Regulation of extracellular calcium entry in endothelial cells: role of intracellular calcium pool.
We have investigated the role of the intracellular Ca2+ pool in regulating Ca2+ entry into vascular endothelial cells. The intracellular Ca2+ pool was mobilized using either thapsigargin (TG) or 2',5'-di(tert-butyl)-1,4-benzohydroquinone (BHQ), inhibitors of the endoplasmic reticulum Ca(2+)-adenosinetriphosphatase (ATPase). Mobilization of intracellular Ca2+ stores with either inhibitor depleted intracellular Ca2+ and greatly reduced subsequent mobilization of the inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ pool by bradykinin. However, bradykinin-induced mobilization of the IP3-sensitive intracellular Ca2+ pool only partially reduced the subsequent response of cells to TG and BHQ. Mobilization of the intracellular Ca2+ pool by either TG or BHQ led to a concentration-dependent elevation of cytosolic Ca2+ concentrations ([Ca2+]i) without initiating inositol polyphosphate formation. In contrast to the rapidly developing, transient rise in Ca2+ concentration initiated by bradykinin, maximal concentrations of TG and BHQ stimulated a slowly developing, prolonged elevation of [Ca2+]i that required extracellular Ca2+ and could be blocked by extracellular Ni2+. Extracellular Ca2+ entered the cell through an activated cation entry pathway, since bradykinin, TG, and BHQ stimulated Mn2+ and 45Ca2+ entry. Bradykinin-stimulated 45Ca2+ uptake reached a peak within 2 min, whereas 45Ca2+ influx initiated by TG or BHQ continued for at least 8 min. Importantly, the [Ca2+]i response after low concentrations of BHQ was more transient than that seen after TG. The return of [Ca2+]i to basal values after low concentrations of BHQ was associated with reversal of Ca(2+)-ATPase inhibition and refilling of the IP3-sensitive Ca2+ pool. The continued elevation of [Ca2+]i and prolonged Ca2+ entry seen with TG was associated with continued Ca(2+)-ATPase inhibition and an empty IP3-sensitive Ca2+ pool. We conclude that mobilization of intracellular Ca2+ stores induces Ca2+ entry in endothelial cells which continues until the intracellular Ca2+ pool is refilled.
Dolor
R J
RJ
Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710.
Hurwitz
L M
LM
Mirza
Z
Z
Strauss
H C
HC
Whorton
A R
AR
D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D009994:Osmolar Concentration; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D016227:Benzoquinones; D013729:Terpenes; C067344:2,5-di-tert-butylbenzoquinone; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D001920:Bradykinin; D002118:Calcium