Physical stimulation of airway surfaces evokes liquid secretion, but the events that mediate this vital protective function are not understood. When cystic fibrosis transmembrane conductance regulator (CFTR) channel activity was used as a functional readout, we found signaling elements compartmentalized at both extracellular and intracellular surfaces of the apical cell membrane that activate apical Cl ؊ conductance in Calu-3 cells. At the outer surface, ATP was released by physical stimuli, locally converted to adenosine, and sensed by A2B adenosine receptors. These receptors couple to G proteins, adenylyl cyclase, and protein kinase A, at the intracellular face of the apical membrane to activate colocalized CFTR. Thus, airways have evolved highly efficient mechanisms to ''flush'' noxious stimuli from airway surfaces by selective activation of apical membrane signal transduction and effector systems.A irways continuously remove noxious materials through a mucociliary clearance process that requires liquid secretion (1, 2). cAMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl Ϫ channels (3, 4) are expected to participate in liquid secretion in airways, but the two key events in the activation of CFTR by local physical stimuli remain puzzling. First, how do physical stimuli initiate the classic cAMP signaling cascade, a process that is tightly regulated by Gprotein-coupled receptors (5)? Second, how does cAMP reach CFTR in the apical membrane? The dogma of G-proteincoupled receptors and adenylyl cyclase (AC) restricted to the basolateral membrane of epithelia does not adequately explain how these events occur (6).Two ongoing areas of research suggest a potentially more relevant, but as yet not fully tested model for activation of apical CFTR by a local physical stimulus. Airway surface epithelia are poorly innervated, suggesting that mucociliary clearance is subject to autocrine͞paracrine control. A leading candidate for mediating mucociliary clearance is the release of cellular nucleotides, because release occurs in response to physical stimuli and luminal nucleotide receptors stimulate apical Cl Ϫ conductance, mucus secretion, and ciliary beating (7,8). The action of ectonucleotidases extends the signaling potential of released ATP on luminal surface by producing adenosine (Ado), a ligand for A2 receptors that couple to AC (9, 10). Recent reports indicate that receptors, intracellular signaling pathways, and scaffolding molecules can form complexes that locally regulate functions in subcellular compartments (11,12). Thus, a model linking a luminal physical stimulus to activation of CFTR requires specific elements, including ATP release, ectonucleotidases, Ado receptors, G proteins, and AC, to be intimately associated with the apical cell membrane. The goal of the present study was to test this hypothesis in polarized airway epithelial cells. MethodsCells. Human Calu-3 cells were grown as previously described (13) on Costar clear transwells (for HPLC and cAMP assay) or homemade permeable ...

We report a facile intracellular manipulation of fluorescent magnetic Fe3O4-CdSe nanoparticles using magnetic force. The growth of CdSe quantum dots on Fe3O4 nanoparticles produces Fe3O4-CdSe nanoparticles with two distinct properties, fluorescence and superparamagnetism. After nonspecific surface modification using glutathione (GSH), the hydrophilic Fe3O4-CdSe@GSH nanoparticles can be easily uptaken by an HEK293T cell line. Confocal images indicate that the uptaken nanoparticles can be manipulated using a small magnet. The successful intracellular manipulation of magnetic nanoparticles may offer a new strategy for studying polarized cells.

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.

We demonstrated previously that Calu-3 airway epithelial cells sense adenosine on their luminal surface through adenosine A2B receptors coupled to adenylyl cyclase. Occupancy of these receptors leads to activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel through protein kinase A (PKA) anchored at the apical membrane. Because luminal A2B receptor activation does not raise total cellular cAMP levels, we hypothesized that activation of phosphodiesterases (PDEs) confines cAMP generated by apical A2B receptors to a microdomain that includes the CFTR channel. Using reverse transcription-PCR, Western blotting, and activity measurements, PDE4D was identified as the major PDE species in airway epithelia. Consistent with these results, inhibitors of PDE4, but not PDE3, selectively abolished the lateral confinement of cAMP signaling in apical membrane patches during cell-attached recordings. Furthermore, stimulation of the CFTR in excised apical patches by rolipram and RS25344 indicated that PDE4 is localized in close proximity to the CFTR channel. Indeed, immunohistochemistry of human airway sections revealed that PDE4D is localized in the apical domain of the cell. PDE4 was activated after luminal adenosine exposure in a PKA-dependent manner. Because PDE4 activity is positively regulated by PKA, our results support a model whereby the PDE diffusion barrier is proportional to the degree of receptor stimulation. These findings underscore the concept that subcellular localization of individual PDE isozymes is an important mechanism for confining cAMP signaling to functional domains within cells.