Molecular identification, immunolocalization, and functional activity of a vacuolar-type H+-ATPase in bovine rumen epithelium |
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Authors: | Elke Albrecht Martin Kolisek Torsten Viergutz Rudolf Zitnan Monika Schweigel |
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Institution: | (1) Muscle Biology and Growth, Research Institute for the Biology of Farm Animals (FBN), Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany;(2) Department of Veterinary Physiology, Faculty of Veterinary Medicine, Free University of Berlin, Oertzenweg 19b, 14163 Berlin, Germany;(3) Reproductive Biology, Research Institute for the Biology of Farm Animals (FBN), Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany;(4) Research Institute of Animal Production, Slovak Agricultural Research Centre, Nitra, Komenskeho 73, 041 81 Kosice, Slovakia;(5) Research Institute for the Biology of Farm Animals (FBN), Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany;(6) Department of Nutritional Physiology “Oskar Kellner”, Research Institute for the Biology of Farm Animals (FBN), Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany |
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Abstract: | In this study, we have studied the expression, localization, and functionality of vacuolar-type H+-ATPase (vH+-ATPase) and Na+/K+-ATPase in the bovine rumen epithelium. Compared with the intracellular pH (pHi) of control rumen epithelial cells (REC; 7.06 ± 0.07), application of inhibitors selective for vH+-ATPase (foliomycin) and Na+/K+-ATPase (ouabain) reduced pHi by 0.10 ± 0.03 and 0.18 ± 0.03 pH-units, respectively, thereby verifying the existence of both functional proteins. Results
from qRT-PCR and immunoblotting clearly confirm the expression of vH+-ATPase B subunit in REC. However, the amount of Na+/K+-ATPase mRNA and protein is tenfold and 11-fold of those of vH+-ATPase subunit B, respectively, reflecting a lower overall abundance of the latter in REC. Na+/K+-ATPase immunostaining has revealed the protein in the plasma membrane of all REC from the stratum basale to stratum granulosum,
with the highest abundance in basal cells. In contrast, the vH+-ATPase B subunit has been detected in groups of cells only, mainly localized in the stratum spinosum and stratum granulosum
of the epithelium. Furthermore, vH+-ATPase has been detected in the cell membrane and in intracellular pools. Thus, functional vacuolar-type H+ pumps are expressed in REC and probably play a role in the adaptation of epithelial transport processes. |
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Keywords: | Rumen epithelial cells Immunohistochemistry Proton pump Na+/K+-ATPase BCECF |
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