Copper transport by lobster hepatopancreatic epithelial cells separated by centrifugal elutriation: measurements with the fluorescent dye Phen Green

Abstract

The hepatopancreas of the American lobster (Homarus americanus) possesses four types of epithelial cells arranged along blind-ended tubules. At the distal tips of these tubules, stem cells termed E-cells differentiate into three other cell types, R-cells, F-cells and B-cells, each of which have different absorptive and secretory roles in the biology of the overall organ. This investigation uses centrifugal elutriation to separate the individual hepatopancreatic epithelial cell types of Homarus americanus and to investigate their plasma membrane copper transport properties using the copper-sensitive fluorescent dye Phen Green. Results show highly dissimilar endogenous concentrations of copper in each cell type and within the vacuoles (vesicles) released from these cells during the centrifugation process ([copper] in vacuoles>E-cells>R-cells>F-cells approximately B-cells). All four cell types were able to absorb copper from external concentrations ranging from 0.01 to 8 micromol l(−1), but considerable differences in transport rates occurred between the cell types. External calcium (0--10 mmol l(−1)) stimulated the uptake of external copper in a saturable fashion, suggesting the occurrence of carrier-mediated metal uptake. Addition of the Ca(2+) channel blocker verapamil (30 micromol l(−1)) to the external medium reduced the uptake rate of copper by all four cell types, but to different extents in each type of cell. External zinc (0--1000 nmol l(−1)) was a competitive inhibitor of copper influx in E- and R-cells, suggesting that the two metals shared the same binding and transport mechanism. A model is proposed which suggests that copper may enter all hepatopancreatic epithelial cell types by a divalent cation antiport process that exchanges intracellular Ca(2+) (or other cations) with either external copper or zinc. Verapamil-sensitive Ca(2+) channels may allow access of external calcium to cytoplasmic exchange sites on the antiporter or to activator sites on the same transport protein. The results suggest that elutriation is an excellent technique for the separation of complex invertebrate organ systems into their separate cell types and for analyzing the physiological properties of each cell type in isolation.