Desensitization of the bradykinin-induced rise in intracellular free calcium in cultured endothelial cells.

We studied the cellular mechanism involved in the desensitization of cultured endothelial cells to bradykinin. Bradykinin (10 nmol/l) evoked a rise in the intracellular free calcium concentration [( Cai2+]), measured with the fluorescent probe indo-1, from 137 +/- 30 (+/- SEM) to 623 +/- 101 nmol/l. Cells were desensitized to bradykinin by repetitive stimulation with the peptide over 10 min, after which they no longer responded to bradykinin. However, purinergic stimulation with ATP (10 mumol/l) elicited the same increase in [Cai2+] in endothelial cells desensitized to bradykinin as in cells never exposed to bradykinin. The initial peak of [Cai2+] after stimulation with bradykinin or ATP was not affected by removal of extracellular calcium ions, indicating mobilization of Ca2+ from intracellular stores. Since GTP-binding proteins (G-proteins) are probably involved in the receptor-mediated stimulation of endothelial cells, we also tested the effects of sodium fluoride (NaF), a reported direct stimulator of G-proteins, on endothelial [Cai2+]. NaF (5 mmol/l) increased [Cai2+] to 412 +/- 88 nmol/l in control cells and was equally effective in cells desensitized to bradykinin. We conclude that the homologous desensitization to bradykinin does not occur at the level of intracellular signal transduction but at the level of membrane receptors.