Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca2+ release in renal epithelial LLC-PK1 cells.
We have studied arginine vasopressin (AVP)-, thapsigargin- and inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release in renal epithelial LLC-PK1 cells. AVP-induced changes in the intracellular free calcium concentration ([Ca2+]i) were studied in indo-1 loaded single cells by confocal laser cytometry. AVP-mediated Ca2+ mobilization was also observed in the absence of extracellular Ca2+, but was completely abolished after depletion of the intracellular Ca2+ stores by 2 microM thapsigargin. Using 45Ca2+ fluxes in saponin-permeabilized cell monolayers, we have analysed how InsP3 affected the Ca2+ content of non-mitochondrial Ca2+ pools in different loading and release conditions. Less than 10% of the Ca2+ was taken up in a thapsigargin-insensitive pool when loading was performed in a medium containing 0.1 microM Ca2+. The thapsigargin-insensitive compartment amounted to 35% in the presence of 110 microM Ca2+, but Ca2+ sequestered in this pool could not be released by InsP3. The thapsigargin-sensitive Ca2+ pool, in contrast, was nearly completely InsP3 sensitive. A submaximal [InsP3], however, released only a fraction of the sequestered Ca2+. This fraction was dependent on the cytosolic as well as on the luminal [Ca2+]. The cytosolic free [Ca2+] affected the InsP3-induced Ca2+ release in a biphasic way. Maximal sensitivity toward InsP3 was found at a free cytosolic [Ca2+] between 0.1 and 0.5 microM, whereas higher cytosolic [Ca2+] decreased the InsP3 sensitivity. Other divalent cations or La3+ did not provoke similar inhibitory effects on InsP3-induced Ca2+ release. The luminal free [Ca2+] was manipulated by varying the time of incubation of Ca(2+)-loaded cells in an EGTA-containing medium. Reduction of the Ca2+ content to one-third of its initial value resulted in a fivefold decrease in the InsP3 sensitivity of the Ca2+ release.
Tshipamba
M
M
Department of Physiology, K.U. Leuven, Belgium.
De Smedt
H
H
Missiaen
L
L
Himpens
B
B
Van Den Bosch
L
L
Borghgraef
R
R
D001127:Arginine Vasopressin / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism*
D001127:Arginine Vasopressin; D000001:Calcimycin; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium