Effects of calcium entry blockers on contractions evoked by endothelin-1, [Ala3,11]endothelin-1 and [Ala1,15]endothelin-1 in rat isolated aorta.

1. The aim of the present study was to examine the contractile responses evoked by the recently characterized vasoactive peptide endothelin-1 (ET-1) and by two of its structural analogues, [Ala3,11]ET-1 and [Ala1,15]ET-1 in endothelium-denuded rat isolated aorta, and also to assess the extent of dependence of these responses on extracellular calcium entry. 2. ET-1 (0.3 to 10 nM), [Ala3,11]ET-1 (2.25 to 225 nM) and [Ala1,15]ET-1 (0.04 to 1.36 microM) evoked concentration-dependent contractions in normal, calcium-containing medium with the order of potency: ET-1 greater than [Ala3,11]ET-1 greater than [Ala1,15]ET-1. 3. Preincubation of tissues for 60 min with diltiazem (1 microM) induced a significant 3 fold rightward shift of concentration-effect curves to ET-1 without affecting maximal responses elicited by 10 nM of this peptide, whereas the same treatment failed to modify concentration-effect curves to [Ala3,11]ET-1. 4. Preincubation of tissues for 60 min with nifedipine (0.1 or 1 microM) markedly inhibited contractions elicited by either ET-1 (10 nM) or [Ala1,15]ET-1 (0.41 microM). Furthermore, when added cumulatively to tissues maximally contracted by ET-1 (10 nM), nifedipine (3 nM to 1 microM) induced concentration-dependent relaxations with an IC50 value of 21.8 +/- 5.9 nM. Maximal relaxation to nifedipine, 1 microM, amounted to 56.9 +/- 11.5%. 5. Submaximal concentrations of ET-1 (3 nM), [Ala3,11]ET-1 (75 nM) and [Ala1,15]ET-1 (0.41 microM), gave about 85% of maximal contractions elicited by noradrenaline (1 microM) in normal, calcium-containing medium. These contractile responses were all reduced by about 70% in calcium-free medium. These contractile responses were all reduced by about 70% in calcium-free medium. Pretreatment of tissues with diltiazem (1 microM) or with nifedipine (0.1 or 1 microM) did not affect these residual contractions. 6. Upon readdition of calcium (10 microM to 10mM) to tissues in calcium-free medium, preincubated with submaximal concentrations of one or the other of the peptides, concentration-dependent contractions were elicited with EC50 values of 1.21 + 1.1 mm (ET-1-exposed rings), 74.6 + 9.1 microM ([Ala31 ']ET-1-exposed rings) and 102 + 27 microM ([Ala' 15]ET-1-exposed rings). Calcium-induced responses were significantly inhibited by diltiazem (1 microM) in both ET-1- and [Ala3"1 ']ET-1-exposed tissues and by nifedipine (0.1 and 1 microM) in ET-1-exposed tissues. However, nifedipine did not significantly affect calcium induced responses in [Ala"'15]ET-1-exposed rings. 7. Readdition of calcium to a calcium-free medium containing 40mm K+, evoked concentrationdependent responses that were unaffected by the presence of ET-1 (3 nM). 8. Overall, these results indicate that contractions to ET-1 and its analogues in rat aorta are to some extent dependent on extracellular calcium. However, the mode of antagonism by diltiazem and nifedipine of responses to the peptides in normal medium, as well as of contractions induced by readdition of calcium in peptide-exposed tissues in calcium-free medium, argue against a direct activation by endothelin or its analogues of L-type calcium channels. Finally, removal of one or the other of the disulphide bonds of ET-1 reduces the potency of the peptide.