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Institute of Veterinary Physiology, Freie Universität Berlin, D-14163 Berlin, GermanyDepartment of Animal Nutrition, West Bengal University of Animal and Fishery Sciences, 700037 Kolkata, India
Stimulation of Ca2+ absorption can counteract hypocalcemia at the onset of lactation. The plant bioactive lipid compound (PBLC) menthol is an agonist for nonselective cation channels of the transient receptor potential (TRP) family. It acutely stimulated Ca2+ absorption in ruminal epithelia of nonadapted animals ex vivo and caused higher plasma Ca2+ concentrations in cows and sheep in vivo. To elucidate the pathway by which menthol feeding increases plasma Ca2+ level, the present study aimed to investigate the long-term dose-dependent effects of dietary menthol-rich PBLC on Ca2+ absorption and mRNA abundances of TRP channels in both rumen and jejunum. Twenty-four growing Suffolk sheep were equally distributed to a Con, PBLC-L, and PBLC-H group, which received 0, 80, and 160 mg/d of a menthol-rich PBLC. After 4 wk, ruminal and jejunal epithelia were analyzed for mRNA abundances of TRPA1, TRPV3, TRPV5–6, and TRPM6–8 genes. The Ca2+ flux rates and electrophysiological properties of epithelia from rumen and mid-jejunum were measured in Ussing chambers in the presence and absence of mucosal Na+. Acute changes in Ca2+ flux rates were measured after mucosal application of 50 µM menthol. Ruminal epithelia had quantifiable transcripts of TRPV3 = TRPM6 > TRPM7 > TRPA1 with no difference among feeding groups. Jejunum had quantifiable transcripts of TRPM7 > TRPA1 ≥ TRPM6 ≥ TRPV6 > TRPV5, where TRPA1, TRPV5, and TRPV6 tended to decrease linearly with increasing PBLC dose. Absorptive net flux of Ca2+ was detected only in the rumen, whereas jejunum showed a high passive permeability to Ca2+. Net flux rates of Ca2+ in the rumen increased in a quadratic manner (highest in PBLC-L animals) and were systematically decreased with the omission of mucosal Na+. Short-circuit current increased in both PBLC feeding groups compared with Con only in the rumen. Acute application of menthol-stimulated mucosal-to-serosal and net Ca2+ flux rates only in ruminal epithelia with higher stimulation in PBLC-fed animals. We conclude that Ca2+ transport is mainly active and transcellular in the rumen. It most likely involves TRPV3 that can be stimulated by menthol. Pre-feeding of menthol-rich PBLC enhances ruminal Ca2+ absorption and sensitizes it to acute stimulation by menthol. By contrast, intestinal Ca2+ absorption is not sensitive to menthol stimulation. Menthol could be used as a tool to enhance ruminal Ca2+ absorption and to prevent hypocalcemia in dairy cows.
Calcium and magnesium physiology and nutrition in relation to the prevention of milk fever and tetany (dietary management of macrominerals in preventing disease).
Vet. Clin. North Am. Food Anim. Pract.2014; 30 (25245611): 643-670
Milk fever and subclinical hypocalcaemia - An evaluation of parameters on incidence risk, diagnosis, risk factors and biological effects as input for a decision support system for disease control.
). Subclinical hypocalcemia affects 6–25% of heifers, 29–41% of cows in the second lactation, and up to 54–60% of cows in the third lactation and beyond (
One main hypocalcemia control strategy aims to increase gastrointestinal Ca2+ absorption by supplying high doses of oral Ca2+ salts or by injection of vitamin D3 and its metabolites (
). Apical Ca2+ entry by this transporter was thought to be coupled to the extrusion of intracellular H+, the latter in parallel to H+ extrusion by the well-documented Na+/H+ exchanger (
). More recently, a nonselective cation channel, with characteristics of the TRP family, was proposed to mediate the apical entry of Ca2+ into the ruminal epithelium (
). Moreover, the direct application of menthol in concentrations of 10 or 100 µM increased the absorptive Ca2+ flux across bovine ruminal epithelia by 32 to 54% ex vivo (
). It was further demonstrated that the dietary supplementation of a blend of PBLC with menthol as the major active compound increased serum Ca2+ concentration in mid-lactation cows in vivo (
It is an open question whether increased serum Ca2+ concentration after menthol supplementation in vivo originated from increased Ca2+ absorption in the rumen, the jejunum, or both. It is further unknown whether menthol solely acts as an acute agonist of TRP channels or whether it also modifies the expression of TRP channels during long-term supplementation. Finally, the functional role of the putative Ca2+/H+ exchanger in relation to TRP-mediated Ca2+ absorption is unclear in the rumen. To answer these open questions, a model study in sheep was designed to measure the changes in TRP channel expression, Ca2+ absorptive capacity, and menthol responsiveness of ruminal and jejunal epithelia after long-term supplementation with 2 doses of menthol-rich PBLC. To evaluate the possible involvement of a Ca2+/H+ exchanger, Ca2+ absorption was assessed in the presence and absence of luminal Na+. The absence of Na+ should abrogate apical Na+/H+ exchange and thus increase the H+ driving force for apical Ca2+/H+ exchange.
