Regulation and amiloride-binding site of epithelial Na+ channels.
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Amiloride↗
Biomedical subjects
K Kunzelmann
Publications and source records attributed to K Kunzelmann.
Specific blockade of slowly activating I(sK) channels by chromanols -- impact on the role of I(sK) channels in epithelia.
Chromanols, which were recently shown to inhibit cAMP-mediated Cl- secretion in colon crypts via a blockade of a cAMP-activated K+ conductance, were analyzed for their effects on distinct cloned K+ channels expressed in Xenopus oocytes. The lead chromanol 293B specifically inhibited I(sK) channels with an IC50 of 7 micromol/l without affecting the delayed rectifier Kv1.1 or the inward rectifier Kir2.1. Moreover, several other chromanols displayed the same rank order of potency for I(sK) inhibition as demonstrated in colon crypts. Finally, we tested the effects of the previously described I(sK) blocker azimilide on cAMP mediated Cl- secretion in rat colon crypts. Similar to 293B azimilide inhibited the forskolin induced Cl- secretion. These data suggest that I(sK) protein induced K+ conductances are the targets for the chromanol 293B and its analogues, and azimilide.
Animals↗
Effect of isosorbiddinitrate on exogenously expressed slowly activating K+ channels and endogenous K+ channels in Xenopus oocytes.
1. The effects of isosorbiddinitrate (ISDN) were tested on membrane currents and resting potential in Xenopus laevis oocytes which were either uninjected or injected with cRNA encoding for K+ channels from three distinct families (slowly activating IsK channels, delayed-rectifying Kv1.1 or inwardly rectifying IRK1 K+ channels). 2. In uninjected oocytes ISDN (1 mM) resulted in a decrease of the holding current at potentials more positive than -100 mV and in an increase at potentials below -100 mV. Increasing extracellular K+ to 100 mM shifted the reversal potential for ISDN-mediated effects to approximately -12 mV, suggesting an inhibition of a K+ conductance by ISDN. 3. In current clamp studies ISDN (1 mM) and Ba2+ (3 mM) depolarized cell membrane. ISDN and Ba2+ had no additive effects on membrane potential when applied simultaneously. In voltage clamp studies, corresponding results were observed for the effects of ISDN and Ba2+ on the holding current with an apparent K(m) of 0.21 and 0.08 mM, respectively. 4. In contrast to ISDN, the nitric oxide (NO) donors isosorbidmononitrate (ISMN) and S-nitrosocysteine (SNOC) had no effects on the holding currents in Xenopus oocytes. Moreover, the guanylate inhibitor LY 83583 did not affect ISDN-mediated holding current alterations, suggesting that ISDN acts independently of the second messenger NO. 5. ISDN inhibited exogenously expressed IsK channels with an apparent K(m) of 0.15 mM, but at 1 mM only weakly inhibited Kv1.1 and IRK1 channels. 6. It is concluded that ISDN inhibits an endogenous K+ conductance in Xenopus oocytes with a similar potency to that shown by expressed IsK channels. These effects are independent of the second messenger NO.
Aminoquinolines↗
Wild type but not deltaF508 CFTR inhibits Na+ conductance when coexpressed in Xenopus oocytes.
Airway epithelial cells bearing mutations of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) possess an increased Na+ conductance along with their well described defect of cAMP dependent Cl- conductance. Currently it is not clear, how this occurs, and whether it is due to a CFTR control of epithelial Na+ conductances which might be defective in CF patients. In the present study, we have tried to identify possible interactions between both CFTR and the epithelial Na+ conductance by overexpressing respective cRNAs in Xenopus oocytes. The expression of all three (alpha, beta, gamma) subunits of the rat epithelial Na+ channel (rENaC) and wild type (wt) CFTR resulted in the expected amiloride sensitive Na+ and IBMX (1 mmol/l) activated Cl- currents, respectively. The amiloride sensitive Na+ conductance was, however, inhibited when the wt-CFTR Cl- conductance was activated by phosphodiesterase inhibition (IBMX). In contrast, IBMX had no such effect in deltaF508 and Na+ channels coexpressing oocytes. These results suggest that wt-CFTR, but not deltaF508-CFTR, is a cAMP dependent downregulator of epithelial Na+ channels. This may explain the higher Na+ conductance observed in airway epithelial cells of CF patients.
