The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl-channel regulated by protein kinase A. The most common mutation In cystic fibrosis (CF), deletion of Phe-S08 (AF508-CFTR), reduces Cl1 secretion, but the fatal consequences of CF have been difficult to rationalie solely in terms ofthis defect. The aim ofthis study was to determine the role of CFTR in HCO_ transport across cell membranes. HCO perbili was asse from measurements of intracellular pH [pHi; from spectrofluorimetry of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-S-(and -6)carboxyfluorescein] and of channel activity (patch damp; cell attached and isolated, inside-out patches) on NIH 3T3 fibroblasts and C127 mammary epithelial cells transfected with wild-type CFTR (WT-CFTR) or AF5S08-CFTR, and also on mock-transfected cells. When WT-CFTR-transfected cells were acidified (pulsed with NH4CI) and incubated in Na+-free (N-methyl-n-glucmine substitution) solutions (to block Na+-dependent pHI, regulatory mechanisms), pH, remained acidic (pH 6.5) until the cells were treated with 20 IAM forskolin (increases cellular [cAMPI); pHi then increased toward (but not completely to) control level (pH1 7.2) at a rate of 0.055 pH unit/min. Forskolin had no effect on rate of pHi recovery in AF508 and mock-transfected cells. This Na+-independent, forskolin-dependent pHi recovery was not observed in HCO /CO2-free medium. Forskolin-treated WT-CFTRtransfected (but not AF508-CFTR or mock-transfected) cells in Cl0-containing, HCO--free solutions showed Cl-channels with a linear I/V relationship and a conductance of 10.4 ± 0.5 pS in symmetrical 150 mM Cl-. When channeis were incubated with different [Cl-I and [HCOi-on the inside and outside, the Cl-/HCO; permeability ratio (determined from reversal potentials of i/V curves) was 3.8 ± 1.0 (mean ± SEM; n = 9); the ratio of conductances was 3.9 ± 0.5 (at 150 mM Cl-and 127 mM HCO5. We conclude that in acidified cells the WT-CFTR functions as a base loader by allowing a cAMPdependent influx of HCO-through channeis that conduct HCOj about one-quarter as efflciently as it conducts Cl-.Under physiological conditions, the electrochemical gradients for both Cl-and HCO5 are directed outward, so CFTR likely contributes to the epithellal secretion of both ions. HCO-3 secretion may be important for controlling pH of the luminal, but probably not the cytoplasmic, fluid in CFTR-containing epithelia. In CF, a decreased secretion of HCO3 may lead to decreased pH of the luminal fluid.The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl-channel regulated by cAMPdependent protein kinase A (1-3). The most common mutation in CF is deletion of Phe-508 of the CFTR (AF508-CFTR), which reduces the ability ofcells to secrete Cl-(4). However, the fatal consequences in CF have been difficult to rationalize solely as a defect in Cl-secretion (5, 6). If the CFTR were also conductive to HCO-, then movements of HCO-might affect the pH of the luminal solution of some epithelia. For example, i...

A precise pH gradient between organelles of the regulated secretory pathway is required for sorting and processing of prohormones. We studied pH regulation in live endocrine cells by targeting biotin-based pH indicators to cellular organelles expressing avidin-chimera proteins. In AtT-20 cells, we found that steady-state pH decreased from the endoplasmic reticulum (ER) (pH ER ‫؍‬ 7.4 ؎ 0.2, mean ؎ S.D.) to Golgi (pH G ‫؍‬ 6.2 ؎ 0.4) to mature secretory granules (MSGs) (pH MSG

Genistein, a protein tyrosine kinase inhibitor, activates the cystic fibrosis transmembrane conductance regulator (CFTR) in transfected NIH-3T3 fibroblasts that express the CFTR (3T3-CFTR). CFTR activity was assayed by 125I efflux and by patch clamping in the cell-attached mode. Both forskolin and genistein stimulated 125I efflux and activated a 9-10 pS anion channel in 3T3-CFTR cells but failed to activate 125I efflux in mock-transfected NIH-3T3 cells. Genistein, unlike forskolin and 3-isobutyl-1-methylxanthine, did not increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) above control levels. This demonstrates that genistein-dependent activation does not involve inhibition of phosphodiesterase activity and suggests that stimulation does not involve a direct activation of protein kinase A. Genistein stimulated 125I efflux to approximately 50% of the maximal rate with forskolin. Genistein did not increase 125I efflux at saturating forskolin but decreased the concentration of forskolin required for half-maximal stimulation. Orthovanadate (VO4), a phosphotyrosine phosphatase inhibitor, inhibited genistein-induced channel activation with an inhibition constant of approximately 20 microM. These effects suggest that, in addition to activation by protein kinase A, the CFTR is regulated by a tyrosine kinase-dependent pathway.

