Figure 3.

Effects of ATP and A23187 on Asc release by HPLC-based electrochemical assay. (a) PCEC were preincubated with ¹⁴C-Asc and the cells were rinsed with the release solution used in Figure 2. The induced release (Test (ATP, A23187)–Control) was determined for 5 min using scintillation counting (¹⁴C) or HPLC (Asc). Values are mean±s.e.m. of five replicates. The Test–Control value for ATP or A23187 did not depend (P>0.05) on the method of measurement (HPLC or ¹⁴C). (b) Asc release induced by 10 μM A23187 or 1 mM ATP using HPLC from PCEC preincubated for 18 h with 400 μM Asc 2-phosphate. (c) Asc release from PCEC preincubated with 100 μM DHAA for 30 min. Values are mean±s.e.m. of 12 replicates. A23187 and ATP increased the Asc release significantly when the cells were preincubated with Asc 2-phosphate or DHAA (P<0.05). (d) Relationship between total Asc content of cells and A23187-induced Asc release. Different levels of total cell Asc were obtained as in (c) but by incubating the cells in the presence of various concentrations of DHAA. The amount released mg protein⁻¹ min⁻¹ correlated linearly (r²=0.9832, P<0.05) with the total loading (nmol mg protein⁻¹).