Fig. 3. Time courses for fractional efflux of ²²Na⁺ (mean ± s.e.m.) in isolated frog sartorius (T. G. West and R. G. Boutilier, unpublished observations) at 20°C during normoxia (98% O₂:2% CO₂; open symbols) and anoxia (98% N₂:2% CO₂; closed symbols). Fractional efflux was determined using methods modified from Overgaard et al. (Overgaard et al., 1997). Briefly, whole sartorius muscles were first preloaded with ²²Na⁺ (2 µCi ml^–1 for 30 min at 20°C in oxygenated Ringer solution). Then the muscles were washed (4x 10 min) in ice-cold Na-free Ringer; a process expected to remove extracellular ²²Na⁺ (see Overgaard et al., 1997). Finally, the muscles were transferred through 2 ml volumes of normal Ringer at 20°C with the treatments and time courses shown. Fractional efflux was calculated as the amount of ²²Na⁺ released to the bathing medium during each time interval divided by the total amount of ²²Na⁺ loaded into the muscle. The total ²²Na⁺ load was the cumulative sum of ²²Na⁺ c.p.m. released plus the c.p.m. remaining in the muscle at the end of the experiment (measured in trichloroacetic acid extracts). Fractional efflux after 30 min anoxia was significantly lower (t-test; t=4.16, P=0.005) than that of normoxic controls at the same time point (i.e. 70 min into the time courses). The ouabain-sensitive component of total fractional efflux (i.e. giving the portion of efflux attributed specifically to pump activity) could not be assessed in these studies because uncontrolled muscle twitches were always induced within 10 min by the addition of 1 mmol l^–1 ouabain, possibly owing to rising cytosolic Ca²⁺ mediated by increased Na⁺/Ca²⁺ exchange after Na⁺-pump blockade. (From T. G. West and R. G. Boutilier, unpublished observations.)