The purpose of this study was to determine the role of NADPH oxidase in H ؉ secretion by airway epithelia. In whole cell patch clamp recordings primary human tracheal epithelial cells (hTE) and the human serous gland cell line Calu-3 expressed a functionally similar zincblockable plasma membrane H ؉ conductance. However, the rate of H ؉ secretion of confluent epithelial monolayers measured in Ussing chambers was 9-fold larger in hTE compared with Calu-3. In hTE H ؉ secretion was blocked by mucosal ZnCl 2 and the NADPH oxidase blockers acetovanillone and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), whereas these same blockers had no effect in Calu-3. We determined levels of transcripts for the NADPH oxidase transmembrane isoforms (Nox1 through -5, Duox1 and -2, and p22 phox ) and found Duox1, -2, and p22 phox to be highly expressed in hTE, as well as the intracellular subunits p40 phox , p47 phox , and p67 phox . In contrast, Calu-3 lacked transcripts for Duox1, p40 phox , and p47 phox . Anti-Duox antibody staining resulted in prominent apical staining in hTE but no significant staining in Calu-3. When treated with amiloride to block the Na ؉ /H ؉ exchanger, intracellular pH in hTE acidified at significantly higher rates than in Calu-3, and treatment with AEBSF blocked acidification. These data suggest a role for an apically located Duox-based NADPH oxidase during intracellular H ؉ production and H ؉ secretion, but not in H ؉ conduction.

Oregon 97403-1229 (J.R.) Arabidopsis (Arabidopsis thaliana) was transformed with a redox-sensing green fluorescent protein (reduction-oxidationsensitive green fluorescent protein [roGFP]), with expression targeted to either the cytoplasm or to the mitochondria. Both the mitochondrial and cytosolic forms are oxidation-reduction sensitive, as indicated by a change in the ratio of 510 nm light (green light) emitted following alternating illumination with 410 and 474 nm light. The 410/474 fluorescence ratio is related to the redox potential (in millivolts) of the organelle, cell, or tissue. Both forms of roGFP can be reduced with dithiothreitol and oxidized with hydrogen peroxide. The average resting redox potentials for roots are 2318 mV for the cytoplasm and 2362 mV for the mitochondria. The elongation zone of the Arabidopsis root has a more oxidized redox status than either the root cap or meristem. Mitochondria are much better than the cytoplasm, as a whole, at buffering changes in redox. The data show that roGFP is redox sensitive in plant cells and that this sensor makes it possible to monitor, in real time, dynamic changes in redox in vivo.Cellular redox status influences many processes in plants, including apoptosis (Cai and Jones, 1999), oxidative defense mechanisms (Foyer and Noctor, 2005), senescence (Groten et al., 2005), allosteric control of enzyme activities, transcription and translation (Apel and Hirt, 2004), and a variety of signal transduction pathways (Drö ge, 2002;Ermak and Davies, 2002;Neill et al., 2002). Yet, as central as is redox status to these processes, the redox potentials (oxidationreduction potential) of living plant cells have rarely been measured during the occurrence of these activities (Renew et al., 2005). Rather, most often plant tissues are homogenized and the homogenates subsequently assayed, either with redox-sensing electrodes, or, by measuring the ratios of the reduced and oxidized forms of glutathione and ascorbate, the two principal redox regulators in living systems (Foyer and Noctor, 2003). Recently the redox state of plant tissues has also been assessed using the dyes 5-(and 6-) carboxy-2#, 7#-dichlorodihydrofluorescein diacetate (C-400; Jiang et al., 2003) and dihydrofluorescein diacetate (N. Smirnoff, personal communication). While such approaches allow one to sum the oxidized and reduced species, and thereby to infer the overall redox status of a tissue, it is not possible with these approaches to obtain a measure of redox potential at the time the events of interest are occurring. Moreover, whole tissue homogenization does not allow one to more finely resolve redox status within the various compartments and organelles comprising a typical plant cell, nor does this approach allow for an assessment of the redox status of the cell wall. As well, homogenizing a tissue precludes the possibility of monitoring dynamic changes of redox status, including reversibility. As a consequence, plant biologists lack knowledge of the rapidity of redox changes in plant cells.Rece...