Dmd060509 147..153

This study characterized the kinetics, variability, and factors that affect UDP–glucuronic acid (UDP-GlcUA) uptake by human liver microsomes (HLM). Biphasic kinetics were observed for UDP-GlcUA uptake by HLM. Uptake affinities (assessed as Kd) of the highand low-affinity components differed by more than an order of magnitude (13 6 6 vs. 374 6 175 mM), but were comparable in terms of the maximal rate of uptake, with mean Vmax values differing less than 2.3-fold (56 6 26 vs. 131 6 35 pmol/min per mg). Variability in total intrinsic transporter activity (Uint) for microsomal UDP-GlcUA uptake across 12 livers was less than 4-fold. Experiments performed to optimize the conditions for microsomal UDP-GlcUA uptake demonstrated that both components were trans-stimulated by preloading (luminal addition) with an alternate UDP-sugar, and essentially abolished by the thiol-alkylating agent N-ethylmaleimide. Furthermore, interaction studies undertaken with a panel of drugs, alternate UDP-sugars, and glucuronide conjugates, at low (2.5 mM) and high (1000 mM) UDP-GlcUA concentrations, demonstrated that both components were inhibited to varying extents. Notably, the nucleoside analogs zidovudine, stavudine, lamivudine, and acyclovir inhibited both the highand lowaffinity components of microsomal UDP-GlcUA uptake by >45% at an inhibitor concentration of 100 mM. Taken together, these data demonstrate that human liver microsomal UDP-GlcUA uptake involves multiple protein-mediated components, and raises the possibility of impaired in vivo glucuronidation activity resulting from inhibition of UDP-GlcUA uptake into the endoplasmic reticulum membrane by drugs and other compounds.