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

Abstract

1. Luminal membrane vesicles (LMV) were isolated from human and pig colonic tissues. They were characterized in terms of purity and ability to transport [14C]butyrate. 2. The 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. 3. The 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 Km value of 14.8 +/- 3.6 mM and a Vmax of 54 +/- 14 nmol min-1 (mg protein)-1. 4. Butyrate 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). 5. The 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).

Figure 1. Immunodetection of villin in pig and human colonic LMV

LMV were dissolved in sample buffer containing SDS. Samples (25 μg of protein each) were seperated by 8% SDS-PAGE and electrotransferred to a nitrocellulose membrane. Immunoblotting was carried out as described in Methods. Lane a, pig colonic homogenate; lane b, pig colonic LMV; lane c, human colonic homogenate; lane d, human colonic LMV.

Figure 2. Immunodetection of NHE2 and NHE3 in pig and human colonic LMV

Colonic LMV and homogenate (25 μg of protein each) were separated by 8% SDS-PAGE and electrotransferred to a nitrocellulose membrane. Immunoblotting was carried out as described in Methods. A, immunodetection of NHE2; B, immunodetection of NHE3. Lane a, pig colonic homogenate; lane b, pig colonic LMV; lane c, human colonic homogenate; lane d, human colonic LMV.

Figure 3. Immunodetection of Na⁺-K⁺-ATPase

Pig and human colonic cellular homogenates, respective LMVs and pig colonic BLMV (control) were treated as described in Figure 1. Lane a, pig colonic cellular homogenate; lane b, pig colonic LMV; lane c, human colonic cellular homogenate; lane d, human colonic LMV; lane e, pig colonic BLMV. In BLMV (lane e), bands corresponding to 95 kDa, the α-subunit and 40 kDa, the β-subunit of Na⁺-K⁺-ATPase (Tarpley et al. 1995) are concentrated compared with the original cellular homogenate (lane a). In LMV, sample levels of Na⁺-K⁺-ATPase are negligible.

Figure 4. Effect of extravesicular medium pH on butyrate uptake into human and pig colonic LMV

The LMV were loaded with the standard loading buffer containing 100 mm mannitol, 100 mm sodium butyrate, 0.1mm MgSO₄ and 20 mm Hepes-Tris (pH 7.5). LMV (100 μg of protein per assay) were incubated in media containing 100 mm mannitol, 100 mm sodium gluconate, 0.1 mm MgSO₄, 1 mm [U-¹⁴C]butyrate and 20 mm Hepes-Tris, for pH 7.0, 7.5 and 8.0 or Mes-Tris for pH 5.5, 6.0 and 6.5. Initial rate of butyrate uptake was measured as described in Methods, at 37 °C for 5 s. Values are presented as the means ± s.e.m. for three experiments.

Figure 5. Time course of butyrate uptake into pig colonic LMV in the presence and absence of an inward-directed pH gradient

LMV were loaded with either 300 mm mannitol (•) or 100 mm mannitol and 100 mm of either sodium butyrate (▴) or NaHCO₃ (▪), 20 mm Hepes-Tris (pH 7.5) and 0.1 MgSO₄. LMV (100 μg protein per assay) were incubated in media containing 100 mm mannitol, 100 mm sodium gluconate, 20 mm Mes-Tris (pH 5.5; A) or 20 mm Hepes-Tris (pH 5.5; B), 0.1 mm MgSO₄ and 1 mm [U-¹⁴C]butyrate. Uptake was measured at 37 °C as described. Values are presented as the means ± s.e.m. for three experiments.

Figure 6. Effect of osmolarity of the external medium on butyrate uptake by pig colonic LMV

LMV were loaded with the standard loading buffer described in Fig. 4. LMV (100 μg protein per assay) were incubated for 30 min at 37 °C in media containing 100 mm sodium gluconate, 20 mm Mes-Tris (pH 5.5), 0.1 mm MgSO₄, 1 mm [U-¹⁴C]butyrate and mannitol in concentrations to give the indicated osmolarity. Values are presented as the means ± s.e.m. for three experiments. Correlation coefficient r² = 0.92.

Figure 7. Effect of increasing butyrate concentrations on butyrate uptake by pig colonic LMV

Initial rates of butyrate transport with increasing butyrate concentrations over the range 0.5 to 50 mm were determined. LMV were loaded with 100 mm mannitol, 100 mm NaHCO₃, 20 mm Hepes-Tris (pH 7.5), and 0.1 mm MgSO₄. LMV (100 μg protein per assay) were incubated in media containing 100 mm mannitol, 0.1 mm MgSO₄ either 20 mm Mes-Tris (pH 5.5) or 20 mm Hepes-Tris (pH 7.5), and varying concentrations of sodium gluconate and sodium butyrate (to maintain a constant media osmolarity and Na⁺ concentration) and constant tracer amounts of [U-¹⁴C]butyrate. Uptake was measured at 37 °C for 5 s. Initial rates of pH-activated butyrate uptake were determined by subtracting the uptake in the absence of a pH gradient from those with an inwardly directed pH gradient. The data are presented as a Michaelis-Menten curve and (inset) as Eadie-Hofstee plot of V/[S] against V, whereV is the velocity and [S] the concentration of the substrate. Values are presented as the means ±s.e.m. for three experiments.

Figure 8. Investigation into the presence of a band-3 homologue in pig and human colonic LMV

Samples (25 μg of protein each) were treated as outlined before (Fig. 1). Human erythrocyte ghosts were used as a control. Lane a, pig colonic homogenate; lane b, pig colonic LMV; lane c, human colonic homogenate; lane d, human colonic LMV; lane e, human erythrocyte ghosts.

Figure 9. Effect of organic and inorganic anions of the extravesicular media on butyrate uptake

LMV, isolated from pig () and human () colon, were preloaded with the standard loading buffer. LMV (100 μg protein per assay) were then incubated in media containing 100 mm mannitol, 80 mm sodium gluconate (60 mm sodium gluconate when Na₂SO₄ was present in the medium); 20 mm Mes-Tris (pH 5.5), 0.1 mm MgSO₄, 1 mm [U-¹⁴C]butyrate and the following Na⁺ salts (20 mm final concentration) acetate, propionate, pyruvate, l-lactate, Cl⁻, NO₃⁻ and SO₄⁻. Uptake was measured at 37 °C for 5 s as before. Values are presented as means ± s.e.m. for three experiments.

Figure 10. Effect of other potential inhibitors on butyrate transport into pig colonic LMV

LMV, loaded with the standard loading buffer, were incubated for 30 min at 4 °C with indicated compounds. Mercurials, phloretin and NPPB were added to a volume of vesicles to give a final concentration of 0.5 mm, whilst the final concentrations of the DEPC and 4-CHC in LMV amounted to 1 and 5 mm, respectively. LMV (control) were incubated at 4 °C for 30 min. Butyrate uptake by pre-treated and control LMV was measured for 37 °C for 5 s with the uptake buffer having the standard composition described in Table 2. Values are presented as means of triplicates ±s.e.m.