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  • Cl− secretion  (8)
  • 1
    Electronic Resource
    Electronic Resource
    Transmitter-induced changes of the membrane voltage of HT29 cells (1992)
    Springer
    Pflügers Archiv 421 (1992), S. 224-229 
    Details
    ISSN: 1432-2013
    Keywords: Cl− conductance ; HT29 ; P2 receptor ; Colon ; Cl− secretion ; cAMP
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The colonic carcinoma cell line HT29 was used to examine the influence of agonists increasing cytosolic cAMP and Ca2+ activity on the conductances and the cell membrane voltage (V m). HT29 cells were grown on glass cover-slips. Cells were impaled by microelectrodes 4–10 days after seeding, when they had formed large plaques. In 181 impalements V m was −51±1 mV. An increase in bath K+ concentration from 3.6 mmol/l to 18.6 mmol/l or 0.5 mmol/l Ba2+ depolarized the cells by 10±1 mV (n=49) or by 9±2 mV (n=3), respectively. A decrease of bath Cl− concentration from 145 to 30 mmol/l depolarized the cells by 11±1 mV (n=24). Agents increasing intracellular cAMP such as isobutylmethylxanthine (0.1 mmol/l), forskolin (10 μmol/l) or isoprenaline (10 μmol/l) depolarized the cells by 6±1 (n=13), 15±3 (n=5) and 6±2 (n=3) mV, respectively. In hypoosmolar solutions (225 mosmol/l) cells depolarized by 9±1 mV (n=6). Purine and pyrimidine nucleotides depolarized the cells dose-dependently with the following potency sequence: UTP 〉 ATP 〉 ITP 〉 GTP 〉 TIP 〉 CTP = 0. The depolarization by ATP was stronger than that by ADP and adenosine. The muscarinic agonist carbachol led to a sustained depolarization by 27±6 mV (n=5) at 0.1 mmol/l, and to a transient depolarization by 12±4 mV (n=5) at 10 μmol/l. Neurotensin depolarized with a half-maximal effect at around 5 nmol/l. The depolarization induced by nucleotides and neurotensin was transient and followed by a hyperpolarization. We confirm that HT29 cells possess Cl−- and K+-conductive pathways. The Cl− conductance is regulated by intracellular cAMP level, cytosolic Ca2+ activity, and cell swelling. The K+ conductance in HT29 cells is regulated by intracellular Ca2+ activity.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00374831
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  • 2
    Electronic Resource
    Electronic Resource
    Forskolin and PMA pretreatment of HT29 cells alters their chloride conductance induced by cAMP, Ca2+ and hypotonic cell swelling (1994)
    Springer
    Pflügers Archiv 428 (1994), S. 76-83 
    Details
    ISSN: 1432-2013
    Keywords: HT29 ; CFPAC-1 ; Cl− secretion ; CFTR ; CF ; Cl− conductance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract HT29 cells were preincubated with forskolin (10−5 mol/l, FORHT) or phorbol 12-myristate 13-acetate (PMA) (10−7 mol/l, PMAHT) for 20 h, which has been shown previously and is also shown here, to upregulate and downregulate, respectively, the expression of the cystic fibrosis transmembrane conductance regulator (CFTR). CFPAC-1 cells underwent the same protocols. HT29 cells were examined by slow (SWC) and fast (FWC) whole-cell patch-clamp techniques. The results of SWC and FWC were indistinguishable and were pooled. CFPAC-1 cells were examined with FWC. The membrane voltage (V) of FORHT was -41.8±1.4 mV (n=77) and that of PMAHT was -43.6±2.4 mV (n=76). The conductance (G) of FORHT (9.4 ±0.9 nS, n=77) was significantly larger than that of PMAHT (3.7±0.4 nS, n=76). Acute application of forskolin (10−5 mol/l, FOR) plus 0.5 mmol/l 8-(4-chlorophenylthio)-cAMP (cAMP) depolarized V by 12 (FORHT) and 8 (PMAHT) mV, respectively. The acute increase of G by FOR plus cAMP was by 7.6±1.9 nS for FORHT (n=22) and only 2.2±1 nS for PMAHT (n=13). ATP (10−4 mol/l) depolarized V in both types of cells. It enhanced G by 16.7±4.1 nS in FORHT (n=14) and