Identification of Compound 48/80-activated Chloride channel in RBL-2H3

Abstract

Mast cells are present in tissues in the body and participate in many physiological processes including allergy, tissue remodeling, fibrosis, angiogenesis, and autoimmunity. They can be activated by many stimuli, including calcium (Ca2+) ionophores, Compound 48/80, neuropeptides and immune stimulation. When activated, mast cells release several mediators, such as histamine, cytokines and bradykinin. According to many reports, increment of intracellular Ca2+ is the key factor for mast cell degranulation. Compound 48/80 mediated Ca2+ mobilization was regulated by 4,4-diisothiocyanato-stilbene-2,2-disulfonic acid (DIDS), general chloride (Cl-) channel blocker. In this thesis, we identify the Compound 48/80 induced Cl- channel which is involved in increase of intracellular Ca2+ and histamine degranulation in Rat Basophilic Leukemia cell (RBL-2H3) cell. Ion channel activated by Compound 48/80 was examined in RBL-2H3 cells using whole-cell patch clamp. Compound 48/80 evoked robust Cl- current in RBL cells and these currents were inhibited by Cl- channel blockers, including DIDS and 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB). Next, we found that Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is expressed in RBL cells by using RT-PCR. Our data showed that Cyclic AMP, known as CFTR agonist, mediated Cl- current was abolished by DIDS and CFTR-(inh)-172. When CFTR is expressed in HEK293T cells, Compound 48/80 induced CFTR dependent current which was inhibited by ADP substitution and activated by Forskolin and IBMX mixture. These findings suggest that Compound 48/80 mediated Cl- channel is CFTR.