The characterization of butyrate transport across pig and human colonic luminal membrane

Corresponding author S. P. Shirazi-Beechey: Epithelial Function and Development Group, Department of Veterinary PreClinical Sciences, The University of Liverpool, Brownlow Hill and Crown Street, Liverpool L69 3BX, UK. Email: spsb@liverpool.ac.uk

Abstract

1Luminal membrane vesicles (LMV) were isolated from human and pig colonic tissues. They were characterized in terms of purity and ability to transport [¹⁴C]butyrate.
2The activity of cysteine-sensitive alkaline phosphatase, and the abundance of villin, NHE2 and NHE3 proteins, markers of the colonic luminal membrane, were significantly enriched in the LMV compared with the original cellular homogenate. The LMV were free from contamination by other cellular organelles and basolateral membranes, as revealed by the negligible presence of either specific marker enzyme activity or characteristic immunogenic protein.
3The transport of butyrate into the luminal membrane vesicles was enhanced 5-fold at pH 5.5 compared with pH 8.0. Butyrate transport was temperature dependent, and was stimulated in the presence of an outward-directed anion gradient in the order of butyrate > bicarbonate > propionate > chloride. Kinetic analysis of increasing substrate concentration showed saturation kinetics with an apparent K_m value of 14.8 ± 3.6 mM and a V_max of 54 ± 14 nmol min⁻¹ (mg protein)⁻¹.
4Butyrate transport was significantly reduced in the presence of short chain fatty acids (SCFA), acetate, propionate and other monocarboxylates (pyruvate and L-lactate). Butyrate uptake was inhibited by several cysteine group modifying reagents such as p-chloromercuribenzosulphonic acid (pCMBS), p-chloromercuribenzoate (pCMB), mersalyl acid and HgCl₂, but not by the stilbene anion exchange inhibitors, 4,4′-diisothiocyanostilbene-2,2′-disulphonate (DIDS) and 4,4′-dinitrostilbene-2,2′-disulphonate (SITS).
5The described properties of butyrate transport across the luminal pole of the colon suggest the involvement of a carrier protein, in the form of a pH-activated anion exchange process. The transporter is distinct from the erythrocyte band-3 type anion exchanger and may belong to the monocarboxylate-type transport proteins (MCT1).

Short chain fatty acids (SCFA; acetate, propionate and butyrate) are the major anions in the colonic lumen. They are produced as a result of the fermentation of dietary fibre by microflora in the lumen of the large intestine (Cummings, 1984) and are rapidly absorbed (Ruppin, Bar-Meir, Soergel, Wood & Schmitt, 1980; Hatch, 1987; Bergman, 1990). Normal colonic epithelia derive 60–70 % of their energy supply from SCFA, particularly butyrate (Scheppach et al. 1992). Butyrate induces cell differentiation, and regulates the growth and proliferation of normal colonic mucosa (Treem, Ahsan, Shoup & Hyams, 1994), and it reduces the growth rate of colorectal cancer cells in culture (Berry & Paraskeva, 1988). Despite the important role of SCFA in the maintenance of colonic health, the detailed molecular mechanism(s) by which SCFA interact with the colonic mucosa is not known. A number of studies have been performed in recent years aimed at identifying the mechanism of SCFA absorption across the colonic luminal membranes using either: (a) isolated membrane vesicles (Stein, Schröder, Milovic & Caspary, 1995; Harig, Ng, Dudeja, Brasitus & Ramaswamy, 1996) or (b) flux studies with whole epithelial preparations (Holtug, Rasmussen & Mortensen, 1992; Engelhardt, Gros, Burmester, Hansen, Becker & Rechkemmer, 1994) or (c) in vivo perfusion experiments (McNeil, Cummings & James, 1978; Holtug, Hove & Mortensen, 1995). Whilst the existence of a specific transport system has been implicated, there is no common consent regarding the mechanism(s) involved in the movement of butyrate across the colonic luminal membrane. Some workers have proposed an anion exchange process (Mascolo, Rajendran & Binder, 1991; Engelhardt et al. 1994), whilst others have suggested the absorption of the protonated acid across the intact epithelia as mechanisms for butyrate transport (Engelhardt, Burmester, Hansen, Becker & Rechkemmer, 1993; Sellin, DeSoignie & Burlingame, 1993).In this paper we describe the properties of butyrate transport in purified and well characterized human and pig colonic luminal membrane vesicles (LMV). Membrane vesicles are particularly suitable for this study where the substrate, i.e. SCFA is rapidly metabolized by the colonic tissue. We report that the transport of butyrate across the luminal pole of human and pig colon is via a pH-activated anion exchange process. The elucidation of the mechanism of SCFA (butyrate) transport in the colon, and the information on the molecular structure of this transporter would facilitate the identification of the molecular and cellular mechanisms by which butyrate interacts with the colonic epithelia in healthy and diseased tissue.

http://onlinelibrary.wiley.com/doi/10.1111/j.1469-7793.1998.819bs.x/full