Abstract
Ruminal vacuolar H(+)-ATPase (vH(+)-ATPase) activity is regulated by metabolic signals. Thus, we tested whether its localization, expression, and activity were changed by different feeding. Young male sheep (n = 12) were either fed hay ad libitum (h) or hay ad libitum plus additional concentrate (h/c) for 2 wk. The vH(+)-ATPase B subunit signal was predominantly found in the cell membrane and cytosol of rumen epithelial cells (REC) with basal/parabasal phenotype. The elevated number (threefold) of these cells in rumen mucosa of h/c-fed sheep reflects a high proliferative capacity and, explains the 2.3-fold increase of the total number of vH(+)-ATPase-expressing REC. However, in accordance with a 58% reduction of the vH(+)-ATPase B subunit mRNA expression in h/c-fed sheep, its protein amount per single REC was decreased. Using the fluorescent probe BCECF and selective inhibitors (foliomycin, amiloride), the contribution of vH(+)-ATPase and Na(+)/H(+) exchanger to intracellular pH (pH(i)) regulation was investigated. REC isolated from h/c-fed sheep keep their pH(i) at a significantly higher level (6.91 ± 0.03 vs. 6.74 ± 0.05 in h-fed sheep). Foliomycin or amiloride decreased pH(i) by 0.16 ± 0.02 and 0.57 ± 0.04 pH units when applied to REC from h-fed sheep, but the effects were markedly reduced (-88 and -33%) after concentrate feeding. Nevertheless, we found that REC proliferation rate and [cAMP](i) were reduced after foliomycin-induced vH(+)-ATPase inhibition. Our results provide the first evidence for a role of vH(+)-ATPase in regulation of REC proliferation, most probably by linking metabolically induced pH(i) changes to signaling pathways regulating this process.