Cell-based homologous expression system for in-vitro characterization of environmental effects on transmembrane peptide transport in fish.

ABSTRACT

All organisms encounter environmental changes that lead to physiological adjustments that could drive evolutionary adaptations. The ability to adjust performance in order to cope with environmental changes depends on the organism's physiological plasticity. These adjustments can be reflected in behavioral, physiological, and molecular changes, which interact and affect each other. Deciphering the role of molecular adjustments in physiological changes will help to understand how multiple levels of biological organization are synchronized during adaptations. Transmembrane transporters, which facilitate a cell's interaction with its surroundings, are prime targets for molecular studies of the environmental effects on an organism's physiology. Fish are subjected to environmental fluctuations and exhibit different coping mechanisms. To study the molecular adjustments of fish transporters to their external surrounding, suitable experimental systems must be established. The Mozambique tilapia (Oreochromis mossambicus) is an excellent model for environmental stress studies, due to its extreme salinity tolerance. We established a homologous cellular-based expression system and uptake assay that allowed us to study the effects of environmental conditions on transmembrane transport. We applied our expression system to investigate the effects of environmental conditions on the activity of PepT2, a transmembrane transporter critical in the absorption of dietary peptides and drugs. We created a stable, modified fish cell-line, in which we exogenously expressed the tilapia PepT2, and tested the effects of water temperature and salinity on the uptake of a fluorescent di-peptide, ß-Ala-Lys-AMCA. While temperature affected only Vmax, medium salinity had a bi-directional effect, with significantly reduced Vmax in hyposaline conditions and significantly increased Km in hypersaline conditions. These assays demonstrate the importance of suitable experimental systems for fish ecophysiology studies. Furthermore, our in-vitro results show how the effect of hypersaline conditions on the transporter activity can explain expression shifts seen in the intestine of saltwater-acclimated fish, emphasizing the importance of complimentary studies in better understanding environmental physiology. This research highlights the advantages of using homologous expression systems to study environmental effects encountered by fish, in a relevant cellular context. The presented tools and methods can be adapted to study other transporters in-vitro.