AJP - Cell Physiology, Vol 263, Issue 5 C1029-C1039, Copyright © 1992 by American Physiological Society
Calcium signaling in endothelia: cellular heterogeneity and receptor internalization
W. H. Weintraub, P. A. Negulescu and T. E. Machen
Department of Molecular and Cellular Biology, University of California, Berkeley 94720.
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.
