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  • 1
    Electronic Resource
    Electronic Resource
    Biphasic increase of apical Cl− conductance by muscarinic stimulation of ht-29cl.19a human colon carcinoma cell line: Evidence for activation of different cl− conductances by carbachol and forskolin (1992)
    Springer
    The journal of membrane biology 127 (1992), S. 81-94 
    Details
    ISSN: 1432-1424
    Keywords: ionomycin ; phorbol-ester ; basolateral K+ conductance ; Cl− channel ; cystic fibrosis ; secretion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The modulation of ion transport pathways in filtergrown monolayers of the Cl−-secreting subclone (19A) of the human colon carcinoma cell line HT-29 by muscarinic stimulation was studied by combined Ussing chamber and microimpalement experiments. Basolateral addition of 10−4 m carbachol induced a complex poly-phasic change of the cell potential consisting of (i) a fast and short (30-sec) depolarization of 15±1 mV from a resting value of −52±1 mV and an increase of the fractional resistance of the apical membrane (first phase), (ii) a repolarization of 22±1 mV leading to a hyperpolarization of the cell (second phase), (iii) a depolarization of 11±1 mV and a decrease of the fractional resistance of the apical membrane (the third phase), (iv) and sometimes, a hyperpolarization of 6±1 mV and an increase of the fractional resistance of the apical membrane (fourth phase). The transepithelial potential increased with a peak value of 2.4±0.3 mV (basolateral side positive). The transepithelial PD started to increase (serosa positive), coinciding with the start of the second phase of the intracellular potential change, and continued to increase during the third phase. Ion replacements and electrical circuit analyses indicate that the first phase is caused by increase of the Cl− conductance in the apical and basolateral membrane, the second phase by increased K+ conductance of the basolateral membrane, and the third phase and the fourth phase by increase and decrease, respectively, of an apical Cl− conductance. The first and second phase of the carbachol effect could be elicited also by ionomycin. They were strongly reduced by EGTA. Phorbol dibutyrate (PDB) induced a sustained depolarization of the cell and a decrease of the apical fractional resistance. The results suggest that two different types of Cl− channels are involved in the carbachol response: one Ca2+ dependent and a second which may be PKC sensitive. In the presence of a supramaximal concentration of forskolin, carbachol evoked a further increase of the apical Cl− conductance. It is concluded that the short-lasting carbachol/Ca2+-dependent Cl− conductance is different from the forskolin-activated conductance. The increase of the Cl− conductance in the presence of forskolin by carbachol may be due to activation of different Cl− channels or to modulation of the PKA-activated Cl− channels by activated PKC.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233281
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  • 2
    Electronic Resource
    Electronic Resource
    The cystic fibrosis defect approached from different angles — new perspectives on the gene, the chloride channel, diagnosis and therapy (1990)
    Springer
    European journal of pediatrics 149 (1990), S. 670-677 
    Details
    ISSN: 1432-1076
    Keywords: Cystic fibrosis ; Gene ; Genetic counseling ; Chloride channels
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The search for the basic defect in cystic fibrosis (CF) has reached a decisive stage since the recent identification of the responsible gene. Electrophysiological and biochemical research had defined the CF defect as a dysregulation of epithelial chloride channels. The putative protein product of the now identified gene shares properties with other known transport proteins, but it is not necessarily itself a chloride channel protein. Elucidation of the primary cellular defect will certainly have important aetiological and hopefully therapeutic implications. The identification of the major gene mutation already has significant consequences for genetic counselling and prenatal diagnosis. Heterozygote detection at the population level awaits identification of the probably heterogenous mutations on about 30% of the CF chromosomes. At present, about 50% of CF patients are homozygous for the recently identified major CF mutation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01959519
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  • 3
    Electronic Resource
    Electronic Resource
    Electrophysiological studies of forskolin-induced changes in ion transport in the human colon carcinoma cell line HT-29 cl.19A: Lack of evidence for a cAMP-activated basolateral K+ conductance (1991)
    Springer
    The journal of membrane biology 122 (1991), S. 239-250 
    Details
    ISSN: 1432-1424
