Endocytosis of gentamicin in a proximal tubular renal cell line.

The mechanisms by which aminoglycosides are accumulated in renal proximal t ubular cells remain unclear. Adsorptive mediated endocytosis, via a common pathway for cationic proteins, or receptor endocytosis, mediated by the gly coprotein 330/ megalin, have been proposed to be involved in gentamicin tra nsport in renal cells. We used the LLC-PK1 cell line, derived from the pig proximal tubule, to explore further the regulation of gentamicin endocytosi s in these cells and to determine the role of clathrin mediated endocytosis and G proteins in this function. Gentamicin endocytosis was strictly tempe rature dependent, whereas total uptake (endocytosis plus binding) did not s ignificantly differ at 4 or 37 degrees C. Substances that suppress receptor mediated, clathrin dependent endocytosis, such as monensin, phenylarsine o xide and dansylcadaverine, or inhibit caveolae mediated endocytosis, such a s nystatin, did not affect gentamicin entrance in LLC-PK1 cells. Among subs tances that disrupt the actin cytoskeleton, only cytochalasin D, that is ac tive also on fluid phase endocytosis, significantly reduced the intracellul ar concentrations of the aminoglycoside. Other maneuvers that perturb clath rin dependent endocytosis without affecting clathrin independent pathway, s uch as acidification of cytosol or incubation in hypertonic medium, were al so without effect. Mastoparan, a well known stimulator of heterotrimeric G proteins, strongly increased endocytosis of gentamicin, and the same effect was evident with two other G protein stimulators, aluminum fluoride and fl uoride alone; however the effect seems not to be mediated by an activation of adenylyl cyclase. In conclusion, gentamicin endocytosis in LLC-PK1 cells is probably clathrin independent, limited by cytochalasin D, which interac ts with cytoskeleton, and increased by substances like mastoparan and alumi num fluoride, which activate heterotrimeric G proteins.