Docosahexaenoic acid and other fatty acids induce a decrease in pHi in Jurkat T-cells

Abstract

1. Docosahexaenoic acid (DHA) induced rapid (t1/2=33 s) and dose-dependent decreases in pHi in BCECF-loaded human (Jurkat) T-cells. Addition of 5-(N,N-dimethyl)-amiloride, an inhibitor of Na+/H+ exchanger, prolonged DHA-induced acidification as a function of time, indicating that the exchanger is implicated in pHi recovery. 2. Other fatty acids like oleic acid, arachidonic acid, eicosapentaenoic acid, but not palmitic acid, also induced a fall in pHi in these cells. 3. To assess the role of calcium in the DHA-induced acidification, we conducted experiments in Ca2+-free (0% Ca2+) and Ca2+-containing (100% Ca2+) buffer. We observed that there was no difference in the degree of DHA-induced transient acidification in both the experimental conditions, though pHi recovery was faster in 0% Ca2+ medium than that in 100% Ca2+ medium. 4. In the presence of BAPTA, a calcium chelator, a rapid recovery of DHA-induced acidosis was observed. Furthermore, addition of CaCl2 into 0% Ca2+ medium curtailed DHA-evoked rapid pHi recovery. In 0% Ca2+ medium, containing BAPTA, DHA did not evoke increases in [Ca2+]i, though this fatty acid still induced a rapid acidification in these cells. These observations suggest that calcium is implicated in the long-lasting DHA-induced acidosis. 5. DHA-induced rapid acidification may be due to its deprotonation in the plasma membrane (flip-flop model), as suggested by the following observations: (1) DHA with a -COOH group induced intracellular acidification, but this fatty acid with a -COOCH3 group failed to do so, and (2) DHA, but not propionic acid, -induced acidification was completely reversed by addition of fatty acid-free bovine serum albumin in these cells. 6. These results suggest that DHA induces acidosis via deprotonation and Ca2+ mobilization in human T-cells.

Figure 1

Effects of DHA on pH_i. Cells (4 × 10⁶ assay⁻¹) were loaded with the fluorescent dye BCECF/AM, as described in Methods. (a) Intracellular acidosis induced by different concentrations of DHA, that is, 2.5, 5, 10 and 20 μM. (b) ΔpH_i represents the amplitude of the intracellular acidification produced. Values are expressed as mean±s.e.m. of independent experiments (n=30). (c) Acidosis induced by DHA (10 μM), as visualized by confocal microscopy (n=6). NS=insignificant differences.

Figure 2

Effects of DHA and PA on pH_i. Cells (4 × 10⁶ assay⁻¹) were loaded with the fluorescent dye BCECF/AM, as described in Methods. The arrowheads indicate the time when DHA (10 μM) and PA (2.5 mM) were added into the cuvette, without interruption in the recordings. The figure shows single traces of observations, which were reproduced independently (n=8). All experiments were performed in the bicarbonate-free medium, pH 7.4.

Figure 3

Effects of DHA on (Ca²⁺)_i. Cells (4 × 10⁶ assay⁻¹) were loaded with the fluorescent dye Fura-2/AM, as described in Methods, and then resuspended in the 100% or 0% Ca²⁺ buffers. DHA (10 μM) was added into the cuvette without interruptions in the recordings (n=4).

Figure 4

Role of external calcium in the DHA-induced decrease in pH_i. Cells (4 × 10⁶ assay⁻¹) were loaded with the fluorescent dye BCECF/AM, as described in Methods. (a) Cells were resuspended in either 0% or 100% Ca²⁺ buffers. (b) Ca²⁺-free/Ca²⁺-reintroduction experiment in 0% Ca²⁺ medium: CaCl₂ (2 mM) was applied after DHA (10 μM), as indicated in the figure. The figures show single traces of observations, which were reproduced independently (n=10).

Figure 5

Effect of intracellular free calcium chelation on the DHA-induced decrease in pH_i. Cells (4 × 10⁶ assay⁻¹) were loaded with the fluorescent dye BCECF/AM and preincubated with BAPTA/AM (50 μM, 15 min), as described in Methods. DHA (10 μM) was added at the end of the incubation. The figure shows single traces of observations, which were reproduced independently (n=6).

Figure 6

Effect of DMA and bafilomycin A1 on DHA-evoked decrease in pH_i. Cells (4 × 10⁶ assay⁻¹) were loaded with the fluorescent dye BCECF/AM, as described in Methods. The arrowheads indicate the time when the test molecules DHA (10 μM), DMA (10 μM) and bafilomycin A1 (50 nM) were added into the cuvette, without interruptions in the recordings. The figures show single traces of observations that were reproduced independently (n=11). Experiments were performed both in the Ca²⁺-containing (100% Ca²⁺) and Ca²⁺-free (0% Ca²⁺) buffers.

Figure 7

Effect of BSA and DHA methyl ester on pH_i. Cells (4 × 10⁶ assay⁻¹) were loaded with the fluorescent dye BCECF/AM, as described in Methods. The arrowheads indicate the time when the test molecules DHA (10 μM), BSA (0.2% w v⁻¹), PA (2.5 mM) and DHA methyl ester (10 μM), were added into the cuvette, without interruptions in the recordings. The figures show single traces of observations that were reproduced independently (n=4). All experiments were performed in the bicarbonate-free medium, pH 7.4.

Figure 8

Effect of different fatty acids on pH_i. Cells (4 × 10⁶ assay⁻¹) were loaded with the fluorescent dye BCECF/AM, as described in Methods. (a–d) Intracellular acidosis induced by different fatty acids: AA, arachidonic acid, EPA, eicosapentaenoic acid, OA, oleic acid and palmitic acid. All fatty acids were used at 10 μM. (e) ΔpH_i represents the amplitude of the intracellular acidification produced by these agents. All fatty acids, including DHA, were used in the same batches of cells. Values are expressed as mean±s.e.m. of independent experiments (n=5). NS=insignificant differences.