Caffeine and 2-Aminoethoxydiphenyl Borate (2-APB) Have Different Ability to Inhibit Intracellular Calcium Mobilization in Pancreatic Acinar Cell

Abstract

Inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) modulate Ca(2+) release from intracellular Ca(2+) store and are extensively expressed in the membrane of endoplasmic/sarcoplasmic reticulum and Golgi. Although caffeine and 2-aminoethoxydiphenyl borate (2-APB) have been widely used to block InsP(3)Rs, the use of these is limited due to their multiple actions. In the present study, we examined and compared the ability of caffeine and 2-APB as a blocker of Ca(2+) release from intracellular Ca(2+) stores and Ca(2+) entry through store-operated Ca(2+) (SOC) channel in the mouse pancreatic acinar cell. Caffeine did not block the Ca(2+) entry, but significantly inhibited carbamylcholine (CCh)-induced Ca(2+) release. In contrast, 2-APB did not block CCh-induced Ca(2+) release, but remarkably blocked SOC-mediated Ca(2+) entry at lower concentrations. In permeabilized acinar cell, caffeine had an inhibitory effect on InsP(3)-induced Ca(2+) release, but 2-APB at lower concentration, which effectively blocked Ca(2+) entry, had no inhibitory action. At higher concentrations, 2-APB has multiple paradoxical effects including inhibition of InsP(3)-induced Ca(2+) release and direct stimulation of Ca(2+) release. Based on the results, we concluded that caffeine is useful as an inhibitor of InsP(3)R, and 2-APB at lower concentration is considered a blocker of Ca(2+) entry through SOC channels in the pancreatic acinar cell.

Keywords: 2-APB; Acinar; Caffeine; InsP3R; SOC.

Fig. 1

Both caffeine and 2-aminoethoxydiphenyl borate (2-APB) dose-dependently inhibit lower carbamylcholine (CCh)-induced [Ca²⁺]_i oscillation in pancreatic acinar cell clusters. (A) Representative trace shows the effects of caffeine and 2-APB on CCh-induced [Ca²⁺]_i oscillation. The data were obtained from 5 separate experiments. The physiological concentration of CCh (300 nM) evoked [Ca²⁺]_i oscillation in the presence of 1.28 mM Ca²⁺. Addition of 20 mM caffeine or 30 µM 2-APB abolished [Ca²⁺]_i oscillation completely. Representative traces of dose responses to caffeine (B) and 2-APB (C) in CCh-induced [Ca²⁺]_i oscillations obtained from at least 4 separate experiments. A gradual increase in caffeine (1~30 mM) or 2-APB (3~100 µM) effectively blocked the [Ca²⁺]_i oscillation in a dose-dependent manner. The oscillation did not fully recover after washout of 2-APB, but it completely recovered after caffeine removal.

Fig. 2

Caffeine and 2-APB have different effects on higher carbamylcholine (CCh)-induced intracellular Ca²⁺ mobilization in pancreatic acinar cell clusters. (A) Representative trace shows the effect of caffeine on CCh-induced [Ca²⁺]_i rise. The data were obtained from 5 separate experiments. Caffeine was added 100 sec prior to CCh perfusion. CCh (10 µM) evoked initial [Ca²⁺]_i peak followed by sustained plateau in the presence of 1.28 mM Ca²⁺. Caffeine (30 mM) blocked [Ca²⁺]_i rise completely. (B) Representative trace shows the effect of 2-APB on CCh-induced [Ca²⁺]_i rise. The data were obtained from 5 separate experiments. 2-APB had no effect on initial [Ca²⁺]_i peak, but it markedly blocked the sustained plateau. (C) Summarized dose responses to caffeine and 2-APB in CCh-induced initial [Ca²⁺]_i peak. Data were represented as % of initial levels, and expressed as mean±S.E. Asterisks indicate the value is significantly different from the corresponding value of control (p<0.05). Only caffeine inhibited CCh-induced initial [Ca²⁺]_i peak, dose dependently.

Fig. 3

2-APB blocks thapsigargin (TG)-induced Ca²⁺ entry in a dose-dependent manner in pancreatic acinar cell clusters. Representative traces show the effects of caffeine (A) and 2-APB (B) on TG-induced Ca²⁺ entry. The data were obtained from at least 6 separate experiments. TG raised [Ca²⁺]_i transiently in the absence of [Ca²⁺]_o, and reintroduction of 1.28 mM Ca²⁺ resulted in a marked increase of Ca²⁺ entry. The Ca²⁺ entry was not reduced by caffeine, but it was blocked by 2-APB dose-dependently. (C) Summarized dose responses to caffeine and 2-APB in TG-induced Ca²⁺ entry. Data were represented as % of maximal levels. Asterisks indicate the value is significantly different from the corresponding values of control (p<0.05). Only 2-APB inhibited SOC-mediated Ca²⁺ entry, dose-dependently.

Fig. 4

Caffeine inhibits inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ release from internal store in the permeabilized pancreatic acinar cell. The effects of caffeine (A) and 2-APB (B) on IP₃-induced Ca²⁺ release were obtained from at least 5 experiments. Arrows indicate the starting point of IP₃ perfusion. The fluorescence ratio was normalized to an initial 10 sec period prior to IP₃ perfusion. Caffeine (20 mM) inhibited submaximal (0.5 µM) and maximal (3 µM) IP₃-induced Ca²⁺ release, whereas 2-APB (30 µM) failed to inhibit both concentrations of IP₃-induced Ca²⁺ release. (C) Summarized data show the effects of caffeine and 2-APB on Ca²⁺ release rates in the permeabilized cell. Rates of Ca²⁺ release were fitted by a single exponential function. Asterisks indicate the value is significantly different from the corresponding values for control (p<0.05). Only caffeine had an inhibitory effect on submaximal and maximal IP₃-induced Ca²⁺ release.

Fig. 5

Higher 2-APB has multiple effects on Ca²⁺ mobilization. (A) Representative trace shows the effects of higher 2-APB on Ca²⁺ mobilization in intact pancreatic acinar cell clusters. The data were obtained from 4 separate experiments. Gradual increase of 2-APB (30 µM~1 mM) enhanced [Ca²⁺]_i dose-dependently. However, patterns of [Ca²⁺]_i rise were variable according to dosage used. (B) Dual effects of 2-APB on Ca²⁺ release in the permeabilized cells were obtained from 4 separate experiments. 2-APB was perfused (arrow head) 30 sec prior to IP₃ perfusion (arrow). The fluorescence ratio was normalized to initial 10 sec period prior to 2-APB perfusion. 2-APB (100 µM) itself elicited Ca²⁺ release, but reduced the submaximal (0.5 µM) IP₃-induced Ca²⁺ release. (C) Summarized data of 2-APB effect on Ca²⁺ release rates in permeabilized cell. Asterisks indicate the value is significantly different from the corresponding values of control (p<0.05). 2-APB (100 µM) markedly inhibited submaximal (0.5 µM) IP₃-induced Ca²⁺ release.