Effects of endothelin-1 on signal transduction in UMR-106 osteoblastic cells.

Endothelin-1 is now recognized to affect the functions of a number of tissues and to activate calcium/phospholipid second messenger pathways in target cells. In the present study, we characterized its effects on signal transduction in UMR-106 cells. To study calcium transients elicited by endothelin-1, cells were loaded either with fluo-3 (for the measurement of cytosolic free calcium) or chlortetracycline (for the measurement of intracellularly stored calcium) as fluorescent probes. Intracellular production of inositol phosphates and cyclic AMP was also measured. Endothelin-1 elicited dose-dependent cytosolic calcium transients with an ED50 of 20 nM. This effect was also seen in EGTA-containing or calcium-free medium; however, the signals were reduced in magnitude. The dihydropyridine calcium channel antagonist nifedipine did not affect the response. Repeated administration of endothelin-1 resulted in homologous desensitization of the response. A 4 minute pretreatment with phorbol ester reduced the initial response to endothelin-1 in both calcium-containing and calcium-free media. A 24 h pretreatment with indomethacin had no effect on response. Using chlortetracycline as an indicator, a significant reduction in intracellularly stored calcium by endothelin-1 was observed. This was prevented by 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate, a blocker of calcium release from internal stores. Endothelin-1 also stimulated the dose-dependent production of inositol phosphates by UMR-106 cells. Indomethacin was also without effect on this process. The increase in inositol trisphosphates was seen within the same time frame as the increase in cytosolic calcium. Endothelin-1 did not influence cyclic AMP production over 5 minutes in these cells. In conclusion, endothelin-1 has a significant effect on UMR-106 osteoblastic cells to activate the calcium and inositol phosphate second messenger systems. Our findings raise the intriguing question of the physiologic role of endothelin-1 in bone metabolism.