The present study concerns the involvement of the ceramide produced through sphingomyelinase (SMase)-mediated catalysis in airway anion secretion of Calu-3 cells. Short-circuit current (I-sc) measurement revealed that isoproterenol (ISO, 0.1 muM)-induced anion secretion was prevented by pretreatment with SMase (0.3 U/ml, for 30 min) from the basolateral but not the apical side, although basal and 1-ethyl-2-benzimidazolitione (1-EBIO, a Ca2+-activated K+ channel opener)-induced I-sc were unaffected. The effects of SMase were reproduced in responses to forskolin (20 muM) or 8-bromo-cAMP (2 mM). C-2-ceramide, a cell-permeable analog, also repressed the 8-bromo-cAMP-induced responses. Nystatin permeabilization studies confirmed that the SMase- and C-2-ceramide-induced repressions were due to hindrance of augmentation of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated conductance across the apical membrane. Further, SMase failed to influence K+ conductance across the basolateral membrane. These results suggest that the ceramide originating from basolateral sphingomyelin acts on activated CFTR from the cytosolic side, hindering anion secretion. (C) 2004 Elsevier Inc. All rights reserved.