Calcium signaling in endothelia: cellular heterogeneity and receptor internalization

The vasoactive factors thrombin, bradykinin (BK), and ATP are released in response to tissue damage and inflammation and act on endothelium to modulate vascular perfusion. We have investigated the second messenger response of endothelium activated by these agonists and, in particular, the mechanism of desensitization to BK. Fura-2 fluorescence ratio imaging of calf pulmonary artery endothelial cells (CPAE) revealed 5- to 10-fold increases on intracellular Ca (Cai) in response to these agents. Maximal doses caused Cai to increase from 52 to 248 nM (thrombin), 556 nM (BK), and 643 nM (ATP). Agonists elicited a rapid (within 30 s) increase of Cai due to release of Ca from intracellular stores followed by a secondary elevation of Cai dependent on entry of external Ca. The temporal characteristics of the Cai responses to all agonists were heterogeneous from cell to cell, and, interestingly, repeated stimulation gave identical signature responses from individual cells, although the amplitude of the Cai response decreased to thrombin and especially bradykinin but not for ATP. This decrease was agonist specific because ATP elicited large increases of Cai after thrombin or BK desensitization. Maximal desensitization was obtained with BK applied for 5-10 min followed by a rest of < 10 min before restimulation. Although desensitization primarily reduced the elevation of Cai due to the release of the internal store, entry of extracellular Ca was also reduced. Cells responded heterogeneously to desensitization in that those with prominent extracellular Ca entry responded most strongly upon a second stimulation with BK. Because desensitized cells still responded to ATP with an increase of Cai, the desensitization was controlled at a step prior to the activation of phospholipase C. Desensitization occurred by a reduction of BK receptor number; a 10-min BK pretreatment reduced [3H]BK binding to receptors by 70% (from 14,600 receptors/cell, Km = 5 nM, to 5,300). As surface receptor numbers decreased, internalized receptors increased as assayed by an acetic acid wash. The time course of the receptor internalization was similar to the decrease in Cai response to BK. We conclude that the vasoactive agonists thrombin, BK, and ATP increase the second messenger Cai in endothelial cells and that a desensitized Cai response occurs with BK, but not with ATP, due to downregulation and endocytosis of the BK receptor.