Role of the phospholipase C-inositol 1,4,5-trisphosphate pathway in calcium release-activated calcium current and capacitative calcium entry

We investigated the putative roles of phospholipase C, polyphosphoinositides, and inositol 1,4,5-trisphosphate (IP3) in capacitative calcium entry and calcium release-activated calcium current (I-crac) in lacrimal acinar cells, rat basophilic leukemia cells, and DT40 B-lymphocytes, Inhibition of phospholipase C with U73122 blocked calcium entry and I-crac activation whether in response to a phospholipase C-coupled agonist or to calcium store depletion with thapsigargin. Run-down of cellular polyphosphoinositides by concentrations of wortmannin that block phosphatidylinositol 4-kinase completely blocked calcium entry and I-crac. The membrane-permeant IP3 receptor inhibitor, 2-aminoethoxydiphenyl borane, blocked both capacitative calcium entry and I-crac. However, it is likely that 2-aminoethoxydipheny1 borane does not inhibit through an action on the IP3 receptor because the drug was equally effective in wild-type DT40 B-cells and in DT40 B-cells whose genes for all three IP3 receptors had been disrupted. Intracellular application of another potent IP3 receptor antagonist, heparin, failed to inhibit activation of I-crac. Finally, the inhibition of I-crac activation by U73122 or wortmannin was not reversed or prevented by direct intracellular application of IP3. These findings indicate a requirement for phospholipase C and for polyphosphoinositides for activation of capacitative calcium entry. However, the results call into question the previously suggested roles of IP3 and IP3 receptor in this mechanism, at least in these particular cell types.