Increased levels of endothelin ETB receptor mRNA in human omental arteries after organ culture: quantification by competitive reverse transcription-polymerase chain reaction

Abstract

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.