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  • 1
    Electronic Resource
    Electronic Resource
    Voltage-dependent Ca2+ influx in the epithelial cell line HT29: simultaneous use of intracellular Ca2+ measurements and nystatin perforated patch-clamp technique (1994)
    Springer
    Pflügers Archiv 426 (1994), S. 427-432 
    Details
    ISSN: 1432-2013
    Keywords: Ca2+ influx ; Nystatin perforated patchclamp technique ; Fura-2 ; HT29 ; ATP ; Thapsigargin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Indirect evidence has accumulated indicating a voltage dependence of the agonist-stimulated Ca2+ influx into epithelial cells. Manoeuvres expected to depolarise the membrane voltage during agonist stimulation resulted in: (1) a decrease of the sustained phase of the adenosine triphosphate (ATP, 10−5 mol/l)-induced intracellular Ca2+ transient, (2) a reduced fura-2 Mn2+-quenching rate, and (3) prevention of the refilling of the agonist-sensitive store. To quantify the change in intracellular Ca2+ as a function of membrane voltage, we measured simultaneously the intracellular Ca2+ activity ([Ca2+]i) with fura-2 and the electrical properties using the nystatin perforated patch-clamp technique in single HT29 cells. Ca2+ influx was either stimulated by ATP (10−5 mol/l) or thapsigargin (TG, 10−8 mol/l). After [Ca2+]i reached the sustained plateau phase we clamped the membrane voltage in steps of 10 mV in either direction. A stepwise depolarisation resulted in a stepwise reduction of [Ca2+]i. Similarly a stepwise hyperpolarisation resulted in a stepwise increase of [Ca2+]i (ATP: 27.5±10 nmol/l per 10 mV, n=6; TG: 19 ±7.9 nmol/l per 10 mV, n=12). The summarised data show a linear relationship between the Δ fluorescence ratio 340/380 nm change and the applied holding voltage. In unstimulated cells the same voltage-clamp protocol did not change [Ca2+]i (n=9). Under extracellular Ca2+-free conditions [Ca2+]i remained unaltered when changing the membrane voltage. These data provide direct evidence that the Ca2+ influx in epithelial cells is membrane voltage dependent. Our data indicate that small changes in membrane voltage lead to substantial changes in [Ca2+]i. This may be due either to a change of driving force for Ca2+ into the cell, or may reflect voltage-dependent regulation of the respective Ca2+ entry mechanism.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00388306
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  • 2
    Electronic Resource
    Electronic Resource
    The effect of intracellular pH on cytosolic Ca2+ in HT29 cells (1996)
    Springer
    Pflügers Archiv 433 (1996), S. 98-108 
    Details
    ISSN: 1432-2013
    Keywords: Key words CO2/HCO3 ; NH3/NH4+ ; pHi ; [Ca2+]i ; Fura-2 ; BCECF ; Ca2+ store ; Ca2+ influx ; Inositol 1 ; 4 ; 5-trisphosphate ; Epithelia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  The influence of intracellular pH (pHi) on intracellular Ca2+ activity ([Ca2+]i) in HT29 cells was examined microspectrofluorometrically. pHi was changed by replacing phosphate buffer by the diffusible buffers CO2/HCO3 –or NH3/NH4 + (pH 7.4). CO2/HCO3 –buffers at 2,5 or 10% acidified pHi by 0.1, 0.32 and 0.38 pH units, respectively, and increased [Ca2+]i by 8–15 nmol/l. This effect was independent of the extracellular Ca2+ activity and the filling state of thapsigargin-sensitive Ca2+ stores. Removing the CO2/HCO3 –buffer alkalinized pHi by 0.14 (2%), 0.27 (5%), and 0.38 (10%) units and enhanced [Ca2+]i to a peak value of 20, 65, and 143 nmol/l, respectively. Experiments carried out with Ca2+-free solution and with thapsigargin showed that the [Ca2+]i transient was due to release from intracellular pools and stimulated Ca2+ entry. NH3/NH4 + (20 mmol/l) induced a transient intracellular alkalinization by 0.6 pHunits and increased [Ca2+]i to a peak (Δ [Ca2+]i = 164 nmol/l). The peak [Ca2+]i increase was not influenced by removal of external Ca2+, but the decline to basal [Ca2+]i was faster. Neither the phospholipase C inhibitor U73122 nor the inositol 1,4,5-trisphosphate (InsP 3) antagonist theophylline had any influence on the NH3/NH4 +-stimulated [Ca2+]i increase, whereas carbachol-induced [Ca2+]i transients were reduced by more than 80% and 30%, respectively. InsP 3 measurements showed no change of InsP 3 during exposure to NH3/NH4 +, whereas carbachol enhanced the InsP 3 concentration, and this effect was abolished by U73122. The pHi influence on ”capacitative” Ca2+ influx was also examined. An acid pHi attenuated, and an alkaline pHi enhanced, carbachol- and thapsigargin-induced [Ca2+]i influx. We conclude that: (1) an alkaline pHi releases Ca2+ from InsP 3-dependent intracellular stores; (2) the store release is InsP 3 independent and occurs via an as yet unknown mechanism; (3) the store release stimulates capacitative Ca2+ influx; (4) the capacitative Ca2+ influx activated by InsP 3 agonists is decreased by acidic and enhanced by alkaline pHi. The effects of pHi on [Ca2+]i should be of relevance under many physiological conditions.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050254
