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  • 1995-1999  (57)
  • 1
    Electronic Resource
    Electronic Resource
    Regulation of epithelial ion channels by the cystic fibrosis transmembrane conductance regulator (1996)
    Springer
    Journal of molecular medicine 74 (1996), S. 527-534 
    Details
    ISSN: 1432-1440
    Keywords: Cystic fibrosis ; Cl- channel ; K+ channel ; Na+ channel ; Respiratory tract ; Colon
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00204979
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  • 2
    Electronic Resource
    Electronic Resource
    Regulation of epithelial ion channels by the cystic fibrosis transmembrane conductance regulator (1996)
    Springer
    Journal of molecular medicine 74 (1996), S. 527-534 
    Details
    ISSN: 1432-1440
    Keywords: Key words Cystic fibrosis ; Cl ; channel ; K+ channel ; Na+ channel ; Respiratory tract ; Colon
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Abstract: 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s001090050056
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  • 3
    Electronic Resource
    Electronic Resource
    Regulation of the Na+2Cl–K+ cotransporter in in vitro perfused rectal gland tubules of Squalus acanthias (1998)
    Springer
    Pflügers Archiv 436 (1998), S. 521-528 
    Details
    ISSN: 1432-2013
    Keywords: Key words cAMP ; Cl ; channels ; Cl ; secretion ; Exocrine secretion ; K+ channels ; Volume regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Previously it has been shown that the Na+2Cl–K+ cotransporter accepts NH4 + at its K+ binding site. This property can be used to estimate its transport rates by adding NH4 + to the bath and measuring the initial furosemide-dependent rates of change in BCECF fluorescence. We have utilized this technique to determine the regulation of the furosemide-inhibitable Na+2Cl–K+ cotransporter in in vitroperfused rectal gland tubules (RGT) of Squalus acanthias. Addition of NH4 + to the bath (20 mmol/l) led to an initial alkalinization, corresponding to NH3 uptake. This was followed by an acidification, corresponding to NH4 + uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (Δfluorescence/time). This acidification could be completely inhibited by furosemide. In the absence of any secretagogue preincubation of RGT in a low Cl– solution (6 mmol/l, low Cl–) for 10 min enhanced the uptake rate significantly from 4.04±0.51 to 12.7±1.30 (n=5). The addition of urea (200 mmol/l) was without effect, but the addition of 300 mmol/l mannitol (+300 mannitol) enhanced the rate significantly from 7.24±1.33 to 14.7±4.6 (n=6). Stimulation of NaCl secretion by a solution maximizing the cytosolic cAMP concentration (Stim) led to a significant increase in NH4 + uptake rate from 5.00±1.33 to 13.3±1.54 (n=6). Similar results were obtained in the additional presence of Ba2+ (1 mmol/l): the uptake rate was increased significantly from 4.23±0.34 to 15.1±1.86 (n=16). In the presence of Stim low Cl– had no additional effect on the uptake rate: 15.1±3.1 versus 15.2±2.8 in high Cl– (n=6). The uptake rate in Stim containing additional +300 mannitol (22.3±4.0, n=5) was not significantly different from that obtained with Stim or +300 mannitol alone. By whatever mechanism the NH4 + uptake rate was increased furosemide (500 µmol/l) always reduced this rate to control values. Hence three manoeuvres enhanced furosemide-inhibitable uptake rates of the Na+2Cl–K+ cotransporter probably independently: (1) lowering of cytosolic Cl– concentration; (2) cell shrinkage; and (3) activation by cAMP.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050667
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  • 4
    Electronic Resource
    Electronic Resource
    Does stimulation of NaCl secretion in in vitro perfused rectal gland tubules of Squalus acanthias increase membrane capacitance? (1998)
    Springer
    Pflügers Archiv 436 (1998), S. 538-544 
    Details
    ISSN: 1432-2013