MATERIALS AND METHODS
Animals, Diet, and Feeding
All experiments were performed in compliance with the German legislation on the welfare of experimental animals and communicated with the State Office for Health and Social Affairs (LAGeSo) Berlin (reference number G0141/17). The experiment was conducted in 2 runs with 12 growing Suffolk sheep in each run during June through August 2017. Animals were equally divided by A.K.P. into 3 dietary groups in a randomized block design with the aim to achieve equal sex and BW (considered as a block) distribution per group. In each run, 4 blocks of 3 sheep were housed in 4 available pens and received hay and water ad libitum plus 600 g/d of a pelleted concentrate. The concentrate was provided in 3 equal portions per day (at 0700, 1100, and 1500 h) and contained either no PBLC (Con), a low dose of PBLC (PBLC-L; 80 mg/d), or a high dose of PBLC (PBLC-H; 160 mg/d) with menthol as the main active ingredient (900 g/kg). Experimental design, feeding regimen, and chemical composition of hay and concentrate have been described in detail previously (
). Although feeding groups were blinded using color-coded feed during the original experiment, the results of the molecular biology experiments (September 2018 through September 2019) and Ussing chamber data (October 2018 through April 2019) of the present study were evaluated after the blinding was opened to assess already published data of this trial (
After a feeding period of at least 4 wk, animals were slaughtered by exsanguination from the carotid arteries after stunning by captive bolt in accordance with German laws as described previously (
). Samples of ruminal papillae and of the jejunal mucosal layer (without submucosal tissue) were taken from each animal, placed in RNAlater solution (Sigma-Aldrich, Taufkirchen, Germany), stored at 4°C for 12 h and at −20°C thereafter. The Nucleospin RNA II kit was used for total RNA isolation (Macherey-Nagel Nucleospin RNA, Düren, Germany) as described previously (
). Reverse transcription was carried out with 1,000 ng RNA using an iScript cDNA synthesis kit (Bio-Rad, Taufkirchen, Germany) according to the manufacturer's instructions. Reactions were subsequently diluted 1:10.
For amplification steps of the reverse-transcription quantitative PCR, exon-transcending primers, and fluorescence-labeled probes with FAM as a reporter and TAMRA or BHQ as quencher were designed for TRPA1, TRPV3, TRPV5, and TRPM6 through TRPM8 (Table 1). The sequence of TRPV6 was obtained from
. Amplification products were sequenced by Microsynth Seqlab (Göttingen, Germany) and sequence specificity was verified by NCBI Blast database (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Table 1Forward (Fwd) and reverse (Rev) primer sequences, probe sequences, and expected amplicon length for transient receptor potential channels and reference genes
A 40-cycle 2-step reverse-transcription quantitative PCR was performed in triplicates on a ViiA7 thermocycler (Applied Biosystems/Life Technologies, Waltham, MA). An aliquot of 3.7 µL of cDNA was added to the master mix of primers, probe, and iTaq Universal Probes Supermix (Bio-Rad, Feldkirchen, Germany) in total assay volumes of 10 µL.
Thresholds were set by the cycler software. The data analysis software qBasePLUS (Biogazelle NV, Zwijnaarde, Belgium) was used for inter-run calibration and to calculate dilution series-based gene-specific amplification efficiency. Reference genes ACTB, GAPDH, and YWHAZ were tested for their expression stability. Quantification cycle (Cq) values of the target genes were normalized with the reference genes that had the lowest geNorm expression stability value M (rumen, GAPDH and YWHAZ; jejunum, ACTB, GAPDH, and YWHAZ) as described earlier (
) and scaled to the mean value of the Con group. The resulting calibrated normalized relative quantities were used for statistical analysis.
Tissue Preparation for Ussing Chamber Experiments
Immediately after slaughter, ruminal and intestinal tissue was obtained from the ventral sac (∼25 × 25 cm) and the middle of the jejunum (∼1.5 m length), respectively, and rinsed in 38°C NaCl solution (9 g/L). Epithelial tissues were stripped off the muscular layers and transported to the laboratory within 30 min in pre-warmed (38°C) transport solution (pH 7.4, 288 mOsm/L; see Table 2) gassed with carbogen (95% O2 and 5% CO2). Further details of the procedure are described in
Table 2Chemical composition (mM), osmolarity, and pH of incubation solutions used for incubation during epithelial preparation, transport, and during Ussing chamber experiments
Ca2+ Flux Rates and Electrophysiological Measurements
Ruminal and jejunal epithelia were mounted in Ussing chambers with an exposed area of 3.14 and 0.95 cm2, respectively. The chambers mounted with ruminal and jejunal epithelia contained 16 and 12 mL of incubation solution, respectively, on both sides (for composition, see Table 2). Serosal incubation solution was identical for ruminal and jejunal epithelia with pH adjusted to 7.4 and osmolarity adjusted to 288 mOsm/L. The mucosal incubation solution for ruminal epithelia had a pH 6.4 (288 mOsm/L) and contained 40 mmol/L of short-chain fatty acids (SCFA) because luminal SCFA had been shown previously to stimulate ruminal Ca2+ absorption (
). At variance, the mucosal incubation solution for jejunal epithelia had a pH of 7.4 (288 mOsm/L) and was devoid of SCFA. For half of the ruminal and jejunal epithelia, Na+ was replaced by N-methyl-d-glucamine (NMDG+) to create Na+-free mucosal incubation solutions. The absence of mucosal Na+ was intended to shut down H+ extrusion via the Na+/H+ exchanger (
). All mucosal incubation solutions were Mg2+-free to avoid interference of Mg2+ with Ca2+ transport proteins and ion channels.
