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HORMONES AND SIGNALING

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

¹Department of Veterinary Physiology, Free University of Berlin, Berlin; and ²Research Institute for the Biology of Farm Animals (FBN), Department of Nutritional Physiology "Oskar Kellner," Dummsterstorf, Germany

Submitted 2 March 2006 ; accepted in final form 30 June 2006

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 (pH_i) 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 pH_i of 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 pH_i by 0.19 ± 0.03 pH units. On the basis of the observed decreases in pH_i resulting 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^–]_e induced a pH_i decrease (–0.51 ± 0.03 pH units) and impaired pH_i recovery from butyrate-induced acid load. Moreover, reduction of [Cl^–]_e abolished the inhibitory effect of foliomycin and markedly reduced the HOE 694- and S3226-sensitive components of pH_i, 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 pH_i regulation of these cells.

active transport; pH_i regulation; Na⁺/H⁺ exchanger; BCECF