Abstract
We assessed the impact of zonal factors on the hepatic reduced glutathione (GSH) conjugation of ethacrynic acid (EA). Uptake of EA by enriched periportal (PP) and perivenous (PV) rat hepatocytes was characterized by both saturable (Vmaxuptake = 3.4 ± 1.7 and 3.2 ± 0.8 nmol/min/mg protein andKmuptake = 51 ± 13 and 44 ± 15 μM) and nonsaturable (12 ± 5 and 12 ± 3 μl/min/mg protein) components. Values for the overall GSH conjugation rates of EA (200 μM) were similar among the zonal hepatocytes and resembled those for the influx transport rates. In the absence of the hepatocyte membrane, GSH conjugation in PV and PP hepatocyte cytosol was similar, but a higher perivenous GSH conjugation activity toward EA (PV/PP of 2.4) that mirrored the higher PV/PP ratios of immunodetectable GSTs Ya (1.7) and Yb2 (2.5) was found in cell lysates obtained by the dual-digitonin-pulse perfusion technique. The GSH conjugation rates in the subcellular fragments were, however, much greater than those observed for intact hepatocytes. Efflux rates of the glutathione conjugate EA-SG from zonal hepatocytes were similar, as were levels of the immunodetectable multidrug-resistance protein 2/canalicular multispecific organic anion transporter (Mrp2/cMoat) in the 100,000 g pellets. The composite results suggest that the GSTs responsible for EA metabolism are more abundant in the PV region, albeit that the gradient of enzymatic activities is shallow. Despite the existence of zonal metabolic activity, the overall GSH conjugation rate of EA is homogeneous among cells because the reaction is rate limited by uptake, which occurs evenly. Results on EA-SG efflux suggest the acinar homogeneity in Mrp2/cMoat function for canalicular transport.
Footnotes
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Send reprint requests to: Dr. K. Sandy Pang, Faculty of Pharmacy, University of Toronto, 19 Russell St., Toronto, Ontario, Canada M5S 2S2. E-mail: pang{at}phm.utoronto.ca
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↵1 This work was supported by the Medical Research Council of Canada (Grants MA9104 and MT15657). R.G.T. was supported by fellowships from Merck-Frosst Canada, the Pharmaceutical Manufacturers Association of Canada–Health Research Foundation and MRC, and the University of Toronto Open Fellowship. This work was presented in part at the Annual Meeting of the American Association for the Study of Liver Diseases, 1998, Chicago, IL.
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Received for publication June 11, 1999.
- Abbreviations:
- GSH
- reduced glutathione
- GSSG
- oxidized glutathione
- EA
- ethacrynic acid
- EA-SG
- ethacrynic acid-glutathione conjugate
- CDNB
- 1-chloro-2,4-dinitrobenzene
- PP
- periportal
- PV
- perivenous
- ALT
- alanine aminotransferase
- GS
- glutamine synthetase
- CYP
- cytochrome P-450
- Mrp2
- rat multidrug resistance protein 2
- MRP2
- human multidrug resistance protein 2
- cMoat
- rat canalicular multispecific organic anion transporter
- GST
- glutathioneS-transferase
- Accepted September 2, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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