Mucosal and serosal solutions were circulated by a gas lift system operated with carbogen, and temperature was maintained at 38°C by temperature-controlled water jackets surrounding the incubation solution reservoirs. Tissue conductance (Gt) as a measure for passive ion permeability via transcellular and paracellular pathways and short-circuit current (Isc) as a measure for an active transcellular ion transport were continuously recorded as described previously (
) with the exception that clamp current application occurred with platinum electrodes inserted directly into the incubation solution. To determine Isc, all epithelia were switched from open to short-circuit mode ∼10 min after mounting.
For every sheep, 8 Ussing chambers were used for ruminal and jejunal tissues each, which were again divided in 4 chambers with Na+-containing and 4 chambers with Na+-free mucosal incubation solution. Within each of those 4-chamber sets of Ussing chambers, 2 epithelia with similar tissue conductance (Gt) were paired to investigate Ca2+ flux rates in mucosal-to-serosal (Jms) and serosal-to-mucosal (Jsm) directions. Unidirectional Ca2+ flux rates were measured using 50 kBq and 70 kBq of 45Ca2+ as tracer in ruminal and jejunal epithelia, respectively. The 45Ca2+ tracer was added to either the mucosal (Jms) or the serosal (Jsm) side of the epithelium, which will be called the “hot” side hereafter. After equilibration for 50 min, flux measurements were performed over 3 consecutive flux periods (P1 through P3) by taking samples (600 µL) at 45-min intervals from the opposite (“cold”) side. The removed sample volume was immediately replaced by new incubation solution with identical composition. The specific activity of Ca2+ on the hot side was determined as mean of two 100-µL samples taken from the hot side at the start and end of flux measurements. Radioactivity was measured by β-counter (LKB Wallace-Perkin-Elmer, Überlingen, Germany) after volume correction of hot samples (to 600 µL) and the addition of 3 mL of liquid scintillation fluid (Aquasafe 300, Zinsser Analytic GmbH, Frankfurt, Germany) to all samples. Unidirectional flux rates (extrapolated to nmol·cm−2·h−1) were calculated by relating the rate of 45Ca2+ appearance on the cold side to the specific activity of the hot side (
). To determine net flux rates (Jnet), Jsm was subtracted from the corresponding Jms for each tissue pair.
Menthol (50 µM) was added to the mucosal side of all epithelia immediately at the start of the second flux period (P2). After allowing for equilibration of flux rates and electrophysiology to menthol stimulation during period P2, the steady state values of flux rates and electrophysiology under menthol stimulation were measured in period P3. This procedure allowed discriminating between long-term adaptation of ruminal and intestinal epithelia to the menthol pre-feeding, measured in the initial flux period P1, and the acute effects of menthol addition on Ca2+ flux rates and electrophysiology in the differently pre-fed animals. The acute effects of menthol stimulation were determined by subtracting the baseline values of period P1 from the values under menthol stimulation determined in period P3, further referred to as ΔJms, ΔJsm, and ΔJnet for flux rates and ΔIsc and ΔGt for electrophysiological values.
Statistical Analysis
One outlier from the PBLC-L group was removed from all electrophysiological and flux data. Electrophysiological data (Gt and Isc) of each epithelium were pooled arithmetically over each flux period to compute the period means of P1 and P3. Electrophysiological and Ca2+ flux rate data were analyzed using the mixed model procedure of SAS version 8.02 (SAS Institute Inc., Cary, NC) in a 3 (PBLC dose; 0, 80, and 160 mg/d) × 2 (Na+; present or absent in mucosal solution) factorial arrangement. The model contained fixed effects of PBLC dose, Na+, interaction between PBLC dose × Na+, block, sex, and run and random effects of chamber within animal. The gene expression data were analyzed with the same mixed model procedure, but without Na+ and interaction between PBLC dose × Na+ in the model to compare only PBLC doses. Polynomial contrasts were used to identify linear and quadratic effects of PBLC doses (0, 80, and 160 mg/d). The overall effects of the pooled PBLC groups compared with the Con group were also determined (Con vs. PBLC). Variability of data was expressed as SEM. Statistical significance was set at P ≤ 0.05, and a trend was considered at 0.05 < P ≤ 0.10.
RESULTS
Quantitative RT-PCR
In the rumen, mRNA expression of TRPV5, TRPV6, and TRPM8 was undetectable or too weak to be quantified. The mRNA for TRPA1, TRPV3, TRPM6, and TRPM7 was quantifiable in the order TRPV3 ≈ TRPM6 > TRPM7 > TRPA1. The calibrated normalized relative quantities of these gene amplicons were not different among feeding groups (Figure 1).
Figure 1Calibrated normalized relative quantities (CNRQ) of mRNA expression of transient receptor potential channels in (A) ruminal and (B) jejunal epithelia in sheep pre-fed with menthol-rich plant bioactive lipid compounds (PBLC) at concentrations of 0 mg/d (Con), 80 mg/d (PBLC-L), or 160 mg/d (PBLC-H). P-values for linear and quadratic effects and Con versus pooled PBLC-L and PBLC-H groups (Con vs. PBLC) are indicated below each gene transcript. Data are LSM ± SEM of n = 8 sheep per group.
