Dual control of cytosolic metals by lysosomal transporters in lobster hepatopancreas

Abstract

This study describes the membrane transport mechanisms used by lobster (Homarus americanus) hepatopancreatic epithelial lysosomes to accumulate and sequester heavy metals from the cytosol, and thereby aid in the regulation of these ions entering the animal from dietary constituents. The present investigation extends previous work describing lysosomal metal uptake by cation exchange with protons and suggests that a second, parallel, lysosomal transport process involving metal-thiol conjugates may work in conjunction with the cation antiporter to control cytoplasmic metal concentrations. Transport of (65)Zn(2+) by lysosomal membrane vesicles (LMV) incubated in 1 mmol l(-1) glutathione (GSH) was not significantly different from metal transport in the absence of the tripeptide. However, preloading LMV with 1 mmol l(-1) alpha-ketoglutarate (AKG), and then incubating in a medium containing 1 mmol l(-1) GSH, more than doubled metal uptake, compared with vesicles equilibrated with chloride or possessing an outwardly directed chloride gradient. Kinetic analysis of lysosomal (65)Zn(2+) influx as a function of zinc concentration, in vesicles containing 1 mmol l(-1) AKG and incubated in 1 mmol l(-1) GSH, revealed the presence of a sigmoidal, low affinity, high capacity carrier process transporting the metal into the organelle. These data indicated the possible presence of an organic anion exchanger in lobster lysosomal membranes. Western blot analysis of LMV with a rabbit anti-rat OAT1 antibody showed the presence of an orthologous OAT1-like protein (approximate molecular mass of 80 kDa) signal from these membranes. These results, and those published previously, suggest the occurrence of two metal transporters on hepatopancreatic membranes, a high affinity, low capacity cation antiporter and a low affinity, high capacity organic anion exchanger. Together these two systems have the potential to regulate cytoplasmic metals over a wide concentration range.