significantly less (by 5.5±1.2nS, n=14) in PMAHT. Also the G increase lasted longer in FORHT. Neurotensin (NT, 10−8 mol/l) also had a stronger and longer lasting effect in FORHT. Exposure to hypotonic bath solution (160 mosmol/l) depolarized V in both types of cells. The increase in G was by 15±2.2 nS in FORHT (n=18) and by 11±1.3 nS in PMAHT (n=23). After being returned to the normotonic media, the decline of G to the control value was delayed in FORHT when compared to PMAHT. Ionomycin (10−7 mol/l) increased G significantly more (to 47±8.5 nS, n=13) in FORHT when compared to PMAHT (to 28±4 nS, n=13). The present data indicate that a 20-h exposure of HT29 cells to FOR versus PMA alters markedly the CFTR concentration. The cells with high CFTR (FORHT) when compared to those with low CFTR (PMAHT) differ not only in their acute G response to cAMP, but also in that to ATP, NT, hypotonic cell swelling, and ionomycin. In contrast, the same pretreatment of CFPAC-1 cells did not alter the G changes induced by ionomycin or hypotonic cell swelling. These results indicate that changes in CFTR expression correlate with the Cl− conductances induced by cAMP, Ca2+ and hypotonic cell swelling.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00374754
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  • 3
    Electronic Resource
    Electronic Resource
    Increase in cytosolic Ca2+ regulates exocytosis and Cl− conductance in HT29 cells (1993)
    Springer
    Pflügers Archiv 424 (1993), S. 329-334 
    Details
    ISSN: 1432-2013
    Keywords: Exocytosis ; Membrane capacitance ; Cl− channel ; Cl− secretion ; Colon
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Increases of cytosolic Ca2+, as occur with agonists such as ATP, neurotensin (NT), hypotonic cell swelling and ionomycin, enhance the membrane conductance (G M) and hence the input conductance (G I) of HT29 cells. In the present study we have examined whether these increases in G M are paralleled by exocytosis. To this end the membrane capacitance (C M) of HT29 cells was measured by patch clamp techniques. Two methods to monitor C M were used: a direct method (DM) and a phase tracking method (PTM). With the DM the following results were obtained. NT (10−8 mol/l, n=9) increased G M and C m significantly from 2.4±0.3 nS and 23.5±3 pF to 32±8 nS and 27.3±3.1 pF respectively. ATP (10−4 mol/l, n=29) had a very similar effect. G m and C m were increased from 5.7±1 nS and 36±4.4 pF to 111±21 nS and 44±5.4 pF respectively. Hypotonic cell swelling (160 mosmol/l, n=18) had a comparable effect: G M and C M were increased from 4.9±1 nS and 30±4.1 pF to 46±10 nS and 37±4.9 pF respectively. Ionomycin (10−7 mol/l, n=4) gave similar results. With the PTM it was possible to monitor the rapid changes in G M and C M, as they were induced by ATP (n=42) and NT (n=29), with high time resolution. The transient and instantaneous (〈 1 s) increases in G I (from 2.1±0.4 to 21.7±1.7 nS in the case of ATP, and from 2.3±0.4 to 26.6±3.1 nS in the case of NT) were closely paralleled by transient increases in C m (from 17.6±1.4 to 21.1±1.7 pF in the case of ATP, and from 20.6±2.3 to 24.3±2.6 pF in the case of NT). The present data indicate that transient (ATP, NT) or more stable (hypotonic cell swelling, ionomycin) increases in [Ca2+]i produce corresponding increments in G m and C M. The relative changes in both parameters correlate with each other. These findings are compatible with the view that exocytosis is related to the Ca2+-mediated control of Cl− conductance.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00384360
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  • 4
    Electronic Resource
    Electronic Resource
    Small and intermediate conductance chloride channels in HT29 cells (1993)
    Springer
