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Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells

  • Original Article
  • Transport processes, metabolism and endocrinology; kidney, gastrointestinal tract, and exocrine glands
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Abstract

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pH;) 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 (Vm) 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/1) increased pH; 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: pH, 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 triethylamine) 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+]; induced by trimethylamine (20 mmol/1) was 160 ± 15 nmol/1 (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+]j transient. Tetramethylamine led to higher [Ca2+]j changes than the other amines tested and only this transient could be completely blocked by atropine (10-6 mol/1). Trimethylamine (20 mmol/1) hyperpolarized Vm 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.

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Authors and Affiliations

  1. Physiologisches Institut der Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse 7, D-79104, Freiburg, Germany

    N. Benning, J. Leipziger, R. Greger & R. Nitschke

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  1. N. Benning
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  2. J. Leipziger
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  3. R. Greger
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  4. R. Nitschke
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Benning, N., Leipziger, J., Greger, R. et al. Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells. Pflügers Arch — Eur J Physiol 432, 126–133 (1996). https://doi.org/10.1007/s004240050114

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  • DOI: https://doi.org/10.1007/s004240050114

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