CHARACTERIZATION OF BUMETANIDE TRANSPORT IN ISOLATED SKATE HEPATOCYTES

The uptake of [H-3]bumetanide was studied in isolated ''skate hepatocy tes in an albumin-free elasmobranch Ringer solution and compared with the uptake of bile acids in the presence of other cholephilic organic anions. [H-3]bumetanide uptake was energy dependent, temperature sensi tive, and exhibited saturation kinetics. In contrast to taurocholate a nd cholate, which are transported only by Na+-independent mechanisms, removal of Na+ reduced the maximal uptake rate (V(max)) for bumetanide from 404 +/- 80 to 230 +/- 47 pmol.mg-1.min-1 without a change in the apparent Michaelis constant (K(m)). The apparent K(m) for the Na+-dep endent portion of bumetanide uptake was 58 +/- 24 muM, and V(max) was 151 +/- 38 pmol . min-1 . mg-1. Taurocholate (100 and 200 muM) inhibit ed Na+-independent bumetanide transport competitively but was a noncom petitive inhibitor for Na+-dependent bumetanide uptake. Furosemide (10 0 muM) and two bumetanide analogues, PF-3034 (500 muM) and PF-2203 (50 0 muM), preferentially inhibited the Na+-dependent bumetanide uptake s ystem, whereas cholate (100 muM) and probenecid (100 muM) preferential ly inhibited Na+-independent bumetanide transport. The sulfhydryl (SH) reagents N-ethylmaleimide, 2,2'-dithio-bis(5-nitropyridine), and p-ch loromercuribenzenesulfonic acid (PCMBS) inhibited both bile acid and b umetanide uptake. Dithiothreitol (500 muM) completely reversed the PCM BS-induced inhibition of bumetanide uptake. These results indicate tha t bumetanide is transported into hepatocytes of the small skate, Raja erinacea, by both Na+-dependent and Na+-independent mechanisms; the la tter is shared by bile acids and probably sulfobromophthalein and othe r organic anions. Their uptake requires free SH groups.