Mechanism of 2-aminoethoxydiphenylboronic acid in acetylcholine-inducing Ca2+ signals in acute dissociated mouse pancreatic acinar cells

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Objective To reveal the mechanism of 2-aminoethoxydiphenyl borate (2APB) modulates intracellular Ca2+ oscillations. Methods Intracellular Ca2+ signals was monitored directly using Ca2+ -dimensional dye (fluo-4) imaging technique in acute dissociated mouse pancreatic acinar cells. The effect and mechanism of 2APB on acetylcholine (Ach)-induced Ca2+ oscillations were examined by patch-clamp whole cell recording. Results (1) 2APB-induced modulations in acinar cell Ca2+ signals were dependent on 2APB concentrations, with increase of 2APB concentrations (1-100 μmol/L), the amplitude and width of Ca2+ oscillations were increased. (2) 2APB enhanced ACh-induced Ca2+ oscillations were also dependent on the concentrations of ACh. When ACh was less than 5 nmol/L, 2APB did not show effect, between ACh 10-30 nmol/L, 2APB potentiated ACh-induced oscillations, while ACh was higher than 100 nmol/L, 2APB inhibited Ca2+ responses. (3) 2APB enhanced ACh-induced Ca2+ oscillations through extracellular rather than intracellular targets because no effect was found by intracellular application of 100 μmol/L 2APB on 10 nmol/L ACh-induced Ca2+ oscillations (P=0.378, n=6); (4) 2APB significantly blocked SOCE, which can reduced the 3 μmol/L TG-induced Ca2+ oscillations current to (3.0±4.8)% (P=0.000, n=6), suggesting that SOCE may be the target of 2APB to enhance Ca2+ oscillations. (5) SOCE blocker GSK can mimic 2APB’s effect to enhance Ca2+ oscillations (P=0.000, n=7). (6) Using thapsigargin to empty Ca2+ pools, the Ca2+ oscillations current become (93.0±9.5)% (P=0.427, n=7) when 2APB added. In other words, 2APB’s effect was eliminated, suggesting that 2APB-induced Ca2+ oscillations is dependent on Ca2+ pool’s conditions. Conclusion At low ACh concentrations (10-30 nmol/L) (where Ca2+ pool is partially emptied and SOCE is open), 2APB blocks SOCC and promotes Ca2+ further release from Ca2+ pool, resulting in Ca2+ oscillation enhancement. Wherease at higher ACh concentrations (>100 nmol/L), 2APB blocks SOCC and prevents Ca2+ pool refilling from extracellular Ca2+ , thus reduces Ca2+ signal response. Key words: 2-aminoethoxydiphenyl borate; Calcium signals; Pancreatic acinar cells; Patch clam