GENTAMICIN UPTAKE BY LLCPK1 CELLS - EFFECT OF INTRACELLULAR AND EXTRACELLULAR PH CHANGES

The mechanisms by which aminoglycosides are transported across the lum inal membrane of renal proximal tubular cells remain unclear. A lumina l organic cation/H+ exchange as well as an adsorptive endocytosis memb rane process has been proposed to be involved in gentamicin renal accu mulation. The objectives of this work were to explore further the effe cts of intracellular and extracellular pH changes on gentamicin uptake . [H-3]Gentamicin uptake by a continuous renal epithelial cell line, L LCPK1, grown as a monolayer on an impermeable surface was measured at different temperatures and pH conditions and in the presence of variou s inhibitors. Uptake of gentamicin was found to be carrier mediated (K -m = 1.26 +/- 0.22 mM, V-max = 289 +/- 27 pmol.mg(-1).min(-1)), energy dependent (inhibited in part by sodium azide), and temperature depend ent (37 degrees C > 4 degrees C). Fifteen-minute gentamicin (10 mu M) uptake was inhibited by 1 mM of the organic cations cimetidine (61.0%) , quinidine (73.5%), quinine (68.6%), and verapamil (61.5%). More impo rtantly, while an outwardly directed proton gradient did not have a si gnificant effect on gentamicin uptake, extracellular acidification (pH 6.5), which leads to a higher degree of gentamicin ionization, signif icantly enhanced gentamicin uptake by LLCPK1 monolayer cells. These re sults suggest that the luminal organic cation/H+ exchanger is not invo lved in gentamicin uptake by renal cultured epithelial cells. Rather, the cationic charge of gentamicin appears to be one of the primary det erminants for renal luminal uptake.