1-Methyl-3-isobutylxanthine↗
Regulation of epithelial ion channels by the cystic fibrosis transmembrane conductance regulator.
In most epithelia ion transport is tightly regulated. One major primary target of such regulation is the modulation of ion channels. The present brief review focuses on one specific example of ion channel regulation by the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR functions as a cAMP-regulated Cl- channel. Its defect leads to the variable clinical pictures of cystic fibrosis (CF), which today is understood as a primary defect of epithelial Cl- channels in a variety of tissues such as the respiratory tract, intestine, pancreas, skin, epididymis, fallopian tube, and others. Most recent findings suggest that CFTR also acts as a channel regulator. Three examples are discussed by which CFTR regulates other Cl- channels, K+ channels, and epithelial Na+ channels. From this perspective it is evident that CFTR may play a major role in the integration of cellular function.
Animals↗
N-Acetyl-L-cysteine and its derivatives activate a Cl- conductance in epithelial cells.
N-Acetyl-L-cysteine (NAC) is a widely used mucolytic drug in patients with a variety of respiratory disorders including cystic fibrosis (CF). The beneficial effects of NAC are empirical and the exact mechanism of action in the airways remains obscure. In the present study we examined the effects on whole-cell (wc) conductance (Gm) and voltage (Vm) of NAC and the congeners S-carboxymethyl-L-cysteine (CMC) and S-carbamyl-L-cysteine (CAC) and L-cysteine in normal and CF airway epithelial cells. L-Cysteine (1 mmol/l) had no detectable effect. The increase in Gm (delta Gm) by the other compounds was concentration dependent and was (all substances at 1 mmol/l) 3.8 +/- 1.4 nS (NAC; n = 11), 4.2 +/- 1.0 nS (CMC; n = 16) and 3.8 +/- 1.6 nS (CAC; n = 18), respectively. The changes in Gm were paralleled by an increased depolarization (delta Vm) when extracellular Cl- concentration was reduced to 34 mmol/l: under control conditions = -4.1 +/- 2.1 versus 10.2 +/- 2.1 mV in the presence of NAC, CMC, CAC (n = 36). In the presence of NAC, CMC and CAC, the reduction in Cl- concentration was paralleled by a reduction of Gm by 2.1 +/- 0.4 nS (n = 35), indicating that all substances acted by increasing the Cl- conductance. Analysis of intracellular pH did not reveal any changes by any of the compounds (1 mmol/l). A Cl- conductance was also activated in HT29 colonic carcinoma and CF tracheal epithelial (CFDE) cells but not in CFPAC-1 cells, which do not express detectable levels of delta F508-CFTR, suggesting that the presence of CFTR may be a prerequisite for the induction of Cl- currents. Next we examined the ion currents in Xenopus oocytes microinjected with CFTR-cRNA. Water-injected oocytes did not respond to activation by forskolin and 3-isobutyl-1-methylxanthine (IBMX) (delta Gm = 0.08 +/- 0.04 microS; n = 10) and no current was activated when these oocytes were exposed to NAC or CMC. In contrast, in CFTR-cRNA-injected oocytes Gm was enhanced when intracellular adenosine 3',5'-cyclic monophosphate (cAMP) was increased by forskolin and IBMX (Gm = 4.5 +/- 1.3 microS; n = 8). Gm was significantly increased by 0.74 +/- 0.2 microS (n = 11) and 0.46 +/- 0.1 microS (n = 10) when oocytes were exposed to NAC and CMC, respectively (both 1 mmol/l). In conclusion, NAC and its congeners activate Cl- conductances in normal and CF airway epithelial cells and hence induce electrolyte secretion which may be beneficial in CF patients. CFTR appears to be required for this response in an as yet unknown fashion.
Acetylcysteine↗
Culture-dependent expression of Na+ conductances in airway epithelial cells.
