Evidence for the presence of endothelin ETA receptors in endothelial cells in situ on the aortic side of porcine aortic valve.

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.

Identification of specific intracellular domains of the human ETA receptor required for ligand binding and signal transduction.

We have investigated the function of the C-terminal and the third intracellular domains of the ETA receptor by expressing truncated and mutated ETA receptors in COS-7 and CHO cells. All the C-terminal truncated ETA receptors were produced at a similar expression level and were detected in the cell membrane using indirect immunostaining. The sizes of the truncated ETA receptors were decreased in proportion to the molecular mass of the truncated amino acid sequence. When the ligand binding activities were determined for various truncated ETA receptors, it was found that more than eight amino acid residues at the proximal cytoplasmic tail of the ETA receptor were required for ET-1 binding. In addition, the deletion of 16 C-terminal amino acid residues from the third intracellular loop severely decreased the ligand binding activity. It seems that deletion of these cytoplasmic domains of the ETA receptor influences the three-dimensional structure of the ligand binding site located in the extracellular domains. The ETA receptor required more than 13 amino acid residues in the proximity of C-terminal cytoplasmic tail and 10 amino acid residues in the C-terminal region of the third intracellular loop to induce the ET-1 dependent increase in intracellular calcium concentration. Both regions are possibly coupled with G-protein to transmit the ET-1 signal.