Ascorbic acid efflux from human brain microvascular pericytes: role of re-uptake

Abstract

Microvascular pericytes take up ascorbic acid on the ascorbate transporter SVCT2. Intracellular ascorbate then protects the cells against apoptosis induced by culture at diabetic glucose concentrations. To investigate whether pericytes might also provide ascorbate to the underlying endothelial cells, we studied ascorbate efflux from human pericytes. When loaded with ascorbate to intracellular concentrations of 0.8-1.0 mM, almost two-thirds of intracellular ascorbate effluxed from the cells over 2 H. This efflux was opposed by ascorbate re-uptake from the medium, since preventing re-uptake by destroying extracellular ascorbate with ascorbate oxidase increased ascorbate loss even further. Ascorbate re-uptake occurred on the SVCT2, since its blockade by replacing medium sodium with choline, by the SVCT2 inhibitor sulfinpyrazone, or by extracellular ascorbate accelerated ascorbate loss from the cells. This was supported by finding that net efflux of radiolabeled ascorbate was increased by unlabeled extracellular ascorbate with a half-maximal effect in the range of the high affinity Km of the SVCT2. Intracellular ascorbate did not inhibit its efflux. To assess the mechanism of ascorbate efflux, known inhibitors of volume-regulated anion channels (VRACs) were tested. These potently inhibited ascorbate transport into cells on the SVCT2, but not its efflux. An exception was the anion transport inhibitor DIDS, which, despite inhibition of ascorbate uptake, also inhibited net efflux at 25-50 µM. These results suggest that ascorbate efflux from vascular pericytes occurs on a DIDS-inhibitable transporter or channel different from VRACs. Further, ascorbate efflux is opposed by re-uptake of ascorbate on the SVCT2, providing a potential regulatory mechanism.

Keywords: SVCT2; ascorbate efflux; ascorbic acid; pericytes; voltage-regulated anion channels.

Figure 1. Intracellular ascorbate concentrations

Panel A. Efflux ascorbate from cells loaded with 100 µM ascorbate (circles) or 10 µM [1-¹⁴C]ascorbate (squares) was measured as described under Materials and methods. Results for radiolabeled ascorbate efflux are expressed as a fraction of the time zero value, which was 49 ± 5 pmol/well. N = 6 plates for each form of ascorbate. Panel B. Efflux of unlabeled ascorbate in the absence (circles) or presence (squares) of 1.6 U/ml ascorbate oxidase was measured as described in Materials and methods. Results are shown from 8 plates, with p < 0.05 compared to the corresponding control sample from the same plate lacking ascorbate oxidase.

Figure 2. Effects of SVCT2 and glucose transport inhibition on intracellular ascorbate concentrations

Panel A. Pericytes were incubated at 37 °C with the indicated concentrations of sulfinpyrazone, followed in 10 min by 100 µM ascorbate. After 30 min the cells were rinsed and taken for assay of intracellular ascorbate as described in Materials and methods. Results are shown from 4 experiments with an “*” indicating p < 0.05 compared to the sample not treated with the inhibitor. Panel B. Cells loaded for 60 min with 100 µM ascorbate were treated without (circles) or with 1 mM sulfinpyrazone at 37 °C during efflux and intracellular ascorbate was measured as described in Materials and methods. Results are shown from 6 experiments, with an “*” indicating p < 0.05 compared to the corresponding time point sample without the inhibitor. Panel C. Cells loaded with 100 µM ascorbate for 60 min were rinsed twice in 37 °C KRH containing 5 mM D-glucose and either 128 mM sodium or 128 mM choline chloride. Cells were incubated at 37°C during ascorbate efflux in sodium-free KRH (first bar) or in sodium-containing KRH with 20 mM D-glucose (second bar), 20 mM L-glucose plus 5 mM D-glucose (third bar), or 5 mM D-glucose plus 10 µM cytochalasin B and 1.6 U/ml ascorbate oxidase (fourth bar). After 30 min, the medium was aspirated, the cells were rinsed twice in the respective KRH and were then taken for assay of intracellular ascorbate. Results are shown from 4–5 experiments, expressed as a fraction of the 5 mM D-glucose control that was carried out in each experiment. An “*” indicates p < 0.01 compared to that control.