In the jejunum, mRNA expression of TRPV3 and TRPM8 was undetectable or too weak to be quantified. The mRNA expression of the other TRP channels was quantifiable in the order TRPM7 > TRPA1 ≥ TRPM6 ≥ TRPV6 > TRPV5. Of these, menthol-rich PBLC pre-feeding decreased the mRNA expression of TRPA1 (Con vs. PBLC, P = 0.043) and TRPV5 (Con vs. PBLC, P = 0.027) when comparing the pooled PBLC feeding groups with Con. In addition, TRPV5 mRNA abundance decreased with increasing PBLC dose linearly (P = 0.010). A trend for a similar linear effect was detected for TRPA1 (P = 0.054) and TRPV6 (P = 0.062).
Electrophysiological Data
In the rumen, pre-feeding the menthol-rich PBLC did not affect baseline Gt in period P1, whereas baseline Isc increased quadratically with increasing PBLC dose (P = 0.039) and was higher in the pooled PBLC groups versus Con (P < 0.001). The omission of Na+ decreased Gt and Isc (P < 0.001; Table 3).
Table 3Effect of menthol-rich plant bioactive lipid compound (PBLC) pre-feeding and mucosal Na+ availability on baseline tissue conductance (Gt) and short-circuit current (Isc), as well as changes of these electrophysiological data (ΔGt and ΔIsc) after mucosal menthol addition ex vivo to ruminal epithelia of sheep
Gt and Isc represent baseline values from flux period P1; ΔGt and ΔIsc reflect changes of Gt and Isc between baseline values (period P1, 0–45 min) and values in the mucosal presence of 50 μM menthol (period P3, 90–135 min).
a–c P < 0.05 for individual means within each item.
1 Gt and Isc represent baseline values from flux period P1; ΔGt and ΔIsc reflect changes of Gt and Isc between baseline values (period P1, 0–45 min) and values in the mucosal presence of 50 μM menthol (period P3, 90–135 min).
2 PBLC fed at 0 mg/d (Con; n = 8), 80 mg/d (PBLC-L; n = 7), or 160 mg/d (PBLC-H; n = 8).
3 LSM (Na+) represent LSM of all treatments (Con, PBLC-L, PBLC-H) under either Na+ or NMDG+ incubation; ± SEM.
4 Con vs. PBLC = control versus pooled PBLC-L and PBLC-H groups.
5 LSM (PBLC) represent LSM of both Na+ and N-methyl-d-glucamine (NMDG+) incubation within a PBLC dose; ± SEM.
Changes in tissue conductance (ΔGt; P = 0.043) and changes in short-circuit current (ΔIsc; P < 0.001) after direct menthol addition to the mucosal side increased in a linear manner with increasing PBLC dose. The responsiveness to acute menthol stimulation was also higher in the pooled PBLC groups compared with Con for ΔGt (Con vs. PBLC, P = 0.011) and ΔIsc (Con vs. PBLC, P = 0.015). The level of ΔGt was generally lower (P < 0.001) in the mucosal presence versus absence of Na+, indicating a lesser time-dependent increase in Gt (from P1 to P3) in the mucosal presence of Na+. A significant PBLC dose × Na+ interaction was observed for ΔIsc (P < 0.001) with lowest values for Con under Na+-free conditions and highest values for PBLC-H under Na+-containing conditions on the mucosal side (Table 3).
In the jejunum, PBLC pre-feeding did not affect basal Isc and Gt values (Table 4). The omission of mucosal Na+ decreased baseline Gt and Isc in all groups (P < 0.001, each). Changes in Isc and Gt values after direct menthol application were not different among pre-feeding groups, with only a trend for higher ΔIsc in the PBLC pre-fed groups (Con vs. PBLC, P = 0.056). Time-dependent changes between P1 and P3 were more negative for ΔGt under Na+-containing conditions (P = 0.003) and more negative for ΔIsc under Na+-free conditions on the mucosal side (P < 0.001).
Table 4Effect of menthol-rich plant bioactive lipid compound (PBLC) pre-feeding and mucosal Na+ availability on baseline tissue conductance (Gt) and short-circuit current (Isc), as well as changes of these electrophysiological data (ΔGt and ΔIsc) after mucosal menthol addition ex vivo to jejunal epithelia of sheep
Gt and Isc represent baseline values from flux period P1; ΔGt and ΔIsc reflect changes of Gt and Isc between baseline values (period P1, 0–45 min) and values in the mucosal presence of 50 μM menthol (period P3, 90–135 min).
LSM (PBLC) represent LSM of both Na+ and N-methyl-d-glucamine (NMDG+) incubation within a PBLC dose; ± SEM.
