Cystic fibrosis (CF) is caused by mutations in the CF transmembrane regulator (CFTR) gene. The deletion of Phe 508 (deltaF508), in CFTR is the most common (70%) mutation in CF. One of the major complications of CF is the development of severe inflammation of the airways of these patients. We have previously demonstrated that the epithelial cells derived from CF patients who carry deltaF508 mutation in CFTR release abnormal levels of arachidonic acid when stimulated by agonists. It is not clearly understood how an abnormality in CFTR may cause an abnormality in arachidonate metabolism. There is compelling evidence to suggest that cells carrying the deltaF508 mutation in the homozygous state have altered acidification of intracellular organelles and defective c-AMP-induced membrane recycling. Last year we reported that the regulation of phospholipase A2 (PLA2)-catalyzed arachidonic acid release may be defective in CF lymphocytes and fibroblasts. We demonstrated that calcium -induced arachidonic acid (AA) 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 deltaF508 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). These data indicated that the cell lines which contained a mutant CFTR in their genome and had a defective c-AMP-induced Cl- efflux appeared to have 5-10 fold higher AA release than those in control cells. 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 4alpha phorbol,12,13 didecanoae and is inhibited by staurosporine (a PKC inhibitor). An inhibitor of PLA2, quinacrine (200microM) abolished AA release in these cells. These data suggested that Ca++ and pKC-stimulated AA release, possibly catalyzed by PLA2, is dysregulated in cells carrying the deltaF508 CFTR mutation in CF. These data indicated that an alteration of a Ca++, PKC-regulated AA release raising the possibility that this abnormality may be due to activation of cPLA2. We now report that cPLA2 is not involved in the abnormal release of AA from these cells. It appears that a Ca++-independent PLA2 may be involved in this process. Transcription of several PLA2s in the mutant as well as control cells are now being studied to determine the type of PLA2 that may dysregulated in CF as a result of deltaF508 mutation. A clear understanding of the mechanism of AA release may allow development of rational therapeutic approaches to control inflammation in the airway of these CF patients.
