Ca 2+ dependence of inositol 1,4,5‐trisphosphate‐induced Ca 2+ release in renal epithelial LLC‐PK 1 cells

We have studied arginine vasopressin (AVP)‐, thapsigargin‐ and inositol 1,4,5‐trisphosphate (InsP 3 )‐mediated Ca 2+ release in renal epithelial LLC‐PK 1 cells. AVP‐induced changes in the intracellular free calcium concentration ([Ca 2+ ] i ) were studied in indo‐1 loaded single cells by confocal laser cytometry. AVP‐mediated Ca 2+ mobilization was also observed in the absence of extracellular Ca 2+ , but was completely abolished after depletion of the intracellular Ca 2+ stores by 2 μM thapsigargin. Using 45 Ca 2+ fluxes in saponin‐permeabilized cell monolayers, we have analysed how InsP 3 affected the Ca 2+ content of nonmitochondrial Ca 2+ pools in different loading and release conditions. Less than 10% of the Ca 2+ was taken up in a thapsigargin‐insensitive pool when loading was performed in a medium containing 0.1 μM Ca 2+ . The thapsigargin‐insensitive compartment amounted to 35% in the presence of 110 μM Ca 2+ , but Ca 2+ sequestered in this pool could not be released by InsP 3 . The thapsigargin‐sensitive Ca 2+ pool, in contrast, was nearly completely InsP 3 sensitive. A submaximal [InsP 3 ], however, released only a fraction of the sequestered Ca 2+ . This fraction was dependent on the cytosolic as well as on the luminal [Ca 2+ ]. The cytosolic free [Ca 2+ ] affected the InsP 3 ‐induced Ca 2+ release in a biphasic way. Maximal sensitivity toward InsP 3 was found at a free cytosolic [Ca 2+ ] between 0.1 and 0.5 μM, whereas higher cytosolic [Ca 2+ ] decreased the InsP 3 sensitivity. Other divalent cations or La 3+ did not provoke similar inhibitory effects on InsP 3 ‐induced Ca 2+ release. The luminal free [Ca 2+ ] was manipulated by varying the time of incubation of Ca 2+ ‐loaded cells in an EGTA‐containing medium. Reduction of the Ca 2+ content to one‐third of its initial value resulted in a fivefold decrease in the InsP 3 sensitivity of the Ca 2+ release. © 1993 Wiley‐Liss, Inc.