2-氨基乙氧基二苯基硼酸对乙酰胆碱诱导的急性分离小鼠胰腺腺泡细胞Ca^2＋信号的机制

Mechanism of 2 - aminoethoxydiphenylboronic acid in acetylcholine - inducing Ca^2＋ signals in acute dissociated mouse pancreatic acinar ceils

目的探讨2-氨基乙氧基二苯基硼酸盐（2APB）增强细胞内Ca^2＋振荡的机制。方法在急性分离的小鼠胰腺腺泡细胞中，使用Ca^2＋敏感染料（fluo-4）成像技术，直接监测细胞内Ca^2＋信号。使用膜片钳全细胞记录技术，检查2APB对乙酰胆碱（ACh）诱导的Ca^2＋振荡的作用特征和机制。结果（1）2APB浓度依赖性的增强了ACh诱导的Ca^2＋振荡，随着2APB浓度增高（1～100μmol／L），Ca^2＋振荡幅度及宽度增大。（2）2APB增强ACh诱导的Ca^2＋振荡也受到ACh浓度的影响。在100μmol／L2APB的条件下，当ACh低于5nmol／L时，通常没有作用；在ACh10-30nmol／L时，增强作用显著；而在ACh100～1000nmol／L时，出现抑制作用。（3）细胞内施加100μmol／L2APB对10nmol／LACh诱导的Ca^2＋振荡无影响（P=0．378，n=6），提示2APB增强ACh诱导的Ca^2＋振荡的作用靶点是在细胞外而不是细胞内。（4）2APB可明显阻断SOCE，能将3μmol／LTG引起Ca^2＋振荡的净电流（标准化后）减少至（3．0±4．8）％（P=0．000，n=6），提示SOCE可能是2APB增强Ca^2＋振荡的靶点。（5）SOCE阻断剂GSK可模拟2APB增强Ca^2＋振荡的作用（P=0．000，n=7）。（6）使用毒胡萝卜素使Ca^2＋池排空后，与给药前（为100％）比较，施加2APB引起Ca^2＋振荡的净电流（标准化后）为（93．0±9．5）％（P=0．427，n=7），即2APB增强Ca^2＋振荡作用被消除。结论在ACh低浓度（10～30nmol／L）的条件下，2APB通过阻断SOCE促进Ca^2＋从钙池进一步排空，导致了Ca^2＋振荡的增强，而在高ACh浓度（〉100nmol／L）的条件下，2APB通过阻断SOCE减少钙池从胞外得到Ca^2＋的补充，导致了Ca^2＋信号减弱。

Objective To reveal the mechanism of 2 -aminoethoxydiphenyl borate （2APB） mod- ulates intracellular Ca^2＋ oscillations. Methods Intracellular Ca^2＋ signals was monitored directly using Ca^2＋ -dimensional dye （fluo -4 ） imaging technique in acute dissociated mouse pancreatic acinar cells. The effect and mechanism of 2APB on acetylcholine （Ach） -induced Ca^2＋ oscillations were examined by patch - clamp whole cell recording. Results （ 1 ） 2APB - induced modulations in acinar cell Ca^2＋ signals were dependent on 2APB concentrations, with increase of 2APB concentrations （ 1 - 100 μmol/L） , the amplitude and width of Ca^2＋ oscillations were increased. （ 2 ） 2APB enhanced ACh - induced Ca^2 ＋ oscillations were also dependent on the concentrations of ACh. When ACh was less than 5 umol/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 Ca^2 ＋ responses. （3） 2APB enhanced ACh - induced Ca^2＋ 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 Ca^2＋ oscillations （P = 0. 378, n = 6） ; （4） 2APB significantly blocked SOCE, which can reduced the 3 μmol/L TG - induced C2^＋ oscillations current to （3.0±4. 8）% （P =0. 000, n =6）, suggesting that SOCE may be the target of 2APB to enhance Ca^2＋ oscillations. （5） SOCE blocker GSK can mimic 2APB＇ s effect to enhance Ca^2＋oscillations （P =0. 000, n = 7）. （ 6 ） Using thapsigargin to empty Ca^2＋ pools, the Ca^2＋ 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 Ca^2＋ oscillations is dependent on Ca^2＋ pool＇ s conditions. Conclusion At low ACh concentrations （10 -30 nmol/L） （where Ca^2＋ pool is partially emptied and SOCE is open）, 2APB blocks SOCC and promotes Ca^2＋ further release from Ca^2＋ pool, resulting in Ca^2＋ oscillation enhancement. Wherease at higher ACh concentrations （ 〉 100 nmol/L） , 2APB blocks SOCC and prevents Ca^2＋ pool re- filling from extracellular Ca^2＋ , thus reduces Ca^2＋ signal response.