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Acta chirurgica Austriaca

, Volume 26, Issue 5, pp 320–325 | Cite as

Bedeutung des HCO3-CO2-Puffersystems für die Homöostase des intrazellulären pH in SW620-Kolonkarzinomzellen

  • E. P. Cosentini
  • G. Bischof
  • G. Hamilton
  • B. Teleky
  • M. Riegler
  • W. Feil
  • E. Wenzl
  • R. Schiessel
Chirurgische Forschung
  • 17 Downloads

Zusammenfassung

Hintergrund: Solide Tumoren weisen azidotische Regionen mit durchschnittlichen extrazellulären pH Werten (pHo) von 6,9 bis 7,0 (Normalgewebe: 7,4 bis 7,5) auf. In humanen SW620-Kolonkarzinomzellen konnten in einer früheren Arbeit ein Na/H-Austauscher sowie ein Na/HCO3-Kotransporter als Säureextrusoren nachgewiesen werden, ein Cl/HCO3-Austauscher wurde im Gegensatz zu anderen Tumorlinien nicht gefunden. Diese Studie untersucht die Auswirkungen unterschiedlicher extrazellulärer pH-Bedingungen auf die intrazelluläre pH-Regulation am Modell der SW620-Kolonkarzinomzellen, durch Variation des CO2-Partialdrucks und der HCO3-Konzentration der Inkubationslösungen.

Methodik: SW620-Zellen wurden in HEPES-gepufferter Lösung (pH = 7,4; 25 °C) mit dem pH-sensitiven Fluoreszenzfarbstoff BCECF beladen und pHi-Veränderungen mittels einer computergesteuerten Spektrofluorimetrieanlage aufgezeichnet. Durch Kombination von 5, 15, 25 und 35 mM HCO3 mit 2,5, 5 und 15 Vol.% CO2 (Rest-Vol.% O2) Begasung wurden 12 Versuchsanordnungen mit unterschiedlichem pHo (pHo = 6,30 bis 7,81) gebildet.

Ergebnisse: Der Ruhe-pHi in HEPES-Lösung betrug 7,52 ± 0,01 (n = 72). Nach Wechsel zu HCO3/CO2-Medium kam es zu einer Ansäuerung von 0,25 bis 0,77 pH-Einheiten. 5% CO2/5 mM HCO3 führte nach einer Ruheperiode von 15 min zu einer pHi-Verschiebung um ≥ 0,2 Einheiten in den sauren Bereich und 2,5% CO2/35 mM HCO3 zu einer pHi-Verschiebung um ≥ 0,2 Einheiten in den alkalischen Bereich gegenüber dem Standard-Ruhe-pHi (7,39 bei 5% CO2/25 mM HCO3). Bei 15% CO2/5 mM HCO3 (pHo = 6,26) konnte keine Rückregulation beobachtet werden.

Schlußfolgerungen: Die Ergebnisse im SW620-In-vitro-Modell zeigen, daß extrazelluläre Abweichungen von Standard-CO2- und-Bikarbonatbedingungen zu Störungen der intrazellulären pH-Homöostase führen. Das Ausmaß der intrazellulären Ansäuerung war von der CO2-Spannung abhängig, die H-Extrusionsraten wurden durch den extrazellulären pH bestimmt. Diese Resultate demonstrieren die prinzipielle Möglichkeit, eine selektive Beeinträchtigung der intrazellulären pH-Homöostase und damit der Zellfunktion von Tumorzellen durch Veränderung des extrazellulären pH zu erzielen.

Schlüsselwörter

Kolonkarzinom Na/H Austauscher Na/HCO3-Kotransporter BCECF intrazellulärer pHi-Regulation intrazelluläre Azidose intrazelluläre Alkalose 

Influence of the HCO3-CO2-buffer-system on the homeostasis of intracellular pH in SW620 colonic carcinoma cells

Summary

Background: Extracellular pH (pHo) in malignant tumors often reaches levels below 7.0 (mean values 0.5 pH units lower compared to normal tissues). This study was designed to investigate regulation and maintainance of intracellular pH (pHi) of a human colonic carcinoma derived cell-line (SW620) under different extracellular pH conditions by modulating CO2-tensions and bicarbonate concentrations. Previously we characterized a Na/H exchanger and a Na/HCO3-cotransporter to account for acid extrusion in these cells, whereas no evidence of the presence of a Cl/HCO3 exchanger was found.

Methods: pHi changes of cells were recorded by spectrofluorimetric monitoring of the pH-sensitive, fluorescent dye BCECF. Dye loaded cells were incubated in air-equilibrated HEPES-buffered solution (pH = 7.4, 25 °C), then bathing solutions were switched to HCO3/CO2-buffered media to simulate different conditions. 12 groups were designed by matching bicarbonate concentrations of 5, 15, 25, and 35 mM with 2.5, 5, or 15% CO2 resulting in different pH (pHo = 6.30 to 7.81).

Results: Steady state pHi of SW620 cells in HEPES-buffered solution was 7.52 ± 0.01 (n = 72). When cells were exposed to HCO3/CO2-buffered solutions with different pHo they rapidly acidified by 0.25 to 0.77 pH units. After a period of 15 min 5% CO2/5 mM HCO3 led to a pHi-change of ≥ 0.2 pHi units towards acidic pHi and 2.5% CO2/35 mM HCO3 to a pHi-change of ≥ 0.2 units towards alkaline pHi, compared to standard resting pHi (7.39 at 5% CO2/25 mM HCO3). At 15% CO2/5 mM HCO3 (pHo = 6.26) no pHi-recovery was observed.

Conclusions: Deviations from standard extracellular CO2 and HCO3 concentrations provide a challenge to cellular homeostasis. Cytoplasmic acidification was dependent on CO2-tensions, H-extrusion rates were determined by extracellular pH (pHo). Manipulation of pHi-regulation in tumor cells by varying extracellular pH could lead to intracellular conditions which might affect cell-function and therefore contribute to anti-tumor strategies.

Key-words

Colonic carcinoma Na/H exchange Na/HCO3 cotransporter BCECF intracellular pH intracellular acidosis intracellular alkalosis 

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Copyright information

© Blackwell Scientific Publications Ltd 1994

Authors and Affiliations

  • E. P. Cosentini
    • 1
  • G. Bischof
    • 1
  • G. Hamilton
    • 1
  • B. Teleky
    • 1
  • M. Riegler
    • 1
  • W. Feil
    • 1
  • E. Wenzl
    • 1
  • R. Schiessel
    • 1
  1. 1.Klinische Abteilung für AllgemeinchirurgieUniversitätsklinik für ChirurgieWien

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