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  • BCECF  (4)
  • K+ channel  (3)
  • 1
    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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  • 2
    Electronic Resource
    Electronic Resource
    Ca2+ regulated K+ and non-selective cation channels in the basolateral membrane of rat colonic crypt base cells (1996)
    Springer
    Pflügers Archiv 432 (1996), S. 1011-1022 
    Details
    ISSN: 1432-2013
    Keywords: Key words Cl ; secretion ; Carbachol ; K+ channel ; cAMP ; Exocrine secretion ; Non-selective cation channel ; Cl ; channel
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  We have previously shown that a new type of K+ channel, present in the basolateral membrane of the colonic crypt base (blm), is necessary for cAMP-activated Cl- secretion. Under basal conditions, and when stimulated by carbachol (CCH) alone, this channel is absent. In the present patch clamp-study we examined the ion channels present in the blm under cell-attached and in cell-excised conditions. In cell-attached recordings with NaCl-type solution in the pipette we measured activity of a K+ channel of 16 ± 0.3 pS (n = 168). The activity of this channel was sharply increased by CCH (0.1 mmol/l, n = 26). Reduction of extracellular Ca2+ to 0.1 mmol/l (n = 34) led to a reversible reduction of activity of this small channel (SKCa). It was also inactivated by forskolin (5 μmol/l, n = 38), whilst the K+ channel noise caused by the very small K+ channel increased. Activity of non-selective cation channels (NScat) was rarely observed immediately prior to the loss of attached basolateral patches and routinely in excised patches. The NScat, with a mean conductance of 49 ± 1.0 pS (n = 96), was Ca2+ activated and required 〉10 μmol/l Ca2+ (cytosolic side = cs). It was reversibly inhibited by ATP (〈1 mmol/l, n = 13) and by 3′,5-dichloro-diphenylamine-2-carboxylate (10–100 μmol/l, n = 5). SKCa was also Ca2+ dependent in excised inside-out basolateral patches. Its activity stayed almost unaltered down to 1 μmol/l (cs) and then fell sharply to almost zero at 0.1 μmol/l Ca2+ (cs, n = 12). SKCa was inhibited by Ba2+ (n = 31) and was charybdotoxin sensitive (1 nmol/l) in outside-out basolateral patches (n = 3). Measurements of the Ca2+ activity ([Ca2+]i) in these cells using fura-2 indicated that forskolin and depolarization, induced by an increase in bath K+ concentration to 30 mmol/l, reduced [Ca2+]i markedly (n = 8–10). Hyperpolarization had the opposite effect. The present data indicate that the blm of these cells contains a small-conductance Ca2+-sensitive K+ channel. This channel is activated promptly by very small increments in [Ca2+]i and is inactivated by a fall in [Ca2+]i induced by forskolin.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050229
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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
    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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  • 5
    Electronic Resource
    Electronic Resource
    pH dependence of K+ conductances of rat cortical collecting duct principal cells (1994)
    Springer
    Pflügers Archiv 428 (1994), S. 631-640 
    Details
    ISSN: 1432-2013
    Keywords: Intracellular pH ; K+ channel ; NH4 +/NH3 Patch clamp ; BCECF
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The K+ channels of the principal cells of rat cortical collecting duct (CCD) are pH sensitive in excised membranes. K+ secretion is decreased with increased H+ secretion during acidosis. We examined whether the pH sensitivity of these K+ channels is present also in the intact cell and thus could explain the coupling between K+ and H+ secretion. Membrane voltages (V m), whole-cell conductances (g c), and single-channel currents of K+ channels were recorded from freshly isolated CCD cells or isolated CCD segments with the patch-clamp method. Intracellular pH (pHi) was measured using the pH-sensitive fluorescent dye 2′-7′-bis(carboxyethyl)-5-6-carboxyfluorescein (BCECF). Acetate (20 mmol/l) had no effect on V m, g c, or the activity of the K+ channels in these cells. Acetate, however, acidified pHi slightly by 0.17±0.04 pH units (n=19). V m depolarized by 12±3 mV (n=26) and by 23±2 mV (n=66) and g c decreased by 26±5% (n=13) and by 55±5% (n=12) with 3–5 or 8–10% CO2, respectively. The same CO2 concentrations decreased pHi by 0.49±0.07 (n=15) and 0.73±0.11 pH units (n=12), respectively. Open probability (P o) of all four K+ channels in the intact rat CCD cells was reversibly inhibited by 8–10% CO2. pHi increased with the addition of 20 mmol/l NH4 +/NH3 by a maximum of 0.64±0.08 pH units (n=33) and acidified transiently by 0.37±0.05 pH units (n=33) upon NH4 +/NH3 removal. In the presence of NH4 +/NH3 V m depolarized by 16±2 mV (n=66) and g c decreased by 26±7% (n=16). The activity of all four K+ channels was also strongly inhibited in the presence of NH4 +/NH3. The effect of NH4 +/NH3 on V m and g c was markedly increased when the pH of the NH4 +/NH3-containing solution was set to 8.5 or 9.2. From these data we conclude that cellular acidification in rat CCD principal cells down-regulates K+ conductances, thus reduces K+ secretion by direct inhibition of K+ channel activity. This pH dependence is present in all four K+ channels of the rat CCD. The inhibition of K+ channels by NH4 +/NH3 is independent of changes in pHi and rather involves an effect of NH3.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00374587
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  • 6
    Electronic Resource
    Electronic Resource
    Whole-cell conductive properties of rat pancreatic acini (1996)
    Springer
    Pflügers Archiv 432 (1996), S. 112-120 
    Details
    ISSN: 1432-2013
    Keywords: Key words Cl ; secretion ; K+ channel ; Carbachol ; Exocrine secretion ; Pancreatic acini
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Acetylcholine-controlled exocrine secretion by pancreatic acini has been explained by two hypotheses. One suggests that NaCl secretion occurs by secondary active secretion as has been originally described for the rectal gland of Squalus acanthias. The other is based on a “push-pull” model whereby Cl− is extruded luminally and sequentially taken up basolaterally. In the former model Cl− uptake is coupled to Na+ and basolateral K+ conductances play a crucial role, in the latter model, Na+ uptake supposedly occurs via basolateral non-selective cation channels. The present whole-cell patch-clamp studies were designed to further explore the conductive properties of rat pancreatic acini. Pilot studies in approximately 300 cells revealed that viable cells usually had a membrane voltage (V m) more hyperpolarized than −30 mV. In all further studies V m had to meet this criterion. Under control conditions V m was −49 ± 1 mV (n = 149). The fractional K+ conductance (f K) was 0.13 ± 0.1 (n = 49). Carbachol (CCH, 0.5 μmol/l) depolarized to −19 ± 1.1 mV (n = 63) and increased the membrane conductance (G m) by a factor of 2–3. In the seeming absence of Na+ [replacement by N-methyl-D-glucamine (NMDG+)] V m hyperpolarized slowly to −59 ± 2 mV (n = 90) and CCH still induced depolarizations to −24 ± 2 mV (n = 34). The hyperpolarization induced by NMDG+ was accompanied by a fall in cytosolic pH by 0.4 units, and a very slow and slight increase in cytosolic Ca2+. f K increased to 0.34. The effect of NMDG+ on V m was mimicked by the acidifying agents propionate and acetate (10 mmol/l) added to the bath. The present study suggests that f K makes a substantial contribution to G m under control conditions. The NMDG+ experiments indicate that the non- selective cation conductance contributes little to V m in the presence of CCH. Hence the present data in rat pancreatic acinar cells do not support the push-pull model.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050112
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