According to previous studies, amiloride-sensitive (Amil+) Na+ channels are present in apical membranes of airway epithelial cells. When isolated from intact tissue and grown in primary culture or as immortalized cell lines, these cells tend to lose these Amil+ Na+ channels. The present study examines this issue in immortalized human bronchial epithelial cells (16HBE14o- cell line). The mRNA of one subunit of the Na+ channel alphahENaC) was semi-quantified by polymerase chain reaction of reverse transcribed RNA. Transcripts were significantly increased when cells were exposed to aldosterone and dexamethasone irrespective of whether grown on permeable supports or plastic. When grown on plastic dishes 16HBE14o-cells showed cAMP-dependent Cl- currents in whole-cell (WC) patch-clamp experiments, corresponding to expression of the cystic fibrosis transmembrane conductance regulator (CFTR). Na+ currents could not be detected although cells expressed significant amounts of alphahENaC as demonstrated by Northern blot analysis. In contrast, when cells were grown on permeable supports or cultured in the presence of butyrate (5 mmol/l, plastic or permeable support) or aldosterone and dexamethasone (both 1 micromol/l, plastic or permeable support), amiloride (10 micromol/l) hyperpolarized the membrane voltage (deltaVm) by 2-9 mV, paralleled by small reductions of WC conductances (deltaGm) of 0.4-4.0 nS. The effects of amiloride on deltaVm were gnerally more pronounced (up to 12 mV) when cells were grown on permeable supports. The amiloride effect (deltaVm) was concentration dependent with an inhibitory constant, Ki, of about 0.1 micromol/l. We further examined whether the induction of an Amil+ Na+ conductance was paralleled by additional changes in membrane conductance. In fact, the cAMP-activated Cl- conductance was significantly attenuated by approximately 80% (n=35) in cells responding to amiloride, whilst the ATP-activated K+ conductance remained unaffected. The present data suggest that cellular mechanisms determining differentiation control the function expression of Na+ and Cl- conductances in human airway epithelial cells.
Adenosine Triphosphate↗
cAMP stimulation of CFTR-expressing Xenopus oocytes activates a chromanol-inhibitable K+ conductance.
Cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel in a large variety of cells expressing this protein. Recently evidence has accumulated that it also regulates other ion channels. A coordinated increase in Cl- and K+ conductances is necessary in many Cl--secreting epithelia. This has, for example, recently been demonstrated for the colonic crypt, for which a new type of K+ channel and a specific inhibitor of this channel, the chromanol 293B, have been described. In the present study we have examined whether the cAMP-evoked activation of CFTR, overexpressed in Xenopus oocytes, in addition to its known activation of a Cl- conductance, also upregulates endogenous K+ channels. It is shown that CFTR-cRNA-injected but not water-injected oocytes possess a cAMP-activated Cl- conductance. Of the cAMP-induced whole-cell current increase, 15-25% was due to a 293B-, Ba2+and TEA+-inhibitable K+ conductance. The cRNA of the mutated CFTR (DeltaF508 CFTR) had no such effect. We conclude that cAMP activated CFTR and an endogenous IsK-type and 293B-sensitive K+ conductance. Similar events, occurring, for example, in the colonic crypt possessing CFTR and 293B-sensitive K+ channels, might explain the coordinated cAMP-mediated increase in Cl- and K+ conductances.
1-Methyl-3-isobutylxanthine↗
Blockade of epithelial Na+ channels by triamterenes - underlying mechanisms and molecular basis.
The three subunits (alpha, beta, gamma) encoding for the rat epithelial Na+ channel (rENaC) were expressed in Xenopus oocytes, and the induced Na+ conductance was tested for its sensitivity to various triamterene derivatives. Triamterene blocked rENaC in a voltage-dependent manner, and was 100-fold less potent than amiloride at pH 7.5. At -90 mV and -40 mV, the IC50 values were 5 microM and 10 microM, respectively. The blockage by triamterene, which is a weak base with a pKa of 6.2, was dependent on the extracellular pH. The IC50 was 1 microM at pH 6.5 and only 17 microM at pH 8.5, suggesting that the protonated compound is more potent than the unprotonated one. According to a simple kinetic analysis, the apparent inhibition constants at -90 mV were 0.74 microM for the charged and 100.6 microM for the uncharged triamterene. The main metabolite of triamterene, p-hydroxytriamterene sulfuric acid ester, inhibited rENaC with an approximately twofold lower affinity. Derivatives of triamterene, in which the p-position of the phenylmoiety was substituted by acidic or basic residues, inhibited rENaC with IC50 values in the range of 0.1-20 microM. Acidic and basic triamterenes produced a rENaC blockade with a similar voltage and pH dependence as the parent compound, suggesting that the pteridinemoiety of triamterene is responsible for that characteristic. Expression of the rENaC alpha-subunit-deletion mutant, Delta278-283, which lacks a putative amiloride-binding site, induced a Na+ channel with a greatly reduced affinity for both triamterene and amiloride. In summary, rENaC is a molecular target for triamterene that binds to its binding site within the electrical field, preferably as a positively charged molecule in a voltage- and pH-dependent fashion. We propose that amiloride and triamterene bind to rENaC using very similar mechanisms.
