Transcriptional down-regulation of the rabbit pulmonary artery endothelin B receptor during phenotypic modulation.
1. We confirmed that endothelium-independent contraction of the rabbit pulmonary artery (RPA) is mediated through both an endothelin A (ET(A)R) and endothelin B (ET(B2)R) 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 microM), an ET(A)R antagonist. 3. Pretreatment with 1 nM sarafotoxin S6c (S6c), an ET(B)R agonist, desensitized the ET(B2)R 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 microM) 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 ET(A)R (BQ123 pIC50 = 8.73 +/- 0.05) or ET(B2)R (S6c pIC50 = 10.15). 6. Competitive displacement experiments using membranes from late passage VSMC confirmed only the presence of the ET(A)R (ET-1 pIC50 = 9.3, BQ123 pIC50 = 8.0, S6c pIC50 < 6.0). 7. The ET(A)R 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 ET(B2)R, we demonstrated the absence of mRNA expression in phenotypically modified VSMC. 9. We conclude that the ET(B2)R expressed by VSMC which mediates contraction of RPA is rapidly down-regulated at the transcriptional level during phenotypic modulation in vitro.
Owe-Young
R
R
Centre for Immunology, St. Vincent's Hospital, Darlinghurst, NSW, Australia. robert@cfi.unsw.edu.au
Schyvens
C G
CG
Qasabian
R A
RA
Conigrave
A D
AD
Macdonald
P S
PS
Williamson
D J
DJ
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1 / Q000494:pharmacology; D019334:Endothelin-3 / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008297:Male; D008970:Molecular Weight; D009119:Muscle Contraction / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D010456:Peptides, Cyclic / Q000494:pharmacology; D010641:Phenotype; D011651:Pulmonary Artery / Q000737:chemistry* / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D002148:Calmodulin-Binding Proteins; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1; D019334:Endothelin-3; D010456:Peptides, Cyclic; D012333:RNA, Messenger; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; C072247:cyclo(Trp-Asp-Pro-Val-Leu); D002118:Calcium