A(2B) adenosine receptor coupled activation of mutant CFTR: cAMP and cPLA(2) signaling

The work reported in this dissertation involves two separate studies designed to (1) characterize the signaling pathways used by adenosine (Ado) and A_2B Ado receptors to regulate the cystic fibrosis transmembrane conductance regulator (CFTR) activity, and (2) utilize native signaling pathways to functionally enhance the activity of CFTR and surface localizing CFTR mutants. Also included in this work is a comprehensive review regarding the molecular biology of Ado receptors. Ultimately, the intent of these studies is to better understand CFTR regulation, which may lead to studies that improve the treatment of individuals with Cystic Fibrosis (CF).; In the first study, A₂ receptor signaling pathways were examined. Stimulation with Ado activated CFTR and Cl− secretion and the apical release of arachidonic acid (AA). Inhibiting cytoplasmic phospholipase A₂ (cPLA₂) activity blocked Ado-stimulated halide efflux in Cos-7 cells transiently expressing CFTR, Calu-3 cells that endogenously express CFTR, and Cl− secretion in vivo across the murine nasal mucosa. Ado alone activated ΔF508 CFTR, while activation of G551D CFTR required Ado stimulation combined with AA and phosphodiesterase (PDE) inhibition. Our studies implicate both CAMP/protein kinase A (PKA) and AA/cPLA₂ signaling pathways in A₂ receptor regulation of CFTR and indicate that Ado can be used in strategies to activate mutant CFTR molecules that cause disease.; In the second study, we further explored regulation of CFTR by PDEs. Earlier experiments have demonstrated a role for the PDE inhibitor (PDEi) milrinone in activating CFTR across the nasal mucosa of mice and in Cl − efflux studies using airway cell lines. However, few studies have examined clinically used PDEis, particularly in the context of polarized airway cell monolayers. Several clinically used PDEis were evaluated, including papaverine, theophylline, rolipram, milrinone, cilostazol, and sildenafil. Each PDEi (excluding theophylline) acutely activated Cl− secretion in Calu-3 cells, and several of these compounds also activated R117H CFTR transiently expressed in CF airway cells. Pretreatment with papaverine, cilostazol, and to a lesser extent rolipram increased the magnitude and duration of low dose Ado-stimulated short circuit current (I _sc) in CFTR-expressing airway cell monolayers. Cumulatively, these studies suggest that PDEs provide tonic regulation of CFTR and that PDEis may represent an additional strategy to maximize wildtype and mutant CFTR activity.