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Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. However, the exact molecular nature of this regulation is still largely unknown. In this report human epithelial CFTR was expressed in human melanoma cells genetically devoid of the filamin homologue actin-cross-linking protein ABP-280 [ABP(-)]. cAMP stimulation of ABP(-) cells or cells genetically rescued with ABP-280 cDNA [ABP(+)] was without effect on whole cell Cl(-) currents. In ABP(-) cells expressing CFTR, cAMP was also without effect on Cl(-) conductance. In contrast, cAMP induced a 10-fold increase in the diphenylamine-2-carboxylate (DPC)-sensitive whole cell Cl(-) currents of ABP(+)/CFTR(+) cells. Further, in cells expressing both CFTR and a truncated form of ABP-280 unable to cross-link actin filaments, cAMP was also without effect on CFTR activation. Dialysis of ABP-280 or filamin through the patch pipette, however, resulted in a DPC-inhibitable increase in the whole cell currents of ABP(-)/CFTR(+) cells. At the single-channel level, protein kinase A plus ATP activated single Cl(-) channels only in excised patches from ABP(+)/CFTR(+) cells. Furthermore, filamin alone also induced Cl(-) channel activity in excised patches of ABP(-)/CFTR(+) cells. The present data indicate that an organized actin cytoskeleton is required for cAMP-dependent activation of CFTR.

Trafficking, activation, and kinetics of delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR were compared in stably transduced C127I mouse mammary epithelial cells. Western blots detected a small amount of fully glycosylated delta F508-CFTR Efflux of 125I was stimulated by forskolin with the same mean effective concentration (EC50; approximately 0.5 microM) for CFTR and delta F508-CFTR cells, but the maximum response was reduced more than fivefold and its latency increased approximately threefold in delta F508-CFTR cells. In delta F508-CFTR cells, 3-isobutyl-1-methylxanthine (IBMX; EC50 = 1.45 microM) and 8-cyclopentyl-1,3-dipropylxanthine (CPX; EC50 = 58 microM) increased the peak forskolin-stimulated efflux rate approximately 2.5-fold and decreased the time to peak. A sevenfold increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels accompanied potentiation of forskolin-induced 125I efflux by IBMX but not by CPX. Elevation of intracellular cAMP increased linear voltage-independent whole cell currents 30-fold in CFTR and 4-fold in delta F508-CFTR cells; the response rate in delta F508-CFTR cells was much slower. Single-channel currents were detected in 57 of 68 cell-attached patches from forskolin-prestimulated CFTR cells vs. 6 of 35 patches in delta F508-CFTR cells. Mean number of active channels per patch was 4.1 for CFTR [open probability (Po) = 0.34] and 0.2 for delta F508-CFTR (Po = 0.11). The lower Po of delta F508-CFTR resulted from an approximately threefold longer mean interburst interval. We estimate that forskolin-stimulated chloride conductance of delta F508-CFTR C127I cells is < 5% of CFTR cells. CPX is approximately 25-fold more potent than IBMX in potentiating delta F508-CFTR and may operate by a mechanism other than elevation of cAMP.

Thyroid-stimulating hormone (TSH; thyrotropin) produces a pleiotropic response in the thyroid gland, accelerating nearly every aspect of metabolic turnover within the follicular epithelia. We examined the effects of TSH on expression of Na(+)-K(+)-ATPase in FRTL-5 cells, a cell line derived from rat thyroid. TSH (10 mU/ml) produced a nearly twofold increase in abundance of the mRNA encoding the catalytic alpha 1-subunit within 6 h of treatment. With the four mRNAs encoding the beta 1-subunit, TSH produced a striking increase in abundance, but this regulation was discoordinate, and some species increased more than others. Similar increases in mRNA abundance were elicited by activators of the adenosine 3',5'-cyclic monophosphate second messenger system. In contrast to the alpha 1- and beta 1-mRNAs, the abundance of the mRNA encoding the beta 2-subunit was unchanged with TSH after 6 h, indicating that the effects of thyrotropin were not universal or indiscriminate. Thyrotropin also caused a 76% increase in Na(+)-K(+)-ATPase activity and a 46% increase in pump-mediated transport after 48 h. These studies suggest that the changes in metabolic turnover initiated by TSH during hormone synthesis include upregulation of the N(+)-K+ pump.

