Activation of wild type and ΔF508-CFTR by phosphodiesterase inhibitors through cAMP-dependent and -independent mechanisms

The cAMP-dependent activation of the cystic fibrosis transmembrane conductance regulator (CFTR) and its modulation through inhibition of phosphodiesterases (PDE) were studied with the cell-attached patchclamp technique in Calu-3 cells (expressing endogenous CFTR) and NIH3T3 cells [expressing either wild-type (Wt)-CFTR or Delta F508-CFTR]. In Calu-3 cells, CFTR current was augmented by increasing concentrations of 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) and reached a saturating level at greater than or equal to 60 mu M. Varying the forskolin concentration also modulated CFTR activity; 10 mu M was maximally effective since supplemental application of 200 mu M CPT-cAMP had no additional effect. Activation of CFTR by increasing the cAMP concentration occurs through an increase of the NPo (product of the number of functional channels and the open probability) since the single-channel amplitude remains unchanged. In Calu-3 and NIH3T3-Wt cells, PDE inhibitors, milrinone (100 mu M), 8-cyclopentyl-1,3-dipropylxanthine (CPX, 25 mu M), and 3-isobutyl-1-methylxanthine (IBMX, 200 mu M), did not enhance CFTR current initially activated with 10 mu M forskolin, but each potentiated CFTR activity elicited with a submaximal forskolin concentration (e.g., 100 nM) and prolonged the deactivation of CFTR channel current upon removal of forskolin. Millimolar IBMX increased the NPo of both Wt- and Delta F508-CFTR even tinder maximal cAMP stimulation. Quantitatively, these effects of millimolar IBMX on NPo approximate those of genistein, which potentiates the cAMP-dependent CFTR activity via a mechanism that does not involve increases in cellular cAMP. Thus, depending on the concentration, PDE inhibitors may affect CFTR through different mechanisms.