EFFECT OF SECRETIN ON INTRACELLULAR PH REGULATION IN ISOLATED RAT BILE-DUCT EPITHELIAL-CELLS
EFFECT OF SECRETIN ON INTRACELLULAR PH REGULATION IN ISOLATED RAT BILE-DUCT EPITHELIAL-CELLS
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DOI：
10.1172/jci116705
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发表时间：
1993-09-01
影响因子：
15.9
通讯作者：
BOYER, JL
中科院分区：
文献类型：
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作者：
ALVARO, D;CHO, WK;BOYER, JL
在从正常大鼠肝脏分离的胆管上皮 (BDE) 细胞中表征了促胰液素对涉及细胞内 pH (pH(i)) 和 HCO3- 排泄调节的离子转运机制的影响。 pH(i) 使用微量荧光法用 2,7-双(羧乙基)-5(6)-羧基-荧光素-乙酰甲基酯 (BCECFAM) 进行测量。 Hepes 中 BDE 的基础 pH(i) 为 7.04+/-0.06，KRB 中 BDE 的基础 pH(i) 为 7.16+/-0.10，并且不受促胰液素 (50-200 nM) 的影响。 Hepes 或 KRB 培养基（含有和不含阿米洛利）中酸负荷的回收率也不会被促胰液素改变，表明 Na+/H+ 交换和 Na+/HCO3- 共转运不受该激素的影响。急性 Cl- 去除后，pH(i) 以 0.125+/-0.06 pHU/min 的最大速率上升 0.24+/-0.08 PHU（H+ 通量速率 = 6.02+/-3.27 mM/min），并在 Cl- 重新接纳后恢复（0.188+/-0.08 pHU/min；H+ 通量速率 = 11.82+/-5.34 mM/min）。用 1 mM DIDS 预处理抑制了 Cl- 去除的效果，而诱导细胞去极化的缬氨霉素可能通过刺激生电 HCO3- 流入而增强了这些效果。促胰液素显着增加Cl-去除后碱化的最大速率(P < 0.012)和Cl-重新接纳后pH(i)恢复的最大速率(P < 0.025)，表明刺激了Cl-/HCO3-交换活性。这些发现用 N6,2'-O-二丁酰腺苷-3'-5'-环单磷酸 (DBcAMP) 重现。 Cl-通道阻滞剂 5-硝基-2'-(3-苯基丙氨基)-苯甲酸酯 (NPPB, 10 muM) 显着降低促胰液素和 DBcAMP 对急性 Cl-去除/再入院促进的 pH(i) 变化的影响。这些发现证实，促胰液素可能通过细胞内信使 cAMP 激活 Cl- 通道来刺激 BDE 细胞中 Cl-/HCO3- 交换器的活性。这反过来又使细胞去极化，刺激生电 Na+/HCO3- 同向运动。 Cl- 通道激活诱导的细胞去极化应通过生电 Na+/HCO3- 同向增强 HCO3- 进入，进而刺激 Cl-/HCO3- 交换。这些机制可以解释促胰液素刺激胆汁中碳酸氢盐的分泌。
The effects of secretin on ion transport mechanisms involved in regulation of intracellular pH (pH(i)) and HCO3- excretion were characterized in bile duct epithelial (BDE) cells isolated from normal rat liver. pH(i) was measured with 2,7-bis(carboxyethyl)-5(6)-carboxy-fluorescein-acetomethylester (BCECFAM) using a microfluorimetric method. Basal pH(i) of BDE was 7.04+/-0.06 in Hepes and 7.16+/-0.10 in KRB and was unaffected by secretin (50-200 nM). Recovery rates from an acid load in Hepes or in KRB media (with and without amiloride) were also not altered by secretin, indicating that Na+/H+ exchange and Na+/HCO3- cotransport were not affected by this hormone. After acute Cl- removal, pH(i) rose 0.24+/-0.08 pHU at a maximal rate of 0.125+/-0.06 pHU/min (H+ flux rates = 6.02+/-3.27 mM/min) and recovered after Cl- readmission (0.188+/-0.08 pHU/min; H+ flux rates = 11.82+/-5.34 mM/min). Pretreatment with 1 mM DIDS inhibited the effects of Cl- removal, while valinomycin, which induces cell depolarization, enhanced these effects, probably by stimulating electrogenic HCO3- influx. Secretin significantly increased both the maximal rate of alkalinization after Cl- removal (P < 0.012) and of pH(i) recovery after Cl- readmission (P < 0.025), indicating stimulation of Cl-/HCO3- exchange activity. These findings were reproduced with N6,2'-O-Dibutyryladenosine-3'-5'-cyclicmonophosphate (DBcAMP). The Cl- channel blocker 5-nitro-2'-(3-phenylpropylamino)-benzoate (NPPB, 10 muM) significantly decreased the effects of secretin and DBcAMP on the pH(i) changes promoted by acute Cl- removal/readmission. These findings establish that secretin stimulates the activity of the Cl-/HCO3- exchanger in BDE cells, probably by activating Cl- channels via the intracellular messenger cAMP. This in turn depolarizes the cell, stimulating electrogenic Na+/HCO3- symport. The cell depolarization induced by Cl- channel activation should enhance HCO3- entrance through electrogenic Na+/HCO3- symport, which in turn stimulates the Cl-/HCO3- exchange. These mechanisms could account for secretin stimulated bicarbonate secretion in bile.