Plasticity of contractile endothelin‐B receptors in human arteries after organ culture

Abstract

1 The pharmacology and mRNA expression of endothelin (ET) receptors in human omental arteries were characterized by use of functional contractile assays and the reverse transcriptase-polymerase chain reaction (RT-PCR). 2 In freshly obtained segments of human omental arteries, ET-1 and ET-3 induced concentration-dependent contractions which were normalized to the response produced by 60 mM K⁺. ET-1 produced a maximum contraction (E_max) amounting to 151 ± 17% of the K⁺ response. The pEC₅₀ for this agonist was 8.64 ± 0.17. The effect of ET-3 was less pronounced (E_max: 71 ± 22% and pEC₅₀: 6.69 ± 0.17) than that of ET-1. The ET receptors involved were characterized with FR139317 (a selective ET_A receptor antagonist), PD 145065 (a mixed ET_A and ET_B receptor antagonist) and BQ 788 (an ET_B receptor antagonist). A high concentration of these antagonists (10 μm) abolished the contractile responses to ET-3, and produced a parallel rightward shift of the ET-1 concentration-response curve without changing the maximal effect. FR 139317 and PD 145065 were equally effective while BQ 788 was much less effective. This is consistent with ET_A receptors mediating contraction in human omental arteries. 3 Arterial segments cultured for 5 days in serum-free Dulbecco's medium at 37°C under sterile and humidified conditions retained contractility although responses to 60 mM K⁺ were somewhat reduced. ET-3 was significantly more potent in the cultured arteries (pEC₅₀: 8.56 ± 0.15) and achieved a greater maximum effect (E_max: 116 ± 19%). Responses were not antagonised by FR 139317 but were competitively blocked by PD 145065 and BQ 788 with the latter antagonist being the more potent. In contrast E_max (179 ± 17%) and pEC₅₀ (8.66 ± 0.23) values for ET-1 were not significantly different from those obtained with fresh arteries. PD 145065 still demonstrated a rightward shift of the ET-1-induced concentration-response curve, whereas FR 139317 and BQ 788 caused non-significant shifts. These findings suggest that functional ET_B receptors contribute significantly to the endothelin contractile response in cultured arteries. 4 Two-site analysis of the ET-1 induced concentration-response curve from cultured arteries suggests that ET_B receptors, at the high potency component, and ET_A receptors, at the low potency component, contribute both to the contractile response in relative proportion of 70% and 30%, respectively. Further analysis suggested that the ET_A receptor would be capable of evoking at least 75% of the ET-1 contraction in the absence of ET_B receptors, although with a lower potency as compared to fresh arteries. 5 Electrophoresis of RT-PCR products from the smooth muscle layer of freshly obtained human arteries indicated the presence of mRNA for both ET_A and ET_B receptors. Arteries cultured for 1 and 5 days demonstrated an increase of mRNA for the ET_B receptor as compared to the ET_A receptor. The identities of the PCR products were verified by restriction enzyme digestion. 6 In freshly obtained human omental arteries, the contractile effects of endothelins appear to be mediated predominantly by the ET_A receptor subtype, with a negligible contribution by ET_B receptors. Cultured arterial segments, however, exhibited a substantial ET_B receptor mediated contractile response and an increase in ET_B receptor mRNA content, consistent with an upregulation of functional ET_Breceptors. These in vitro data suggest plasticity in the smooth muscle cell expression of contractile ET_Breceptors.