SUMMARY:We have previously shown that, in normal human airway tissue, localization of the cystic fibrosis transmembrane conductance regulator (CFTR) can be affected by epithelial maturation, polarity, and differentiation and that CFTR trafficking and apical localization depend on the integrity of the airway epithelium. In this study, we addressed the question of whether the three-dimensional (3-D) organization of adult human airway epithelial cells in suspension culture under rotation, leading to spheroid-like structures, could mimic the in vivo phenomenon of differentiation and polarization. The kinetics of the differentiation, polarity, and formation of the CFTR-ZO-1-ezrin complex was analyzed by transmission, scanning, and immunofluorescence microscopy. Functional activity of the airway surface epithelium was assessed by monitoring the degree of cAMP-stimulated chloride efflux from cultured cells. Our results show that after the initial step of dedifferentiation, characterized by a loss of ciliated cells and disappearance of epithelial subapical CFTR-ezrin-ZO-1 complex, the isolated cells formed 3-D spheroid structures within 24 hours. After 15 days, progressive ciliogenesis was observed and secretory cells could be identified. After 35 days of 3-D culture, ZO-1, CFTR, ezrin, and CD59 were apically or subapically located, and well-differentiated secretory and ciliated cells were identified. CFTR functionality was assessed by analyzing the Cl Ϫ secretion after amiloride and forskolin perfusion. After 35 days of culture of spheroids in suspension, a significant increase in Cl Ϫ efflux was observed in well-differentiated ciliated cells. (Lab Invest 2002, 82:989 -998).
PSD-95/Dlg-A/ZO-1 (PDZ) domains play an essential role in determining cell polarity. The Na ؉ /H ؉ exchanger regulatory factor (NHERF), also known as EBP50, contains two PDZ domains that mediate the assembly of transmembrane and cytosolic proteins into functional signal transduction complexes. Moreover, it has been shown that cystic fibrosis transmembrane conductance regulator (CFTR) and  2 -adrenergic receptor ( 2 AR) bind equally well to the PDZ1 domain of EBP50. We hypothesized that  2 AR activation may regulate CFTR protein expression. To verify this, we evaluated the effects of a pharmacologically relevant concentration of salmeterol (2.10 ؊7 M), a long acting  2 AR agonist, on CFTR expression in primary human airway epithelial cells (HAEC).  2 AR stimulation induced a time-dependent increase in apical CFTR protein expression, with a maximal response reached after treatment for 24 h. This effect was post-transcriptional, dependent upon the  2 AR agonist binding to  2 AR and independent of the known  2 AR agonist-mediated cAMP/PKA pathway. We demonstrated by immunohistochemistry that CFTR,  2 AR, and EBP50 localize to the apical membrane of HAEC. Analyses of anti-EBP50 protein immunoprecipitate showed that salmeterol induced an increase in the amount of CFTR that binds to EBP50. These data suggest that  2 AR activation regulates the association of CFTR with EBP50 in polarized HAEC.
The effect of cell swelling on intracellular calcium concentration ([Ca2+]i) was studied in newborn rat cardiomyocytes. Hypotonic cell swelling induced a fast and transient [Ca2+]i increase (hypotonically induced calcium increase, HICI; 388±47 nM, n=14). HICI was not inhibited by cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic Ca2+-ATPase, nor ryanodine (an inhibitor of calcium-induced calcium release), whereas it was abolished (11±19 nM, n=5) in the absence of external calcium. Thus, HICI appeared to depend exclusively on entry of external calcium. Gadolinium ion (Gd3+), a generic inhibitor of stretch-activated cation channels (SACs), was unable to affect HICI (353±79 nM, n=6). Similarly, HICI was unaffected by internal Na+ depletion and external Na+ omission. These results suggest that neither Gd3+-sensitive SACs nor Na+-Ca2+ exchange is responsible for HICI. Conversely, HICI was inhibited by diltiazem (42±4 nM, n=3) and by membrane predepolarization (40±18 nM, n=5), suggesting an involvement of L-type voltage-activated calciumchannels. Cardiomyocyte swelling was followed by a regulatory volume decrease (RVD). The putative role of HICI in volume regulation was studied by removal of external calcium. This procedure significantly slowed RVD but did not abolish it. In conclusion, newborn rat cardiomyocytes exhibit an external-calcium-dependent HICI which contributes partially to the RVD.
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