Library

X
feed icon rss

Your email was sent successfully. Check your inbox.

An error occurred while sending the email. Please try again.

Proceed reservation?

Export
  • 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
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
    Paper (German National Licenses)
    Fulltext
  • 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
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
    Paper (German National Licenses)
    Fulltext
  • 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
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
    Paper (German National Licenses)
    Fulltext
  • 4
    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
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
    Paper (German National Licenses)
    Fulltext
  • 5
    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
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
    Paper (German National Licenses)
    Fulltext
Close ⊗
This website uses cookies and the analysis tool Matomo. More information can be found here...