Radiation inactivation studies of hepatic sinusoidal reduced glutathione transport system

AbstractSinusoidal transport of reduced glutathione (GSH) is a carrier-mediated process. Perfused liver and isolated hepatocyte models revealed a low-affinity transporter with sigmoidal kinetics (Km∼3.2–12 mM), while studies with sinusoidal membrane vesicles (SMV) revealed a high-affinity unit (Km∼0.34 mM) besides a low-affinity one (Km∼3.5–7 mM). However, in SMV, both the high- and low-affinity units manifested Michaelis–Menten kinetics of GSH transport. We have now established the sigmoidicity of the low-affinity unit (Km∼9) in SMV, consistent with other models, while the high-affinity unit has been retained intact with Michaelis–Menten kinetics (Km∼0.13 mM). We capitalized on the negligible cross-contributions of the two units to total transport at the low and high ends of GSH concentrations and investigated their characteristics separately, using radiation inactivation, as we did in canalicular GSH transport (Am. J. Physiol. 274 (1998) G923–G930). We studied the functional sizes of the proteins that mediate high- and low-affinity GSH transport in SMV by inactivation of transport at low (trace and 0.02 mM) and high (25 and 50 mM) concentrations of GSH. The low-affinity unit in SMV was much less affected by radiation than in canalicular membrane vesicles (CMV). The target size of the low-affinity sinusoidal GSH transporter appeared to be considerably smaller than both the canalicular low- and high-affinity transporters. The high-affinity unit in SMV was markedly inactivated upon irradiation, revealing a single protein structure with a functional size of ∼70 kDa. This size is indistinguishable from that of the high-affinity GSH transporter in CMV reported earlier

Novel properties of hepatic canalicular reduced glutathione transport revealed by radiation inactivation

Transport of GSH at the canalicular pole of hepatocytes occurs by a facilitative carrier and can account for ∼50% of total hepatocyte GSH efflux. A low-affinity unit with sigmoidal kinetics accounts for 90% of canalicular transport at physiological GSH concentrations. A low-capacity transporter with high affinity for GSH has also been reported. It is not known whether the same or different proteins mediate low- and high-affinity GSH transport, although they do differ in inhibitor specificity. The bile of rats with a mutation in the canalicular multispecific organic anion transporter (cMOAT or MRP-2, a 170-kDa protein) is deficient in GSH, implying that cMOAT may transport GSH. However, transport of GSH in canalicular membrane vesicles (CMV) from these mutant rats remains intact. We examined the functional size of the two kinetic components of GSH transport by radiation inactivation of GSH uptake in rat hepatic CMV. High-affinity transport of GSH was inactivated as a single exponential function of radiation dose, yielding a functional size of ∼70 kDa. In contrast, low-affinity canalicular GSH transport exhibited a complex biexponential response to irradiation, characterized by an initial increase followed by a decrease in GSH transport. Inactivation analysis yielded a ∼76-kDa size for the low-affinity transporter. The complex inactivation indicated that the low-affinity transporter is associated with a larger protein of ∼141 kDa, which masked ∼80% of the potential transport activity in CMV. Additional studies, using inactivation of leukotriene C4transport, yielded a functional size of ∼302 kDa for cMOAT, indicating that it functions as a dimer.</jats:p

Pharmacokinetics, distribution, metabolism, and excretion of the dual reuptake inhibitor [¹⁴C]-nefopam in rats

1. This study examined the pharmacokinetics, distribution, metabolism, and excretion of [14C] nefopam in rats after a single oral administration. Blood, plasma, and excreta were analyzed for total radioactivity, nefopam, and metabolites. Metabolites were profiled and identified. Radioactivity distribution was determined by quantitative whole-body autoradiography. 2. The pharmacokinetic profiles of total radioactivity and nefopam were similar in male and female rats. Radioactivity partitioned approximately equally between plasma and red blood cells. A majority of the radioactivity was excreted in urine within 24 hours and mass balance was achieved within 7 days. 3. Intact nefopam was a minor component in plasma and excreta. Numerous metabolites were identified in plasma and urine generated by multiple pathways including: hydroxylation/oxidation metabolites (M11, M22a and M22b, M16, M20), some of which were further glucuronidated (M6a to M6c, M7a to M7c, M8a and M8b, M3a to M3d); N-demethylation of nefopam to metabolite M21, which additionally undergoes single or multiple hydroxylations or sulfation (M9, M14, M23), with some of the hydroxylated metabolites further glucuronidated (M2a to M2d). 4. Total radioactivity rapidly distributed with highest concentrations found in the urinary bladder, stomach, liver, kidney medulla, small intestine, uveal tract, and kidney cortex without significant accumulation or persistence. Radioactivity reversibly associated with melanin-containing tissues.</p