    Keywords: Cl− transport ; K+ conductance ; forskolin ; cAMP ; HT-29 cl.19A ; electrophysiology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Forskolin (i.e, cAMP)-modulation of ion transport pathways in filter-grown monolayers of the Cl−-secreting subclone (19A) of the human colon carcinoma cell line HT29 was studied by combined Ussing chamber and microimpalement experiments. Changes in electrophysiological parameters provoked by serosal addition of 10−5 m forskolin included: (i) a sustained increase in the transepithelial potential difference (3.9±0.4 mV). (ii) a transient decrease in transepithelial resistance with 26±3 Ω · cm2 from a mean value of 138±13 Ω · cm2 before forskolin addition, (iii) a depolarization of the cell membrane potential by 24±1 mV from a resting value of −50±1 mV and (iv) a decrease in the fractional resistance of the apical membrane from 0.80±0.02 to 0.22±0.01. Both, the changes in cell potential and the fractional resistance, persisted for at least 10 min and were dependent on the presence of Cl− in the medium. Subsequent addition of bumetanide (10−4 m), an inhibitor of Na/K/2Cl cotransport, reduced the transepithelial potential, induced a repolarization of the cell potential and provoked a small increase of the transepithelial resistance and fractional apical resistance. Serosal Ba2+ (1mm), a known inhibitor of basolateral K+ conductance, strongly reduced the electrical effects of forskolin. No evidence was found for a forskolin (cAMP)-induced modulation of basolateral K+ conductance. The results suggest that forskolin-induced Cl− secretion in the HT-29 cl.19A colonic cell line results mainly from a cAMP-provoked increase in the Cl− conductance of the apical membrane but does not affect K+ or Cl− conductance pathways at the basolateral pole of the cell. The sustained potential changes indicate that the capacity of the basolateral transport mechanism for Cl− and the basal Ba2+-sensitive K+ conductance are sufficiently large to maintain the Cl− efflux across the apical membrane. Furthermore, evidence is presented for an anomalous inhibitory action of the putative Cl− channel blockers NPPB and DPC on basolateral conductance rather than apical Cl− conductance.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01871424
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  • 4
    Electronic Resource
    Electronic Resource
    Characterization of swelling-induced ion transport in HT-29Cl.19A cells. Role of inorganic and organic osmolytes during regulatory volume decrease (1996)
    Springer
    Pflügers Archiv 433 (1996), S. 276-286 
    Details
    ISSN: 1432-2013
    Keywords: Key words HT-29Cl.19A ; Cell volume regulation ; Chloride secretion ; Amino acids ; Fura-2 ; [Ca2+]i ; Thapsigargin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Combined intracellular and transepithelial potential and resistance measurements were performed to localize the ion conductances activated by hypo-osmotic shock of cultured human colonic carcinoma cells (HT-29Cl.19A). Furthermore, the effect of cell swelling induced by a hypo-osmotic solution on the intracellular Ca2+ activity [Ca2+]i and release of amino acids into the extracellular solution was examined. Application of a 40% hypo-osmotic solution on both sides of confluent monolayers induced a hyperpolarization of the intracellular potential caused by increased K+ conductance of the basolateral membrane, followed by a sustained depolarization due to increased Cl–conductance in the apical and basolateral membranes. Usually no transepithelial current occurred, presumably because of random distribution of Cl–channels. However, in some monolayers cell swelling induced a transepithelial Cl–current because of a more pronounced expression of volume-sensitive Cl–channels in the apical membrane. Exposure to hypo-osmotic solution increased [Ca2+]i transiently. The increase of [Ca2+]i was also observed to occur in the presence of the muscarinic receptor agonist carbachol or the inhibitor of the microsomal Ca2+-ATPase thapsigargin (TG), which prevented carbachol-induced Ca2+ release, suggesting that cell swelling recruits Ca2+ from a different source compared to carbachol or TG. Following incubations with hypo-osmotic solutions, about 60% of the intracellular free amino acids including aspartate, glutamate, glycine and taurine was released. It is concluded that the regulatory volume decrease (RVD) in HT-29Cl.19A colonocytes is achieved by activation of K+ and Cl–conductances, resulting in net loss of salt, as well by extrusion of amino acids.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050278
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  • 5
    Electronic Resource
    Electronic Resource
    Activation of ion transport by combined effects of ionomycin, forskolin and phorbol ester on cultured HT-29cl.19A human colonocytes (1993)
    Springer
    Pflügers Archiv 425 (1993), S. 90-99 
    Details
    ISSN: 1432-2013
