Altered Na+/K+-ATPase expression plays a role in rumen epithelium adaptation in sheep fed hay ad libitum or a mixed hay/concentrate diet

Abstract

In this study we investigated rumen papillae morphology and the localization and expression of the<br />Na^+/K⁺-ATPase&nbsp;in eight sheep fed hay ad libitum (h) or hay ad libitum plus additional concentrate (h/c). Four sheep were provided with the ad libitum h-diet for the complete three-week experimental period. The second group of four sheep received the h-diet for only one week and was fed the mixed hay/concentrate (h/c) diet for another two weeks. The amount of concentrate supplement was stepwise increased from 150 to 1000 g/day and given in two meals. Following slaughter rumen papillae from the atrium ruminis (AR), the rumen ventralis (RV) and the ventral blind sac (BSV) were fixed and examined for morphological changes and Na⁺/K⁺-ATPase localization by morphometric methods and immunohistochemistry. Ruminal epithelial cells (REC) originating from the strata basale to granulosum were also isolated. Cellular Na⁺/K⁺-ATPase expression (mRNA and protein) and differentiation state were determined by RT-PCR, Western blot, and flow cytometry. Compared with data from h-fed sheep, morphometric analysis revealed an increased length and width of rumen papillae in h/c-fed sheep, resulting in a marked 41% and 62% increase in rumen papillae surface in AR and RV, respectively. The rumen mucosa of h/c-fed sheep was characterized by a predominant stratum corneum (42 &plusmn; 0.7 &micro;m vs. 28 &plusmn; 0.5 &micro;m), but the thickness of the metabolically active cell layers remained unchanged. REC suspensions from sheep fed the h/c diet generally contained more cells (7.30 &plusmn; 0.83 vs. 3.49 &plusmn; 0.52 &times; 10⁷/ml; P &lt; 0.001) and an increased proportion of REC positive for basal cytokeratin and for the differentiation marker cytokeratin 10 (P &lt; 0.05). Cellular (cell membrane) and epithelial (stratum basale to stratum granulosum) Na⁺/K⁺-ATPase localization was similar between rumen regions and was not changed by concentrate feeding. After two weeks on the h/c-diet, a 96% increase in the absolute number of Na⁺/K⁺-ATPase-positive REC (6.56 &plusmn; 0.84 vs. 3.35 &plusmn; 0.51 &times; 10⁷/ml; P = 0.003) and a 61% elevation (P = 0.043) in Na⁺/K⁺-ATPase protein expression in REC from the upper third of the suprabasal cell layers were found. Moreover, a two-fold (P = 0.001) elevation in cell membrane surface area accompanied by a reduction (1.19 &times; 10^&ndash;7 &plusmn; 1.72 &times; 10^&ndash;9 arbitrary units (AU)/cm2 vs. 1.73 &times; 10^&ndash;7 &plusmn; 8.16 &times; 10^&ndash;9 AU/cm² in the h-group; P &lt; 0.001) in specific Na⁺/K⁺-ATPase fluorescence per cm² of cell membrane surface area was observed after h/c-feeding. Na⁺/K⁺-ATPase &alpha; subunit mRNA expression was also reduced (P &lt; 0.0001) from 0.154 &plusmn; 0.013 to 0.057 &plusmn; 0.004 pg per pg S18 mRNA control in the h/c-compared with the h-group. Thus, the h/c-diet led to a rapid increase in REC number and total cell membrane surface area in metabolically active and resorptive cell layers and was accompanied by a reduction in Na⁺/K⁺-ATPase mRNA expression and abundance per cell membrane surface area.