    Pflügers Archiv 424 (1993), S. 456-464 
    Details
    ISSN: 1432-2013
    Keywords: Cl− channels ; Cl− secretion ; HT29 ; Ca2+ ; cAMP ; Protein kinase A ; Cytosolic inhibitor ; Cystic fibrosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Recently, it has been shown that intermediate conductance outwardly rectifying chloride channels (ICOR) are blocked by cytosolic inhibitor (C. I.) found in the cytosol of human placenta and epithelial cells. C. I. also reduced the baseline current in excised membrane patches of HT29 cells. In the present study, this effect of C. I. was characterized further. Heat treated human placental cytosol was extracted in organic solvents and dissolved in different electrolyte solutions. It is shown that the reduction of baseline conductance (g o) is caused by inhibition of small non-resolvable channels, which are impermeable to Na+ and SO4 2−, but permeable to Cl−. The regulation of these small Cl−-conducting channels (g o) and of ICOR was examined further. First, no activating effects of protein kinase A (PKA) on the open probability (P o) of the ICOR or on the go) were observed. The Po of the ICOR was reduced by 22% in a Ca2+-free solution. g o was insensitive to changes in the Ca2+ activity. The effects of C. I. from a cystic fibrosis (CF) placenta and the CF pancreatic duct cell line CFPAC-1 were compared with the effects of corresponding control cytosols, and no significant differences between CF and control cytosols were found. We conclude that the excised patches of HT29 cells contain ICOR and small non-resolvable Cl−-conducting channels which are similarly inhibited by C. I. Apart from a weak effect of Ca2+ on the ICOR, g o and the ICOR do not seem to be directly controlled by Ca2+ or PKA. C. I. of normal and CF epithelia have a similar inhibitory potency on Cl− channels.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00374908
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  • 5
    Electronic Resource
    Electronic Resource
    A new class of inhibitors of cAMP-mediated Cl− secretion in rabbit colon, acting by the reduction of cAMP-activated K+ conductance (1995)
    Springer
    Pflügers Archiv 429 (1995), S. 517-530 
    Details
    ISSN: 1432-2013
    Keywords: Cl− secretion ; Diarrhoea ; K+ conductance ; K+ channel blocker
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Previously we have shown that arylamino-benzoates like 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), which are very potent inhibitors of NaCl absorption in the thick ascending limb of the loop of Henle, are only poor inhibitors of the cAMP-mediated secretion of NaCl in rat colon. This has prompted our search for more potent inhibitors of NaCl secretion in the latter system. The chromanole compound 293 B inhibited the equivalent short-circuit current (I sc) induced by prostaglandin E2 (n=7), vasoactive intestinal polypeptide (VIP,n=5), adenosine (n=3), cholera toxin (n=4) and cAMP (n=6), but not by ionomycin (n=5) in distal rabbit colon half maximally (IC50) at 2 μmol/l from the mucosal and at 0.7 μmol/l from the serosal side. The inhibition was reversible and paralleled by a significant increase in transepithelial membrane resistance [e.g. in the VIP series from 116±16 Ω·cm2 to 136±21 Ω·cm2 (n=5)]. A total of 25 derivatives of 293 B were examined and structure activity relations were obtained. It was shown that the racemate 293 B was the most potent compound with-in this group and that its effect was due to the enantiomer 434 B which acted half maximally at 0.25 μmol/l. Further studies in isolated in vitro perfused colonic crypts revealed that 10 μmol/l 293 B had no effect on the membrane voltage across the basolateral membrane (V bl) in non-stimulated crypt cells: −69±3 mV versus −67±3 mV (n=10), whilst in the same cells 1 mmol/l Ba2+ depolarised (V bl) significantly. However, 293 B depolarised (V bl) significantly in the presence of 1 μmol/l forskolin: −45±4mV versus −39±5 mV (n=7). Similar results were obtained with 0.1 mmol/l adenosine. 