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Address correspondence to Seiko F. Okada, Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. Fax: (919) 966-5178; email: seiko_okada{at}med.unc.edu
Extracellular ATP regulates several elements of the mucus clearance process important for pulmonary host defense. However, the mechanisms mediating ATP release onto airway surfaces remain unknown. Mitochondrial voltage-dependent anion channels (mt-VDACs) translocate a variety of metabolites, including ATP and ADP, across the mitochondrial outer membrane, and a plasmalemmal splice variant (pl-VDAC-1) has been proposed to mediate ATP translocation across the plasma membrane. We tested the involvement of VDAC-1 in ATP release in a series of studies in murine cells. First, the full-length coding sequence was cloned from a mouse airway epithelial cell line (MTE7b–) and transfected into NIH 3T3 cells, and pl-VDAC-1-transfected cells exhibited higher rates of ATP release in response to medium change compared with mock-transfected cells. Second, ATP release was compared in cells isolated from VDAC-1 knockout [VDAC-1 (–/–)] and wild-type (WT) mice. Fibroblasts from VDAC-1 (–/–) mice released less ATP than WT mice in response to a medium change. Well-differentiated cultures from nasal and tracheal epithelia of VDAC-1 (–/–) mice exhibited less ATP release in response to luminal hypotonic challenge than WT mice. Confocal microscopy studies revealed that cell volume acutely increased in airway epithelia from both VDAC-1 (–/–) and WT mice after luminal hypotonic challenge, but VDAC-1 (–/–) cells exhibited a slower regulatory volume decrease (RVD) than WT cells. Addition of ATP or apyrase to the luminal surface of VDAC-1 (–/–) or WT cultures with hypotonic challenge produced similar initial cell height responses and RVD kinetics in both cell types, suggesting that involvement of VDAC-1 in RVD is through ATP release. Taken together, these studies suggest that VDAC-1, directly or indirectly, contributes to ATP release from murine cells. However, the observation that VDAC-1 knockout cells released a significant amount of ATP suggests that other molecules also play a role in this function.
Key Words: voltage-dependent anion channel • ATP release • osmotic cell swelling • regulatory volume decrease • airway epithelia
Abbreviations used in this paper: CFTR, cystic fibrosis transmembrane conductance regulator; DMEM, Dulbecco's modified eagles medium; mt, mitochondrial; pl, plasmalemmal; RVD, regulatory volume decrease; TCA, trichloroacetic acid; VDAC, voltage-dependent anion channel; WT, wild type.
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