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In order to stabilize the intraruminal pH, bicarbonate secretion by the ruminal epithelium seems to be an important prerequisite. The present study therefore focussed on the characterization of bicarbonate exporting systems in ruminal epithelial cells. Intracellular pH (pHi) was measured spectrofluorometrically in primary cultured ruminal epithelial cells loaded with the pH-sensitive fluorescent dye, 2,7-bis(carboxyethyl)-5(6′)-carboxyfluorescein acetomethyl ester. Switching from CO2/HCO-buffered to HEPES-buffered solution caused a rapid intracellular alkalinization followed by a counter-regulation towards initial pHi. The recovery of pHi was dependent upon extracellular chloride, but independent of extracellular sodium. Adding 500 μM H2DIDS significantly reduced the increase of pHi. For further characterization of the bicarbonate exporting systems, we tested the ability to reverse the direction from HCO export to import in the absence of sodium and chloride. Under sodium and chloride-free conditions, counter-regulation after CO2-induced pHi decrease did not differ from pHi recovery in the presence of sodium and chloride. Existence of bicarbonate exporting systems in cultured ruminal epithelial cells and intact ruminal epithelium was verified by reverse transcription polymerase chain reaction (RT-PCR). Using RT-PCR and subsequent sequencing, expression of mRNA encoding for AE2, DRA and PAT1 could be found. Bicarbonate exporting systems could therefore be detected both on the functional and structural level. [ABSTRACT FROM AUTHOR]

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 μM 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). [ABSTRACT FROM AUTHOR]

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