Transport of N1-methylnicotinamide by organic cation-proton exchange in rat liver membrane vesicles

Abstract

The characteristics of hepatic organic cation transport were examined in basolateral (blLPM) and canalicular (cLPM) rat liver plasma membrane vesicles, using the naturally occurring organic cation, N1-methylnicotinamide (NMN). In blLPM vesicles, an outwardly directed H+ gradient (pHin 5.9/pHout 7.9) stimulated [3H]NMN uptake compared with [3H]NMN uptake under pH-equilibrated conditions. The time course of [3H]NMN uptake exhibited a transient "over-shoot" phenomenon, consistent with active transport. The proton ionophore, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, had no effect on [3H]NMN uptake, demonstrating that pH-dependent [3H]NMN uptake was not the result of a H+ diffusion potential. An outwardly directed H+ gradient also stimulated [3H]NMN uptake under voltage-clamped conditions, consistent with electroneutral NMN-H+ exchange. Under conditions that effectively dissipated the H+ gradient, no active transport of [3H]NMN was observed. In the absence of a pH gradient, the intravesicular presence of NMN trans-stimulated the uptake of [3H]NMN. NMN-H+ exchange was differentiated from sinusoidal Na(+)-H+ exchange by determining sensitivity to amiloride. The substrate specificity of NMN-H+ exchange in blLPM vesicles was examined by determining the cis-inhibitory effects of typical endogenous and exogenous substrates of other epithelial organic cation-H+ exchangers. Kinetic analysis of initial rates of carrier-mediated [3H]NMN uptake over a NMN concentration range of 0.05-15 mM demonstrated that uptake occurred via two saturable transport systems, one a high-affinity low-capacity process and the other a low-affinity high-capacity type. In contrast, in cLPM vesicles, no pH gradient-dependent [3H]NMN uptake was demonstrated. These findings are consistent with the presence of an organic cation-H+ antiport on the sinusoidal membrane, with features distinct from the renal antiport, such as substrate specificity and membrane localization, that may account for differences in drug disposition by these two organs.