Amiloride↗
Gene targeting of CFTR DNA in CF epithelial cells.
A goal of cystic fibrosis (CF) gene therapy is correction of the mutant CF transmembrane conductance regulator (CFTR) gene with wild-type (wt) DNA sequences to restore normal CFTR protein and function. Experiments with wtCFTR cDNA expression vectors have shown that the Cl ion transport phenotype associated with CF can be corrected to resemble that in normal cells. An alternative to cDNA-based gene therapy strategies is one that corrects endogenous mutant sequences by targeted replacement with the wt homologue. To test whether such a strategy was feasible, a small fragment homologous replacement (SFHR) strategy was used to replace specific genomic sequences in human epithelial cells. Small fragments of genomic wtCFTR DNA were transfected into transformed CF epithelial cells. Replacement by exogenous CFTR DNA at the appropriate genomic locus and its expression as mRNA was indicated by: (1) allele-specific polymerase chain reaction (PCR) amplification of genomic DNA and mRNA-derived cDNA; and (2) hybridization of PCR products with allele-specific probes. In addition, the functional activity of CFTR protein was determined by whole cell patch clamp. Southern hybridization and patch clamp analyses suggested that approximately 1 in 100 CF cells underwent a homologous replacement event that resulted in intact Cl transport.
Cell Line↗
Episomal expression of wild-type CFTR corrects cAMP-dependent chloride transport in respiratory epithelial cells.
The isolation of the gene responsible for the Cl- ion transport defect in cystic fibrosis (CF) has provided important information about the relationship between the disease pathology and the underlying genetic and biochemical mechanisms. In addition, new areas of investigation and therapy are now possible. Most notably, the isolation of the CF gene, the cystic fibrosis transmembrane conductance regulator (CFTR) has been led to the development of different gene therapy strategies. To circumvent possible complications due to insertional mutagenesis and virally induced immune responses, we have employed Epstein-Barr virus (EBV)-based expression vectors for correction of the cAMP-dependent Cl- transport defect associated with CF. A CFTR-containing expression construct under the regulation of the Rous sarcoma virus (RSV) long terminal repeat (LTR) (pREP5/CFTR) was introduced into transformed human airway epithelial cells defective in cAMP-dependent Cl-transport. Transfected cells were assayed for Cl- ion transport by (36)Cl- efflux, SPQ, and patch clamp and showed restoration of intact cAMP-dependent transport. CFTR transcription from pREP5/CFTR was detected by Northern hybridization. The level of response to agonists appeared to be dependent on the level of CFTR expression. When cells were tested for functional expression of CFTR after removal of selection pressure, they showed a continuous decrease in responsiveness to forskolin as a function of time after removal of selection. This decrease correlated with a loss of CFTR mRNA in the loss of the PREP5/CFTR. After 12 to 15 weeks growth without selection both cAMP-dependent Cl- transport and plasmid-derived CFTR mRNA were not detectable. However, it was still possible to rescue cAMP-dependent Cl- transport in these transfected cells by reselection suggesting the presence of the CFTR containing plasmid in a portion of the cells. Analysis of DNA indicated that the pREP5/CFTR vector copy number was reduced from 30 copies per cell with continuous selection, to approximately 0.3 copies per cell after 20 weeks without hygromycin B.
Cells, Cultured↗
Mutations in the putative pore-forming domain of CFTR do not change anion selectivity of the cAMP activated Cl- conductance.
