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  • 1
    Electronic Resource
    Electronic Resource
    The cystic fibrosis transmembrane conductance regulator attenuates the endogenous Ca2+ activated Cl– conductance of Xenopus oocytes (1997)
    Springer
    Pflügers Archiv 435 (1997), S. 178-181 
    Details
    ISSN: 1432-2013
    Keywords: Key words CFTR ; Ca2+ ; Chloride channels ; Ionomycin ; Xenopus oocytes ; CF
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Oocytes from Xenopus laevis activate a Ca2+ dependent Cl– conductance when exposed to the Ca2+ ionophore ionomycin. This Ca2+ activated Cl– conductance (CaCC) is strongly outwardly rectifying and has a halide conductivity ratio (GI– / GCl–) of about 4.4. This is in contrast to the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl– conductance, which produces more linear I/V curves with a GI– / GCl– ratio of about 0.52. Ionomycin enhanced CaCC (ΔG) in water injected and CFTR expressing ooyctes in the absence of 3-isobutyl-1-methylxanthine (IBMX, 1 mmol/l) by (μS) 23 ± 1.9 (n=9) and 23.6 ± 2.3 (n=11). Stimulation by IBMX did not change CaCC in water injected oocytes. CaCC was inhibited in CFTR-expressing ooyctes after stimulation with IBMX or a membrane permeable form of cAMP and was only 5.1 ± 0.48 μS (n=18) and 6.9 ± 0.6 (n=3), respectively. Inhibition of CaCC was correlated to the amount of CFTR-current activated by IBMX. ΔF508-CFTR which demonstrates only a small residual function in activating a cAMP dependent Cl– channel in oocytes inhibited CaCC to a lesser degree (ΔG=12.1 ± 1.1 μS; n=7). Changes of CFTR and CaCC-Cl– whole cell conductances were also measured when extracellular Cl– was replaced by I–. The results confirmed the reduced activation of CaCC in the presence of activated CFTR. No evidence was found for inhibition of CFTR-currents by increase of intracellular Ca2+. Moreover, intracellular cAMP was not changed by ionomycin and stimulation by IBMX did not change the ionomycin induced Ca2+ increase in Xenopus oocytes. Taken together, these results suggest that activation of CFTR-Cl– currents is paralleled by an inhibition of Ca2+ activated Cl– currents in ooyctes of Xenopus laevis. These results provide another example for CFTR-dependent regulation of membrane conductances other than cAMP-dependent Cl– conductance. They might explain previous findings in epithelial tissues of CF-knockout mice.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050498
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  • 2
    Electronic Resource
    Electronic Resource
    Culture-dependent expression of Na+ conductances in airway epithelial cells (1996)
    Springer
    Pflügers Archiv 431 (1996), S. 578-586 
    Details
    ISSN: 1432-2013
    Keywords: αhENaC ; Airway epithelial cells Amiloride ; CFTR ; Patch clamp ; RT-PCR
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract According t0 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 0r as immortalized cell lines, these cells tend t0 lose these Amil+ Na+ channels. The present study examines this issue in immortalized human bronchial epithelial cells (16HBE140- cell line). The mRNA of one subunit of the Na+ channel (αhENaC) was semi-quantified by polymerase chain reaction of reverse transcribed RNA. Transcripts were significantly increased when cells were exposed t0 aldosterone and dexamethasone irrespective of whether grown on permeable supports 0r plastic. When grown on plastic dishes 16HBE140-cells showed cAMP-dependent Cl− currents in whole-cell (WC) patch-clamp experiments, corresponding t0 expression of the cystic fibrosis transmembrane conductance regulator (CFTR). Na+ currents could not be detected although cells expressed significant amounts ofαhENaC as demonstrated by Northern blot analysis. In contrast, when cells were grown on permeable supports 0r cultured in the presence of butyrate (5 mmol/l, plastic 0r permeable support) 0r aldosterone and dexamethasone (both 1 μol/l, plastic 0r permeable support), amiloride (10 μmol/1) hyperpolarized the membrane voltage (ΔVm) by 2–9 mV paralleled by small reductions of WC conductances (ΔGm) of 0.4-4.0 nS. The effects of amiloride on ΔVm were generally more pronounced (up t0 12 mV) when cells were grown on permeable supports. The amiloride effect (ΔVm) was concentration dependent with an inhibitory constant, Ki, of about 0.1 μmol/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 t0 amiloride, whilst the ATP-activated K+ conductance remained unaffected. The present data suggest that cellular mechanisms determining differentiation control the functional expression of Na+ and Cl− conductances in human airway epithelial cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02191906
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  • 3
    Electronic Resource
    Electronic Resource
    Culture-dependent expression of Na+ conductances in airway epithelial cells (1996)
    Springer
    Pflügers Archiv 431 (1996), S. 578-586 
    Details
    ISSN: 1432-2013
    Keywords: Key wordsαhENaC ; Airway epithelial cells ; Amiloride ; CFTR ; Patch clamp ; RT-PCR
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract 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 (αhENaC) 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 αhENaC 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 aldo-sterone and dexamethasone (both 1 μmol/l, plastic or permeable support), amiloride (10 μmol/l) hyperpolarized the membrane voltage (ΔV m) by 2–9 mV, paralleled by small reductions of WC conductances (ΔG m) of 0.4–4.0 nS. The effects of amiloride on ΔVm were generally more pronounced (up to 12 mV) when cells were grown on permeable supports. The amiloride effect (ΔV m) was concentration dependent with an inhibitory constant, K i, of about 0.1 μmol/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 functional expression of Na+ and Cl− conductances in human airway epithelial cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050038
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  • 4
    Electronic Resource
    Electronic Resource
    cAMP stimulation of CFTR-expressing Xenopus oocytes activates a chromanol-inhibitable K+ conductance (1996)
    Springer
    Pflügers Archiv 432 (1996), S. 516-522 
    Details
    ISSN: 1432-2013
    Keywords: Key words Cl ; channel ; K+ channel ; Cellular homeostasis ; Cystic fibrosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  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 (ΔF508 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050164
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  • 5
    Electronic Resource
    Electronic Resource
    Blockade of epithelial Na+ channels by triamterenes – underlying mechanisms and molecular basis (1996)
    Springer
    Pflügers Archiv 432 (1996), S. 760-766 
    Details
    ISSN: 1432-2013
    Keywords: Key words Triamterene ; Amiloride ; Na+ channel ; Epithelia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The three subunits (α, β, γ) 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 μM and 10 μM, respectively. The blockage by triamterene, which is a weak base with a pK a of 6.2, was dependent on the extracellular pH. The IC50 was 1 μM at pH 6.5 and only 17 μM 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 μM for the charged and 100.6 μM 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 μM. 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 α-subunit-deletion mutant, Δ278–283, 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050196
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