Cellular hypercalcemia is an early event in deoxycholate injury of rabbit gastric mucosal cells

Ca²⁺ entry into the cell may be an early event in the pathophysiology of bile salt-induced gastric mucosal injury. The aim of this study was to characterize the rise in cytosolic free Ca²⁺ associated with bile salt injury and its association with cell injury and death. Rabbit gastric mucosal cells were preloaded with the Ca²⁺ indicator fura 2-acetoxymethyl ester (fura 2-AM) for 20 min at 37°C and then exposed to graded concentrations of the bile salt deoxycholate (DC). Cytosolic free Ca²⁺ concentration ([Ca²⁺](i)) was estimated by spectrofluorometry. The resting [Ca²⁺](i) in gastric cells was 177 ± 15 nM (n = 6). When cells were subjected to 0.5 mM DC, there was a time-dependent rise in [Ca²⁺](i). An increase in [Ca²⁺](i) was observed within 2 min, at which time [Ca²⁺](i) rose from 177 ± 15 to 486 ± 30 nM. The maximal increase in [Ca²⁺](i) was observed after 20 min of exposure to 0.5 mM DC (639 ± 49 nM), and [Ca²⁺](i) remained unchanged for at least 2 h. The increase in [Ca²⁺](i) depended on the concentration of DC. The minimum effective dose of DC was 0.2 mM, with which [Ca²⁺](i) was increased by 1.6-fold (from 177 to 285 nM). At 0.5 mM DC also caused a rise in ⁴⁵Ca²⁺ influx into the cells and reduced the viability of gastric cells from 96% to 58% at 2 h. The DC-induced rise in cytosolic free Ca²⁺ depended on the presence of extracellular Ca²⁺. In the absence of extracellular Ca²⁺ there was no rise in cytosolic Ca²⁺ and gastric cells were protected from cell death caused by DC. The DC-induced cell death was reduced from 26% to 10% and from 37% to 16% at 60 and 90 min, respectively, by removal of extracellular Ca²⁺. The association of DC with gastric calls was not altered by removing extracellular Ca²⁺. This suggests decreased DC-induced injury in the absence of extracellular Ca²⁺ is due to the protection from cellular hypercalcemia rather than some other mechanism related to reduced binding and/or association of DC to gastric cells. These experiments show that rising [Ca²⁺](i) appears to be an early pathophysiological event in bile salt-induced cellular injury and that extracellular Ca²⁺ is critical to produce this effect.