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-I and ET-3 induced concentration-dependent contractions which were normalized to the response produced by 60 mM Kf. ET-I produced a maximum contraction (E(max)) amounting to 151+/-17% of the K+ response. The pEC(50) for this agonist was 8.64+/-0.17. The effect of ET-3 was less pronounced (E(max): 71+/-22% and pEC(50): 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 mu M) abolished the contractile responses to ET-3, and produced a parallel rightward shift of the ET-I concentration-response curve without changing the maximal effect. FR139317 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 degrees 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 (pEC(50): 8.56+/-0.15) and achieved a greater maximum effect (E(max): 116+/-19%). Responses were not antagonised by FR139317 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 pEC(50) (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-l-induced concentration-response curve, whereas FR139317 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-I 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 I 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(B) receptors. These in vitro data suggest plasticity in the smooth muscle cell expression of contractile ET(B) receptors.
