Molecular and functional evidence for a Na(+)-HCO3(-)-cotransporter in sheep ruminal epithelium

The present study aimed to identify the HCO3(-)-dependent mechanisms contributing to the homeostasis of the intracellular pH (pHi) in ruminal epithelial cells of sheep. Therefore, pHi was measured spectrofluorometrically in primary cultured ruminal epithelial cells loaded with the pH-sensitive fluor...

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Bibliographic Details
Published in:Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology Biochemical, systemic, and environmental physiology, 2003-06, Vol.173 (4), p.277-284
Main Authors: Huhn, K, Müller, F, Honscha, K U, Pfannkuche, H, Gäbel, G
Format: Article
Language:English
Subjects:
Animals
Base Sequence - genetics
Cells
Cells, Cultured
Epithelium - metabolism
Hydrogen-Ion Concentration
Intracellular Membranes - metabolism
Molecular Sequence Data
Reverse Transcriptase Polymerase Chain Reaction
Rumen - metabolism
Sequence Homology, Nucleic Acid
Sheep - metabolism
Sodium-Bicarbonate Symporters - genetics
Sodium-Bicarbonate Symporters - metabolism
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Summary:The present study aimed to identify the HCO3(-)-dependent mechanisms contributing to the homeostasis of the intracellular pH (pHi) in ruminal epithelial cells of sheep. Therefore, pHi was measured spectrofluorometrically in primary cultured ruminal epithelial cells loaded with the pH-sensitive fluorescent dye, 2',7'-bis(carboxyethyl)-5(6')-carboxyfluorescein acetoxymethyl ester. Switching from a HEPES-buffered to a CO2/HCO3(-)-buffered solution caused a rapid intracellular acidification followed by a counter-regulation towards alkaline levels. The counter-regulation was totally dependent upon extracellular Na+, but independent of intracellular Cl-. Adding 30 microM EIPA to the solutions did not affect the pHi counter-regulation following the acidification. Presence of 500 M H2DIDS inhibited the counter-regulation of pHi by 67%. These results pointed to a Na(+)-HCO3(-)-cotransporter (NBC) as the main pHi regulatory mechanism in the presence of HCO3-. Existence of an NBC in both cultured ruminal epithelial cells and intact ruminal epithelium was verified by reverse transcription polymerase chain reaction (RT-PCR) studies. RT-PCR yielded a band of the expected molecular size of 333 bp in both cultured cells and intact epithelium. The mRNA sequences were identical and shared a homology of 62% with human kidney NBC (Genebank accession number AF007216), of 66% with rat kidney NBC (AF004017) and of 65% with mouse duodenal NBC (AF141934).
ISSN:0174-1578
1432-136X
DOI:10.1007/s00360-003-0333-0