Cystic fibrosis transmembrane conductance regulator (CFTR) apparently forms Cl- channels in apical membranes of secretory epithelial cells. A detailed model describes molecular structure and biophysical properties of CFTR and the impact of various mutations as they occur in cystic fibrosis. In the present report mutations were introduced into the putative 6th alpha-helical transmembrane pore forming domain of CFTR. The mutants were subsequently expressed in Xenopus oocytes by injection of the respective cRNAs. Whole cell (wc) conductances could be reversibly activated by IBMX (1 nmol/l) only in oocytes injected with wild-type (wt) or mutant CFTR but not in oocytes injected with water or antisense CFTR. The activated conductance was partially inhibited by (each 100 mumol/l) DIDS (27%) and glibenclamide (77%), but not by 10 mumol/l NPPB. The following mutations were examined: K335E, R347E, R334E, K335H, R347H, R334H. They did not measurably change the wt-CFTR anion permeability (P) and we conductance (G) sequence of: PCl- > PBr- > P1- and GCl- > GBr- > G1-, respectively. Moreover, anomalous mole fraction behavior for the cAMP activated current could not be detected: neither in wt-CFTR nor in R347E-CFTR. Various mutants for which positively charged amino acids were replaced by histidines (K335H, R347H, R334H) did not show pH sensitivity of the IBMX activated wc conductance. We, therefore, cannot confirm previous results. CFTR might have a different molecular structure than previously suggested or it might act as a regulator of ion conductances.
1-Methyl-3-isobutylxanthine↗
Actin-dependent activation of ion conductances in bronchial epithelial cells.
Activation of Cl- and K+ channels is necessary to drive ion secretion in epithelia. There is substantial evidence from previous reports that vesicular transport and exocytosis are involved in the regulation of ion channels. In the present study we examined the role of cytoskeletal elements and components of intracellular vesicle transport on ion channel activation in bronchial epithelial cells. To this end, cells were incubated with a number of different compounds which interact with either microtubules or actin microfilaments, or which interfere with vesicle transport in the Golgi apparatus. The effectiveness of these agents was verified by fluorescence staining of cellular microtubules and actin. The function was examined in 36Cl- efflux studies as well as in whole-cell (WC) patch-clamp and cell-attached studies. The cells were studied under control conditions and after exposure to (in mmol/l) ATP (0.1), forskolin (0.01), histamine (0.01) and hypotonic bath solution (HBS, NaCl 72.5). In untreated control cells, ATP primarily activated a K+ conductance whilst histamine and forskolin induced mainly a Cl- conductance. HBS activated both K+ and Cl- conductances. Incubation of the cells with brefeldin A (up to 100 mumol/l) did not inhibit WC current activation and 36Cl- efflux. Nocodazole (up to 170 mumol/l) reduced the ATP-induced WC current, and mevastatin (up to 100 mumol/l) the cell-swelling-induced WC current. Neither had any effect on the WC current induced by forskolin and histamine. Also 36Cl- efflux induced by HBS, ATP, forskolin and histamine was unaltered by these compounds.(ABSTRACT TRUNCATED AT 250 WORDS)
Actins↗
Na+ and Cl- conductances in airway epithelial cells: increased Na+ conductance in cystic fibrosis.
Na+ and Cl- conductances in the apical membrane of respiratory epithelial cells are essential for electrolyte and water transport in the airways. Apart from the well described defect in adenosine 3' : 5' cyclic monophosphate-(cAMP-) dependent activation of Cl- conductances in cystic fibrosis (CF), an increased Na+ conductance has also been reported from transepithelial measurements. In the present experiments we tried to identify these conductances in nasal epithelial cells using patch-clamp and microelectrode techniques. With these methods we found identical and relatively low membrane voltages of about -36 mV in both freshly isolated and primary cultured normal and CF nasal epithelial cells. A Cl- conductance could be activated by cAMP in normal (deltaG = 0.3 +/- 0.8 nS, n = 10) but not in CF (deltaG = 0.3 +/- 0.1 nS, n = 11) cells, whereas Ca2+-dependent Cl- currents activated by adenosine 5'-triphosphate (ATP) and bradykinin were present in both types of cells. Cell-attached membrane patches from stimulated cells did not reveal discernible single-channel events when activated with any of the agonists. A Na+ conductance was also detected in freshly isolated ciliated respiratory cells in impalement studies, as evidenced by the hyperpolarization induced by 10 micromol/l amiloride (deltaV = -5.2 +/- 0.6 mV, n = 56) and when Na+ was replaced in the bath by N-methyl-D-glucamine (NMDG) (deltaV = -5.7 +/- 0.9 mV, n = 14). In whole-cell patch-clamp experiments, the amiloride-induced hyperpolarization was significantly larger in CF (deltaV = 9.7 +/- 2.4 mV, n = 22) when compared to normal (deltaV = -3.3 +/- 0.9 mV, n = 27) cells in short-term culture. Reverse transcriptase polymerase chain reaction analysis of normal respiratory cells identified messenger RNA of both the cystic fibrosis transmembrane conductance regulator (CFTR) as well as the human epithelial Na+ channel (hNaCh). The present experiments confirm the absence of a cAMP-dependent Cl- conductance in CF respiratory epithelial cells and support previous findings obtained in transepithelial and microelectrode studies which indicate an increased Na+ conductance in respiratory epithelial cells from CF patients.