    Keywords: Key words CFTR ; Cl ; channels ; Cl ; secretion ; Endocytosis ; Exocrine secretion ; Exocytosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  NaCl secretion in rectal gland tubules (RGT) of Squalus acanthias requires the activation of Cl– channels in the luminal membrane. The RGT and its mechanism of activation are an early evolutionary paradigm of exocrine secretion. The respective Cl– channels probably resemble the shark equivalent of the cystic fibrosis transmembrane conductance regulator (CFTR). Activation of these Cl– channels occurs via cAMP. It has been hypothesized that the activation of CFTR occurs via exocytosis or inhibited endocytosis. To examine this question directly by electrical measurements we have performed whole-cell patch-clamp analyses of in vitro perfused RGT. NaCl secretion was stimulated by a solution (Stim) containing forskolin (10 µmol/l), dibutyryl-cAMP (0.5 mmol/l) and adenosine (0.5 mmol/l). This led to the expected strong depolarization and an increase in membrane conductance (G m). The membrane capacitance (C m) was measured by a newly devised two-frequency synchronous detector method. It was increased by Stim significantly from 5.00±0.22 to 5.17±0.21 pF (n=50). The increase in C m correlated with the increase in G m with a slope of 51 fF/nS. Next the effect of furosemide (500 µmol/l) was examined in previously stimulated RGT. Furosemide was supposed to inhibit coupled Na+2Cl–K+ uptake and to reduce cell volume but not membrane trafficking of Cl– channels. Furosemide reduced G m slightly (due to the fall in cytosolic Cl– concentration) and C m to the same extent by which Stim had increased it. Both changes were statistically significant, and the slope of ΔC m/ΔG m was similar to that caused by Stim. Inhibitors of microtubules or actin (colchicine, phalloidin and cytochalasin D added at 10 µmol/l to the pipette solution and dialysed for 〉10 min) did not alter cell voltage, G m or C m, nor did these inhibitors abolish the stimulatory effect of cAMP. These data suggest that the small C m changes observed with Stim reflect a minor cell volume increase and an ”unfolding” of the plasma membrane. The present data do not support the exocytosis/endocytosis hypothesis of cAMP-mediated activation of Cl– channels in these cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050669
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  • 5
    Electronic Resource
    Electronic Resource
    Luminal ATP induces K+ secretion via a P2Y2 receptor in rat distal colonic mucosa (1998)
    Springer
    Pflügers Archiv 436 (1998), S. 712-716 
    Details
    ISSN: 1432-2013
    Keywords: Key words ATP ; Distal colon ; Exocrine secretion ; K+ secretion ; Luminal receptors ; P2Y2 receptor ; Rat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  We have previously investigated, in studies of rat distal colonic mucosa, the effect of ATP added to the basolateral side on ion transport and [Ca2+]i. It was demonstrated that ATP acts via a P2Y1 receptor to increase [Ca2+]i and NaCl secretion. In the present study we investigated the effect of luminally added nucleotides (ATP, UTP) on transepithelial voltage (V te) and resistance (R te) in Ussing chamber experiments on rat distal colonic mucosa. Both nucleotides induced a rapid and transient (within 30 s) change of V te to lumen-positive values (resting V te: –2±1 mV; peak V te after 100 µmol/l ATP: +2.4±1.1 mV) and a decrease of R te from 89.9±10.3 to 83.8±9.1 Ωcm2 (n=10). Similar values were obtained with luminal UTP (n=15). The estimated EC50 values for both nucleotides were approximately 6 µmol/l. The ATP-induced V te effect was nearly completely sensitive to Ba2+. Addition of the K+ channel blocker Ba2+ (1 mmol/l) to the luminal solution reversibly inhibited 77±4% (n=5) of the ATP-induced V te effect. Experiments to identify the respective P2 receptor subtype revealed the following rank order of potency at 500 µmol/l agonist: UTP≥ATP〉〉2-methylthio-ATP=ADP〉〉adenosine〉 AMP〉β,γ-methylene-ATP (n=5). This closely resembles the published rank order for the P2Y2 receptor. Using the reverse-transcriptase polymerase chain reaction (RT-PCR) technique P2Y2 receptor-specific mRNA was detected in total RNA extracted from isolated crypts. In summary these data indicate that luminal ATP and UTP act via a P2Y2 receptor in the luminal membrane of colonic mucosa to elicit a transient K+ secretion.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050693
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  • 6
    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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  • 7
    Electronic Resource
    Electronic Resource
    A modified confocal laser scanning microscope allows fast ultraviolet ratio imaging of intracellular Ca2+ activity using Fura-2 (1997)
    Springer
    Pflügers Archiv 433 (1997), S. 653-663 
    Details
    ISSN: 1432-2013