13.9 ± 0.40
12.9 ± 0.39
13.4 ± 0.41
Isc, μEq·cm−2·h−1
Na+
9.49
8.05
4.84
7.46 ± 1.63
0.72
0.32
0.91
<0.001
0.079
NMDG+
−12.9
−14.7
−6.05
−11.2 ± 1.66
LSM (PBLC)
−1.69 ± 2.02
−3.35 ± 1.68
0.60 ± 2.29
ΔGt, mS·cm−2
Na+
−1.51
−0.86
−1.32
−1.23 ± 0.26
0.30
0.39
0.19
0.003
0.70
NMDG+
−0.55
−0.075
0.19
−0.14 ± 0.26
LSM (PBLC)
−1.03 ± 0.32
−0.47 ± 0.31
−0.56 ± 0.33
ΔIsc, μEq·cm−2·h−1
Na+
−0.37
2.59
2.77
1.66 ± 0.77
0.079
0.20
0.056
<0.001
0.85
NMDG+
−8.01
−5.81
−6.33
−6.72 ± 0.76
LSM (PBLC)
−4.19 ± 1.18
−1.61 ± 0.84
−1.78 ± 0.71
1 Gt and Isc represent baseline values from flux period P1; ΔGt and ΔIsc reflect changes of Gt and Isc between baseline values (period P1, 0–45 min) and values in the mucosal presence of 50 μM menthol (period P3, 90–135 min).
2 PBLC fed at 0 mg/d (Con; n = 8), 80 mg/d (PBLC-L; n = 7), or 160 mg/d (PBLC-H; n = 8).
3 LSM (Na+) represent LSM of all treatments (Con, PBLC-L, PBLC-H) under either Na+ or NMDG+ incubation; ± SEM.
4 Con vs. PBLC = control versus pooled PBLC-L and PBLC-H groups.
5 LSM (PBLC) represent LSM of both Na+ and N-methyl-d-glucamine (NMDG+) incubation within a PBLC dose; ± SEM.
In the rumen, the basal Ca2+ flux rates measured in the initial flux period P1 were higher (P < 0.001) for Jms than for Jsm, resulting in a positive Jnet (Figure 2). The latter increased in a quadratic manner (P = 0.043) from 41.0 ± 1.82 nmol·cm−2·h−1 in Con to 46.7 ± 2.05 in PBLC-L and 39.5 ± 1.91 nmol·cm−2·h−1 in PBLC-H. The quadratic response of Jnet was largely mirroring a similar trend for a quadratic response in Jms (P = 0.067), whereas Jsm tended to increase linearly (P = 0.053). The replacement of mucosal Na+ by NMDG+ decreased the basal Jms and Jnet (P < 0.001, each) by approximately 30% in all feeding groups, whereas Jsm increased (P < 0.001; Figure 2).
Figure 2Effect of long-term menthol-rich plant bioactive lipid compound (PBLC) pre-feeding in a dose range of 0 mg/d (Con), 80 mg/d (PBLC-L), and 160 mg/d (PBLC-H) on baseline values in period P1 for (A) mucosal-to-serosal (Jms), (B) serosal-to-mucosal (Jsm), and (C) resulting net (Jnet) flux rates of Ca2+ in the presence (left) and absence (right) of mucosal Na+ (replaced by N-methyl-d-glucamine, NMDG+) in the rumen. Jms tended to increase in a quadratic manner (Q, P = 0.067), whereas Jsm tended to increase linearly (L, P = 0.053), resulting in a quadratic effect for Jnet (Q, P = 0.043). The omission of Na+ decreased Jms and Jnet, and increased Jsm at P < 0.001, each (***). P-values for linear (L) and quadratic effects (Q) and Con versus pooled PBLC-L and PBLC-H groups (Con vs. PBLC) are indicated for each graph. Data are given as LSM ± SEM of n = 8, 7, and 8 sheep in Con, PBLC-L, and PBLC-H, respectively.
In the jejunum, Jms and Jsm did not differ statistically across feeding groups (Figure 3); hence, no significant Jnet could be calculated. Pre-feeding of PBLC or replacement of mucosal Na+ by NMDG+ did not affect any of the measured Ca2+ flux rates in the jejunum (Figure 3).
Figure 3Effect of long-term menthol-rich plant bioactive lipid compound (PBLC) pre-feeding) in a dose range of 0 mg/d (Con), 80 mg/d (PBLC-L), and 160 mg/d (PBLC-H) on baseline values in period P1 for (A) mucosal-to-serosal (Jms) and (B) serosal-to-mucosal (Jsm) flux rates of Ca2+ in the presence (left) and absence (right) of mucosal Na+ (replaced by N-methyl-d-glucamine, NMDG+) in the jejunum. Jms and Jsm did not differ statistically; hence, no significant Jnet could be calculated. Menthol-rich PBLC pre-feeding had no influence on Jms and Jsm fux rates. No significant effect of Na+ could be observed in the jejunum. P-values for linear (L) and quadratic effects (Q) and Con versus pooled PBLC-L and PBLC-H groups (Con vs. PBLC) are indicated for each graph. Data are given as LSM ± SEM of n = 8, 7, and 8 sheep in Con, PBLC-L, and PBLC-H, respectively.
After direct menthol application on the mucosal side of the epithelium, we subtracted the Ca2+ flux rates after menthol (P3) addition from the baseline Ca2+ flux rates before menthol addition (P1) to detect changes of Ca2+ flux rates (ΔJ).
