This project investigates the mutation of the gene coding for a cAMP- regulated anion channel, the cystic fibrosis transmembrane regulator (CFTR) protein causes Cystic Fibrosis (CF). The deletion, Phe 508 (deltaF508), in CFTR is the most common mutation in CF. A major complication of CF is extensive and destructive inflammation of the airways. The secondary biochemical and functional alterations caused in CF epithelial cells by a defect in CFTR function are not yet completely understood. Recent evidence suggests that cells carrying the deltaF508 mutation in the homozygote state have altered acidification of intracellular organelles and defective c-AMP-induced membrane recycling. It has been suggested that the regulation of phospholipase A2 (PLA2)- catalyzed arachidonic acid release may be defective in CF lymphocytes and fibroblasts. Last year the group described the results of calcium ionophore A23187-induced arachidonic acid release in the following cell lines: T84 cells (a colon carcinoma line which expresses high level of normal CFTR; CFPAC-1 cells (a pancreatic carcinoma line from a delta F508 CF patient) and clones derived from CFPAC-1 cells after transfection with a retroviral vector containing the normal CFTR gene (CFPAC-PLJ-CFTR) or with vector only (CFPAC-PLJ). The project's data indicated that those lines containing a mutant CFTR and showing defective c-AMP-induced Cl- efflux have a 5-10 fold higher arachidonate release than that in the controls. This increased release is dependent upon extracellular Ca++ and is further enhanced by phorbol-12-myristate, 13-acetate(PMA, stimulator of PKC) but not by the inactive derivative 4` phorbol,12,13 didecanoae and is inhibited by staurosporine (a PKC inhibitor). An inhibitor of PLA2, quinacrine (200micromoles) abolishes arachidonate release in these cells. These data suggested that Ca++ and pKC-stimulated arachidonate release, possibly catalyzed by PLA2, is disregulated in cells carrying the delta F508 CFTR mutation in CF. These data indicated that an alteration of a Ca++, PKC-regulated arachidonate release raising the possibility that this abnormality may be due to activation of cPLA2. Although the basal activity of cPLA2 is equivalent in the control as well as in CF cells, there may be agonist induced enhancement of activity of this enzyme in CF cells. Should this be the case cPLA2 inhibition may regulate the abnormal arachidonate release and thereby controlling the severe inflammation commonly found in the respiratory tracts of CF patients.
