Fig. 1. Structural formulae of ascorbic acid (AA), 6-Br-ascorbate (BrAA), nipecotic, kynurenic and diclofenamic acids. The conjugates of these three last drugs with AA (AA–Nipec, AA–Kynur and AA–Diclo) and BrAA (BrAA–Nipec, BrAA–Kynur and BrAA–Diclo) are also shown.

Fig. 2. Kinetics of ascorbate (AA) uptake in HRPE cells. Uptake of AA was measured by 60 min incubation at 37 °C, pH 7.5. AA concentrations were in the range 2.5–1000 μM. The concentration of [¹⁴C]AA was kept constant at 50 μM. Inset: Eadie–Hofstee transformation of the same data. V, AA transport (nmol/(10⁶cells per 60 min)); S, AA concentration (μM). This is a single representative experiment performed in duplicate.

Fig. 3. Inhibition of 50 μM [¹⁴C]AA uptake by ascorbic acid (AA), 6-bromoascorbate (BrAA), nipecotic (A), kynurenic (B), diclofenamic (C) acids and their conjugates with ascorbate and 6-bromoascorbate (A–C, respectively). [¹⁴C]AA uptake in the presence of inhibitors was measured at 37 °C. These are single representative experiments performed in duplicate.

Fig. 4. Inhibition of [¹⁴C]AA transport by (A) 75 μM 6-bromoascorbate (BrAA) and increasing concentrations of the AA-conjugates of nipecotic and kynurenic acids. Inhibition of [¹⁴C]AA transport by (B) increasing concentrations of the BrAA-conjugates of nipecotic and kynurenic acids. HRPE cells were incubated for 60 min at 37 °C. The compounds inhibit in a competitive manner the uptake of [¹⁴C]AA in HRPE. Double reciprocal plots of a representative determination are shown.

Fig. 5. Kinetics of 6-bromoascorbate (BrAA) uptake in HRPE cells, obtained by HPLC analysis. Uptake of BrAA was measured by 60 min incubation at 37 °C, pH 7.5. BrAA concentrations were in the range 0.25–100 μM. Inset: Eadie–Hofstee transformation of the same data. V, BrAA transport (nmol/(10⁶cells per 60 min)); S, BrAA concentration (μM). This is a single representative experiment performed in duplicate.

Fig. 6. HPLC chromatograms for 500 μM BrAA–Kynur in extracellular fluid of HRPE cells (A) and for the related intracellular fluid after incubation at 37° C for 60 min (B). Any uptake of BrAA–Kynur has been registered.

Fig. 7. HPLC chromatograms for 200 nM BrAA–Nipec (A) and for the difference between chromatogram of the intracellular fluids of HRPE cells incubated with 5 mM BrAA–Nipec and buffer solution (B). The peak at 10.8 min is referred to BrAA. The difference has been performed using the Chem Station software (Agilent).

Inhibition constant values (K_i) of AA, BrAA, nipecotic, kynurenic, diclofenamic acids and their conjugates with AA and BrAA, obtained by inhibition of 50 μM [¹⁴C]AA uptake on HRPE cells

Values ± S.E.M. are from three independent experiments performed in duplicate.

K_t and V_max values obtained by [¹⁴C]AA kinetic experiments performed in the absence and in the presence of increasing concentrations of inhibitors

Values ± S.E.M. are from three independent experiments performed in duplicate.

Effect of i.p. injection of nipecotic acid (0.75 mmol/kg), AA–Nipec (0.75 mmol/kg) and BrAA–Nipec (0.75 mmol/kg) in the absence and in the presence of diclofenamic acid (3.75 mmol/kg) on pentylenetetrazole (PTZ)-induced convulsion in mice