Characterization of bumetanide transport in isolated skate hepatocytes.
The uptake of [3H]bumetanide was studied in isolated skate hepatocytes in an albumin-free elasmobranch Ringer solution and compared with the uptake of bile acids in the presence of other cholephilic organic anions. [3H]bumetanide uptake was energy dependent, temperature sensitive, and exhibited saturation kinetics. In contrast to taurocholate and cholate, which are transported only by Na(+)-independent mechanisms, removal of Na+ reduced the maximal uptake rate (Vmax) for bumetanide from 404 +/- 80 to 230 +/- 47 pmol.mg-1 x min-1 without a change in the apparent Michaelis constant (Km). The apparent Km for the Na(+)-dependent portion of bumetanide uptake was 58 +/- 24 microM, and Vmax was 151 +/- 38 pmol.min-1 x mg-1. Taurocholate (100 and 200 microM) inhibited Na(+)-independent bumetanide transport competitively but was a noncompetitive inhibitor for Na(+)-dependent bumetanide uptake. Furosemide (100 microM) and two bumetanide analogues, PF-3034 (500 microM) and PF-2203 (500 microM), preferentially inhibited the Na(+)-dependent bumetanide uptake system, whereas cholate (100 microM) and probenecid (100 microM) preferentially inhibited Na(+)-independent bumetanide transport. The sulfhydryl (SH) reagents N-ethylmaleimide, 2,2'-dithio-bis(5-nitropyridine), and p-chloromercuribenzenesulfonic acid (PCMBS) inhibited both bile acid and bumetanide uptake. Dithiothreitol (500 microM) completely reversed the PCMBS-induced inhibition of bumetanide uptake. These results indicate that bumetanide is transported into hepatocytes of the small skate, Raja erinacea, by both Na(+)-dependent and Na(+)-independent mechanisms; the latter is shared by bile acids and probably sulfobromophthalein and other organic anions. Their uptake requires free SH groups.
Blumrich
M
M
Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672.
Petzinger
E
E
Boyer
J L
JL
D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D001667:Binding, Competitive; D001692:Biological Transport / Q000187:drug effects; D002034:Bumetanide / Q000378:metabolism*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002478:Cells, Cultured; D019826:Cholic Acid; D002793:Cholic Acids / Q000378:metabolism; D003486:Cyanides / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D009840:Oligomycins / Q000494:pharmacology; D012860:Skates, Fish; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013656:Taurocholic Acid / Q000378:metabolism; D013997:Time Factors
D002793:Cholic Acids; D003486:Cyanides; D009840:Oligomycins; D013439:Sulfhydryl Reagents; D002034:Bumetanide; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D013656:Taurocholic Acid; D000968:Antimycin A; D019826:Cholic Acid