Differential stimulation of cytosolic phospholipase A(2) by bradykinin in human cystic fibrosis cell lines

Tracheal epithelial cells and skin fibroblasts from different cystic fibrosis (CF) patients bearing the Delta F508 mutation of cystic fibrosis transmembrane conductance regulator (CFTR) released more arachidonic acid in response to bradykinin than do other CF and normal cells. immortalized tracheal epithelial cell lines were used as models to study the mechanisms of this dysregulation. An 85 kD cytosolic phospholipase A(2) (cPLA(2)) was found in these cells and bradykinin increased its binding to membranes of Delta F508 cells (CFT-2) but not to those of a double heterozygous CF cells (CFT-1), or of control cells (NT-I). The expression of G alpha q/11 protein was also increased in Delta F508 cells, with increased stimulation of phosphatidylinositol diphosphate-specific phospholipase C (PLC) by bradykinin, and an early, transient activation of mitogen-activated protein (MAP) kinase. As the binding of cPLA(2) to membranes is Ca2+-dependent, the increased coupling to PLC could cause the hypersensitivity to bradykinin. Comparison of the effects of bradykinin to those observed with thapsigargin, an inhibitor of calcium reuptake, suggests that the increase of intracellular calcium is not the only mechanism involved in arachidonic acid release by bradykinin in Delta F508 cells. The lack of effect of calcium ionophore A23187 or TPA on arachidonic acid release from any of the cell lines suggested that activation needs a PKC-independent cPLA(2) phosphorylation step, perhaps via MAP kinase activation. The binding of cPLA, to membranes after bradykinin stimulation still occurred in CFT2 cells (Delta F508) homogenized in EDTA, suggesting that a membrane component plus increased intracellular calcium influenced cPLA(2) anchoring to membranes. The defective processing of Delta F508 CFTR seems to increase cPLA(2) stimulation by bradykinin, since the bradykinin-stimulated release of arachidonic acid is reversed by growing cells at 28 degrees C for 48 h. The Delta F508 mutation of CFTR appears to increase the stimulation of cPLA(2) by Gq-mediated receptors in a PKC-independent and MAP kinase-dependent manner. Hence normal CFTR, or normally processed Delta F508 CFTR, inhibit cPLA(2) stimulation. The greater reactivity of Delta F508 CFTR cells to inflammatory mediators might be part of the increased sensitivity of CF patients to lung inflammation.