293 B depolarised (V bl) from −40±5 mV to −30±4 mV (n=19). This was paralleled by an increase in the fractional resistance of the basolateral membrane. VIP had a comparable effect. The hyperpolarisation induced by 0.1 mmol ATP was not influenced by 10 μmol/l 293 B: −75±6 mV versus −75±6 mV (n=6). Also 293 B had no effect on basal K+ conductance (n=4). Hence, we conclude that 293 B inhibits the K+ conductance induced by cAMP. This conductance is apparently relevant for Cl− secretion and the basal K+ conductance is insufficient to support secretion.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00704157
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  • 6
    Electronic Resource
    Electronic Resource
    The amiloride inhibitable Na+ conductance of rat colonic crypt cells is suppressed by forskolin (1996)
    Springer
    Pflügers Archiv 431 (1996), S. 984-986 
    Details
    ISSN: 1432-2013
    Keywords: Cl− secretion ; Na+ absorption ; CAMP ; exocrine secretion ; Cl− channel ; Na+ channel ; colon
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Previously we have shown that mid crypt cells of corticoid treated rats possess an amiloride inhibitable Na+ conductance (NAC) and show an increased Cl− conductance when stimulated by prostaglandin or the second messenger CAMP. The NAC is supposed to determine the magnitude of NaCl absorption. The Cl− conductance defines the magnitude of NaCl secretion. In the present whole cell (WC) patch clamp study we have examined whether the amiloride (3 μmol/l) inhibitable NAC is downregulated when the Cl− conductance is increased by forskolin (5 μmol/l, n = 20) or the phosphodiesterase inhibitor IBMX (1 mmol/l, n = 5). Under control conditions the amiloride inhibitable NAC was 2.7 ± 0.4 nS. Forskolin depolarized the voltage from -58 ± 2.0 to-48 ± 1.9 mV and enhanced the WC conductance by 3.25 ± 0.6 nS in these cells. The amiloride inhibitable NAC was reduced to 0.38 ± 0.2 nS. These data confirm that forskolin enhances the Cl− conductance in these cells and they show for the first time that the Na+ conductance is reduced simultaneously. Thus the cells are able to change the direction of NaCl transport from absorption to secretion.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02332187
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  • 7
    Electronic Resource
    Electronic Resource
    The effect of secretagogues on ion conductances of in vitro perfused, isolated rabbit colonic crypts (1995)
    Springer
    Pflügers Archiv 429 (1995), S. 494-502 
    Details
    ISSN: 1432-2013
    Keywords: Cl− secretion ; cAMP ; K+ conductance ; Cl− conductance ; Diarrhoea
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Several secretagogues were used in this study, including those which enhance intracellular cyclic adenosine monophosphate (cAMP) production, as well as others which elevate intracellular Ca2+ activity and are known to increase Cl− secretion in the intact colon and in colonic carcinoma cell lines. They were examined with respect to their effects on electrophysiological properties in isolated rabbit distal colonic crypts. Crypts were dissected manually and perfused in vitro. Transepithelial voltage (V te), transepithelial resistance (R te), membrane voltage across the basolateral membrane (V bl), and fractional basolateral membrane resistance (FR bl), were estimated. Basolateral prostaglandin E2 (PGE2, ⩾0.1 μmol/l), vasoactive intestinal peptide (VIP, 1 nmol/l) and adenosine (0.1 mmol/l) induced an initial depolarisation and a secondary partial repolarisation of (V bl). In the case of adenosine, the