Differential incorporation of docosahexaenoic acid into distinct cholesterol-rich membrane raft domains

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Abstract

We investigated the influence of docosahexaenoic acid (DHA) on the fatty acid and protein compositions of two populations of membrane rafts present in Caco-2 cells. DHA (100 μM) had no significant influence on the fatty acid or protein compositions of tight junction-associated, Lubrol insoluble, membrane rafts. However, DHA did significantly alter the fatty acid and protein compositions of “archetypal” Triton X-100 insoluble membrane rafts. The DHA content of the raft lipids increased 25-fold and was accompanied by a redistribution of src and fyn out of the rafts. DHA also increased Caco-2 cell monolayer permeability producing a 95% drop in transepithelial electrical resistance and a 8.56-fold increase in the flux of dextran. In conclusion, the data demonstrate that DHA does not increase permeability through modifying the TJ-associated rafts. The data do, however, show that DHA is differentially incorporated into different classes of membrane rafts, which has significant implications to our understanding of how omega-3 PUFAs modulate plasma membrane organization and cell function.

Section snippets

Materials and methods

Materials. Tissue culture media and supplements were purchased from Invitrogen (Paisley, UK). All protease inhibitors were obtained from Calbiochem (Nottingham, UK) and the Lubrol WX was from Serva (Oxford, UK). The claudin and occludin antibodies were purchased from Zymed (San Francisco, USA) and the src antibody from Upstate (Chandler’s Ford, UK) whereas the horseradish peroxidase conjugated secondary antibodies were from Bio-Rad (Hemel Hempstead, UK). All other reagents were obtained from

DHA is incorporated into Caco-2 cell membranes

To establish that feeding the Caco-2 cell monolayers DHA did result in the incorporation of the PUFA into cell membrane lipids, the fatty acid composition of total cell membranes was determined 4 and 8 days after the introduction of DHA (100 μM) (Table 1). DHA produced significant alterations in the fatty acid profile of the cell membranes. By day 8, the amount of DHA present in the membrane lipids had increased nearly tenfold and was accompanied by a compensatory decrease in mainly oleic acid

Discussion

One of the primary ways omega-3 PUFAs, such as EPA and DHA, influence cell function is through modulating the lipid and protein composition, and therefore the function, of membrane rafts. This phenomenon and has been documented in a number of tissues, including the intestine, [4], [5], [14], [15], [16], [17]. EPA and DHA both increase the permeability of Caco-2 cell monolayers [7], [8] by “loosening” the epithelial TJs. Currently the exact mechanism whereby omega-3s influence TJ integrity is

Acknowledgments

This work was supported by a project grant awarded by the Biotechnology and Biological Sciences Research Council, UK.

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