Relationship between agonist- and thapsigargin-sensitive calcium pools in adrenal glomerulosa cells. Thapsigargin-induced Ca2+ mobilization and entry.
The relationships between agonist-sensitive calcium pools and those discharged by the Ca(2+)-ATPase inhibitor thapsigargin were studied in intact bovine adrenal glomerulosa cells and a subcellular adrenocortical membrane fraction. In Fura-2-loaded glomerulosa cells, angiotensin II (AII) stimulated a rapid increase in cytoplasmic Ca2+ concentration ([Ca2+]i) followed by a smaller plateau phase that was dependent on extra-cellular Ca2+. In such cells thapsigargin caused a sustained and dose-dependent increase in [Ca2+]i which was diminished in Ca(2+)-deficient medium. The contribution of an influx component to the thapsigargin-induced [Ca2+]i response was demonstrated by measurement of 45Ca influx rate in glomerulosa cells. Thapsigargin-induced Ca2+ entry was significantly less than that evoked by AII, and its kinetics were similar to those of the concomitant increase in [Ca2+]i. The rate of emptying of the agonist-responsive Ca2+ pool after thapsigargin treatment, as indicated by the progressive decrease in the size of the AII-induced Ca2+ transient, showed a rapid initial (t1/2 = 1.7 min) component that accounted for about 80% of the response and a slowly decreasing phase with t1/2 = 112 min. The latter thapsigargin-resistant component was abolished by the removal of extracellular Ca2+. Pretreatment with AII dose-dependently attenuated but did not abolish the subsequent Ca2+ response to thapsigargin and also increased the rate of the Ca2+ rise induced by thapsigargin. In bovine adrenocortical microsomes, thapsigargin inhibited the ATP-dependent filling of Ca2+ pools and caused a dose-dependent rise in extravesicular Ca2+ levels when added to previously loaded microsomes. The thapsigargin-releasable Ca2+ pool in adrenal microsomes was larger than the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-sensitive Ca2+ pool but only slightly greater than the GTP-releasable pool. Ins(1,4,5)P3-induced Ca2+ release was reduced markedly when ATP-dependent Ca2+ loading of the microsomes was prevented by prior addition of thapsigargin. However, the subsequent Ca2+ response to Ins(1,4,5)P3 was consistently better preserved after the addition of thapsigargin to microsomes preloaded with Ca2+. This difference suggests that although Ca2+ uptake by the Ins(1,4,5)P3-responsive pool is also sensitive to thapsigargin, once filled, this pool shows a slower passive leakage than other thapsigargin-sensitive pools. These findings indicate that thapsigargin increases [Ca2+]i by inhibiting Ca2+ uptake into multiple intracellular Ca2+ pools and by also promoting entry of extracellular Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)
Ely
J A
JA
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Ambroz
C
C
Baukal
A J
AJ
Christensen
S B
SB
Balla
T
T
Catt
K J
KJ
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin; D015384:Zona Glomerulosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D007295:Inositol Phosphates; D013729:Terpenes; D000804:Angiotensin II; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium