Role and mechanisms of action of acetylcholine in the regulation of rat cholangiocyte secretory functions
Role and mechanisms of action of acetylcholine in the regulation of rat cholangiocyte secretory functions
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DOI：
10.1172/jci119655
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发表时间：
1997-09-15
影响因子：
15.9
通讯作者：
Benedetti, A
中科院分区：
文献类型：
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作者：
Alvaro, D;Alpini, G;Benedetti, A
我们研究了从正常大鼠肝脏分离的离体胆管单位 (IBDU) 和胆管细胞中乙酰胆碱 (ACh) 受体的出现情况，以及 ACh 在调节 Cl-/HCO3- 交换活性中的作用和机制。通过测量急性 Cl- 去除/重新接纳引起的细胞内 pH 值的变化来评估 Cl-/HCO3- 交换活性。通过免疫荧光、免疫电镜和逆转录酶 PCR 检测 IBDU 和分离的胆管细胞中的 M3 亚型 ACh 受体。逆转录酶PCR未检测到M1亚型ACh受体mRNA，免疫荧光检测M2亚型为阴性。 ACh (10 μM) 对 Cl-/HCO3- 交换剂的基础活性没有影响。当 IBDU 暴露于 ACh 加促胰液素时，与单独使用促胰液素 (50 nM) 相比，ACh 显着 (P < 0.03) 增加了 Cl- 去除后碱化的最大速率和 Cl- 重新接纳后的最大恢复率，表明 ACh 增强了促胰液素对 Cl-/HCO3- 交换活性的刺激作用。 ACh 的这种作用可被 M3 ACh 受体拮抗剂 4-二苯基乙酰氧基-N-(2-氯乙基)-哌啶 (40 nM)、细胞内 Ca2+ 螯合剂 1,2-双 (2-氨基苯氧基)-乙烷-N,N,N',N'-四乙酸乙酰氧基甲酯 (50 μM) 阻断，但不能被蛋白激酶 C 拮抗剂星形孢菌素阻断（0.1μM）。离体大鼠胆管细胞中的细胞内 cAMP 水平不受单独 ACh 的影响，但与单独的促胰液素相比，接触促胰液素加 ACh 后细胞内 cAMP 水平显着升高（P < 0.01）。两种钙调神经磷酸酶抑制剂 FK-506 和环孢菌素 A (100 nM) 分别消除了 ACh 诱导的促胰液素对细胞内 cAMP 水平和 Cl-/HCO3- 交换活性的增强作用。结论：M3 ACh 受体在大鼠胆管细胞中显着且分散。 ACh 不影响 Cl-/HCO3- 交换器的基础活性，但通过 Ca2+ 依赖性、蛋白激酶 C 不敏感途径增强促胰液素对该阴离子交换器的刺激，从而增强促胰液素对腺苷酸环化酶的刺激。钙调神经磷酸酶很可能介导钙和腺苷酸环化酶途径之间的串扰。由于促胰液素在副交感神经占主导地位期间以胆管细胞为目标，因此当肠道中碳酸氢盐需求最大时，促胰液素 (cAMP) 和 ACh (Ca2+) 对 Cl-/HCO3- 交换器的协调调节可能在胆汁中导管碳酸氢盐排泄的调节中发挥重要作用。
We investigated, in isolated bile duct units (IBDU) and cholangiocytes isolated from normal rat liver, the occurrence of acetylcholine (ACh) receptors, and the role and mechanisms of ACh in the regulation of the Cl-/HCO3- exchanger activity. The Cl-/HCO3- exchanger activity was evaluated measuring changes in intracellular pH induced by acute Cl- removal/readmission. M3 subtype ACh receptors were detected in IBDU and isolated cholangiocytes by immunofluorescence, immunoelectron microscopy, and reverse transcriptase PCR. M1 subtype ACh receptor mRNA was not detected by reverse transcriptase PCR and M2 subtype was negative by immunofluorescence. ACh (10 mu M) showed no effect on the basal activity of the Cl-/HCO3- exchanger. When IBDU were exposed to ACh plus secretin, ACh significantly (P < 0.03) increased the maximal rate of alkalinization after Cl- removal and the maximal rate of recovery after Cl- readmission compared with secretin alone (50 nM), indicating that ACh potentiates the stimulatory effect of secretin on the Cl-/HCO3- exchanger activity. This effect of ACh was blocked by the M3 ACh receptor antagonist, 4-diphenyl-acetoxy-N-(2-chloroethyl)-piperidine (40 nM), by the intracellular Ca2+ chelator, 1,2-bis (2-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid acetoxymethylester (50 mu M), but not by the protein kinase C antagonist, staurosporine (0.1 mu M). Intracellular cAMP levels, in isolated rat cholangiocytes, were unaffected by ACh alone, but were markedly higher after exposure to secretin plus ACh compared with secretin alone (P < 0.01). The ACh-induced potentiation of the secretin effect on both intracellular cAMP levels and the Cl-/HCO3- exchanger activity was individually abolished by two calcineurin inhibitors, FK-506 and cyclosporin A (100 nM).Conclusions: M3 ACh receptors are markedly and diffusively represented in rat cholangiocytes. ACh did not influence the basal activity of the Cl-/HCO3- exchanger, but enhanced the stimulation by secretin of this anion exchanger by a Ca2+-dependent, protein kinase C-insensitive pathway that potentiates the secretin stimulation of adenylyl cyclase. Calcineurin most likely mediates the cross-talk between the calcium and adenylyl cyclase pathways. Since secretin targets cholangiocytes during parasympathetic predominance, coordinated regulation of Cl-/HCO3- exchanger by secretin (cAMP) and ACh (Ca2+) could play a major role in the regulation of ductal bicarbonate excretion in bile just when the bicarbonate requirement in the intestine is maximal.