The cloning and characterization of the gene coding for CFTR (Cystic Fibrosis Transmembrane Regulator) has identified the target for the genetic lesion(s) causing the vast majority of the cases of Cystic Fibrosis (CF). A number of questions, however, remain unanswered about the biochemical and molecular chain of events starting from mutations in the CFTR gene to exert a physiological effect as CF. The possible link(s) between an alteration in cAMP stimulated Cl- afflux and the other biochemical defects described in CF are still unclear. An altered regulation of phospholipase A2 activity in CF fibroblasts was described and it is suggested that this could significantly contribute to the physiological manifestations of CF. Thus, we have studied the regulation of arachidonate (Ara) release in CF and control cells, in order to clarify the possible connections between Ara metabolism and CFTR gene mutation. Initial studies with CFPAC-1 (derived from ductal adenocarcinoma of a CF patient) and T84 (human colon carcinoma cells which express very high levels of wild type CFTR) showed that both cell lines released Cl- with an identical rate in response to 1OuM Ca++ ionophore A23187. Since in several secretory epithelia A23187-stimulated Cl- efflux is mediated through Ara cascade, we measured A23187 stimulated Ara release in CFPAC-1 and T84 cells. We observed a significantly and reproducibly higher A23187 inducible Ara release in CFPAC-1 cells as compared to T84 cells. This response is Ca++ dependent and chloride channel blocking agents increased Ara release by A23187 in CFPAC-1 cells. Replacing extracellular Cl- with gluconate, which deplete intracellular Cl- rapidly, reduced Ara release and depolarization of cells while extracellular KCl abolished it. A23187-induced ARA release in CFPAC-1 cells is further enhanced by PMA, a protein kinase C stimulator and inhibited by staurosporin, a protein kinase C inhibitor. These results were reproducibly repeated with two other cell lines, PLJ6 CFPAC-1 cells transfected with a mammalian retroviral vector only and CFPAC-20 where the same cells were transfected with the vector containing wild type CFTR cDNA inserts. These data suggest that the CF genotype or at least the most common mutation in this disease, DF508, is accompanied by an alteration in Ara release. The higher Ara release in CFTR-defective cells may represent a compensatory mechanism by which CF cells regulate their Cl- efflux induced by A23187. Alternatively, it is possible that CFTR is indirectly involved in the regulation of Ara release. Since overproduction of Ara may initiate inflammatory reactions via eicosanoid production, it may be responsible for several, if not all of the pulmonary complications in CF.
