Na+-dependent transport of pyruvate in erythrocytes of the Pacific hagfish (Eptatretus stouti)

Abstract

We investigated the mechanisms of pyruvate transport in the erythrocytes of an ancient marine agnathan, the Pacific hagfish (Eptatretus stouti), and a sedentary euryhaline teleost, the starry flounder (Platichthys stellatus). Uptake of [14C]pyruvate (50 µM, 10°C) by flounder erythrocytes was slow (t1/2(half-life) ~ 30 min), nonconcentrative, and mediated by the band 3 Cl-/HCO-3exchanger in combination with a process similar to the H+/monocarboxylate symporter present in freshwater teleosts and mammalian erythrocytes. In contrast, pyruvate uptake by hagfish erythrocytes (50 µM, 10°C) was rapid (t1/2~ 1.5 min) and, in 10 min, reached an intracellular concentration more than 20-fold higher than that present in the extracellular medium. Pyruvate accounted for almost 90% of the accumulated intracellular radioactivity, the remaining label being incorporated into tricarboxylic acid cycle intermediates and glutamate. Influx of pyruvate was saturable (apparent Km= 12 mM) and inhibited by p-chloromercuriphenylsulphonate (PCMBS) (Ki= 71 µM) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) (Ki= 0.49 mM). Transport was inhibited poorly by α-cyano-4-hydroxycinnamate (CIN) (Ki> 4 mM) and was not coupled to the movement of protons. Instead, the influx of pyruvate was Na+dependent. A sigmoidal relationship between pyruvate transport and extracellular Na+concentration was observed, suggesting a Na+:pyruvate coupling ratio greater than 1:1. In contrast with previously described Na+-dependent monocarboxylate transport activities in mammalian renal and intestinal epithelia, the hagfish erythrocyte system did not transport lactate.