Alvaro D, Alpini G, Jezequel A M, Bassotti C, Francia C, Fraioli F, Romeo R, Marucci L, Le Sage G, Glaser S S, Benedetti A
Division of Gastroenterology, Department of Clinical Medicine, University of Rome, "La Sapienza," Viale dell'Universita' 37, 00185 Rome, Italy.
J Clin Invest. 1997 Sep 15;100(6):1349-62. doi: 10.1172/JCI119655.
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 microM) 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 microM), but not by the protein kinase C antagonist, staurosporine (0.1 microM). 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).
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.
我们在从正常大鼠肝脏分离出的胆管单位（IBDU）和胆管细胞中，研究了乙酰胆碱（ACh）受体的存在情况，以及ACh在调节Cl⁻/HCO₃⁻交换体活性中的作用和机制。通过测量急性去除/重新引入Cl⁻诱导的细胞内pH变化来评估Cl⁻/HCO₃⁻交换体活性。通过免疫荧光、免疫电子显微镜和逆转录聚合酶链反应（RT-PCR）在IBDU和分离的胆管细胞中检测到M3亚型ACh受体。通过RT-PCR未检测到M1亚型ACh受体mRNA，通过免疫荧光检测M2亚型为阴性。ACh（10微摩尔）对Cl⁻/HCO₃⁻交换体的基础活性无影响。当IBDU暴露于ACh加促胰液素时，与单独使用促胰液素（50纳摩尔）相比，ACh显著（P < 0.03）提高了去除Cl⁻后的最大碱化速率和重新引入Cl⁻后的最大恢复速率，表明ACh增强了促胰液素对Cl⁻/HCO₃⁻交换体活性的刺激作用。ACh的这种作用被M3 ACh受体拮抗剂4-二苯基乙酰氧基-N-（2-氯乙基）-哌啶（40纳摩尔）、细胞内Ca²⁺螯合剂1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸乙酰甲酯（50微摩尔）阻断，但未被蛋白激酶C拮抗剂星形孢菌素（0.1微摩尔）阻断。在分离的大鼠胆管细胞中，细胞内cAMP水平单独不受ACh影响，但与单独使用促胰液素相比，暴露于促胰液素加ACh后显著更高（P < 0.01）。两种钙调神经磷酸酶抑制剂FK-506和环孢素A（100纳摩尔）分别消除了ACh诱导的促胰液素对细胞内cAMP水平和Cl⁻/HCO₃⁻交换体活性的增强作用。
M3 ACh受体在大鼠胆管细胞中显著且广泛存在。ACh不影响Cl⁻/HCO₃⁻交换体的基础活性，但通过一条Ca²⁺依赖性、对蛋白激酶C不敏感的途径增强促胰液素对该阴离子交换体的刺激作用，该途径增强了促胰液素对腺苷酸环化酶的刺激作用。钙调神经磷酸酶很可能介导了钙和腺苷酸环化酶途径之间的相互作用。由于促胰液素在副交感神经占优势时作用于胆管细胞，促胰液素（cAMP）和ACh（Ca²⁺）对Cl⁻/HCO₃⁻交换体的协同调节可能在肠道对碳酸氢盐需求最大时胆汁中导管碳酸氢盐排泄的调节中起主要作用。
