Articles in PresS. Am J Physiol Regul Integr Comp Physiol (September 12, 2012). doi:10.1152/ajpregu.00597.2011
Modulation of vH+-ATPase is part of the functional adaptation of sheep rumen
epithelium to high-energy diet
Judith Kuzinski1, Rudolf Zitnan2, Elke Albrecht3, Torsten Viergutz4, Monika SchweigelRöntgen1
1
Research Unit Nutritional Physiology “Oskar Kellner”, Leibniz Institute for Farm Animal
Biology (FBN), Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany
2
Animal Production Research Centre Nitra, Institute of Nutrition, Division Kosice,
Komenskeho 73, 041 81 Kosice, Slovakia
3
Research Unit Muscle Biology and Growth, Leibniz Institute for Farm Animal Biology
(FBN), Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany
4
Research Unit Reproductive Biology, Leibniz Institute for Farm Animal Biology (FBN),
Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany
Running head
Modulation of vH+-ATPase
Author for correspondence:
Monika Röntgen (formerly Schweigel)
Research Unit Nutritional Physiology “Oskar Kellner”,
Leibniz Institute for Farm Animal Biology (FBN),
Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany
E-mail: roentgen@fbn-dummerstorf.de
Tel.: ++49-38208-68682
Fax: ++49-38208-68652
Copyright © 2012 by the American Physiological Society.
Abstract
Ruminal vacuolar H+-ATPase (vH+-ATPase) activity is regulated by metabolic signals. Thus,
we tested if its localization, expression and activity were changed by different feeding. Young
male sheep (n = 12) were either fed hay ad libitum (h) or hay ad libitum plus additional
concentrate (h/c) for two weeks. The vH+-ATPase B subunit signal was predominantly found
in the cell membrane and cytosol of rumen epithelial cells (REC) with basal/parabasal
phenotype. The elevated number (3-fold) of these cells in rumen mucosa of h/c-fed sheep
reflects a high proliferative capacity and, explains the 2.3-fold increase of the total number of
vH+-ATPase-expressing REC. However, in accordance with a 58% reduction of the vH+ATPase B subunit mRNA expression in h/c-fed sheep, its protein amount per single REC was
decreased. Using the fluorescent probe 2´,7´-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein
and selective inhibitors (foliomycin, amiloride), the contribution of vH+-ATPase and Na+/H+
exchanger to intracellular pH (pHi) regulation was investigated. REC isolated from h/c-fed
sheep keep their pHi at a significantly higher level (6.91 ± 0.03 vs. 6.74 ± 0.05 in h-fed
sheep). Foliomycin or amiloride decreased pHi by 0.16 ± 0.02 and 0.57 ± 0.04 pH units when
applied to REC from h-fed sheep, but the effects were markedly reduced (-88 and -33%) after
concentrate feeding. Nevertheless, we found that REC proliferation rate and [cAMP]i were
reduced after foliomycin-induced vH+-ATPase inhibition. Our results provide first evidence
for a role of vH+-ATPase in regulation of REC proliferation most probably by linking
metabolically induced pHi changes to signaling pathways regulating this process.
Keywords
Epithelial cells, proton pump, intracellular pH regulation, active transport, proliferation,
BCECF
1
INTRODUCTION
The transport capacity of the rumen epithelium for short chain fatty acids (SCFA), the main
energy source for ruminants (39), and electrolytes is known to adapt to the level of energy
intake and type of nutrition (44). The adaptation processes include morphological
transformations such as alterations of the rumen epithelium papillae number and size (44). In
addition, changes occur in the metabolic properties of rumen epithelial cells (REC) (3) and the
expression and activity of cellular transport proteins (26, 43, 44).
Ruminal transport processes are known to be energized by a highly expressed Na+/K+ATPase and modulation of the pump expression and activity by concentrate feeding and by
forage types inducing different rumen fermentation patterns have been observed (24, 25). In
addition to the Na+/K+-ATPase, the existence of functional vacuolar-type H+-ATPases (vH+ATPase) in REC has been shown by our group (1, 9). The vH+-ATPase is a multi-subunit
enzyme complex composed of the V1 sector (subunits A to H) responsible for the ATPase
activity, and the membrane inserted Vo sector (with at least subunits a to e), which functions
in H+ translocation across limiting membranes (51). The pump is present in various
intracellular compartments and required for a variety of processes, including transcytosis of
receptor-ligand complexes and other molecules, e.g., NH3/NH4+, the coupled transport of
neurotransmitters and protein breakdown (35, 42, 50). In specific cell systems (monocytes,
osteoclasts) and epithelia (renal, epididymis) a link between electrogenic H+ secretion by vH+ATPases localized on the cell membrane and ion transport and/or the regulation of cytosolic
pH (pHi) has been found (6, 19).
