Increased levels of endothelin ETB receptor mRNA in human omental arteries after organ culture: quantification by competitive reverse transcription-polymerase chain reaction.
1. Using competitive reverse transcription-polymerase chain reaction (RT-PCR) and in vitro pharmacology, smooth muscle endothelin ETB receptor expression was studied in segments of human omental artery, fresh and after organ culture for 1 and 5 days. 2. The competitive RT-PCR assay used in the present study uses an internal RNA standard bearing a 69 b.p. deletion in order to control all steps of the reaction, including the RT step. Control experiments showed linearity over five subsequent 1:10 dilutions and a wide range of cycle numbers. The assay was able to quantify subattomolar concentrations in samples under 1 microgram total RNA, making it possible to measure mRNA expression even in small tissue biopsies. 3. In fresh arteries, ETB mRNA levels were 0.19 +/- 0.05 amol/microgram total RNA (range 0.03-0.42 amol/microgram; n = 8). After organ culture, an increase in ETB mRNA levels by 317 +/- 28 and 288 +/- 12% was found at days 1 and 5, compared with fresh arteries, respectively. 4. In vitro pharmacology showed that endothelin (ET)-1 induced a strong and potent contraction in fresh arteries, whereas the selective ETB receptor agonist IRL 1620 failed to induce a significant contraction. The ET-1-induced contraction was not altered in potency or Emax after organ culture for 1 and 5 days. In contrast, IRL 1620 induced a clear contraction after 1 day, which increased further in both Emax and potency after 5 days organ culture. 5. Our results indicate that a massive new transcription of ETB receptor mRNA is induced by organ culture, resulting in functional contractile ETB receptors on the smooth muscle layer.
Möller
S
S
Department of Internal Medicine, Lund University Hospital, Sweden. sebastian.moller@med.lu.se
Adner
M
M
Edvinsson
L
L
D001158:Arteries / Q000187:drug effects / Q000378:metabolism; D001483:Base Sequence; D019332:Endothelin-1 / Q000494:pharmacology; D016232:Endothelins / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D009852:Omentum / Q000098:blood supply; D009924:Organ Culture Techniques; D010446:Peptide Fragments / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis*; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D018401:Sample Size; D013997:Time Factors; D014158:Transcription, Genetic
D019332:Endothelin-1; D016232:Endothelins; D010446:Peptide Fragments; D012333:RNA, Messenger; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; C074308:sovateltide