Characterization of the bradykinin-stimulated calcium influx pathway of cultured vascular endothelial cells. Saturability, selectivity, and kinetics.
Measurement of fura-2 fluorescence and 45Ca2+ uptake was used to evaluate Ca2+ influx in cultured bovine aortic endothelial cells (BAECs) stimulated by bradykinin (BK). The BK-stimulated influx pathway was characterized with respect to its 1) sensitivity to extracellular Ca2+, 2) inhibition by membrane depolarization, and 3) permeability to Ba2+ and Sr2+. The results indicate that the activity of the influx pathway is a saturable function of extracellular Ca2+ and that membrane depolarization inhibits Ca2+ influx by changing the apparent affinity and maximal capacity of the pathway for Ca2+. Fura-2 fluorescence was used to compare the profiles for BK-stimulated changes in cytosolic Ca2+, Sr2+, and Ba2+ (Ca2+i, Ba2+i, and Sr2+i). Addition of Ca2+ and Sr2+ to Ca2+-depleted cells in the presence of BK produced a transient increase in Ca2+i and Sr2+i. Following the peak of the response, Ca2+i and Sr2+i declined within 2 min to a steady elevated level. Blockade of influx by the addition of La3+ at the peak of the response to Ca2+ and Sr2+ immediately reduced Ca2+i and Sr2+i to basal levels. Addition of Ba2+ to Ca2+-depleted cells in the presence of BK produced an increase in Ba2+i which continued to rise with time to a steady level. Addition of La3+ after Ba2+, however, did not reduce Ba2+i. These results suggest that 1) Ca2+ and Sr2+ (but not Ba2+) are sequestered by intracellular mechanisms and that the declining phase of the Ca2+ and Sr2+ response reflects a time and divalent cation-dependent inactivation of the influx pathway. The inactivation of the influx pathway was further demonstrated by measuring the kinetics of BK-stimulated 45Ca2+ uptake into BAECs. The results of these experiments demonstrate that BK stimulates a 100- to 150-fold increase in Ca2+ permeability of the BAEC but that the influx pathway turns off or inactivates within 2 min. The magnitude of the flux, the voltage sensitivity, and the ability to conduct Ca2+, Sr2+, and Ba2+ are suggestive of a channel mechanism.
Schilling
W P
WP
Department of Physiology and Molecular Biophysics, Baylor College of Medicine, Houston, Texas 77030.
Rajan
L
L
Strobl-Jager
E
E
D000818:Animals; D001464:Barium / Q000502:physiology; D001667:Binding, Competitive; D001920:Bradykinin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002118:Calcium / Q000502:physiology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D002417:Cattle; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D007700:Kinetics; D007811:Lanthanum / Q000494:pharmacology; D008564:Membrane Potentials; D013324:Strontium / Q000502:physiology
D015220:Calcium Channels; D001464:Barium; D007811:Lanthanum; D001920:Bradykinin; D002118:Calcium; D013324:Strontium