Uptake and glutathione conjugation of ethacrynic acid and efflux of the glutathione adduct by periportal and perivenous rat hepatocytes

We assessed the impact of zonal factors on the hepatic reduced glutathione (GSH) conjugation of ethacrynic acid (EA). Uptake of EA by enriched peripor tal (PP) and perivenous (PV) rat hepatocytes was characterized by both satu rable (V-max(uptake) = 3.4 +/- 1.7 and 3.2 +/- 0.8 nmol/min/mg protein and K-m(uptake) = 51 +/- 13 and 44 +/- 15 mu M) and nonsaturable (12 +/- 5 and 12 +/- 3 mu l/min/mg protein) components. Values for the overall GSH conjug ation rates of EA (200 mu M) were similar among the zonal hepatocytes and r esembled those for the influx transport rates. In the absence of the hepato cyte 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 p erfusion technique. The GSH conjugation rates in the subcellular fragments were, however, much greater than those observed for intact hepatocytes. Eff lux rates of the glutathione conjugate EA-SG from zonal hepatocytes were si milar, as were levels of the immunodetectable multidrug-resistance protein 2/canalicular multispecific organic anion transporter (Mrp2/cMoat) in the 1 00,000g pellets. The composite results suggest that the GSTs responsible fo r EA metabolism are more abundant in the PV region, albeit that the gradien t of enzymatic activities is shallow. Despite the existence of zonal metabo lic activity, the overall GSH conjugation rate of EA is homogeneous among c ells because the reaction is rate limited by uptake, which occurs evenly. R esults on EA-SG efflux suggest the acinar homogeneity in Mrp2/cMoat functio n for canalicular transport.