In the rumen (Figure 4), menthol addition did not acutely affect ΔJsm but increased ΔJms flux rates linearly in all feeding groups (P = 0.008). This resulted in a linear increase of ΔJnet (P = 0.038). When comparing the pooled PBLC-L and PBLC-H feeding groups with the Con group, responsiveness of ΔJnet to menthol was higher in PBLC-prefed animals (Con vs. PBLC, P = 0.008). In line with the smaller increases in ΔGt mentioned earlier, ΔJsm were also smaller in the presence of mucosal Na+ (P = 0.002), most likely indicating smaller time-dependent increases in passive epithelial conductance to ions, including Ca2+, when mucosal Na+ is present. The ΔJms and ΔJnet were not affected by the omission of mucosal Na+.
Figure 4Acute effects of mucosal menthol application on ruminal epithelia of sheep pre-fed with menthol-rich plant bioactive lipid compounds (PBLC) in a dose range of 0 mg/d (Con), 80 mg/d (PBLC-L), and 160 mg/d (PBLC-H) with regard to changes of (A) mucosal-to-serosal (ΔJms), (B) serosal-to-mucosal (ΔJsm), and (C) resulting net (ΔJnet) flux rates of Ca2+ in the presence (left) and absence (right) of mucosal Na+ (replaced by N-methyl-d-glucamine, NMDG+). The PBLC pre-fed animals responded to acute menthol application with higher ΔJms stimulation than Con animals (Con vs. PBLC, P = 0.001), resulting also in a higher increase of ΔJnet for PBLC pre-fed animals (Con vs. PBLC, P = 0.008). The absence of mucosal Na+ did not affect ΔJms and ΔJnet, albeit ΔJsm increased at P = 0.002 (**). P-values for linear (L) and quadratic effects (Q) and Con versus pooled PBLC-L and PBLC-H groups (Con vs. PBLC) are indicated for each graph. Data were computed from the difference of values in period P3 minus values in period P1 and are presented as LSM ± SEM of n = 8, 7, and 8 sheep in Con, PBLC-L, and PBLC-H, respectively.
In the jejunum, menthol-rich PBLC pre-feeding had no effect on changes of Ca2+ flux rates after direct menthol application (Figure 5). The omission of Na+ increased ΔJms (P = 0.025) and ΔJsm (P = 0.015) independent of PBLC pre-feeding.
Figure 5Acute effects of mucosal menthol application on jejunal epithelia of sheep pre-fed with menthol-rich plant bioactive lipid compounds (PBLC) in a dose range of 0 mg/d (Con), 80 mg/d (PBLC-L), and 160 mg/d (PBLC-H) with regard to changes of (A) mucosal-to-serosal (ΔJms) and (B) serosal-to-mucosal flux rates (ΔJsm) of Ca2+ in the presence (left) and absence (right) of mucosal Na+ (replaced by N-methyl-d-glucamine, NMDG+). Menthol-rich PBLC pre-feeding had no influence on ΔJms and ΔJsm flux rates. The omission of Na+ increased ΔJms (*P = 0.025) and ΔJsm (*P = 0.015). P-values for linear (L) and quadratic effects (Q) and Con versus pooled PBLC-L and PBLC-H groups (Con vs. PBLC) are indicated for each graph. Data were computed from the difference of values in period P3 minus values in period P1 and are presented as LSM ± SEM of n = 8, 7, and 8 sheep in Con, PBLC-L, and PBLC-H, respectively.
). Hypocalcemia results from delayed and partially insufficient activation of endogenous Ca2+-homeostatic mechanisms, which are the release of Ca2+ from internal stores (e.g., bones) and an enforcement of gastrointestinal Ca2+ absorption (
), an effective support of Ca2+-homeostatic mechanisms by management strategies is of utmost importance. One such strategy can be to take advantage of the Ca2+-absorptive capacity of the rumen (
Mechanisms and regulation of calcium absorption from the gastrointestinal tract in pigs and ruminants: comparative aspects with special emphasis on hypocalcemia in dairy cows.
) in previous studies, the main aim of the present study was to identify the mechanisms behind it. In the present report, we demonstrate that these previous findings can be explained mainly by long-term sensitization of Ca2+ absorption to acute stimulation by menthol solely in the rumen, which was not accompanied by altered mRNA expression of ruminal TRP ion channels. The study further extended our general knowledge on Ca2+ transport mechanisms in the ruminant forestomach and small intestine and expanded the current knowledge on hypocalcemia prevention measures (
The Ca2+ flux data of the present study showed much greater Jms than Jsm, indicating a high capacity for active transcellular absorption of Ca2+ from the rumen. This is in line with various studies on ruminal Ca2+ transport (
Mechanisms and regulation of calcium absorption from the gastrointestinal tract in pigs and ruminants: comparative aspects with special emphasis on hypocalcemia in dairy cows.
A Ca2+/H+ exchanger of unknown molecular identity has been postulated as a key apical entry mechanism for Ca2+ in the ruminal epithelium because Ca2+ absorption is highly responsive to the luminal presence of SCFA (
). The entry of protonated SCFA into ruminal epithelial cells is thought to provide the H+ driving force for the putative Ca2+/H+ exchanger due to acidification of the intracellular space (
). A similar explanation is established for the stimulation of ruminal Na+ absorption by the apical Na+/H+ exchanger of the ruminal epithelium, which has been identified as NHE3 on the molecular level (
). However, if apical Ca2+/H+ exchange and Na+/H+ exchange were really stimulated in parallel by SCFA via intracellular acidification, an omission of Na+ from the mucosal SCFA-containing solution should shut down H+ extrusion via NHE3 and thereby stimulate H+ extrusion via the putative Ca2+/H+ exchanger, resulting in elevated Jms and Jnet of Ca2+. By sharp contrast, we observed a decrease in Jms and Jnet after replacing Na+ with NMDG+ in the mucosal solution. The latter calls into question the functional involvement of a Ca2+/H+ exchange mechanism in ruminal Ca2+ transport.