Amiloride↗
Epithelial cell specific properties and genetic complementation in a delta F508 cystic fibrosis nasal polyp cell line.
Analysis of vectorial ion transport and protein trafficking in transformed cystic fibrosis (CF) epithelial cells has been limited because the cells tend to lose their tight junctions with multiple subcultures. To elucidate ion transport and protein trafficking in CF epithelial cells, a polar cell line with apical and basolateral compartments will facilitate analysis of the efficacy of different gene therapy strategies in a "tight epithelium" in vitro. This study investigates the genotypic and phenotypic properties of a CF nasal polyp epithelial, delta F508 homozygote, cell line that has tight junctions pre-crisis. The cells (sigma CFNPE14o-) were transformed with an origin-of-replication defective SV40 plasmid. They develop transepithelial resistance in Ussing chambers and are defective in cAMP-dependent Cl- transport as measured by efflux of radioactive Cl-, short circuit current (Isc), or whole-cell patch clamp. Stimulation of the cells by bradykinin, histamine, or ATP seems to activate both K(+)- and Ca(+2)-dependent Cl- transport. Measurement of 36Cl- efflux following stimulation with A23187 and ionomycin indicate a Ca(+2)-dependent Cl- transport. Volume regulatory capacity of the cells is indicated by cell swelling conductance. Expression of the CF transmembrane conductance regulator mRNA was indicated by RT-PCR amplification. When cells are grown at 26 degrees C for 48 h there is no indication of cAMP-dependent Cl- as has been previously indicated in heterologous expression systems. Antibodies specific for secretory cell antigens indicate the presence of antigens found in goblet, serous, and mucous cells; in goblet and serous cells; or in goblet and mucous cells; but not antigens found exclusively in mucous or serous cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗
Effects of P-glycoprotein expression on cyclic AMP and volume-activated ion fluxes and conductances in HT-29 colon adenocarcinoma cells.
The tissue distribution of P-glycoprotein (Pgp) and the structurally related cystic fibrosis transmembrane conductance regulator (CFTR) is apparently mutually exclusive, particularly in epithelial; where one protein is expressed the other is not. To study the possible function(s) of Pgp and its potential effects on CFTR expression in epithelia, HT-29 colon adenocarcinoma cells, which constitutively express CFTR, were pharmacologically adapted to express the classical multidrug resistance (MDR) phenotype (Pgp+). Concomitant with the appearance of Pgp and MDR phenotype (drug resistance, reduced drug accumulation and increased drug efflux), CFTR levels and cAMP-stimulated Cl conductances were markedly decreased compared to wild-type HT-29 (Pgp-) cells (as shown using the whole cell patch clamp technique). Removal of drug pressure led to the gradual decrease in Pgp levels and MDR phenotype, as evidenced by increased rhodamine 123 accumulation (Pgp-Rev). Concomitantly, CFTR levels and cAMP-stimulated Cl- conductances increased. The cell responses of Pgp/Rev cells were heterogeneous with respect to both Pgp and CFTR functions. We also studied the possible contribution to Pgp to hypotonically activated (HCS) ion conductances. K+ and Cl- effluxes from Pgp- cells were markedly increased by HCS. This increase was twice as high as that induced by the cation ionophore gramicidin; it was blocked by the Cl- channel blocker DIDS (4,4'-disothiocyano-2,2'-disulfonic stilbene) and required extracellular Ca2+. In Pgp+ cells, the HCS-induced fluxes were not significantly different from those of Pgp- cells. Verapamil (10 microM), which caused 80% reversal of Pgp-associated drug extrusion, failed to inhibit the HCS-evoked Cl- efflux of Pgp+ cells. Similarly, HCS increased Cl- conductance to the same extent in Pgp-, Pgp+ and Pgp-Rev cells. Verapamil (100 microM), but not 1,9-dideoxyforskolin (50 and 100 microM), partially inhibited the HCS-evoked whole cell current (WCC) in all three lines. Since the inhibition by verapamil was not detected in the presence of the K+ channel blocker Ba2+ (3 mM), it is suggested that verapamil affects K+ and not Cl- conductance. We conclude that hypotonically activated Cl- and K+ conductances are similar in HT-29 cells irrespective of Pgp expression. Expression of high levels of Pgp in HT-29 cells confers no physiologically significant capacity for cell volume regulation.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
cAMP-dependent activation of ion conductances in bronchial epithelial cells.
