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.
Department of Internal Medicine, Lund University Hospital, Sweden. sebastian.moller@med.lu.se
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Möller
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Adner
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Edvinsson
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