A vH+-ATPase is present in cultured sheep ruminal epithelial cells

Abstract

In this study, the existence and functional activity of a vacuolar-type H+-ATPase (vH+-ATPase) was explored in primary cultures of sheep ruminal epithelial cells (REC). The mRNA transcripts of the E and B subunits of vH+-ATPase were detectable in RNA from REC samples by RT-PCR. Immunoblotting of REC protein extractions with antibodies directed against the B subunit of yeast vH+-ATPase revealed a protein band of the expected size (60 kDa). Using the fluorescent indicator BCECF and selective inhibitors (foliomycin, HOE 694, S3226), the contribution of vH+-ATPase and Na+/H+exchanger (NHE) subtype 1 and 3 activity to the regulation of intracellular pH (pHi) was determined in nominally HCO3−-free, HEPES-buffered NaCl medium containing 20 mM of the short-chain fatty acid butyrate as well as after reduction of the extracellular Cl−concentration ([Cl−]e) from 136 to 36 mM. The initial pHiof REC was 7.4 ± 0.1 in nominally HCO3−-free, HEPES-buffered NaCl medium and 7.0 ± 0.1 after acid loading with butyrate. Selective inhibition of the vH+-ATPase with foliomycin decreased pHiby 0.19 ± 0.03 pH units. On the basis of the observed decreases in pHiresulting from inhibition of vH+-ATPase as well as of subtypes 1 and 3 of NHE, vH+-ATPase activity appears to account for ∼30% of H+extrusion, whereas the activities of NHE subtypes 3 and 1 account for 20 and 50% of H+extrusion, respectively. Lowering of [Cl−]einduced a pHidecrease (−0.51 ± 0.03 pH units) and impaired pHirecovery from butyrate-induced acid load. Moreover, reduction of [Cl−]eabolished the inhibitory effect of foliomycin and markedly reduced the HOE 694- and S3226-sensitive components of pHi, indicating a role of Cl−in the function of these H+extrusion mechanisms. We conclude that a vH+-ATPase is expressed in ovine REC and plays a considerable role in the pHiregulation of these cells.