    Keywords: Key words Confocal microscopy ; Acousto-optic tunable filter ; Fura-2 ; Ratio imaging ; HT29 cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  A confocal, ultraviolet laser scanning microscope (LSM) for reliable ratio measurements of localized intracellular Ca2+ gradients using the Ca2+-sensitive dye Fura-2 was developed. In a commercial LSM, the filter wheels for the excitation band-pass filters and the grey filters were replaced by acousto-optic tunable filters (AOTF) for rapid switching (≤1.5 μs) of the ultraviolet (351 and 364 nm) and the visible (457, 476, 488, 514 nm) excitation light. This enabled dual wavelength excitation of Fura-2, or 2’7’-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) for pH measurements. Changing to a transmitted-light detector of high sensitivity allowed for simultaneous recording of differential interference contrast images of the preparation with the excitation light. The AOTF fine control of the intensity of the excitation light and improvements in the emission detector sensitivity enabled the acquisition of up to 120 ratio pairs of high-quality images from a single cell. The optical capabilities and limitations of the instrument were evaluated with fluorescent beads and dye-loaded cultured cells. Agonist-induced intracellular Ca2+ transients in HT29 cells were recorded to test for the instrument’s ability to measure changes in [Ca2+]i. Ratio z-sections from Fura-2-loaded cells showed an inhomogeneity of the Fura-2 loading with an accumulation of the dye mostly in the mitochondria. We show, as an example of the microscope’s achievable resolution, the spatial and temporal heterogeneity of [Ca2+]i signals in mitochondria and the cytosol in response to agonist-evoked stimulation of HT29 cells. In addition, we show that the lipophilic, membrane-bound Fura-2 derivative Fura-C18, for measurements of near-membrane Ca2+ changes, can be used with this confocal microscope. This new LSM is expected to deepen our understanding of localized [Ca2+]i signals; for example, the nuclear Ca2+ signalling or the [Ca2+]i changes that occur during stimulation of ion secretion in polarized epithelial cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050327
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  • 8
    Electronic Resource
    Electronic Resource
    Voltage-dependent, slowly activating K+ current (I Ks) and its augmentation by carbachol in rat pancreatic acini (1999)
    Springer
    Pflügers Archiv 438 (1999), S. 604-611 
    Details
    ISSN: 1432-2013
    Keywords: Carbachol Exocrine secretion Pancreatic acinus Potassium current (IKs)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract. Acetylcholine-stimulated exocrine secretion of Cl– and water requires the concomitant activation of K+ channels. However, there has not been much investigation of the carbachol- (CCH-) activated K+ channel of rodent pancreatic acini. Here, in a study of rat pancreatic acini, we characterize a voltage-dependent, slowly activating outward current (I Ks) that is augmented by CCH. Intact acini were obtained by enzymatic digestion and fast-whole-cell patch-clamp was applied. With symmetrical [Cl–] (32 mmol/l) in the pipette and bath solution, acinar cells had resting membrane voltages of –45±0.8 mV (n=97) under current-clamp conditions. CCH (10 µmol/l), which is known to activate Cl– channels via a Ca2+-mediated pathway, sharply depolarized the membrane to –4±0.5 mV, which was more negative than E Cl (0 mV), and reversed it to –41±0.9 mV (n=83) by washout. A clamp voltage of 0 mV activated I Ks under control conditions (91±8.6 pA, n=83). During CCH application an increase of outward current was observed at 0 mV, and at –50 mV a marked increase of inward Cl– current occurred. In the presence of CCH the slow activation of I Ks was rarely distinguishable because of interference by the huge Cl– conductance. During CCH washout and decrease of inward current, a persistent augmentation of I Ks was revealed (486±36.3 pA, n=83). I Ks and its augmentation were abolished by substituting K+ in the pipette solution with Cs+. Augmentation of I Ks was mimicked by applying ionomycin (0.1 µmol/l), a Ca2+ ionophore. Pharmacological blockers were tested. The chromanol 293B and clotrimazole blocked I Ks at micromolar concentrations (IC50=3 µmol/l and 9 µmol/l, respectively) and Ba2+ was a poor blocker (IC50=3 mmol/l). In the presence of CCH (0.2 µmol/l), the membrane was depolarized to around –20 mV and the addition of 293B (10 µmol/l) further depolarized the membrane by 11±3 mV (n=5). These data suggest the presence of I Ks channels in rat pancreatic acini and their muscarinic activation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004249900071
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  • 9
    Electronic Resource
    Electronic Resource
    Carbachol activates a K+ channel of very small conductance in the basolateral membrane of rat pancreatic acinar cells (1999)