Nonselective cation channels provide an accepted alternative for the apical entry mechanism of Ca2+ into the ruminal epithelium (
). They have been functionally identified as members of the TRP family based on the responsiveness of ruminal Ca2+ currents or flux rates to stimulation by menthol and few other PBLC (
). In the present study, application of 50 µM menthol also stimulated transepithelial Jms and Jnet of Ca2+, thus supporting the concept of TRP-mediated Ca2+ absorption from the rumen. As many of these TRP channels are also conductive for Na+ (
), replacement of Na+ by NMDG+ expectedly reduced the transepithelial current and conductance of ruminal epithelia.
The menthol-sensitive Ca2+-conducting TRPM8 was not detectable in ruminal tissue on the mRNA level in the present study on the ovine rumen, as well as in a previous study on the bovine and ovine rumen (
). Therefore, it seems unlikely that TRPM8 is directly involved in ruminal Ca2+ transport, albeit it may have a role in mechano-sensory transduction, nociception, and inflammation in the gastrointestinal tract (
). Similarly, our results exclude a role of TRPV5 and TRPV6 in ruminal Ca2+ transport. Their mRNA was not detected in the present study, which confirmed earlier studies showing no or weak mRNA abundance of TRPV5 and TRPV6 in ruminal epithelia of sheep, goat, and cattle (
suggested TRPV3 as the potential candidate for menthol-stimulated Ca2+ transport in ruminal epithelial cells. In the present study, we detected mRNA signals for the Ca2+ conductive TRPV3 and TRPA1 in the ruminal epithelium, which are both known to be stimulated by PBLC such as menthol (
). Of these, TRPV3 had the strongest mRNA expression among all investigated TRP channels. Furthermore, a recent study identified appreciable TRPV3 protein expression in the bovine ruminal epithelium (
), which could explain the stimulatory effect of SCFA on Ca2+ flux rates without postulation of a Ca2+/H+ exchanger. Because TRPV3 channel currents are inhibited by NMDG+ (
), the postulation of TRPV3 as the major apical influx mechanism for Ca2+ could also explain the decreased Jms and Jnet of Ca2+ observed after replacing Na+ by NMDG+ in the present study.
A final argument for a major involvement of TRPV3 in ruminal Ca2+ absorption is the finding that Jms and Jnet were notably higher in the present study compared with all previous studies (
). The key difference to those previous studies is that we omitted Mg2+ from all mucosal incubation solutions. It is well established that Ca2+ currents via TRPV3 can be inhibited by Mg2+ due to interferences in the pore region (
). Therefore, a higher Ca2+ absorption in the absence of Mg2+ ions is highly suggestive of TRP-mediated permeation. Admittedly, the lowering of mucosal pH to 6.4 (instead of pH 7.4) should also contribute to high Jms and Jnet due to an increased proportion of ionized Ca2+. However, a mucosal pH value of 6.4 had already been used in the previous study of
, with similar Jsm but much smaller Jms and Jnet compared with the present study. The latter underlines that avoidance of Mg2+ blockage of TRPV3 by omission of mucosal Mg2+ is the most suitable explanation for the very high Jms and Jnet in the present study.
Collectively, our results together with previous findings provide several lines of indirect support for a major role for TPRV3 in Ca2+ absorption from rumen and its stimulation by menthol, although an additional involvement of TRPA1 seems possible. As an adjunct finding, we also identified ruminal expressions of TRPM6 and TRPM7, which apparently play a central role in ruminal Mg2+ transport (
Expression and functional activity of the Na/Mg exchanger, TRPM7 and MagT1 are changed to regulate Mg homeostasis and transport in rumen epithelial cells.