    Keywords: Forskolin ; Carbachol ; Ionomycin ; PDB ; Protein kinase C ; Cl− channels ; K+ channels ; Intestinal secretion ; Cystic fibrosis ; T84 cell line
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The differentiated clone 19A of the HT-29 human colon carcinoma cell line was used as a model to study the intracellular electrophysiological effects of interaction of the cAMP, the protein kinase C (PKC) and the Ca2+ pathways, (a) A synergistic effect between ionomycin and forskolin was observed. From intracellular responses it was concluded that the synergistic effect is caused by activation of an apical Cl− conductance by protein kinase A and a basolateral K+ conductance by Ca2+. (b) A transient synergistic effect of ionomycin and the phorbol ester phorbol dibutyrate (PDB) was found. The decrease of the response appeared to be due to PKC-dependent inactivation of the basolateral K+ conductance. The synergism is caused by PKC-dependent increase of the apical Cl− conductance and Ca2+-dependent increase of the basolateral K+ conductance. (c) The effects of carbachol and PDB were not fully additive presumably because of their convergence on PKC activation, (d) Forskolin and PDB, when added in this order, had a less than additive effect. Results of cell-attached patch-clamp studies, presented in the accompanying paper, showed a synergistic effect of forskolin and PDB on non-rectifying small-conductance Cl− channels. Assuming that these channels are involved in the transepithelial responses it is suggested that forskolin and PDB induce a modulatory, synergistic increase of the apical Cl− conductance when both pathways are activated simultaneously. (e) The HT-29cl.19A cells differ from T84 cells in that the latter did not respond with an increase of the short-circuit current to addition of phorbol ester. This may be due to a very low expression of PKCα.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00374508
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  • 6
    Electronic Resource
    Electronic Resource
    Synergistic activation of non-rectifying small-conductance chloride channels by forskolin and phorbol esters in cell-attached patches of the human colon carcinoma cell line HT-29cl.19A (1993)
    Springer
    Pflügers Archiv 425 (1993), S. 100-108 
    Details
    ISSN: 1432-2013
    Keywords: Non-rectifying small-conductance Cl− channels ; Cell-attached patch-clamp ; Protein kinase A ; Protein kinase C ; Forskolin ; Phorbol dibutyrate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Cell-attached patch-clamp studies with the human colon carcinoma HT-29cl.19A cells revealed a small chloride channel with a unitary conductance of 6.5 pS at 70 mV and 4.6 pS at −70 mV clamp potential after cAMP was increased by activation of adenylyl cyclase by forskolin. Usually channels inactivated upon patch excision, but in a few excised patches the channels stayed active and displayed a linear I/V relation in symmetrical (150 mmol/l) chloride solutions with a conductance of 7.5 pS. A 16-fold increase in channel incidence was observed when forskolin and phorbol 12,13-dibutyrate (PDB) were present together. The open probability was voltage-independent and was not different in the presence of forskolin plus PDB or with forskolin alone. The conductance sequence of the channel as deduced from outward currents carried by five different anions including chloride was: Cl−〉Br−〉NO3 −〉gluconate 〉 I−. The permeability sequence deduced from the reversal potentials was NO3 −≥Br−〉Cl−〉I−〉gluconate. With iodide in the pipette the conductance decreased strongly. Moreover, the inward current was reduced by 61%, indicating a strong inhibition of the chloride efflux by iodide. Similarly, the forskolin-induced increase of the short-circuit current (I sc) in confluent filter-grown monolayers was strongly reduced by iodide in the apical perfusate. Iodide also increased the fractional resistance of the apical membrane and repolarized the membrane potential, indicating an inhibitory action on the forskolin-induced increase of the apical chloride conductance. The PDB-induced I sc was also reduced by iodide, suggesting that the same chloride conductance is involved in the forskolin and in the PDB response. The results suggest that forskolin via cAMP-dependent protein kinase and PDB via protein kinase C regulate the same non-rectifying small-conductance chloride channels in the HT-29cl.19A cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00374509
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  • 7
    Electronic Resource
    Electronic Resource
    Chloride secretion induced by phorbol dibutyrate and forskolin in the human colonic carcinoma cell line HT-29Cl.19A is regulated by different mechanisms (1995)
    Springer
    Pflügers Archiv 430 (1995), S. 705-712 
    Details
    ISSN: 1432-2013
    Keywords: HT-29cl.19A ; Human colon cell line ; Temperature ; Brefeldin A Nocodazole ; Phorbolester ; PDB ; Forskolin ; Nystatin ; CFTR