The cAMP-dependent activation of Cl- channels was studied in a bronchial epithelial cell line (16HBE14o-) in fast and slow whole-cell, and cell-attached patch-clamp experiments. The cells are known to express high levels of cystic fibrosis transmembrane conductance regulator mRNA and protein. Isoproterenol, forskolin and histamine (all 10 mumol/l) reversibly and significantly depolarized the membrane voltage (Vm) and increased the whole-cell Cl- conductance significantly by 34.0 +/- 0.9 (n = 3), 18.1 +/- 2.7 (n = 50), and 25 +/- 4.5 (n = 37) nS respectively. The effect of histamine was blocked by cimetidine (10 mumol, n = 5) but not by diphenhydramine (10 mumol/l, n = 4), which suggests binding of histamine to H2 receptors. The forskolin-induced current was not inhibited significantly by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (0.5 mmol/l, n = 9) nor glibenclamide (10 mumol/l, n = 3) and had an anion-permeability sequence of Cl = Br- > I- (n = 9). In cell-attached recordings forskolin (10 mumol/l) increased the conductance of the patched membrane from 65.5 +/- 13.6 pS to 150.8 +/- 33.2 pS (n = 30). Although the conductance was increased significantly, clear ion channel events occurring in parallel with the current activation were not detected in the cell-attached membrane. In 4 out of 30 cell-attached recordings single-channel currents were observed. These channels, with a single-channel conductance of about 6 pS, were already active before forskolin was added. No effect of forskolin on the channel amplitude, open probability or kinetics of these channels was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Bronchi↗
Ca(2+)- and swelling-induced activation of ion conductances in bronchial epithelial cells.
The present study was performed to examine Ca(2+)-dependent and cell-swelling-induced ion conductances in a polarized bronchial epithelial cell line (16HBE14o-). Whole-cell currents were measured in fast and slow whole-cell patch-clamp experiments in cells grown either on filters or on coated plastic dishes. In addition the transepithelial voltage (Vte) and resistance (Rte) were measured in confluent monolayers. Resting cells had a membrane voltage (Vm) of -36 +/- 1.1 mV (n = 137) which was mainly caused by K+ and Cl- conductances and to a lesser extent by a Na+ conductance. Vte was apical-side-negative after stimulation. Equivalent short-circuit (Isc = Vte/Rte) was increased by the secretagogues histamine (0.1 mmol/l), bradykinin (0.1 - 10 mumol/l). and ATP (0.1 - mumol/l). The histamine-induced Is was blocked by either basolateral diphenhydramine (0.1 mmol/l, n = 4) or apical cimetidine (0.1 mmol/l, n = 4). In fast and slow whole-cell recordings ATP and bradykinin primarily activated a transient K+ conductance and hyperpolarized Vm. This effect was mimicked by the Ca2+ ionophore ionomycin (1 mumol/l, n = 11). Inhibition of the bradykinin-induced Isc by the blocker HOE140 (1 mumol/l, n = 3) suggested the presence of a BK2 receptor. The potency sequence of different nucleotide agonists on the purinergic receptor was UTP approximately ATP > ITP > GTP approximately CTP approximately [beta, gamma-methylene] ATP approximately 2-methylthio-ATP = 0 and was obtained in Isc measurements and patch-clamp recordings. This suggests the presence of a P2u receptor. Hypotonic cell swelling activated both Cl- and K+ conductances.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine Triphosphate↗