Effects of butyrate(−) on ruminal Ca(2+) transport: evidence for the involvement of apically expressed TRPV3 and TRPV4 channels

The ruminal epithelium absorbs large quantities of NH(4)(+) and Ca(2+). A role for TRPV3 has emerged, but data on TRPV4 are lacking. Furthermore, short-chain fatty acids (SCFA) stimulate ruminal Ca(2+) and NH(4)(+) uptake in vivo and in vitro, but the pathway is unclear. Sequencing of the bovine homologue (bTRPV4) revealed 96.79% homology to human TRPV4. Two commercial antibodies were tested using HEK-293 cells overexpressing bTRPV4, which in ruminal protein detected a weak band at the expected ~ 100 kDa and several bands ≤ 60 kDa. Immunofluorescence imaging revealed staining of the apical membrane of the stratum granulosum for bTRPV3 and bTRPV4, with cytosolic staining in other layers of the ruminal epithelium. A similar expression pattern was observed in a multilayered ruminal cell culture which developed resistances of > 700 Ω · cm(2) with expression of zonula occludens-1 and claudin-4. In Ussing chambers, 2-APB and the TRPV4 agonist GSK1016790A stimulated the short-circuit current across native bovine ruminal epithelia. In whole-cell patch-clamp recordings on HEK-293 cells, bTRPV4 was shown to be permeable to NH(4)(+), K(+), and Na(+) and highly sensitive to GSK1016790A, while effects of butyrate(−) were insignificant. Conversely, bTRPV3 was strongly stimulated by 2-APB and by butyrate(−) (pH 6.4 > pH 7.4), but not by GSK1016790A. Fluorescence calcium imaging experiments suggest that butyrate(−) stimulates both bTRPV3 and bTRPV4. While expression of bTRPV4 appears to be weaker, both channels are candidates for the ruminal transport of NH(4)(+) and Ca(2+). Stimulation by SCFA may involve cytosolic acidification (bTRPV3) and cell swelling (bTRPV4). SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00424-021-02647-7.