Martin D. Bootman

The synergistic action of cytosolic Ca2+ and inositol 1,4,5-trisphosphate (InsP3) in releasing intracellular Ca2+ stores has been suggested to be responsible for the complex intracellular Ca2 signals observed during hormonal stimulation of many cell types. However, the ability of cytosolic Ca2+ to potentiate Ca2+ release has recently been questioned because of the observed inhibitory effects of Ca2+ chelators used in previous studies. In the present study, EGTA and BAPTA [1,2-bis-(2-amino-phenoxy)ethane- NNN'N'-tetra-acetic acid] poorly inhibited InsP3-induced Ca2+ release from permeabilized A7r5 smooth-muscle cells. Additionally, stimulatory effects of cytosolic and luminal Ca2+ were observed either in the complete absence of Ca2+ chelator or at constant Ca(2+)-free chelator concentration. These data suggest that potentiation of InsP3-induced Ca2+ release by Ca2+ in A7r5 cells reflects an interaction between Ca2+ and InsP3 receptors, rather than a decrease in chelator-dependent inhibition. The EC50 for activation of InsP3-induced Ca2+ release by cytosolic Ca2+ was unaffected by ATP, or by changing InsP3 concentration, although InsP3-induced Ca2+ release became less sensitive to the inhibitory effects of cytosolic Ca2+ as the InsP3 concentration was elevated. Increasing H+ or Mg2+ concentration shifted the Ca(2+)-activation curve towards higher Ca2+ concentrations. These data suggest that, in addition to the InsP3-binding site, the affinity of the Ca(2+)-binding site(s) on InsP3 receptors can be modulated by intracellular cations.

ABSTRACT Since its introduction to Ca 2+ signaling in 1997, 2‐aminoethoxydiphenyl borate (2‐APB) has been used in many studies to probe for the involvement of inositol 1,4,5‐trisphosphate receptors in the generation of Ca 2+ signals. Due to reports of some nonspecific actions of 2‐APB, and the fact that its principal antagonistic effect is on Ca 2+ entry rather than Ca 2+ release, this compound may not have the utility first suggested. However, 2‐APB has thrown up some interesting results, particularly with respect to store‐operated Ca 2+ entry in nonexcitable cells. These data indicate that although it must be used with caution, 2‐APB can be useful in probing certain aspects of Ca 2+ signaling.—Bootman, M. D., Collins, T. J., Mackenzie, L., Roderick, H. L., Berridge, M. J., Peppiatt, C. M. 2‐Aminoethoxydiphenyl borate (2‐APB) is a reliable blocker of store‐operated Ca 2+ entry but an inconsistent inhibitor of InsP 3 ‐induced Ca 2+ release. FASEB J . 16, 1145–1150 (2002)