..

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Abstract

1. The effects of endothelin-1 (ET-1), a novel 21 amino acid peptide, were examined on isolated tracheal smooth muscle. Tracheal chains, prepared from the trachea of both rat and guinea-pig, were cumulatively dosed with ET-1 (0.01-1μM) following construction of a cumulative concentration response curve to the reference contractile agonist, carbachol (CCh, 0.1-100μM). The mechanism of action and modulation of the ET-1 induced response on tracheal smooth muscle, by various agents has been examined. 2. On rat tracheal smooth muscle cumulative dosing with ET-1 (0.01-1μM), induced a consistent contractile response with a calculated EC50 value of 0.22±0.085μM and a maximum contractile tension induced by 1μM ET-1 of 0.67±0.090g. In contrast, the response of guinea-pig tracheal smooth muscle to cumulative dosing with ET-1 was variable. ET-1 induced a biphasic response, a transient relaxation which was followed by a contractile response. Calculation of EC50 values (1.59±0.37μM) was only possible in those tissues which displayed concentration response curves >50% whereas in others, the response achieved only 25-30% of the maximum contractile tension induced by CCh and in these tissues the relaxation responses were more prevalent. 3. Incubation of both rat and guinea-pig tracheal smooth muscle with 10uM of bestatin, captopril, phosphoramidon or thiorphan failed to affect the ET-1 induced response, indicating that ET-1 is not a substrate for the aminopeptidase, angiotensin converting and neutral endopeptidase enzymes on tracheal smooth muscle. 4. Incubation of both rat and guinea-pig tracheal smooth muscle in a reduced Ca2+ containing buffer had no effect on the ET-1 induced contractile responses in either preparation, but did potentiate the relaxation responses induced by 0.05μM, 0.5μM and 1μM ET-1 on guinea-pig tracheal smooth muscle. 5. In the presence of 10μM nicardipine, the ET-1 induced response on rat tracheal smooth muscle was attenuated, demonstrating that influx of extracellular Ca2+ via the dihydropyridine Ca2+ channels is necessary for the full expression of constrictor activity. On guinea-pig tracheal smooth muscle however, preincubation with nicardipine had no effect on the ET-1 induced relaxant and contractile responses, suggesting that influx of extracellular Ca2+ via the dihydropyridine sensitive Ca2+ channels is not essential for the constrictor activity of ET-1 in this tissue preparation. 6. Incubation of rat tracheal smooth muscle with 25μM indomethacin prior to exposure to ET-1, had no effect on the ET-1 induced response, indicating that the cyclooxygenase metabolites of arachidonic acid have no part in the response induced by the peptide. In contrast, in the presence of 25 μM indomethacin, the ET-1 induced relaxation responses observed on guinea-pig tracheal smooth muscle were abolished and the contractile response potentiated. It is therefore suggested that the ET-1 induced relaxation responses are mediated by the ET-1 induced release of the relaxant products of the cyclooxygenase enzyme which limit the constrictor activity of the peptide in this tissue preparation. 7. Removal of the tracheal epithelium from the tracheal smooth muscle of the rat and guinea-pig had no effect on the subsequent response to the peptide, indicating that the tracheal epithelium does not modulate the ET-1 induced effects. However, preincubation of epithelium denuded guinea-pig tracheal tissues with indomethacin abolished the ET-1 induced relaxation responses and produced a concentration response curve with a much steeper relationship than that observed on epithelium intact tissues in the presence of indomethacin. These observations suggest that the tracheal smooth muscle releases indomethacin sensitive and the epithelium indomethacin insensitive relaxant factors in response to ET-1, which then limit the constrictor action of the peptide. 8. The response of rat tracheal smooth muscle to ET-1 was unaffected by preincubation with the lipoxygenase enzyme inhibitor nordihydroguaiaretic acid (NDGA) (10μM), indicating the non-involvement of the lipoxygenase metabolites of arachidonic acid in the ET-1 induced response, whereas, preincubation of guinea-pig tracheal smooth muscle with NDGA resulted in a marked inhibition of the ET-1 induced contractile response, indicating that the lipoxygenase metabolites of arachidonic acid are intimately involved in the contractile response. 9. Rat tracheal smooth muscle was insensitive to the actions of platelet activating factor (PAF) and therefore the effects of thePAF antagonist [1-O-Hexadecyl-2-acetyl-sn-glycero-3-phospho(N,N,N-trimethyl)hexanolamine] were not evaluated. On guinea-pig tracheal smooth muscle, PAF induced relaxation responses which were sensitive to the actions of 1nM PAF antagonist. The contractile responses induced by ET-1 were unaffected by preincubation with the PAF antagonist (1nM) indicating the noninvolvement of PAF. In contrast, the ET-1 induced relaxation responses were potentiated at the higher concentrations of the peptide. 10. Repeat cumulative dosing with ET-1 (0.01μM) on both rat and guinea-pig tracheal smooth muscle resulted in a marked attenuation of the contractile response, indicating that ET-1 induces tachyphylaxis on repeat dosing. On guinea-pig tracheal tissues however, the degree of tachyphylaxis was dependent on the initial response to the peptide; those tissues where the initial response ET-1 was less than 50% of the maximum contractile tension induced by CCh, displayed no tachyphylaxis, whereas those tissues which displayed good contractile responses on the initial exposure to the peptide, exhibited marked tachyphylaxis upon repeat dosing. 11. Repeat single dosing with ET-1 (0.05, 0.5 and 1μM) on rat tracheal smooth muscle showed tachyphylaxis, the degree of tachyphylaxis was greatest at the higher concentrations of the peptide. Repeat single dosing with ET-1 (0.05, 0.5 and 1μM) on guinea-pig tracheal smooth muscle induced variable responses. At 0.05μM ET-1 no tachyphylaxis was observed; at 0.5μM ET-1 tachyphylaxis to the ET-1 induced relaxation responses only was observed; at 1uM ET-1 tachyphylaxis to the ET-1 induced contractile responses was observed. In the presence of indomethacin the relaxant responses were abolished and the potentiated contractile responses displayed marked tachyphylaxis, the higher the concentration of ET-1, the greater the degree of tachyphylaxis. 12. Binding studies performed with [125I]ET-1 indicated the presence of saturable binding sites for the peptide on the tracheal smooth muscle of both the rat and the guinea-pig, however, such studies were limited by the high degree of non-specific binding. Increasing the concentration of protein used potentiated the saturable binding and reduced the non-specific binding to an acceptable level in rat tissues, whereas only a slight potentiation of saturable binding was observed with the guinea-pig.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors : Peachey, Julie A.
Date : 1992
Additional Information : Thesis (Ph.D.)--University of Surrey (United Kingdom), 1992.
Depositing User : EPrints Services
Date Deposited : 06 May 2020 14:23
Last Modified : 06 May 2020 14:30
URI: http://epubs.surrey.ac.uk/id/eprint/856177

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