Journal Article

Rescue of defective pancreatic secretion in cystic-fibrosis cells by suppression of a novel isoform of phospholipase C

Li, Jianmin, Tsang, Lai Ling, Jiang, Jian Li, Lam, Sun Yee, Zhu, Hu, Chung, Yiu Wa, Zhou, Zuomin, Zhu, Jin Xia, Yu, Mei Kuen, Leung, Ka Man, Rowlands, Dewi Kenneth, Sha, Jiahao, Lo, Pui Shan, Chan, Hsiao Chang

2003

Overview

Cystic fibrosis is caused by mutations in the gene encoding an ion-transport protein, the cystic-fibrosis transmembrane conductance regulator (CFTR). Defective secretion of anions is the primary cause of many of the clinical manifestations of cystic fibrosis, including pancreatic insufficiency. We aimed to identify a molecular mechanism from which a new method to circumvent defective pancreatic secretion could be derived. Multiple-human-tissue RT-PCR and semiquantitative RT-PCR analyses were used to examine gene expression. An antisense technique was used in conjunction with radioimmunoassay, Fura-2 spectrofluorometry, immunohisto-chemistry, and the short-circuit current technique (Ussing chamber) for elucidation of gene function and its application in rescuing defective pancreatic secretion. We cloned a newly identified gene, NYD-SP27, which has structural similarity to an isoform of phospholipase C. NYD-SP27 was expressed endogenously in human pancreatic-duct cells and upregulated in cystic fibrosis. Suppression of NYD-SP27, by transfection of its antisense into human cystic-fibrosis pancreatic-duct cells, resulted in augmentation of phospholipase-C-coupled calcium-ion release and protein kinase C activity, improvement in the amount of mutated CFTR reaching the plasma membrane, and restoration of cAMP-activated pancreatic anion secretion. NYD-SP27 exerts an inhibitory effect on phospholipase-C-coupled processes that depend on calcium ions and protein kinase C, including CFTR trafficking and function. Its upregulation in pancreatic-duct cells may reveal a previously unsuspected defect in cystic fibrosis contributing to pancreatic insufficiency, and thus represents a new target for pharmacological intervention in cystic fibrosis.

• Publisher: Elsevier Ltd,Lancet,Elsevier Limited
• Subject: Anions / Antisense DNA / Antisense Elements (Genetics) / Biological and medical sciences / Calcium / Calcium - metabolism / Calcium ions / Cell Line / Chloride / Cloning, Molecular / Conductance / Cyclic AMP - metabolism / Cystic fibrosis / Cystic Fibrosis - genetics / Cystic Fibrosis - metabolism / Cystic Fibrosis - physiopathology / Cystic Fibrosis Transmembrane Conductance Regulator - genetics / Cystic Fibrosis Transmembrane Conductance Regulator - metabolism / Exocrine Pancreatic Insufficiency - genetics / Exocrine Pancreatic Insufficiency - metabolism / Exocrine Pancreatic Insufficiency - physiopathology / Experiments / Fluorescence spectroscopy / Fura-2 / Gastroenterology. Liver. Pancreas. Abdomen / Gene expression / Gene Expression - genetics / Gene Expression - physiology / Genes / Humans / Ion Transport - genetics / Kinases / Liver. Biliary tract. Portal circulation. Exocrine pancreas / Medical sciences / Membrane Proteins - metabolism / Molecular chains / Mutation / Mutation - genetics / Organic chemistry / Other diseases. Semiology / Pancreas / Pancreatic Ducts - cytology / Pancreatic Ducts - physiology / Pharmacology / Phospholipase / Phospholipase C / Pneumology / Polymerase chain reaction / Protein Isoforms - genetics / Protein Isoforms - metabolism / Protein kinase C / Protein Kinase C - metabolism / Protein transport / Proteins / Radioimmunoassay / Respiratory system : syndromes and miscellaneous diseases / Restoration / Short circuits / Short-circuit current / Transfection / Transfection - methods / Type C Phospholipases - antagonists & inhibitors / Type C Phospholipases - genetics / Type C Phospholipases - metabolism