Rapid down regulation of Ca2+ signals induced by endothelin-1 in a human bronchial epithelial cell line

Ca2+ transients evoked by endothelin-1 (ET-1) were measured in single cells of an immortalized human tracheal epithelial cell line using Fura-2. ET-induced Ca2+ transients were compared to signals evoked via established phospholipase-C linked receptors (H1 histamine; P2y purinergic, ATP). Saturating concentrations of histamine (100 microM) and ATP (10 microM) caused Ca2+ transients of identical amplitude, whereas a saturating concentration of ET-1 (10 nM) on average resulted in a slightly smaller change in fluorescence ratio (80 +/- 27%). H1 and P2y induced Ca2+ signals caused by brief (10-30 s) application of the agonists were highly reproducible. No desensitization to these ligands was observed, if between two exposures cells were superfused with agonist-free solution for > or = 200 s. A single exposure to ET-1 (10 nM) for > or = 6 s reduced sensitivity of the cell to a second exposure to ET-1. On average, the signal upon a second application of 10 nM ET-1 had an amplitude of 30% of the first ET-1 induced signal in that cell. After two 10 s exposures to the peptide, less than 10% of the initial amplitude was measured. This desensitization did not affect responsiveness to histamine or ATP. No recovery from desensitization to ET-1 was observed for 12 h after a single brief treatment with the peptide. Thereafter, responsiveness to ET-1 re-appeared with a half time of about 5 h and was complete by about 20 h. Ca2+ signals to all three agonists were absent in thapsigargin treated cells. Their amplitude was not affected by superfusion of the cells with Ca(2+)-free solution. Under conditions of store-depletion either by ET-1 or by thapsigargin, a change from Ca(2+)-free to Ca(2+)-containing solution induced a slow rise in [Ca2+]i, suggesting the existence of a capacitative Ca2+ entry pathway. ET-receptors most likely of the ETA subtype are subject to a novel type of desensitization-at least with regard to Ca2+ signalling-which might reflect a signal transduction mechanism specific to ET receptors.