Proceedings of The Physiological Society
Puerto de la Cruz, Tenerife (2003) J Physiol 548P, P179
Isolation and characterisation of enterocytes from the intestine of gilthead seabream (Sparus aurata): a comparative study of enzymatic activities
R. Dópido, C. Rodríguez, T. Gómez and M. Díaz
Department of Animal Biology, University of La Laguna, Tenerife, Spain
A protocol for the isolation and characterisation of highly pure viable enterocytes from Sparus aurata has been established. These enterocytes, are suitable for further physiological studies to elucidate the mechanisms of absorption and transport of nutrients, water and ions as well as the homeostasis and osmoregulation which take place in the intestinal epithelium. Considering that the physiology of the digestive tract varies along its proximal-distal axis, we have isolated cells from the three different intestinal regions, i.e. anterior intestine (AI), posterior intestine (PI) and the pyloric caeca (PC). Finally, it was intended to make a comparison between the activity of some digestive enzymes in both the isolated cells and the complete intestinal preparations, in order to establish the active role that isolated enterocytes play in these processes.
For cell isolation, the fish were killed in accordance with the requirements of the European Convention for the Care and Use of Laboratory Animals. The intestinal sections were incubated in a hyperosmolar, low sodium, high potassium containing (intracellular-like) solutions, to which EDTA and DTT (dithiothreitol) were added as disjunctive agents (Dópido et al. 2001). Results obtained from trypan blue exclusion, oxygen consumption, cellular ATP content and lactate dehydrogenase liberation were all traduced in a high viability rate of the isolated cells.
Morphological identification of isolated cells by light microscopy and image analysis, after differential staining showed that the high-yield epithelial cell preparations were all composed of more than 98 % enterocytes, which were slightly smaller in size in PI. The rest of cells were large mucous cells present in a lower proportions in the PI.
The digestive enzymatic activities measured in isolated enterocytes and complete intestinal preparations from the three intestinal regions were sucrase (S), maltase (M), phosphatase alkaline (PA), 5
Where applicable, experiments conform with Society ethical requirements