Cellular localization of calcium, heavy metals, and metallothionein in lobster (Homarus americanus) hepatopancreas.

This investigation combines confocal microscopy with the cation-specific fluorescent dyes Fluo-3 and BTC-5N to localize calcium and heavy metals along the length of intact lobster (Homarus americanus) hepatopancreatic tubules and isolated cells. A metallothionein-specific antibody, developed in mollusks with cross-reactivity in crustaceans, showed the tissue-specific occurrence of this metal-binding protein in several organ systems in lobster and in single cell types isolated from lobster hepatopancreas. Individual lobster hepatopancreatic epithelial cell types were separated into pure single cell type suspensions for confocal and antibody experiments. Intact hepatopancreatic tubules showed high concentrations of both calcium and heavy metals at the distal tips of tubules where mitotic stem cells (E-cells) are localized. In addition, a concentrated distribution of calcium signal within isolated single premolt E-cells in solution was disclosed that might suggest an endoplasmic reticulum compartmentation of this cation within these stem cells. Both E- and R-cells showed significantly (P < 0.05) greater intracellular calcium concentrations in premolt than intermolt, suggesting the accumulation of this cation in these cells prior to the molt. Antibody studies with lobster tissues indicated that the hepatopancreas possessed 5-10 times the metallothionein concentration as other lobster organ systems and that isolated E-cells from the hepatopancreas displayed more than twice the binding protein concentrations of other cells of this organ or those of blood cells. These results suggest that crustacean hepatopancreatic stem cells (E-cells) and R-cells play significant roles in calcium and heavy metal homeostasis in this tissue. Interactions between the four hepatopancreatic cell types in this regulatory activity remain to be elucidated.

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

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.