Figure 3. Effects of intra- and extracellular unlabeled ascorbate on radiolabeled ascorbate efflux

Pericytes in 12-well plates were incubated for 60 min with 10 µM radiolabeled ascorbate, the medium was aspirated, and the cells were rinsed twice in KRH containing 5 mM D-glucose. The cells were incubated with 1 ml of glucose-KRH containing the indicated concentration of unlabeled ascorbate. After 60 min, radiolabeled ascorbate was measured in the medium (Panel A) or in the cells (Panel B) as described in Materials and methods. Results are shown from 6 experiments, with an “*” indicating p < 0.05 compared to the sample not treated with unlabeled ascorbate. Panel C. Pericytes were loaded with 10 µM radiolabeled ascorbate for 20 min in complete medium, followed by addition of the indicated concentration of DHA. After another 30 min at 37°C, the medium was removed and the cells were rinsed twice in 37°C glucose-KRH. Following addition of 1 ml of glucose-KRH at 37°C, efflux was measured at 30 min as described in Materials and methods. Results are shown from 6 experiments, with an “*” indicating p < 0.05 compared to the sample not treated with DHA.

Figure 4. VRAC inhibitor effects on ascorbate uptake and efflux

Panel A. After 2 rinses in KRH containing 5 mM D-glucose at 37°C, pericytes were treated with the inhibitors as noted for 10 min, followed by addition of 100 µM ascorbate. After 30 min at 37°C, the cells were rinsed and taken for assay of intracellular ascorbate as described in Methods. The inhibitor concentrations were: niflumic acid, 100 µM; DCPIB, 20 µM; clomiphene, 100 µM; NPPB, 100 µM, and IAA-94, 100 µM. Results are shown from 4 experiments, with an “*” indicating p < 0.05 compared to the sample not treated with an inhibitor. Panels B – F. Pericytes were loaded with 100 µM ascorbate for 60 min in medium at 37°C. After medium aspiration and 2 rinses in glucose-KRH, the cells were treated with glucose-KRH containing the inhibitor noted in each panel at the same concentration used in the uptake studies. Efflux of unlabeled ascorbate was measured at each time point indicated. Results are shown from 3–4 experiments, with an “*” indicating p < 0.05 compared to the corresponding control at 20 or 60 min of efflux.

Figure 5. DIDS inhibition of ascorbate uptake and efflux

Panel A. Pericytes were rinsed and treated in KRH containing 5 mM D-glucose with the indicated concentrations of DIDS at 37°C for 30 min before rinsing and assay of intracellular ascorbate. Results are from 6 experiments, with an “*” indicating p < 0.05 compared to a sample not treated with DIDS. Panel B. Pericytes loaded with 100 µM unlabeled ascorbate for 60 min in culture medium at 37°C were rinsed twice in 37°C glucose-KRH and treated without (circles) or with (squares) 100 µM DIDS in 1 ml of glucose-KRH. At the indicated times, the medium was aspirated and the cells were rinsed twice in 2 ml of ice-cold KRH before assay of intracellular ascorbate. Results are from 5 experiments, with an “*” indicating p < 0.05 compared to the sample not treated with DIDS at the same time point. Panel C. Pericytes were loaded with 100 µM unlabeled ascorbate for 60 min, rinsed twice in glucose-KRH, and treated in 37 °C glucose-KRH with the indicated concentration of DIDS. After 30 min, the cells were rinsed again in ice-cold KRH and taken for assay of intracellular ascorbate. Results are shown from 6 experiments with an “*” indicating p < 0.05 compared to the sample not treated with DIDS. Panel D. Pericytes in culture medium were loaded with 10 µM radiolabeled ascorbate for 60 min, rinsed twice in glucose-KRH, and treated with the indicated concentration of DIDS in glucose-KRH. After 30 min, the cells were rinsed and taken for assay of intracellular radiolabeled ascorbate. Results are shown from 7 experiments, with an “*” indicating p < 0.05 compared to the sample not treated with DIDS.