Effects of butyrate\u2212 on ruminal Ca2+ transport: evidence for the involvement of apically expressed TRPV3 and TRPV4 channels

Franziska Liebe,Stefan Mergler,Hendrik Liebe,Friederike Stumpff,Gerhard Sponder

doi:10.1007/s00424-021-02647-7

Abstract

The ruminal epithelium absorbs large quantities of NH4+ and Ca2+. A role for TRPV3 has emerged, but data on TRPV4 are lacking. Furthermore, short-chain fatty acids (SCFA) stimulate ruminal Ca2+ and NH4+ 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 · cm2 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 NH4+, 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 NH4+ and Ca2+. Stimulation by SCFA may involve cytosolic acidification (bTRPV3) and cell swelling (bTRPV4).

Highlights

The era of modern transport physiology began with Ussing’s famous discovery of active transport across amphibian skin [105]
Given that studies in vivo and in vitro have shown a strong stimulatory effect of short-chain fatty acids (SCFA) on the transport of C a2+ [44, 54, 81,82,83, 104, 110, 117] and ammonia [12, 13] across the rumen, we investigated if bTRPV3, bovine homologue of TRPV4 (bTRPV4), or both channels are candidates for this SCFA sensitive pathway for the uptake of cations
HEK-293 cells were transfected with the bTRPV4-streptavidin tag (Strep)-yellow fluorescent protein (YFP) construct in a p5TO vector (Supplement, Part B)

Summary

Introduction

The era of modern transport physiology began with Ussing’s famous discovery of active transport across amphibian skin [105]. Over 70 years later, we still know very little about transport processes across stratified squamous epithelia of mammalian species. A classic example for a transporting cornified stratified epithelium in mammals is the ruminal epithelium of cattle and sheep [37, 93]. Interest in this tissue has historically been high since insufficient transport capacity of the rumen may lead to malnutrition and disease in ruminants [6, 63]. There is a direct interest in understanding more about the underlying ruminal transport processes, with potential repercussions for understanding

Methods

Results

Conclusion