In many cell types, depletion of intracellular calcium stores promotes the opening of plasma membrane channels resulting in capacitative calcium entry (CCE). There is growing evidence for non-capacitative calcium entry (NCCE) pathways activated by lipid messengers including arachidonic acid (AA). CCE and NCCE cannot be simultaneously activated (Luo et al. 2001; Mignen et al. 2001; Moneer & Taylor, 2002), although it is not clear how this mutual antagonism is achieved.

Calcium signals were monitored in fura-2-loaded HEK-293 and Saos-2 cells. All experiments were performed at room temperature (~20 °C). AA (30 µM) and the calcium-ATPase inhibitor thapsigargin (2 µM) were applied exogenously to activate NCCE and CCE, respectively.

With both HEK-293 and Saos-2 cells, thapsigargin and AA generated biphasic responses resulting from mobilization of a common intracellular calcium store followed by a prolonged calcium influx phase. Despite mobilization of a common calcium pool, these agents did not activate the same calcium influx pathway. Thapsigargin activated a classical CCE pathway that was inhibited by 2-aminoethoxydiphenyl borate (2-APB; 100 µM) (HEK-293, n = 20 cells; Saos-2, n = 12 cells) and gadolinium (Gd³⁺; 1 µM) (HEK-293, n = 20 cells; Saos-2, n = 14). The calcium influx activated by AA was not affected by 2-APB or Gd³⁺, but was blocked by LOE-908, a diagnostic for NCCE (Moneer & Taylor, 2002) (HEK-293, n = 12 cells; Saos-2, n = 14 cells).

Stimulation of cells with thapsigargin in the presence of Gd³⁺ produces a transient response reflecting only the mobilization of intracellular stores. Subsequent addition of AA does not cause any further change in calcium, indicating that under these conditions NCCE was not activatable. Similar observations have been suggested to indicate that activation of CCE is solely sufficient to prevent NCCE (Luo et al. 2001). However, addition of AA to cells during an ongoing CCE signal (i.e. no Gd³⁺ present) caused a rapid and transient decline of the intracellular calcium concentration, followed by a progressive increase in calcium that was fully inhibited by LOE-908 (HEK-293, n = 20 cells; Saos-2, n = 13 cells). This pattern of response essentially represented the switch from CCE to NCCE. In our hands, AA appears to be a dominant signal for calcium influx over CCE – it rapidly inhibits ongoing CCE and steadily promotes NCCE. The presence of Gd³⁺ during CCE activation may cause sequestration of channel subunits also required for NCCE, therefore inhibiting subsequent AA responses.