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The human colonic carcinoma cell line HT29cl.19A responds to the protein kinase C activator PDB (4-β-phorbol 12,13-dibutyrate), as it does to forskolin (an activator of adenylyl cyclase), with a secretory response when the cells are grown on filters and studied at 36 °C. Previously, we showed that when cells were grown on Petri dishes and studied at about 25 °C with the cell-attached patch-clamp technique, forskolin, but not PDB, could activate 8-pS chloride channels (cystic fibrosis transmembrane conductance regulator, CFTR, channels). The present work was carried out to study this discrepancy. Experiments in Ussing chambers, at different temperatures, showed that the responses to PDB and forskolin differ in their temperature sensitivity. This was also found following conventional microelectrode and Ussing chamber studies with nystatin-permeabilized epithelial layers carried out at 25 °C and at 36 °C. Pre-incubation with the microtubular disruptive agents nocodazole or colcemid did not affect the response to PDB or forskolin, suggesting that chloride secretion induced by these agonists in these cells is independent of the microtubular structure. Pre-incubation with brefeldin A strongly inhibited the response to PDB, but the response to forskolin was hardly affected. The differing effect of temperature and brefeldin A on the responses to forskolin and PDB may be due to the activation of two distinct mechanisms by protein kinases A and C.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00386165
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  • 8
    Electronic Resource
    Electronic Resource
    LLC-PK1 cells as a model system to study proximal tubule transport of water and other compounds relevant for renal stone disease (1999)
    Springer
    Urological research 27 (1999), S. 109-115 
    Details
    ISSN: 1434-0879
    Keywords: Key words LLC-PK1 ; Water transport ; Oxalate Stone salts ; ; Nephrolithiasis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract LLC-PK1 cells were cultured on a permeable support in a two-compartment culture system. Confluent monolayers received an ultrafiltrate-like solution at the apical side and a plasma-like solution at the basolateral side. The distribution of various solutes, including phosphate, calcium, and oxalate over both compartments was measured in time. The transport of water was monitored by alterations in fluid concentrations of radiolabeled inulin. Bicarbonate, glucose, and phosphate were transported rapidly from the apical to basolateral side of the monolayer. Sodium and chloride were reabsorbed without major consequences for the osmolality in the apical and basal fluid. Calcium and potassium were also reabsorbed, but to a smaller extent than sodium. The luminal concentration of oxalate gradually increased to values that were at least three times higher (12.0 ± 0.4 μmol/l) than those in the contraluminal fluid (3.8 ± 0.1 μmol/l). However, since the luminal rise of oxalate completely matched the rise of inulin in the apical fluid this appeared to be the passive consequence of active water reabsorption rather than of net directed oxalate transport. The LLC-PK1 model could prove useful to study the regulation of proximal tubule water transport and its effect on luminal stone salt concentrations under different physiological conditions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002400050096
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  • 9
    Electronic Resource
    Electronic Resource
    Calcium ionophore plus excision induce a large conductance chloride channel in membrane patches of human colon carcinoma cells HT-29cl.19A (1993)
    Springer
    Cellular and molecular life sciences 49 (1993), S. 313-316 
    Details
    ISSN: 1420-9071
    Keywords: Maxi-chloride channel ; HT-29 ; intestinal epithelium ; electrolyte transport ; Ca2+-ionophore
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract In excised inside-out membrane patches of the human colon carcinoma HT-29cl.19A cells a large conductance (373±10 pS) chloride channel was found. Channel activity could only be observed after excision of patches from cells incubated with calcium ionophore. The channel was never observed in cell-attached patches. The channel was strongly voltage dependent, being open only between +30 and −30 mV clamp potentials. The selectivity sequence among anions, deduced from reversal potentials, was I〉Br〉Cl〉F〉gluconate. The PNa/PCl was 0.09. Although a similar type of channel, has been described earlier, this is the first report stating its appearance in patches of intestinal epithelial cells requiring the combined action of Ca2+ ionophore and excision, suggesting its control by an intracellular compound.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01923409
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