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

The hepatopancreas of the American lobster (Homarus americanus) possesses f our types of epithelial cells arranged along blind-ended tubules, At the di stal tips of these tubules, stem cells termed E-cells differentiate into th ree other cell types, a-cells, F-cells and B-cells, each of which have diff erent absorptive and secretory roles in the biology of the overall organ, T his investigation uses centrifugal elutriation to separate the individual h epatopancreatic epithelial cell types of Homarus americanus and to investig ate their plasma membrane copper transport properties using the copper-sens itive fluorescent dye Phen Green, Results show highly dissimilar endogenous concentrations of copper in each cell type and within the vacuoles (vesicl es) released from these cells during the centrifugation process ([copper] i n vacuoles >E-cells >R-cells >F-cells approximate toB-cells). All four cell types were able to absorb copper from external concentrations ranging from 0.01 to 8 mu mol l(-1), but considerable differences in transport rates oc curred between the cell types, External calcium (0-10 mmol l(-1)) stimulate d the uptake of external copper in a saturable fashion, suggesting the occu rrence of carrier-mediated metal uptake. Addition of the Ca2+ channel block er verapamil (30 mu mol l(-1)) to the external medium reduced the uptake ra te 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 c opper influx in E- and R-cells, suggesting that the two metals shared the s ame binding and transport mechanism. A model is proposed which suggests tha t copper mag enter all hepatopancreatic epithelial cell types by a divalent cation antiport process that exchanges intracellular Ca2+ (or other cation s) with either external copper or zinc. Verapamil-sensitive Ca2+ channels m ag allow access of external calcium to cytoplasmic exchange sites on the an tiporter or to activator sites on the same transport protein. The results s uggest that elutriation is an excellent technique for the separation of com plex invertebrate organ systems into their separate cell types and for anal yzing the physiological properties of each cell type in isolation.