Using the whole cell configuration of the patch-clamp technique, we have identified an adenosine 3',5'-cyclic monophosphate (cAMP)-regulated chloride conductance in pancreatic duct cells. Basal whole cell currents in single isolated cells were very low (approximately 5 pA/pF) but could be stimulated 17-fold by elevation of intracellular cAMP. The cAMP-activated currents exhibited 1) a high chloride selectivity, 2) a near linear current-voltage relationship, 3) time and voltage independence, 4) block by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and 5) an anion selectivity sequence based on permeability ratios of SCN > NO3 > Br > Cl > I > HCO3 > F > ClO4 > gluconate. Currents in single cells ran down within a few minutes; however, stable chloride currents could be recorded from duct cell clusters in which four or five cells were in electrical communication. We present evidence suggesting that these cAMP-regulated currents are carried by cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. Physiologically, these CFTR channels act in parallel with chloride-bicarbonate exchangers to facilitate bicarbonate secretion across the apical plasma membrane of the duct cell.

In this study we investigated the properties of adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated Cl- efflux in Caco-2 monolayers by measuring 125I efflux rates from preloaded cells and using patch-clamp electrophysiology. The addition of a cocktail containing 100 microM dibutyryl cAMP (DBcAMP), 10 microM forskolin, and 1 mM 3-isobutyl-1-methylxanthine caused a significant (P less than 0.05) increase in the rate of 125I efflux. Dissipation of cell potential by adding valinomycin (4.5 microM) with 135 mM extracellular KCl reduced the cAMP-evoked 125I efflux. These results suggest that cAMP-stimulated anion efflux occurs through a conductive pore or channel. Whole cell currents evoked with DBcAMP or forskolin were anion selective, PCl greater than PI greater than Pgluconate, and exhibited a linear current-voltage (I-V) relationship. Currents evoked with depolarizing or hyperpolarizing voltage steps showed no evidence of time-dependent activation or inactivation. Single Cl- channels were stimulated in cell-attached patches after treatment with cAMP. Onset of channel activity occurred after 20-30s of cAMP treatment, and the response was long lasting. The I-V relationship for the channel activated in cell-attached patches by cAMP was best fit using two linear regressions. The slope conductance of the channel was 3.2 +/- 0.6 and 7.4 +/- 0.3 pS at hyperpolarizing and depolarizing potentials, respectively. Substitution of 140 mM NaCl with 70 mM NaCl in the patch pipette resulted in a positive shift in reversal potential, indicating that the channel is anion selective.(ABSTRACT TRUNCATED AT 250 WORDS)

The outwardly rectified chloride channel of secretory epithelial cells is inhibited by disulfonic stilbene (DS) compounds such as 4,4'-diisothiostilbene-2,2'-disulfonic acid (DIDS) [R. J. Bridges, R. T. Worrell, R. A. Frizzell, and D. J. Benos, Am. J. Physiol. 256 (Cell Physiol. 25): C902-C912, 1989]. A 13-amino acid peptide (P49) corresponding to the putative DS binding site region of the murine anion exchange protein was synthesized, and polyclonal antibodies were generated against it and then purified over a P49 affinity column. The resulting monospecific antibodies reacted on Western blots with a 95- to 100-kDa protein from human erythrocytes and a 55- to 60-kDa protein from the human colonic tumor cell line, T84. The reaction with T84 protein did not appear to represent recognition of an anion exchanger because anion efflux from T84 cells was independent of external Cl-. In addition, monoclonal antibodies raised against human band 3 recognized the band 3 protein in human red cell ghost preparations but recognized nothing in T84 cell membrane preparations. In T84 cells, DIDS protected the 60-kDa protein from antibody binding. The anti-P49 antibody blocked outwardly rectified Cl- channels incorporated into planar lipid bilayer membranes from rat colon. Immunocytochemical data reveal specific binding of the anti-P49 antibody to perinuclear cytoplasmic vesicles. Forskolin caused these antibody-labeled vesicles to migrate from the perinuclear region to the plasma membrane under conditions and with a time course identical to that seen for stimulation of Cl- transport in these cells. Our results suggest that the protein may be a part of a chloride channel complex of secretory epithelial cells.