Benedetti A, Strazzabosco M, Ng O C, Boyer J L
Department of Internal Medicine and Liver Center, Yale University, School of Medicine, New Haven, CT 06510.
Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):792-6. doi: 10.1073/pnas.91.2.792.
To test the hypothesis that rat hepatocyte canalicular Cl-/HCO3- exchange activity might be regulated by HCO3- or protein kinase-induced changes in the apical targeting of vesicles, isolated rat hepatocytes were cultured in the presence or absence of HCO3-/CO2.Cl-/HCO3- exchange activity increased in cells cultured in the presence of HCO3-/CO2 or when stimulated by dibutyryl cAMP. Both of these effects were blocked by either colchicine or the protein kinase C agonist phorbol 12,13-dibutyrate. Fluorescence and confocal microscopy, respectively, revealed increased pericanalicular-apical membrane localization of two canalicular markers, peanut agglutinin and a 110-kDa canalicular ecto-ATPase, when hepatocyte couplets were preincubated in HCO3-/CO2-containing medium, an effect that was again blocked by colchicine. Dibutyryl cAMP also stimulated canalicular localization of the 110-kDa protein. These findings suggest that hepatocyte Cl-/HCO3- exchange activity is regulated by HCO3-/CO2 and by protein kinase A and protein kinase C agonists through microtubule-dependent targeting of vesicles containing this exchanger to the canalicular domain.
为了验证大鼠肝细胞胆小管 Cl⁻/HCO₃⁻交换活性可能受 HCO₃⁻或蛋白激酶诱导的囊泡顶端靶向变化调控这一假说，将分离的大鼠肝细胞在有或无 HCO₃⁻/CO₂的情况下进行培养。在有 HCO₃⁻/CO₂培养的细胞中或用二丁酰环磷腺苷（dibutyryl cAMP）刺激时，Cl⁻/HCO₃⁻交换活性增加。秋水仙碱或蛋白激酶 C 激动剂佛波醇 12,13 - 二丁酸（phorbol 12,13 - dibutyrate）均可阻断这两种效应。当肝细胞膜偶联物在含 HCO₃⁻/CO₂的培养基中预孵育时，荧光显微镜和共聚焦显微镜分别显示两种胆小管标志物——花生凝集素和一种 110 kDa 的胆小管外源性 ATP 酶在胆小管周围 - 顶端膜的定位增加，而秋水仙碱再次阻断了这一效应。二丁酰环磷腺苷也刺激了 110 kDa 蛋白的胆小管定位。这些发现表明，肝细胞 Cl⁻/HCO₃⁻交换活性受 HCO₃⁻/CO₂以及蛋白激酶 A 和蛋白激酶 C 激动剂的调控，其机制是通过微管依赖性地将含有这种交换体的囊泡靶向运输到胆小管区域。