initial depolarization of (V bl) was by 31±2 mV (n=47).R te fell significantly from 16.4±3.6 to 14.2±3.7 Ω·cm2 (n= 6), andFR blincreased significantly from 0.11±0.02 to 0.51±0.10 (n=6). In the second phase the repolarisation of (V bl) amounted 11±2 mV (n=47) and a steadystate (V bl) of −51±2 mV (n=47) was reached.R te fell further and significantly to a steady-state value of 12.4±3.8 Ω·cm2 (n=6) andFR bl fell significantly to 0.42±0.13 (n=6). In 30% of the experiments, a transient hyperpolarisation of (V bl) by 8±2 mV (n=14) was seen during wash out of adenosine. In the presence of adenosine, but not under control conditions, lowering of luminal Cl− concentration from 120 to 32 mmol/l depolarised (V bl) significantly by 8±1 mV (n=9). Basolateral ATP and ADP (0.1 mmol/l) led to a short initial depolarisation followed by a sustained and significant hyperpolarisation by 6±2 mV (n=27) and 5±4 mV (n=8), respectively. Carbachol (CCH) hyperpolarised (V bl) in a concentration-dependent manner. At 100 μmol/l (bath) the hyperpolarisation was by 14±2 mV (n=11) andFR bl fell slightly. Neurotensin (⩾10 nmol/l), isoproterenol (⩾10 μmol/l) and uridine 5′-triphosphate (UTP, 0.1 mmol/l) had no effect. It is concluded that PGE2, VIP and adenosine upregulate sequentially a luminal Cl− conductance and a basolateral K+ conductance by increasing intracellular cAMP concentration. Ca2+ mobilising hormones such as ATP, ADP, and CCH increase the basolateral K+ conductance, while the effect on luminal Cl− conductance appears to be very limited.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00704154
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  • 8
    Electronic Resource
    Electronic Resource
    Isolated perfused rabbit colon crypts: stimulation of Cl− secretion by forskolin (1993)
    Springer
    Pflügers Archiv 425 (1993), S. 373-380 
    Details
    ISSN: 1432-2013
    Keywords: Isolated perfused colon crypt ; Basolateral membrane voltage ; Cl− conductance ; Forskolin ; Loop diuretics ; Cl− secretion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The aim of this study was to characterize ion conductances and carrier mechanisms of isolated in vitro perfused rabbit colonic crypts. Crypts were isolated from rabbit colon mucosa and mounted on a pipette system which allowed controlled perfusion of the lumen. In non-stimulated conditions basolateral membrane voltage (V b1) was −65±1 mV (n=240). Bath Ba2+ (1 mmol/ l) and verapamil (0.1 mmol/l) depolarized V b1 by 21±2 mV (n=7) and 31±1 (n=4), respectively. Lowering of bath Cl− concentration hyperpolarized V b1 from −69±3 to −75±3 mV (n=9). Lowering of luminal Cl− concentration did not change V b1. Basolateral application of loop diuretics (furosemide, piretanide, bumetanide) had no influence on V b1 in non-stimulated crypts. Forskolin (10−6 mol/l) in the bath depolarized V b1 by 29±2 mV (n=54) and decreased luminal membrane resistance. In one-third of the experiments a spontaneous partial repolarization of V b1 was seen in the presence of forskolin. During forskolin-induced depolarization basolateral application of loop diuretics hyperpolarized V b1 significantly and concentration dependently with a potency sequence of bumetanide 〉 piretanide ≥ furosemide. Lowering bath Cl− concentration hyperpolarized V b1. Lowering of luminal Cl− concentration from 120 to 32 mmol/l during forskolin-induced depolarization led to a further depolarization of Vb1 by 7±2 mV (n=10). We conclude that Vb1 of rabbit colonic crypt cells is dominated by a K+ conductance. Stimulation of the cells by forskolin opens a luminal Cl− conductance. Basolateral uptake of Cl− occurs via a basolateral Na+ : 2Cl− : K+ cotransport system.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00374861
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