P-glycoprotein in sheep liver and small intestine: gene expression and transport efflux activity.

The role of the transporter P-glycoprotein (P-gp) in the disposition kinetics of different drugs therapeutically used in veterinary medicine has been demonstrated. Considering the anatomo-physiological features of the ruminant species, the constitutive expression of P-gp (ABCB1) along the sheep gastrointestinal tract was studied. Additionally, the effect of repeated dexamethasone (DEX) administrations on the ABCB1 gene expression in the liver and small intestine was also assessed. The ABCB1 mRNA expression was determined by real-time quantitative PCR. P-gp activity was evaluated in diffusion chambers to determine the efflux of rhodamine 123 (Rho 123) in the ileum from experimental sheep. The constitutive ABCB1 expression was 65-fold higher in the liver than in the intestine (ileum). The highest ABCB1 mRNA expression along the small intestine was observed in the ileum (between 6- and 120-fold higher). The treatment with DEX did not elicit a significant effect on the P-gp gene expression levels in any of the investigated gastrointestinal tissues. Consistently, no significant differences were observed in the intestinal secretion of Rho 123, between untreated control (Peff S-M = 3.99 × 10(-6)  ± 2.07 × 10(-6) ) and DEX-treated animals (Peff S-M = 6.00 × 10(-6)  ± 2.5 × 10(-6) ). The understanding of the efflux transporters expression and activity along the digestive tract may help to elucidate clinical implications emerging from drug interactions in livestock.

Expression of urea transporters is affected by dietary nitrogen restriction in goat kidney.

Ruminants are known to be able to very effectively recycle urinary urea and reuse it as a source of N for ruminal microbes. It is presumed that urea recycling is accomplished by specialized urea transporters (UT) which are localized in the kidney. This could be especially important in times of increased N requirement, such as during growth or during reduced dietary N intake. The aim of our study was to characterize and to localize UT in the goat (capra hircus) kidney and to investigate its response to reduced dietary N intake in growing goats. Therefore, 12 growing, male goats were fed either a diet containing high (17% CP in complete diet) or low (9% CP in complete diet) N content for 6 wk. After harvesting, blood and kidney samples were taken and analyzed. The mRNA of the different UT isoforms, UT-A1, UT-A2 and UT-B, were detected semiquantitatively in renal tissue by Northern blot analysis. For UT-A2 and UT-B, no statistically significant effect of dietary N restriction on renal mRNA expression could be detected (UT-A2: P = 0.26, UT-B: P = 0.07). However, renal mRNA abundance of UT-A1 significantly increased in the kidney of low-N-fed goats (P = 0.01). Furthermore, protein amounts of UT-B were verified by western blotting; and the localization of UT-A2 and UT-B protein was demonstrated by immunohistochemistry. No significant differences in protein amounts of UT-B could be observed comparing the 2 feeding groups (P = 0.78). The UT-B was localized in renal medulla and papilla, whereas UT-A2 was only found in renal medulla. In addition, comparison of UT-A and UT-BAA sequences of monogastric animals and ruminants showed a high degree of homology, indicating a similar function of the transporters among these species. In summary, we conclude that in ruminants, urea reabsorption in the kidney is most likely increased in response to a low-N diet via an upregulation of UT-A1 mRNA expression. Hypothetically, the reabsorbed urea can then be returned to the rumen via the bloodstream and thus be reused as a source of N for protein synthesis of ruminal microbial community.

Cytochrome P450 3A expression and function in liver and intestinal mucosa from dexamethasone-treated sheep.

The effects of repeated administrations of dexamethasone (DEX) (3 mg/kg/day by i.m. route for 7 days) on the gene expression profile of a cytochrome P450 (CYP) 3A28-like isoenzyme, on the expression of a CYP3A-immunoreactive protein and on CYP3A-dependent metabolic activities in sheep liver and small intestinal mucosa were evaluated in the current work. CYP 3A-dependent metabolic activities (erythromycin and triacetyl-oleandomycin N-demethylations) were assessed in microsomal fractions. The mRNA expression of CYP3A28-like, glucocorticoid receptor, constitutive androstane receptor, pregnane X receptor and retinoic X receptor alpha (RXRα) was determined by quantitative real-time PCR. The expression of a CYP3A-immunoreactive protein was measured by Western blot analyses. In the liver, DEX treatment increased CYP3A28-like mRNA levels (2.67-fold, P<0.01) and CYP3A apoprotein expression (1.34-fold, P<0.05) and stimulated CYP3A-dependent metabolism. High and significant correlation coefficients between CYP3A-dependent activities and CYP3A28-like gene (r=0.835-0.856, P<0.01) or protein (r=0.728-0.855, P<0.05) expression profiles were observed. Among the transcriptional factors, DEX only stimulated (2.1-fold, P<0.01) the mRNA expression of RXRα. In sheep small intestine, DEX caused a slight increment (34.6%, P<0.05) in erythromycin N-demethylase activity in the jejunal mucosa and a significant enhancement (P<0.05) of CYP3A apoprotein level in the duodenal mucosa.

Dietary nitrogen reduction enhances urea transport across goat rumen epithelium.

Ruminants are very capable of adapting their N homeostasis to a reduced dietary N intake. However, the limits of this physiological adaptation are still unknown. The aim of the present study was to determine the quantity of dietary N intake at which the needs of the animal are still satisfied. A study was performed in young White Saanen goats under conditions of dietary N reduction. Different semisynthetic diets with 19 to 7% CP were fed. Urea transport rates across the rumen epithelium from the blood into the ruminal fluid were quantified by Ussing chamber experiments. Reduced N intake increased urea transport rates across the mucosa, which could be inhibited by phloretin. The role of parietal urease in driving urea transfer across the epithelium was negligible because its activity was inhibited by antibiotics during in vitro incubations of the epithelium. Concentrations of ammonia in the ruminal fluid were decreased by reducing dietary N intake, accompanied by diminished urease activity at the smallest dietary N intake. Over the range of plasma urea concentrations observed in the different feeding groups, salivary urea concentrations were 73% of plasma urea concentrations. By plotting plasma urea concentrations against serosal to mucosal urea flux rates, a threshold at 1.75 mmol of urea/L of plasma could be assessed, below which urea flux was strongly increased. This indicates that rumen urea transfer could be stimulated by decreased plasma urea concentrations via unknown mechanisms. The physiological relevance of this adaptation of the rumen epithelium is that it is considered a central mechanism in the N homeostasis of growing goats under reduced N intake.