INCREASED LEVELS OF ENDOTHELIN ETB RECEPTOR MESSENGER-RNA 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 recept or expression was studied in segments of human omental artery, fresh a nd after organ culture for 1 and 5 days. 2. The competitive RT-PCR ass ay used in the present study uses an internal RNA standard bearing a 6 9 b.p. deletion in order to control all steps of the reaction, includi ng the RT step. Control experiments showed linearity over five subsequ ent 1:10 dilutions and a wide range of cycle numbers. The assay was ab le to quantify subattomolar concentrations in samples under 1 mu g tot al RNA, making it possible to measure mRNA expression even in small ti ssue biopsies. 3. In fresh arteries, ETB mRNA levels were 0.19+/-0.05 amol/mu g total RNA (range 0.03-0.42 amol/mu g; n = 8). After organ cu lture, an increase in ETB mRNA levels by 317+/-28 and 288+/-12% was fo und 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 recep tor agonist IRI, 1620 failed to induce a significant contraction. The ET-1-induced contraction was not altered in potency or E-max after org an culture for 1 and 5 days. In contrast, IRL 1620 induced a clear con traction after 1 day, which increased further in both E-max and potenc y after 5 days organ culture. 5. Our results indicate that a massive n ew transcription of ETB receptor mRNA is induced by organ culture, res ulting in functional contractile ETB receptors on the smooth muscle la yer.