Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells.
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 (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 pKa 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 Delta[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 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.
Benning
N
N
Physiologisches Institut der Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany.
Leipziger
J
J
Greger
R
R
Nitschke
R
R
D000468:Alkalies / Q000494:pharmacology*; D000588:Amines / Q000494:pharmacology*; D001285:Atropine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D016257:Fura-2; D019073:HT29 Cells; D006801:Humans; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism*; D008744:Methylamines / Q000494:pharmacology; D009994:Osmolar Concentration; D018408:Patch-Clamp Techniques
D000468:Alkalies; D000588:Amines; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008744:Methylamines; D001285:Atropine; D006859:Hydrogen; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; C023336:trimethylamine; D002118:Calcium; D016257:Fura-2