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  • 1
    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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  • 2
    Electronic Resource
    Electronic Resource
    ATP increases [Ca2+]i and ion secretion via a basolateral P2Y-receptor in rat distal colonic mucosa (1997)
    Springer
    Pflügers Archiv 434 (1997), S. 77-83 
    Details
    ISSN: 1432-2013
    Keywords: Key words Colon ; Fura-2 ; Rat colonic crypt ; ATP ; P2Y-receptor ; Purinoceptor ; Exocrine secretion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Under resting conditions the mammalian distal colon is a NaCl-absorptive epithelium. NaCl absorption occurs at surface cells in colonic crypts. Intracellular Ca2+ or cAMP are important second messengers that activate NaCl secretion, a function that is most pronounced in crypt bases. In the present study we examined the effect of extracellular ATP on isolated crypts of rat distal colon using the fura-2 technique. Intracellular Ca2+ ([Ca2+]i) was measured spectrofluorimetrically either by photon counting or video imaging. ATP reversibly increased [Ca2+]i in crypt base cells with an EC50 of 4.5 μmol/l (n = 11). This [Ca2+]i increase was composed of an initial peak, reflecting intracellular store release, and a secondary plateau phase reflecting transmembrane influx. Digital video imaging revealed that agonist-induced [Ca2+]i elevations were most marked at the crypt base. In the middle part of the crypt ATP induced smaller increases of [Ca2+]i (peak and plateau) as compared to basal cells and in surface cells this [Ca2+]i transient was even further reduced. Attempts to identify the relevant P2-receptor demonstrated the following rank order of potency: 2MeS-ATP 〉 ADP ≥ ATP 〉〉 AMP 〉 UTP 〉 AMP-PCP 〉 adenosine. In Ussing chamber experiments ATP (1 mmol/l) functioned as a secretagogue, increasing transepithelial voltage (V te) and equivalent short-circuit current (I sc): ΔI sc = –36.4 ± 5.4 μA/cm2, n = 17. Adenosine itself (1 mmol/l) induced an increase of I sc of similar magnitude to that induced by ATP: ΔI sc = –55.1 ± 8.4 μA/cm2, n = 9. The effect of adenosine, but not that of ATP, was fully inhibited by the A1/A2-receptor antagonist 8-(p-sulphophenyl)theophylline, 0.5 mmol/l, n = 4. Together these data indicate that: (1) basolateral ATP induces [Ca2+]i in isolated rat colonic crypts and acts as a secretagogue in the distal rat colon; (2) a basolateral P2Y-receptor is responsible for this ATP-induced NaCl secretion; (3) the ability of ATP to increase I sc in Ussing chamber experiments is not mediated via adenosine; and (4) the agonist-induced [Ca2+]i signals are mostly located in the crypt base, which is the secretory part of the colonic crypt.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/PL00008079
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  • 3
    Electronic Resource
    Electronic Resource
    No evidence for cell-to-cell coupling in rat colonic crypts: studies with Lucifer Yellow and with photobleaching (1998)
    Springer
    Pflügers Archiv 436 (1998), S. 83-89 
    Details
    ISSN: 1432-2013
    Keywords: Key words Colon ; Cell-to-cell coupling ; Lucifer Yellow ; Rat colonic crypt ; Gap junction ; Exocrine secretion ; Fluorescence recovery after photobleaching
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Epithelial cells of exocrine glands (pancreas, lacrimal glands, salivary glands, sweat glands and gastric glands) are intimately linked together by gap junctions. Due to this close junctional coupling exocrine secretion occurs as the well concerted effort of a cell population. Colonic crypts have, on the one hand, anatomical and functional properties resembling those of exocrine glands (mostly crypt base cells) and, on the other hand, properties of absorbing cells (mostly surface cells). In the mid-crypt, depending on the functional status, absorption and secretion can occur. The present study was aimed at examining whether rat distal colonic crypt cells co-ordinate their functional status by cell-to-cell coupling. Two types of measurements were performed: as an independent assessment of cell viability the membrane voltage (V m) was measured with the fast whole-cell patch-clamp technique; to investigate cellular coupling simultaneously Lucifer Yellow (LY) (mol. wt. 443) distribution was visualized using digital video imaging. LY (500 μmol/l) was included into the patch pipette filling solution. The recorded V m was –73.4±2.3 mV in crypt base cells (n=15), –63.7±2.1 mV in mid-crypt cells (n=17) and –52.3±2.9 mV in crypt surface cells. All cells tested reversibly responded to carbachol (100 μmol/l) with a persistent hyperpolarization, as previously shown. Activation of Cl- secretion by elevation of the cAMP concentration with forskolin (5 μmol/l) led to a reversible depolarization. Throughout the duration of each individual experiment [mean experimental time in basal cells: 18.3±2.5 min (n=15), in mid-crypt cells: 19.6±3.4 min (n=17) and in crypt surface cells: 11.7±3.4 min (n=13)] LY dye distribution was solely confined to the patched cell. In addition bleaching of calcein fluorescence in laser scan microscopy was not followed by dye back diffusion, whereas this was clearly the case in pancreatic acini (n=5). These data indicate that colonic crypt cells are not coupled by gap junctions under resting conditions or in the presence of secretagogues.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050607
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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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