Glucocorticoids differentially regulate Na-bile acid cotransport in normal and chronically inflamed rabbit ileal villus cells

Abstract

Previous studies have demonstrated that apical Na-bile acid cotransport (ASBT) is inhibited during chronic ileitis by both a decrease in the affinity as well as a decrease in the number of cotransporters. Methylprednisolone (MP), a commonly used treatment for inflammatory bowel disease (IBD, e.g., Crohn's disease), has been shown to reverse the inhibition of several other Na-solute cotransporters during chronic enteritis. However, the effect of MP on ASBT in the chronically inflamed ileum is not known. MP stimulated ASBT in villus cells from the normal rabbit ileum by increasing the cotransporter expression without a change in the affinity of the cotransporter for bile acid. Western blot studies demonstrated an increase in cotransporter expression. MP reversed the inhibition of ASBT in villus cells from the chronically inflamed ileum. Kinetic studies demonstrated that the mechanism of MP-mediated reversal of ASBT inhibition was secondary to a restoration of both affinity as well as cotransporter numbers. Western blot analysis demonstrated restoration of cotransporter numbers after MP treatment of rabbits with chronic ileitis. Thus MP stimulates ASBT in the normal ileum by increasing cotransporter numbers. MP reverses the inhibition of ASBT during chronic ileitis. However, MP restores the diminished affinity as well as cotransporter expression levels during chronic ileitis. Thus MP differentially regulates ASBT in the normal and in the chronically inflamed ileum.

Fig. 1.

Effect of methylprednisolone (MP) treatment on (ASBT) in villus cells from the normal and chronically inflamed ileum. Na-dependent taurocholate (tauro) uptake is defined as taurocholate uptake in the presence of extracellular Na minus that in the absence of Na. Y-axis labeling is the same for A and B. Statistical comparisons are made of uptakes of different conditions for each time point. A: Na-dependent bile acid uptake as a function of time in villus cells from control rabbits. This uptake is significantly increased at all time points measured in villus cells from MP-treated normal rabbits. B: in villus cells from the chronically inflamed ileum, Na-dependent taurocholate uptake was significantly inhibited. Treatment of rabbits with chronic ileal inflammation with MP almost completely reversed the inhibition of Na-dependent taurocholate uptake. prot, protein.

Fig. 2.

Effect of MP treatment on Na-K-ATPase activity in villus cells from the normal and chronically inflamed ileum. Villus cell Na-K-ATPase activity is expressed as nanomoles of P_i released per milligram protein per minute. MP has no effect on Na-K-ATPase activity in normal villus cells. However, Na-K-ATPase activity inhibition in the chronically inflamed ileum is reversed nearly back to normal levels by MP treatment.

Fig. 3.

Effect of MP treatment on ASBT in villus cell brush-border membrane vesicle (BBMV) isolated from the normal and chronically inflamed ileum. Na-dependent taurocholate uptake (pmol/mg of protein) is defined as taurocholate uptake in the presence of extravesicular Na minus that in the absence of Na. Y-axis labeling is the same for A and B. Statistical comparisons are made of uptakes of different conditions for each time point. All uptakes were done in triplicate, and each n represents BBMV preparations from different animals. Statistical comparisons are made of uptakes of different conditions for each time point. A: Na-dependent taurocholate uptake as a function of time in villus cell BBMV from control rabbits. This uptake was significantly increased at all time points measured in villus cell BBMV from MP-treated normal rabbits. B: in villus cell BBMV from the chronically inflamed ileum, Na-dependent taurocholate uptake was significantly reduced. Treatment of rabbits with chronic ileitis with MP almost completely reversed the inhibition of Na-dependent taurocholate uptake.

Fig. 4.

Kinetics of Na-dependent bile acid uptake in villus cell BBMV from control and MP-treated ileum. A: ³H-taurocholate uptake is shown as a function of varying concentrations of extravesicular taurocholate. Isosmolarity was maintained by adjusting the concentration of mannitol. Uptake for all concentrations was determined at 6 s. As the concentration of extravesicular taurocholate was increased, uptake of taurocholate was stimulated and subsequently became saturated in villus cell BBMV in all conditions. B: Lineweaver-Burk plot of these data with Enzfiter yielded kinetic parameters. Lineweaver Burk plot showed that the K_m remains constant, whereas the V_max changes significantly. The maximal rate of uptake of ³H-taurocholate (V_max) was stimulated by MP. However, the affinity for ³H-taurocholate uptake in BBMV was unaffected in the MP-treated ileum. The data shown are an average of 3 experiments, and each uptake was done in triplicate.

Fig. 5.

Kinetics of Na-dependent bile acid uptake in villus cell BBMV from chronically inflamed and MP treated ileum. A) ³H-taurocholate uptake is shown as a function of varying concentrations of extravesicular taurocholate. Isosmolarity was maintained by adjusting the concentration of mannitol. Uptake for all concentrations was determined at 6 s. As the concentration of extravesicular taurocholate was increased, uptake of taurocholate was stimulated and subsequently became saturated in villus cell BBMV in all conditions. B: Lineweaver-Burk plot of these data with Enzfiter yielded kinetic parameters. The diminished maximal rate of uptake of ³H-taurocholate (V_max) in the chronically inflamed ileum was reversed by MP treatment. Affinity (1/Michaelis constant) for taurocholate uptake, which was significantly diminished during chronic ileitis, was also reversed by MP in chronically inflamed ileum. The data shown are an average of 3 experiments, and each uptake was done in triplicate.

Fig. 6.

Real-time PCR analysis of the ASBT in the normal, inflamed, and MP-treated rabbit ileum. Four experiments each with different animals are shown. RTQ-PCR demonstrated that the message for ASBT is increased significantly in the normal when treated with MP. Treatment of rabbits with chronic ileitis with MP partially restored the message levels of ASBT.

Fig. 7.

Immunoreactive levels of ASBT in the normal, inflamed, and MP-treated rabbit ileum. Representative data of 4 experiments each with different animals are shown. Top: Western blot analysis demonstrated that the amounts of immunoreactive ASBT protein were increased in normal ileum treated with MP. Western blot analysis also demonstrated that the MP treatment reversed the amounts of immunoreactive ASBT protein nearly restored back to normal levels. Bottom: densitometry demonstrated the relative quantitation of ASBT, which confirmed both Western blot findings.