The Mechanisms Underlying Anion Transport Mediated By CFTR In Epithelial Cells

Electrolyte transport across the epithelia of different organs is an important function that maintains the stability of the body internal environment and immunologic defenses, and it plays vital role among organ systems for human health.Ion transport in airway epithelium is a focal point of studying non-breathing functions. Many diseases such as Chronic Obstructive Pulmonary Disease(COPD), asthma, cystic fibrosis (CF), originate from small airway and pulmonary alveoli. However, since most previous researches on ion transport using trachea and bronchi, study on electrolyte transport in distal airways appears to be absent. Meanwhile, the function of electrolyte transport across colonic epithelium plays a formidable role in the process of intestinal tract and immune defenses and it has been kept a lot of unexplored aspects in cellular mechansms of anion transport.In this study, primary culture of small airway epithelial cells in vitro is explored. The culture condition is optimized and the air-liquid interface primary culture system is established. The property of anion transport across polarized monolayer of epithelial cells is studied using electrophysiolgical technique. The agonists such as cAMP-evoking agent forskolin and phosphodiesterase inhibitor IBMX were applied to stimulate cells. Ion substitution and pharmacological blocking agent were applied to study the current composition and pathway of ion transport.The results suggest that forskolin/IBMX could stimulate Cl-, HCO3- secretion which is mediated by apical located cystic fibrosis transmembrane conductance ergulator (CFTR) channel. Basolateral located Na+-K+-2Cl--cotransporter (NKCC) transports anions across the basolateral membrane into the cells and it provide anion source for the secretion. The sodium concentration gradient between the cell membrane is the necessary driving force for the anion secretion. By further study on anion secretion, we found that the source of HCO3- mainly produced by the action of intracellular carbonic anhydrase (CA) and transported across basolateral membrane by Na+-HCO3- cotransporter (NBC). The main pathway of HCO3- secretion across apical membrane is CFTR channel.In this study, the properties of anion transport also explored in freshly isolated colonic epithelium of rat and human epithelial cell line Caco-2 and HT-29. The adrenoceptor agonist adrenalin can stimulate the anion secretion through CFTR channel in isolated rat colonic epithelium. The current induced by adrenerlin is lower than the current induced by forskolin. ATP as an Ca+2-dependent Cl- channel agonist also activated ion secretion. In addition, adrenalin and IBMX can activate converse current responses. In Caco-2 and HT-29 cell monolayer, both adrenalin and ATP can stimulate anion current, demonstrating the different signal pathways of the two agonists.In brief, the study established primary culture system of porcine distal airway epithelial cells and investigated its anion secretion mechanism, particularly, HCO3-secretion using the cultured cell monolayer. We also preliminary explored the anion secretion stimulated by adrenalin and ATP. The results of our research provide an experimental basis for related physiological and pathological mechanism, and a theoretical reference for drug development and clinical care in respiratory tract disease therapy. The preliminary result in freshly isolated rat colonic epithelium, human colonic cell line Caco-2 and HT-29 provided a reference for further research on electrolyte transport and physiological and pathological mechanism and clinical treatment.