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  • 3
    Electronic Resource
    Electronic Resource
    Flufenamate and Gd3+ inhibit stimulated Ca2+ influx in the epithelial cell line CFPAC-1 (1994)
    Springer
    Pflügers Archiv 428 (1994), S. 583-589 
    Details
    ISSN: 1432-2013
    Keywords: Ca2+ influx ; Fura-2 ; CFPAC-1 ; Flufenamate ; Gd3+ ; ATP ; Thapsigargin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The relevant influx pathway for stimulated Ca2+ entry into epithelial cells is largely unknown. Using flufenamate (Flu) and Gd3+, both known pharmacological blockers of non-selective cation currents in other epithelial preparations, we tested whether the stimulated Ca2+ entry in CFPAC-1 cells was inhibited by these agents. Transmembraneous Ca2+ influx into CFPAC-1 cells was stimulated by either ATP (10−4 and 10−5 mol/l), carbachol (CCH, 10−4 mol/l) or thapsigargin (TG, 10−8 mol/l). Three different experimental approaches were used. (1) Because the plateau phase of an agonist-induced [Ca2+]i transient reflects Ca2+ influx into these cells, we investigated the influence of Flu and Gd3+ on the level of the stimulated [Ca2+]i plateau. (2) The fura-2 Mn2+-quenching technique was used to visualise divalent cation entry and monitor its inhibition. (3) During the “refilling period” after agonist-induced discharge of the intracellular pools the putative influx inhibitors Flu and Gd3+ were given and subsequently the filling state of the agonist-sensitive intracellular stores tested. The results from the first experimental approach showed that both Flu and Gd3+ were potent inhibitors of the stimulated Ca2+ entry in CFPAC-1 cells. Flu reversibly decreased the ATP-induced [Ca2+]i plateau in a concentration dependent manner, with an IC50 value of 33 μmol/l (n = 6). Similar results were obtained for the CCH-(n = 5) and the TG-induced (n = 5) [Ca2+]i plateau. Gd3+ concentration dependently inhibited the stimulated Ca2+ plateau. A complete block of the ATP-induced [Ca2+]i plateau was seen at 0.5 μmol/l (ATP 10−5 mol/l, n = 8). The second approach showed that Flu (10−4 mol/l) completely inhibited the ATP- (10−5 mol/l, n = 3), CCH-(10−4 mol/l, n = 4) and TG-(10−8 mol/l, n = 3)-induced fura-2 Mn2+ quench. Gd3+ also inhibited the fura-2 Mn2+-quenching rate (n = 9). The third approach showed that Flu (n = 6) and Gd3+ (n = 8) inhibited the refilling of the ATP-sensitive intracellular Ca2+ store. These results show that inhibitors of non-selective cation currents in other epithelial preparations are potent inhibitors of stimulated Ca2+ influx in CFPAC-1 cells. Whether this inhibitory effect concerns a non-selective cation channel remains to be established.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00374581
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  • 4
    Electronic Resource
    Electronic Resource
    Agonist-induced intracellular Ca2+ transients in HT29 cells (1993)
    Springer
    Pflügers Archiv 423 (1993), S. 519-526 
    Details
    ISSN: 1432-2013
    Keywords: Carbachol ; Adenosine triphosphate ; Neurotensin ; Fura-2 ; Intracellular Ca2+ ; Ca2+ influx ; Mn2+ ; Verapamil ; Ni2+
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In the present study we have investigated the mechanism of intracellular Ca2+ activity ([Ca2+]i) changes in HT29 cells induced by adenosine triphosphate (ATP), carbachol (CCH), and neurotensin (NT). [Ca2+]i was measured with the fluorescent Ca2+ indicator fura-2 at the single-cell level or in small cell plaques with high time resolution (1–40Hz). ATP and CCH induced not only a dose-dependent [Ca2+]i peak response, but also changes of the plateau phase. The [Ca2+]i plateau was inversely dependent on the ATP concentration, whereas the CCH-induced [Ca2+]i plateau increased at higher CCH concentrations. NT showed (from 10−10 to 10−7 mol/l) in most cases only a [Ca2+]i spike lasting 2–3 min. The [Ca2+]i plateau induced by ATP (10−6 mol/l) and CCH (10−5 mol/l) was abolished by reducing the Ca2+ activity in the bath from 10−3 to 10−4 mol/l (n=7). In Ca2+-free bathing solution the [Ca2+]i peak value for all three agonists was not altered. Using fura-2 quenching by Mn2+ as an indicator of Ca2+ influx the [Ca2+]i peak was always reached before Mn2+ influx started. Every agonist showed this delayed stimulation of the Ca2+ influx with a lag time of 23±1.5 s (n=15) indicating a similar mechanism in each case. Verapamil (10−6–10−4 mol/l) blocked dose dependently both phases (peak and plateau) of the CCH-induced [Ca2+]i increase. Short pre-incubation with verapamil augmented the effect on the [Ca2+]i peak, whereas no further influence on the plateau was observed. Ni2+ (10−3 mol/l) reduced the plateau value by 70%.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00374950
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    Paper (German National Licenses)
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