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

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). Supplementary Information The online version contains supplementary material available at 10.1007/s00424-021-02647-7.


C) Immunoblotting
For protein extraction, solvents and samples were cooled throughout the experiments to minimize protein degradation.
For HEK-293 cells, after washing with phosphate-buffered saline (PBS), cells were harvested mechanically by scraping in PBS. After centrifugation (500 g, 5 min), the cell pellet was resuspended in PBS (1 mL) and transferred into a new tube. PBS was removed via centrifugation (700 g, 4 min). The cell pellet was lysed in RIPA buffer (100 μL) for 30 min with intermediate gentle agitation. Finally, tube was put in an ultrasound bath (5 min) and centrifuged by a clarifying spin (20 min, 15 000 g, 4 °C). The supernatant contained the protein.

D) Modulators and solutions for patch-clamping and calcium imaging
Experimental solutions were stored at -20 °C (for weeks) or +4 °C (for days). Glucose was added to the buffer on the experimental day. TRP-modulators were obtained from Sigma-Aldrich, diluted in DMSO, stored at -20 °C, and added to the reservoir with corresponding bath solution immediately before each experiment at a ratio of ≤ 1:1000.  *) Solutions designated as "NaCl 6.4" and "NaBu 6.4" were buffered to pH 6.4 with MES.

E) Whole-cell patch-clamp experiments
Patch-clamp experiments were performed in a continuously perfused bath chamber at 23 °C [6,9,12,13]. A DMZ Universal Puller (Zeitz Instruments, Munich, Germany) was used to pull the pipettes. An EPC9 patch-clamp amplifier (HEKA Electronic, Lambrecht, Germany) recorded the currents using Patchmaster Software (HEKA Electronic). Agar bridges to ground the bath and for correction of the initial offset potential were made using the initial NaCl solution (Supplement, Part D). After seal formation and establishment of the whole-cell configuration, HEK-293 cells were clamped at a resting potential of -40 mV, from which the potential was stepped to values between +100 and -120 mV in 10 mV steps for 80 ms each at 5 kHz (Pulse protocol I), allowing analysis of current kinetics and determination of the reversal potential. Afterwards, a continuous pulse protocol in 20 mV steps with a low sampling rate (100 Hz) was applied to monitor solution changes (Pulse protocol II), alternating between these two pulse protocols automatically. The continuous pulse protocols were subsequently merged using Igor Pro 6.37 (WaveMetrics Inc., Lake Oswego, USA). After each overexpressing HEK-293 cell, a control cell was measured.

F) Data analysis
Data evaluation was performed using Igor Pro 6.37 (WaveMetrics Inc., Lake Oswego, USA). The Ussing chamber data were recorded at 10 points · min -1 and were averaged via binomial smoothing using Igor Pro Software before further analysis. The data analysis of patch-clamp experiments was essentially performed as described in Schrapers et al. [13]. Incomplete measurements or those with a series resistance ≤ 2 MOhm or an unstable current level were excluded. The reversal potentials (Vrev) were calculated by linear interpolation between the values above and below a current of zero in the corresponding IV-curve and corrected for liquid junction potentials using JPCalcWin software (School of Medical Sciences, Sydney, Australia) [1]. The relative permeability ratio p(K + )/p(Na + ) was estimated from the reversal potentials measured in cells filled with KGlu and superfused with NaGlu solution (Supplement, Part D, III) using the Goldman-Hodgkin-Katz equation [7,13]: From this, it follows that: Here, T is the temperature (296.15 K), F the Faraday constant (~ 96485 C · mol -1 ), and R the universal gas constant (~ 8.3 J ⋅ K -1 ⋅ mol -1 ). Note that the concentrations of [Na + ]in and [K + ]out were small (6.8 and 5 mmol · L -1 , respectively). Furthermore, the positive reversal potential in NaCl solution suggests that p(Cl -) was low, while [Cl -]out = [Cl -]in = 20 mmol · L -1 , so that the contribution of Clcould be neglected. The relative permeability ratio p(NH4 + )/p(Na + ) was calculated from the difference of Vrev in NaCl and NH4Cl solution (Supplement, Part D, II) using the standard relationship [7]: