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  • 1
    Electronic Resource
    Electronic Resource
    Effect of intracellular pH on agonist-induced [Ca2+]i transients in HT29 cells (1997)
    Springer
    Pflügers Archiv 434 (1997), S. 466-474 
    Details
    ISSN: 1432-2013
    Keywords: Key words BCECF ; Fura-2 ; pHi ; [Ca2+]i ; HT29 ; Carbachol ; Neurotensin ; ATP ; InsP3 ; Cell volume ; Calcein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  In this study we examined the influence of intracellular pH (pHi) on agonist-induced changes of intracellular Ca2+ activity ([Ca2+]i) in HT29 cells. pHi and [Ca2+]i were measured microspectrofluorimetrically using BCECF and fura-2, respectively. Buffers containing trimethylamine (TriMA), NH3/NH4 + and acetate were used to clamp pHi to defined values. The magnitudes of the peak and plateau of [Ca2+]i transients induced by carbachol (CCH, 10–6 mol/l) were greatly enhanced by an acidic pHi and nearly abolished by an alkaline pHi. The relationship between pHi and the [Ca2+]i peak was nearly linear from pHi 7.0 to 7.8. This effect of pHi was also observed at higher CCH concentrations (10–4 and 10–5 mol/l), at which the inhibitory effect of an alkaline pHi was more pronounced than the stimulatory effect of an acidic pHi. An acidic pHi shifted the CCH concentration/response curve to the left, whereas an alkaline pHi led to a rightward shift. The influence of pHi on [Ca2+]i transients induced by neurotensin (10–8 mol/l) or ATP (5 × 10–7 mol/l) was similar to its influence on those induced by CCH, but generally not as pronounced. Measurements of cellular inositol 1,4,5-trisphosphate (InsP 3) showed no changes in response to acidification with acetate (20 mmol/l) or alkalinization with TriMA (20 mmol/l). The InsP 3 increase induced by CCH was unaltered at an acidic pHi, but was augmented at an alkaline pHi. Confocal measurements of cell volume showed no significant changes induced by TriMA or acetate. Slow-whole-cell patch-clamp experiments showed no additional effect of CCH on the membrane voltage (V m) measured after TriMA or acetate application. We conclude that pHi is a physiological modulator of hormonal effects in HT29 cells, as the [Ca2+]i responses to agonists were significantly changed at already slightly altered pHi. The measurements of InsP 3, cell volume and V m show that pHi must act distally to the InsP 3 production, and not via changes of cell volume or V m.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050422
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  • 2
    Electronic Resource
    Electronic Resource
    Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells (1996)
    Springer
    Pflügers Archiv 432 (1996), S. 126-133 
    Details
    ISSN: 1432-2013
    Keywords: Key words Trimethylamine ; pHi ; [Ca2+]i ; Membrane voltage ; BCECF ; Fura-2 ; Ca2+ store ; Capacitative Ca2+ influx
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pHi) and intracellular Ca2+ activity ([Ca2+]i) of HT29 cells was investigated microspectrofluorimetrically using pH- and Ca2+- sensitive fluorescent indicators, [i.e. 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (V m) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pK a value. Trimethylamine (20 mmol/l) increased pHi by 0.78 ± 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pHi and a slow and incomplete recovery: pHi stayed 0.26 ± 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pHi. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca2+]i transient which reached a peak value within 10–25 s and then slowly declined to a [Ca2+]i plateau. The initial Δ[Ca2+]i induced by trimethylamine (20 mmol/l) was 160 ± 15 nmol/l (n = 17). The [Ca2+]i peak was independent of the Ca2+ activity in the bath solution, but the [Ca2+]i plateau was significantly lower under Ca2+-free conditions and could be immediately interrupted by application of CO2 (10%; n = 6), a manoeuvre to acidify pHi in HT29 cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca2+ stores completely abolished this [Ca2+]i transient. Tetramethylamine led to higher [Ca2+]i changes than the other amines tested and only this transient could be completely blocked by atropine (10−6 mol/l). Trimethylamine (20 mmol/l) hyperpolarized V m by 22.5 ± 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 ± 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pHi changes, release Ca2+ from intracellular, inositol-1,4,5-trisphosphate-sensitive Ca2+ stores and increase Ca2+ influx into HT29 cells. The latter may be related to both the store depletion and the hyperpolarization.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050114
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  • 3
    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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  • 4
    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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  • 5
    Electronic Resource
    Electronic Resource
    Hypertonic cell shrinkage reduces the K+ conductance of rat colonic crypts (1998)
    Springer
    Pflügers Archiv 436 (1998), S. 227-232 
    Details
    ISSN: 1432-2013
    Keywords: Key words K+ channel ; Nonselective cation channel ; Volume regulation ; Calcium ; Ca2+
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  It has previously been shown in studies of a renal epithelial cell line that nonselective cation (NSC) channels are activated by exposure to hypertonic solution. We have also found such channels in excised patches of colonic crypt cells. They require high Ca2+ activities on the cytosolic side and a low ATP concentration for their activation and have not been recorded from cell-attached patches of colonic crypts. We examine here whether this type of channel is activated by hypertonic cell shrinkage. Bath osmolality was increased by addition of 25, 50 or 100 mmol/l mannitol. Cell-attached and whole-cell patch recordings were obtained from rat base and mid-crypt cells. In whole-cell recordings we found that addition of 50 or 100 mmol/l mannitol depolarized these cells significantly from –78±2.0 to –66±3.8 mV (n=22) and from –78±1.3 to –56±2.6 mV (n=61), respectively, and reduced the whole-cell conductance from 20±8.0 to 14±6.6 nS (n=7) and from 20±3.0 to 9.8±1.6 nS (n=19), respectively. In cell-attached patches K+ channels with a single-channel conductance of ≈16 pS were found in most recordings. The activity of these channels (N×P o, N=number, P o=open channel probability) was reduced from 2.08±0.37 to 0.98±0.23 (n=15) by the addition of 50 mmol/l mannitol and from 1.75±0.26 to 0.77±0.20 (n=12) by 100 mmol/l mannitol. No NSC channel activity was apparent in any of these recordings. Previously we have shown that the 16-pS K+ channel is controlled by cytosolic Ca2+ ([Ca2+]i). Therefore we measured [Ca2+]i by the fura-2 method and found that hypertonic solution reduced [Ca2+]i significantly (n=16). These data indicate that exposure of rat colonic crypts to hypertonic solutions does not activate NSC channels; [Ca2+]i falls in hypertonic solution leading to a reduction in the value of K+ channel N×Po, a reduced whole-cell conductance and depolarization of mid-crypt cells. These processes probably assist volume regulation inasmuch as they reduce KCl losses from the cell.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050626
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  • 6
    Electronic Resource
    Electronic Resource
    pH regulation in HT29 colon carcinoma cells (1994)
    Springer
    Pflügers Archiv 428 (1994), S. 179-185 
    Details
    ISSN: 1432-2013
    Keywords: BCECF ; Na+/H+ exchanger ; HCO 3 − /Cl− exchanger ; Na+-dependent HCO 3 − transporter ; DIDS ; HOE-694
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The pH regulation in HT29 colon carcinoma cells has been investigated using the pH-sensitive fluorescent indicator 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Under control conditions, intracellular pH (pHi) was 7.21±0.07 (n=22) in HCO 3 − -containing and 7.21±0.09 (n=12) in HCO 3 − -free solution. HOE-694 (10 μmol/l), a potent inhibitor of the Na+/H+ exchanger, did not affect control pHi. As a means to acidify cells we used the NH 4 + /NH3 (20 mmol/l) prepulse technique. The mean peak acidification was 0.37±0.07 pH units (n=6). In HCC 3 − -free solutions recovery from acid load was completely blocked by HOE-694 (1 μmol/l), whereas in HCO3 3 − -containing solutions a combination of HOE-694 and 4,4′-diisothiocyanatostilbene-2, 2′-disulphonate (DIDS, 0.5 mmol/l) was necessary to show the same effect. Recovery from acid load was Na+-dependent in HCO 3 − -containing and HCO 3 − -free solutions. Removal of external Cl− caused a rapid, DIDS-blockable alkalinization of 0.33±0.03 pH units (n=15) and of 0.20±0.006 pH units (n=5), when external Na+ was removed together with Cl−. This alkalinization was faster in HCO 3 − -containing than in HCO 3 − -free solutions. The present observations demonstrate three distinct mechanisms of pH regulation in HT29 cells: (a) a Na+/H+ exchanger, (b) a HCO 3 − /Cl− exchanger and (c) a Na+-dependent HCC 3 − transporter, probably the Na+-HCO 3 − /Cl− antiporter. Under HCO 3 − — free conditions the Na+/H+ exchanger fully accounts for recovery from acid load, whereas in HCO 3 − -containing solutions this is accomplished by the Na+/H+ exchanger and a Na+-dependent mechanism, which imports HCO 3 − . Recovery from alkaline load is caused by the HCO 3 − /Cl− exchanger.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00374856
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