THE EFFECT OF BOVINE MILK EXOSOMES ON THE SODIUM POTASSIUM ATPASE OF HUMAN CACO-2 CELLS

Abstract

Given the species-specific formulation of milk, it is no surprise to find the increase in bovine dairy consumption by humans implicated in the development of several modern diseases of civilization. However, the milk constituents involved in chronic diseases have not been entirely identified. Within the last decade, increasing evidence is shedding light on a potential causative agent: exosomes. Exosomes are the smallest category of extracellular vesicles secreted by cells into body fluids like milk. They transport intact bioactive components to distant cells within an organism as well as to external recipients, including the mother’s own offspring and human consumers. The recent identification of enclosed and displayed inflammatory cytokines implicates exosomes in inflammatory chronic diseases, like inflammatory bowel disease (IBD). One of the major targeted proteins in this condition is the sodium potassium ATPase, which is responsible for actively pumping 3 sodium ions to the outside and 2 potassium ions to the inside of the intestinal cells producing thus a sodium gradient used to drive secondary active transport processes and water movements by osmosis. The ATPase maintains consequently water homeostasis, and perturbations in its activity may result in diarrhea and/or constipation which are typical of IBD. Many cytokines are elevated in patients with IBD, such as Tumor Necrosis Factor alpha (TNFα), Interleukinβ and Transforming growth factor β (TGFβ), some of which have been identified on exosomes. These cytokines have also been shown to endogenously affect the pump. This work was conducted to investigate if bovine milk due to exosomal inflammatory cargo, may alter the activity of the pump in in the colon, using Caco-2 cells as a model. Exosomes were extracted from fat free milk by a series of ultra-centrifugation steps and filtration. Cells were treated with different concentrations of bovine exosome proteins and for different time intervals, and the activity of the pump was assayed by measuring the amount of inorganic phosphate liberated in the presence and absence of ouabain. A time response study revealed, at a dose of 550 µg exosome proteins, stimulation of the ATPase up to 6 hours with a maximal effect at one hour, followed by inhibition at later time points. Both stimulation and inhibition were found to be dose dependent. This work was restricted to the study of the stimulatory effect induced at one hour, at a chosen exosome concentration of 225 µg. Because PGE2 was previously demonstrated to exert a similar dual and opposite effect on the pump, it was considered as a potential effector. The suspected involvement of the prostaglandin in the action of exosomes was confirmed when their effect was abrogated by indomethacin and imitated by exogenous PGE2. The exosomes’ induced stimulation still appeared in presence of a blocker of EP1 and EP2, was enhanced further when EP4 was blocked, and changed to an inhibition in presence of an EP3 antagonist, suggesting an involvement of both EP3 and EP4. The activation of the Na+/K+ ATPase was significantly enhanced or reduced when cells were treated respectively with an inhibitor or an activator of PKA. Since EP4 and EP3 act via Gs and Gi respectively, they exert opposite effects on PKA and consequently on the ATPase. The stimulatory effect of EP3 on the ATPase predominates over that of EP4 resulting in a net activation of the pump. To our knowledge this study highlights for the first time a molecular mechanism potentially linking dairy consumption to the Na+/K+ ATPase activity and may be chronic diseases like IBD.