Long-Term Effect of Menthol-Rich PBLC on Ruminal Ca2+ Transport
Pre-feeding of menthol-rich PBLC increased baseline Isc in the rumen quadratically with increasing PBLC dose. A similar finding was reported in a previous study (
). An increasing Isc suggests either enhanced cation absorption or enhanced anion secretion, which is essentially active and transcellular. Results could thus fit to an increased opening of TRP cation channels with subsequently increased cation absorption (
). Analyses of mRNA expression revealed no influence of PBLC dose on the expression of all investigated TRP channels in the ruminal epithelium. This does not preclude an altered expression of TRP proteins because discordances between mRNA and protein expression are sometimes noted (
). Alternatively, it could suggest that permanent exposure of the ruminal epithelium to agonists such as menthol could lead to more prominent translocation of TRP channels into the apical membrane or sustained stimulation (or both), with or without affecting their expressions. The quadratic increase in Isc with increasing PBLC concentrations was paralleled by a trend for a quadratic increase of Jms and a quadratic increase of Jnet, further supporting an increased basal TRP channel activity after 4 wk of PBLC pre-feeding. The magnitude of this increase, however, was so small that it may not quantitatively explain the increase in serum Ca2+ level in sheep (
Strikingly, menthol pre-fed individuals reacted with higher increases in tissue conductivity (ΔGt) and lesser time-dependent decreases in short-circuit currents (ΔIsc) after direct menthol application in the Ussing chamber. An increase in tissue conductivity accompanied by higher short-circuit currents, the latter being essentially transcellular, support an increased ion channel opening after menthol application and fit increased channel-mediated cation absorption. This linearly links to the finding of increased absorptive Ca2+ flux rates (ΔJms and ΔJnet) after direct menthol application in the Ussing chambers. Absorptive Ca2+ flux rates were almost doubled in both groups pre-fed with PBLC. This strongly suggests that an increased responsiveness of ruminal Ca2+ absorption to menthol stimulation is the main mechanism by which prolonged PBLC pre-feeding increases Ca2+ supply to the blood. It will have to be elucidated in further studies whether this increased responsiveness of ruminal Ca2+ absorption to acute menthol stimulation is based on increased TRP protein translation, increased integration of translated TRP channels into the apical membrane, more sustained stimulation of the TRP channel population in the apical membrane, or any combination of these possibilities.
Jejunal Ca2+ Transport
In jejunum, we detected no significant net transport of Ca2+ in any of the feeding groups. The latter agrees with 2 previous studies (
We chose jejunum as a representative intestinal segment because jejunum appears to be most important for Ca2+ absorption among all intestinal segments of ruminants (
), including the present study. By contrast, we detected no signals for the menthol-sensitive channels TRPV3 and TRPM8 on the mRNA level in jejunum. The mRNA of menthol-sensitive TRPA1 was expressed. However, menthol did not affect electrophysiological properties and jejunal Ca2+ flux rates, thereby calling into question the functional significance of TRPA1 in the jejunum of our animals. Moreover, the functional significance of TRPV5 and TRPV6 is equally questionable under the experimental conditions applied because no net Ca2+ transport from the mucosal to the serosal side was observed.
Pre-feeding the menthol-rich PBLC tended to decrease the mRNA expression of TRPA1, TRPV5, and TRPV6 linearly, with lower mRNA expression of TRPA1 and TRPV5 in the pooled PBLC groups versus Con. Given the fact that these channels did not functionally trigger any net flux of Ca2+ under control conditions, their downregulation after PBLC pre-feeding also appears functionally not important. Teleologically, however, such downregulation makes sense. Because pre-feeding of menthol-rich PBLC tends to increase Ca2+ serum levels in sheep and increases Ca2+ levels in cows (
), a surplus of Ca2+ in PBLC pre-fed sheep should lead to reduced activation of vitamin D3. This may, in turn, lower the expression of, at least, TRPV5 and TRPV6 because their expressions are under control of vitamin D3 and its metabolites (
The present study substantiated a major role of the rumen in active transcellular absorption of Ca2+. The involvement of a previously suggested Ca2+/H+ exchanger for apical Ca2+ entry is challenged, because the replacement of mucosal Na+ did not stimulate but decreased Ca2+ absorption in ruminal epithelia. Alternatively, TRP channels allow apical Ca2+ entry and, hence, transcellular absorption in the rumen. Among these, a dominant role of TRPV3 is suggested from several lines of indirect evidence, including its strong mRNA expression, sustained stimulation of Ca2+ flux rates by menthol and low pH, as well as their inhibition by NMDG+ and Mg2+. Pre-feeding a menthol-rich PBLC for several weeks had no effects on the mRNA expression of ruminal TRP channels. Nonetheless, baseline Ca2+ absorption increased and, most importantly, the acute responsiveness of ruminal epithelia to menthol stimulation almost doubled. This unique finding provides a rationale to use menthol-containing feed additives as a prophylactic strategy to prevent hypocalcemia in dairy cows. Future studies will need to fine-tune this strategy for dairy cows and prove the clinical effectiveness in the transition phase, taking into account the possibility of concurrent suppression of calcitriol-sensitive absorption pathways in the jejunum (e.g., TRPV5 and TRPV6).
ACKNOWLEDGMENTS
We acknowledge the financial support of this project from Alexander von Humboldt-Stiftung, Bonn, Germany (to A.K.P. and J.R.A.), PerfomaNat GmbH, Berlin, Germany (to J.R.A.), and an Elsa Neumann stipend of the State of Berlin, Germany (to S.G.). We gratefully thank the barn staff of the animal facility of the Freie Universität Berlin, as well as J. N. Schulte, M. Grunau, and K. Soellig (all from Freie Universität Berlin, Germany) for their assistance, and F. Stumpff (Freie Universität Berlin, Germany) for expert discussions on this experiment. K.T.S and H.S.B. are or were employed by PerformaNat GmbH. J.R.A. is co-author of patent WO2014020067A1 that relates to the subject of the study. These facts did not affect the collection and interpretation of data. Other authors have no conflict of interest.
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Mechanisms and regulation of calcium absorption from the gastrointestinal tract in pigs and ruminants: comparative aspects with special emphasis on hypocalcemia in dairy cows.
Expression and functional activity of the Na/Mg exchanger, TRPM7 and MagT1 are changed to regulate Mg homeostasis and transport in rumen epithelial cells.
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