β3-Adrenoceptor control the cystic fibrosis transmembrane conductance regulator through a cAMP/protein kinase A-independent pathway

Abstract

In human cardiac myocytes, we have previously identified a functional β3-adrenoceptor in which stimulation reduces action potential duration. Surprisingly, in cardiac biopsies obtained from cystic fibrosis patients, β3-adrenoceptor agonists produced no effects on action potential duration. This result suggests the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) chloride current in the electrophysiological effects of β3-adrenoceptor stimulation in non-cystic fibrosis tissues. We therefore investigated the control of CFTR activity by human β3- adrenoceptors in a recombinant system: A549 human cells were intranuclearly injected with plasmids encoding CFTR and β3-adrenoceptors. CFTR activity was functionally assayed using the 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescent probe and the patch-clamp technique. Injection of CFTR-cDNA alone led to the expression of a functional CFTR protein activated by cAMP or cGMP. Co-expression of CFTR (but not of mutated ΔF508-CFTR) with high levels of β3-adrenoceptor produced an increased halide permeability under base-line conditions that was not further sensitive to cAMP or β3-adrenoceptor stimulation. Patch-clamp experiments confirmed that CFTR channels were permanently activated in cells co-expressing CFTR and a high level of β3- adrenoceptor. Permanent CFTR activation was not associated with elevated intracellular cAMP or cGMP levels. When the expression level of β3- adrenoceptor was lowered, CFTR was not activated under base-line conditions but became sensitive to β3-adrenoceptor stimulation (isoproterenol plus nadolol, SR 58611, or CGP 12177). This later effect was not prevented by protein kinase A inhibitors. Our results provide molecular evidence that CFTR but not mutated ΔF508-CFTR is regulated by β3-adrenoceptors expression through a protein kinase A-independent pathway.