Role of Purinergic Receptor in Alpha Fodrin degradation in Par C5 Cells

Autoantibodies specific for alpha-fodrin fragments are found in the tissues of persons afflicted with Sjogren's syndrome (SS). However, the mechanism for alpha-fodrin degradation remains elusive. The following experiments utilized Par C5 cells to examine the role of P2X7 receptor (P2X7R) in apoptosis, particularly in the cleavage and release of alpha-fodrin, an apparent SS autoantigen. Five mM ATP stimulation induced apoptotic cell death with a sustained Ca2+ influx, which was mimicked in HEK cells transfected with P2X7R. ATP also induced cleavage of alpha-fodrin mediated by caspase-3 and calpain, releasing alpha-fodrin fragments through membrane blebs. However, both apoptotic cell death and alpha-fodrin cleavage were inhibited in the presence of 300 mu M oxidized-ATP (ox-ATP), an irreversible blocker of P2X7R, or in Ca2+-free solution. We concluded that P2X7R plays an important role in apoptosis and alpha-fodrin degradation in salivary epithelial cells, providing an important clue elucidating the presence of alpha-fodrin fragments in SS tissues. Abbreviations: ATP, adenosine triphosphate; ROCK, Rho-associated kinase; MTT, methyl tetrazolium; PI, propidium iodide; [Ca2+] (i), intracellular Ca2+ concentration; ox-ATP, oxidized ATP; RT-PCR, reverse-transcriptase polymerase chain-reaction; Ab, antibody; SS, Sjogren's syndrome; HEK, human embryonic kidney; HEK-P2X7R, HEK293 cells expressing recombinant P2X7R; FACS, fluorescence-activated cell-sorting.