PLASTICITY OF CONTRACTILE ENDOTHELIN-B RECEPTORS IN HUMAN ARTERIES AFTER ORGAN-CULTURE

1 The pharmacology and mRNA expression of endothelin (ET) receptors in human omental arteries were characterized by use of functional contra ctile 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 nor malized 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 pE C(50) for this agonist was 8.64+/-0.17. The effect of ET-3 was less pr onounced (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 select ive ET(A) receptor antagonist), PD 145065 (a mixed ET(A) and ET(B) rec eptor antagonist) and BQ 788 (an ET(B) receptor antagonist). A high co ncentration of these antagonists (10 mu M) abolished the contractile r esponses to ET-3, and produced a parallel rightward shift of the ET-I concentration-response curve without changing the maximal effect. FR13 9317 and PD 145065 were equally effective while BQ 788 was much less e ffective. This is consistent with ET(A) receptors mediating contractio n in human omental arteries. 3 Arterial segments cultured for 5 days i n serum-free Dulbecco's medium at 37 degrees C under sterile and humid ified conditions retained contractility although responses to 60 mM K were somewhat reduced. ET-3 was significantly more potent in the cult ured arteries (pEC(50): 8.56+/-0.15) and achieved a greater maximum ef fect (E(max): 116+/-19%). Responses were not antagonised by FR139317 b ut were competitively blocked by PD 145065 and BQ 788 with the latter antagonist being the more potent. In contrast E(max) (179+/-17%) and p EC(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, wh ereas FR139317 and BQ 788 caused non-significant shifts. These finding s suggest that functional ET(B) receptors contribute significantly to the endothelin contractile response in cultured arteries. 4 Two-site a nalysis 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 t o the contractile response in relative proportion of 70% and 30%, resp ectively. 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 fres h arteries. 5 Electrophoresis of RT-PCR products from the smooth muscl e layer of freshly obtained human arteries indicated the presence of m RNA 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 compar ed to the ET(A) receptor. The identities of the PCR products were veri fied by restriction enzyme digestion. 6 In freshly obtained human omen tal arteries, the contractile effects of endothelins appear to be medi ated predominantly by the ET(A) receptor subtype, with a negligible co ntribution by ET(B) receptors. Cultured arterial segments, however, ex hibited a substantial ET(B) receptor mediated contractile response and an increase in ET(B) receptor mRNA content, consistent with an upregu lation of functional ET(B) receptors. These in vitro data suggest plas ticity in the smooth muscle cell expression of contractile ET(B) recep tors.