January 1999, Volume 126, Issue 1, Pages 103 - 110

Original Article

Transcriptional down-regulation of the rabbit pulmonary artery endothelin B receptor during phenotypic modulation R. Owe-Young1,4, C.G. Schyvens2, R.A. Qasabian1, A.D. Conigrave3, P.S. Macdonald2 & D.J. Williamson1,5 1Centre for Immunology, St. Vincent's Hospital, Darlinghurst, NSW 2010, Australia     2Cardiopulmonary Transplant Unit, St. Vincent's Hospital, Darlinghurst, NSW 2010, Australia     3Department of Biochemistry, University of Sydney, NSW 2006, Australia     4Author for correspondence: E-mail: robert@cfi.unsw.edu.au 5Current address: Department of General Medicine, Sir Charles Gairdner Hospital, Verdun St. Nedlands WA 6009, Australia

Keywords

endothelin receptor;   pulmonary artery;   vascular smooth muscle;   phenotypic modulation;   intracellular calcium;   mRNA

Abstract

1   We confirmed that endothelium-independent contraction of the rabbit pulmonary artery (RPA) is mediated through both an endothelin A (ETAR) and endothelin B (ETB2R) receptor. 2   The response of endothelium-denuded RPA rings to endothelin-1 (ET-1, pD2=7.84±0.03) was only partially inhibited by BQ123 (10 µM), an ETAR antagonist. 3   Pretreatment with 1 nM sarafotoxin S6c (S6c), an ETBR agonist, desensitized the ETB2R and significantly attenuated the response to ET-3 (pD2=7.40±0.02 before, <6.50 after S6c). 4   Pretreatment with S6c had little effect on the response to ET-1, but BQ123 (10 µM) caused a parallel shift to the right of the residual ETAR-mediated response to ET-1 (pD2=7.84±0.03 before S6c, 7.93±0.03 after S6c, 6.81±0.05 after BQ123). 5   Binding of radiolabelled ET-1 to early passage cultures of RPA vascular smooth muscle cells (VSMC) displayed two patterns of competitive displacement characteristic of the ETAR (BQ123 pIC50=8.73±0.05) or ETB2R (S6c pIC50=10.15). 6   Competitive displacement experiments using membranes from late passage VSMC confirmed only the presence of the ETAR (ET-1 pIC50=9.3, BQ123 pIC50=8.0, S6c pIC50<6.0). 7   The ETAR was functionally active and coupled to rises in intracellular calcium which exhibited prolonged homologous desensitization. 8   Using a reverse transcriptase polymerase chain reaction for the rabbit ETB2R, we demonstrated the absence of mRNA expression in phenotypically modified VSMC. 9   We conclude that the ETB2R expressed by VSMC which mediates contraction of RPA is rapidly down-regulated at the transcriptional level during phenotypic modulation in vitro.

Received 7 September 1998; Revised 5 October 1998; Accepted 8 October 1998

^© Macmillan Publishers Ltd 1999