Coupling of CFTR-mediated anion secretion to nucleoside transporters and adenosine homeostasis in calu-3 cells

The purpose of this study was to characterize the role of adenosine-dependent regulation of anion secretion in Calu-3 cells. RT-PCR studies showed that Calu-3 cells expressed mRNA for A2a and A2B but not A1 or A3 receptors, and for hENTl, hENT2 and hCNT3 but not hCNTl or hCNT2 nucleoside transporters. Short-circuit current measurements indicated that A2b receptors were present in both apical and basolateral membranes, whereas A2a receptors were detected only in basolateral membranes. Uptake studies demonstrated that the majority of adenosine transport was mediated by hENTl, which was localized to both apical and basolateral membranes, with a smaller hENT2-mediated component in basolateral membranes. Whole-cell current measurements showed that application of extracellular nitrobenzylmercaptopurine ribonucleoside (NBMPR), a selective inhibitor of hENTl-mediated transport, had similar effects on whole-cell currents as the application of exogenous adenosine. Inhibitors of adenosine kinase and 5′-nucleotidase increased and decreased, respectively, whole-cell currents, whereas inhibition of adenosine deaminase had no effect. Single-channel studies showed that NBMPR and adenosine kinase inhibitors activated CFTR Cl- channels. These results suggested that the equilibrative nucleoside transporters (hENTl, hENT2) together with adenosine kinase and 5′-nucleotidase play a crucial role in the regulation of CFTR through an adenosine-dependent pathway in human airway epithelia.