Carrier-mediated urea transport across the mitochondrial membrane of an elasmobranch (Raja erinacea) and a teleost (Oncorhynchus mykiss) fish

In osmoregulating teleost fish, urea is a minor nitrogen excretory product, whereas in osmoconforming marine elasmobranchs it serves as the major tissue organic solute and is retained at relatively high concentrations (∼400 mmol/l). We tested the hypothesis that urea transport across liver mitochondria is carrier mediated in both teleost and elasmobranch fishes. Intact liver mitochondria in rainbow trout ( Oncorhynchus mykiss) demonstrated two components of urea uptake, a linear component at high concentrations and a phloretin-sensitive saturable component [Michaelis constant ( Km) = 0.58 mmol/l; maximal velocity ( Vmax) = 0.12 μmol·h−1·mg protein−1] at lower urea concentrations (<5 mmol/l). Similarly, analysis of urea uptake in mitochondria from the little skate ( Raja erinacea) revealed a phloretin-sensitive saturable transport ( Km= 0.34 mmol/l; Vmax= 0.054 μmol·h−1·mg protein−1) at low urea concentrations (<5 mmol/l). Surprisingly, urea transport in skate, but not trout, was sensitive to a variety of classic ionophores and respiration inhibitors, suggesting cation sensitivity. Hence, urea transport was measured in the reverse direction using submitochondrial particles in skate. Transport kinetics, inhibitor response, and pH sensitivity were very similar in skate submitochondrial particle submitochondrial particles ( Km= 0.65 mmol/l, Vmax= 0.058 μmol·h−1·mg protein−1) relative to intact mitochondria. We conclude that urea influx and efflux in skate mitochondria is dependent, in part, on a bidirectional proton-sensitive mechanism similar to bacterial urea transporters and reminiscent of their ancestral origins. Rapid equilibration of urea across the mitochondrial membrane may be vital for cell osmoregulation (elasmobranch) or nitrogen waste excretion (teleost).

Lack of biliary lipid excretion in the little skate, Raja erinacea, indicates the absence of functional Mdr2, Abcg5, and Abcg8 transporters

The ABC transporters bile salt export pump (BSEP; encoded by the ABCB11 gene), MDR3 P-glycoprotein ( ABCB4), and sterolin 1 and 2 ( ABCG5 and ABCG8) are crucial for the excretion of bile salt, phospholipid, and cholesterol, respectively, into the bile of mammals. The current paradigm is that phospholipid excretion mainly serves to protect membranes of the biliary tree against bile salt micelles. Bile salt composition and cytotoxicity, however, differ greatly between species. We investigated whether biliary phospholipid and cholesterol excretion occurs in a primitive species, the little skate, which almost exclusively excretes the sulphated bile alcohol scymnolsulphate. We observed no phospholipid and very little cholesterol excretion into bile of these animals. Conversely, when scymnolsulphate was added to the perfusate of isolated mouse liver perfusions, it was very well capable of driving biliary phospholipid and cholesterol excretion. Furthermore, in an erythrocyte cytolysis assay, scymnolsulphate was found to be at least as cytotoxic as taurocholate. These results demonstrate that the little skate does not have a system for the excretion of phospholipid and cholesterol and that both the MDR3 and the two half-transporter genes, ABCG5 and ABCG8, have evolved relatively late in evolution to mediate biliary lipid excretion. Little skate plasma membranes may be protected against bile salt micelles mainly by their high sphingomyelin content.