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

Abstract

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.