    Springer
    Pflügers Archiv 438 (1999), S. 597-603 
    Details
    ISSN: 1432-2013
    Keywords: Acetylcholine Chromanol Exocrine pancreas Exocrine secretion KVLQT1
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract. Secretion of Cl– requires the presence of a K+ conductance to hyperpolarize the cell, and to provide the driving force for Cl– exit via luminal Cl– channels. In the exocrine pancreas Cl– secretion is mediated by an increase in cytosolic Ca2+ ([Ca2+]i). Two types of Ca2+-activated K+ channels could be shown in pancreatic acinar cells of different species. However, there are no data on Ca2+-activated K+ channels in rat pancreatic acini. Here we examine the basolateral K+ conductance of freshly isolated rat pancreatic acinar cells in cell-attached and cell-excised patch-clamp experiments. Addition of carbachol (CCH, 1 µmol/l) to the bath led to the activation of very small conductance K+ channels in cell-attached patches (n=27), producing a noisy macroscopic outward current. The respective outward conductance increased significantly by a factor of 2.1±0.1 (n=27). Noise analysis revealed a Lorentzian noise component with a corner frequency (f c) of 30.3±3.5 Hz (n=19), consistent with channel activity in these patches. The estimated single-channel conductance was 1.5±0.4 pS (n=19). In cell-excised patches (inside out) from cells previously stimulated with CCH, channel activity was only observed in the presence of K+ in the bath solution. Under these conditions f c was 47.6±11.9 Hz (estimated single-channel conductance 1.1±0.2 pS, n=20). The current/voltage relationship of the noise showed weak inward rectification and the reversal potential shifted towards E K + when Na+ in the bath was replaced by K+. Channel activity in cell-excised patches was slightly reduced by 10 mmol/l Ba2+ (23.6±2.1% of the total outward current) and was completely absent when K+ in the bath was replaced by Na+. Reduction of the [Ca2+]i from 1 mmol/l to 1 µmol/l in cell-excised experiments decreased the current by 52.3±12.3% (n=5). Expression of KVLQT1, one of the possible candidates for a small-conductance K+ channel in rat pancreatic acinar cells, was shown by reverse transcriptase polymerase chain reaction (RT-PCR). In fact, a KVLQT-blocker (chromanol 293B) reduced channel activity in seven excised patches. These data suggest that CCH activates very small conductance K+ channels in rat pancreatic acinar cells, most likely via an increase in [Ca2+]i.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004249900070
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  • 10
    Electronic Resource
    Electronic Resource
    Evidence for Na+/Ca2+ exchange in the rectal gland of Squalus acanthias (1999)
    Springer
    Pflügers Archiv 439 (1999), S. 49-51 
    Details
    ISSN: 1432-2013
    Keywords: Cl– secretion cAMP Cytosolic Ca2+ Exocrine secretion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract. Previously we have shown that stimulation of in vitro perfused rectal gland tubules (RGT) of the dogfish Squalus acanthias by adenosine 3',5'-cyclic monophosphate (cAMP), (as a cocktail comprising 0.1 mmol/l dibutyryl-cAMP, 10 µmol/l forskolin and 0.1 mmol/l adenosine, hereafter termed STIM) leads to an increase in cytosolic Ca2+ ([Ca2+]i) and that this assists Cl– secretion by enhancing basolateral K+ conductance. In the present study we examined the mechanism of the cAMP-induced increase in [Ca2+]i. [Ca2+]i was measured using the fura-2 technique in isolated in vitro perfused RGT. As before, STIM enhanced [Ca2+]i. This elevation of [Ca2+]i was prevented completely when STIM was added in the presence of the Na+2Cl–K+ cotransport inhibitor furosemide (0.5 mmol/l). This suggests that the increase in [Ca2+]i induced by STIM is caused by a concomitant increase in cytosolic Na+ ([Na+]i) and not by the activation of second messenger cascades. Furosemide prevents this increase in [Na+]i and hence the elevation of [Ca2+]i. Moreover, the plateau phase of the [Ca2+]i transient produced by carbachol (CCH, 0.1 mmol/l) was augmented strongly when bath Na+ was reduced to 5 mmol/l. These data suggest that the level of [Ca2+]i is determined by Na+-dependent Ca2+ export, most likely via a Na+/Ca2+ exchanger. The increase in [Na+]i accompanying stimulation of Cl– secretion reduces the rate of Ca2+ export leading to an elevation of [Ca2+]i, as does a reduction in bath Na+ which augments the [Ca2+]i plateau produced by CCH.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004249900163
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