Fanjul Marjorie, Alvarez Laetitia, Salvador Christel, Gmyr Valéry, Kerr-Conte Julie, Pattou François, Carter Nicholas, Hollande Etienne
Laboratoire de Biologie Cellulaire et Moléculaire des Epithéliums (EA 3032), Université Paul Sabatier, 38 rue des 36 Ponts, 31400 Toulouse, France.
Histochem Cell Biol. 2004 Feb;121(2):91-9. doi: 10.1007/s00418-003-0616-2. Epub 2004 Jan 22.
The high concentration of HCO(3)(-) ions (150 mM) in the human pancreatic ducts raises the question of the membrane proteins responsible for their secretion in addition to the Cl(-)/HCO(3)(-) exchanger. In this study, we investigated the expression of carbonic anhydrase IV (CA IV), a possible candidate. Experiments were carried out on specimens of normal human pancreas obtained from brain-dead donors ( n=9) as well as on isolated human ductal cells. Two antibodies were generated: CA IV NH(2) antibody directed against the NH(2) terminal of human glycosyl phosphatidylinositol (GPI)-anchored CA IV and CA IV COOH antibody directed against the COOH terminal of the same protein before its association with a GPI in the rough endoplasmic reticulum. A 35-kDa CA IV was detected in the homogenates of human pancreas. Immunocytochemistry demonstrated the expression of CA IV in centroacinar cells and in intercalated, intralobular, and interlobular ductal cells. The immunoreactivity observed with the CA IV COOH antibody was mainly localized on luminal membranes of ductal cells. Treatment of purified plasma membranes with phosphatidylinositol-phospholipase C indicated that the CA IV expressed in pancreatic ducts was not GPI-anchored. Its detection in the same extracts by the CA IV COOH antibody indicated that it was anchored by a hydrophobic segment at the carboxy terminal. Taken together, these results suggest that normal human pancreatic ductal cells express a 35-kDa CA IV anchored in their luminal plasma membrane by a hydrophobic segment of the COOH terminus. In view of its localization and its mode of anchorage in luminal plasma membranes, this CA IV may participate in the maintenance of luminal pH.
人胰管中高浓度的HCO(3)(-)离子（150 mM）除了Cl(-)/HCO(3)(-)交换体外，还引发了对负责其分泌的膜蛋白的疑问。在本研究中，我们调查了碳酸酐酶IV（CA IV）这一可能的候选蛋白的表达情况。实验在从脑死亡供体获取的正常人胰腺标本（n = 9）以及分离的人导管细胞上进行。制备了两种抗体：针对人糖基磷脂酰肌醇（GPI）锚定的CA IV的NH(2)末端的CA IV NH(2)抗体，以及针对同一蛋白在粗面内质网中与GPI结合之前的COOH末端的CA IV COOH抗体。在人胰腺匀浆中检测到了一条35 kDa的CA IV条带。免疫细胞化学显示CA IV在中央腺泡细胞以及闰管、小叶内导管和小叶间导管细胞中表达。用磷脂酰肌醇 - 磷脂酶C处理纯化的质膜表明，胰管中表达的CA IV不是GPI锚定的。CA IV COOH抗体在相同提取物中检测到它，表明它是通过羧基末端的疏水片段锚定的。综上所述，这些结果表明正常人胰管细胞表达一种35 kDa的CA IV，它通过COOH末端的疏水片段锚定在其管腔质膜上。鉴于其定位及其在管腔质膜中的锚定方式，这种CA IV可能参与维持管腔pH值。
