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 (pH(i)) 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 pH(i) by 0.78 +/- 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pH(i) and a slow and incomplete recovery: pH(i) stayed 0.26 +/- 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pH(i). 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 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 pH(i) 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 pH(i) 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.