1. In the present study, we determined whether ETA receptors are present on endothelial cells in situ, by use of front-surface fluorometry of fura-2-loaded porcine aortic valvular strips and reverse transcription polymerase chain reaction (RT-PCR). 2. Although endothelin-1 (ET-1) and endothelin-3 (ET-3) induced maximum elevations of cytosolic Ca2+ concentrations ([Ca2+]i) at 10(-7) M, the peak elevations of [Ca2+]i induced by ET-1 were much greater than those induced by ET-3. 3. The application of ET-1 after ET-3 induced an additional increase in [Ca2+]i, while the application of ET-3 after ET-1 had no effect. A selective ETA receptor antagonist, BQ-123, partially inhibited the ET-1-induced Ca2+ transient but had no effect on ET-3-induced Ca2+ transients. These experiments indicated the presence of functioning ETA receptors in addition to ETB receptors in endothelial cells in situ. 4. The sequence of pig lung ETA receptor complimentary DNA (cDNA) was determined by PCR. RT-PCR, using specific primers for pig ETA receptor sequence and total RNA from endothelial cells on the aortic side of the aortic valve, gave the expected size of band. This PCR product was sequenced and was found to be identical to the sequence of the pig lung ETA receptor. 5. The partial sequence of the pig lung ETB receptor was also determined. RT-PCR for the pig ETB receptor revealed that endothelial cells of the aortic valve express ETB receptor messenger RNA (mRNA). 6. We confirmed that functioning ETA receptors and expression of ETA receptor mRNA exist in the endothelial cells on the aortic side of porcine aortic valves.
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