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Fig. 5. Inhibitors of endocytic vesicular traffic abrogate the protective effect of preconditioning on Na⁺ homeostasis. Intracellular Na⁺ content was assessed in isolated hepatocytes using a fluorescent sodium probe as detailed in Materials and Methods. Two parallel set of samples were prepared in which hepatocytes were preconditioned by a hypoxia-reoxygenation cycle (PC) or by treatment with 1 µM CGS21680 (CGS). In some samples, preconditioning and subsequent incubation under hypoxic conditions were performed in the presence of 250 nM WM, 10 mM 3MA or 20 µM Cyt D, as for the experiments described in Figs 3 and 4. Changes in cellular [Na⁺] were monitored during a 60 minute incubation. WM, 3MA and Cyt D were shown not to influence the intracellular Na⁺ content in hepatocytes incubated under control or hypoxic conditions (A). In control (Co) hepatocytes incubated under control conditions the homeostasis of intracellular Na⁺ was preserved, whereas in hepatocytes incubated under hypoxic conditions an influx of Na⁺ is observed. This influx is limited in preconditioned hepatocytes but it returns to the same values as in non-preconditioned hepatocytes when preconditioning and subsequent incubation are performed in the presence of WM, 3MA or Cyt D (B,C). Data are the means±s.d. of three independent experiments.

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