Uptake of sulfate conjugates by isolated rat hepatocytes.
The uptake of estrone sulfate (E1S; 1 to 400 microM), harmol sulfate (HS; 5 to 900 microM), and 4-methylumbelliferyl sulfate (4MUS; 5 to 1000 microM) was investigated in isolated rat hepatocytes in the presence or absence of inhibitors. Uptake of all of the sulfate conjugates was rapid and exhibited saturation kinetics, best characterized by saturable and nonsaturable (linear transmembrane clearance) transport systems. The KM's were: 16 +/- 6, 123 +/- 28, and 64 +/- 6 microM for E1S, HS, and 4MUS, respectively, with corresponding Vmax's of 0.85 +/- 0.56, 0.48 +/- 14, and 0.42 +/- 0.07 nmol/min/10(6) cells. The nonsaturable uptake clearances, which displayed concentration-independent uptake, were 3 +/- 2, 1 +/- 0.1, 0.5 +/- 0.1 microliter/min/10(6) cells, respectively. Uptake of E1S was inhibited by ouabain (1 mM) and replacement of sodium by choline, whereas HS was insensitive to the addition or substitution. Uptake of both E1S and HS was significantly reduced by metabolic inhibitors (antimycin A, 2.7 microM, rotenone, 30 microM, and KCN, 2 mM) and temperature reduction (from 37 to 27 degrees C). 4,4'-Diisothiocyanostilbene-2-2-'disulfonic acid (2 mM), an inhibitor of anion transport, reduced E1S and HS uptake; E1S uptake was also reduced by HS. HS uptake by both saturable and nonsaturable transport components was depressed by 4MUS (300 microM); the apparent KM was increased by 83% while the Vmax remained unaltered, and the nonsaturable component was decreased by 48%. The data strongly suggest that multiple pathways exist for the uptake of E1S, HS, and 4MUS. E1S uptake is sodium-dependent, requires energy, and is inhibited by anions such as 4,4'diisothiocyanostilbene-2-2'-disulfonic acid and other sulfate conjugates. HS uptake, while being energy dependent, is not sodium dependent, and is inhibited by 4MUS in a competitive fashion. At least one of these pathways is shared.
Hassen
A M
AM
Faculty of Pharmacy, University of Toronto, Ontario, Canada.
Lam
D
D
Chiba
M
M
Tan
E
E
Geng
W
W
Pang
K S
KS
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism; D002794:Choline / Q000494:pharmacology; D004966:Estrogens, Conjugated (USP) / Q000493:pharmacokinetics*; D004970:Estrone / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D006247:Harmine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D066298:In Vitro Techniques; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D010042:Ouabain / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley
D002352:Carrier Proteins; D004966:Estrogens, Conjugated (USP); C019492:4-methylumbelliferyl sulfate; C104826:harmol sulfate; D004970:Estrone; D006923:Hymecromone; D006247:Harmine; D010042:Ouabain; D002794:Choline; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; C017296:estrone sulfate