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1 Veterinär-Physiologisches Institut, Universität Leipzig, D-04103 Leipzig; and 2 Physiologisches Institut, Tierärztliche Hochschule Hannover, D-30173 Hannover, Germany
Due to intensive intracellular metabolism of
short-chain fatty acids, ruminal epithelial cells generate large
amounts of D-
-hydroxybutyric acid, acetoacetic acid, and
lactic acid. These acids have to be extruded from the cytosol to avoid
disturbances of intracellular pH (pHi). To evaluate acid
extrusion, pHi was studied in cultured ruminal epithelial
cells of sheep using the pH-sensitive fluorescent dye
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein.
Extracellular addition of D--hydroxybutyrate,
acetoacetate, or lactate (20 mM) resulted in intracellular
acidification. Vice versa, removing extracellular
D--hydroxybutyrate, acetoacetate, or lactate after preincubation with the respective monocarboxylate induced an increase of pHi. Initial rate of pHi decrease as well as
of pHi recovery was strongly inhibited by pCMBS (400 µM)
and phloretin (20 µM). Both cultured cells and intact ruminal
epithelium were tested for the possible presence of proton-linked
monocarboxylate transporter (MCT) on both the mRNA and protein levels.
With the use of RT-PCR, mRNA encoding for MCT1 isoform was demonstrated
in cultured ruminal epithelial cells and the ruminal epithelium.
Immunostaining with MCT1 antibodies intensively labeled cultured
ruminal epithelial cells and cells located in the stratum basale of the
ruminal epithelium. In conclusion, our data indicate that MCT1 is
expressed in the stratum basale of the ruminal epithelium and may
function as a main mechanism for removing ketone bodies and lactate
together with H+ from the cytosol into the blood.
intracellular pH; short-chain fatty acids; intracellular metabolism
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