Normal vH+-ATPase activity has been shown to be essential for cell proliferation, growth,
viability and survival (20, 23, 29, 30, 47, 53) and increasing evidence suggests that the pump
is a main component of signal transduction pathways regulating these processes. Dechant et
al. (7) identified the vH+-ATPase as a sensor of intracellular pH (pHi) changes and novel
2
activator of protein kinase A (PKA) in response to glucose uptake and phosphorylation. The
vH+-ATPase E subunit interacts with the guanine nucleotide exchange factor Son of sevenless
1 protein (Sos1) thereby activating the mSos1-dependent Rac1 signaling pathway which is
important for growth factor receptor-mediated control of cell growth and differentiation (33).
Also, the anti-apoptotic function of the B subunit of vH+-ATPase seems to involve
modulation of the MEK1/ERK (extracellular signal-related kinases) MAP (mitogen activated
protein) kinase pathway (16, 29). On the other hand, vH+-ATPase localization and assembly
of its Vo and V1 sectors is regulated by several signals (pHi, HCO3-, pCO2 and glucose) and
pathways including PKA, AMPK, the actin-based cytoskeleton and the enzyme aldolase, all
known to link cell metabolism, proliferation and transport of substrates and ions (4, 14, 38,
42, 51).
In our previous studies we characterized rumen vH+-ATPase as an important component
for REC pHi regulation (9) and a sensor of substrate and energy availability in vitro (28).
Metabolic inhibition (MI) of REC by glucose substitution with 2-deoxyglucose (2-DOG)
and/or application of antimycin induced a strong pHi reduction (0.44 ± 0.04 pH units)
resulting to about 50% from vH+-ATPase inhibition (28). The ruminal vH+-ATPase B subunit
shows diffuse cytosolic distribution after MI. Furthermore, in sheep with prolonged energy
deficiency an impairment of the glycolytic pathway accompanied by a reduced REC vH+ATPase activity and vH+-ATPase B subunit expression was observed.
Thus, we hypothesize that REC vH+-ATPase activity and expression of the rumen
epithelium underlies metabolic regulation and can be modulated by nutrition-related factors.
This would provide an efficient mechanism to adapt rumen epithelium energy (ATP)
consumption in dependence of substrate and energy availability either directly and/or via vH+ATPase-mediated up or down regulation of energy demanding functions such as growth and
proliferation.
3
As in our previous experiments (28) the effect of substrate and/or energy deficiency has
been investigated, the aim of the present study has been to explore a putative role of the vH+ATPase in the functional adaptation of the rumen epithelium to a concentrate-supplemented
diet. To this purpose, sheep were fed either an ad libitum hay diet (h diet) or a diet consisting
of hay ad libitum and additional concentrate (h/c diet). After two weeks of these two feeding
regimes, the localization, expression and functional activity of the vH+-ATPase were
investigated in both feeding groups. REC were isolated by fractional trypsination (10) and
used to investigate the cellular vH+-ATPase expression (mRNA and protein) and
differentiation state by using quantitative real-time polymerase chain reaction (qRT-PCR),
Western blot and flow cytometry. In addition, vH+-ATPase activity was estimated from
measurements of pHi by using the fluorescence probe 2´,7´-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and by an enzymatic activity assay. Fixed rumen papillae from the
atrium ruminis were used for vH+-ATPase localization by immunohistochemistry and for
morphometric analysis.
MATERIALS AND METHODS
Materials. Medium 199, trypsin, glutamine, antibiotics (gentamycin, nystatin, kanamycin,
penicillin-streptomycin), fetal calf serum (FCS) and Dulbecco`s phosphate-buffered saline
(DPBS) were purchased from PAN Biotech (Aidenbach, Germany). HyQTase was obtained
from Thermo Fisher Scientific (Bonn, Germany). BCECF-AM and pluronic acid were from
Molecular Probes Inc. (Eugene, USA). Foliomycin and amiloride were from Sigma Aldrich
(Munich, Germany). All chemicals for Western blot analysis were purchased from Carl Roth
(Karlsruhe, Germany).
Antibodies. The monoclonal mouse antibody used in this study was specific for the 60-kDa
subunit (B subunit) of the yeast vH+-ATPase (13D11-B2, Molecular Probes, Invitrogen,
4
Darmstadt, Germany) and has been shown to detect the sheep protein specifically (1, 9).
Mouse monoclonal anti-cytokeratin 10 [DE-K10] and anti-basal cell cytokeratin [RCK103]
antibodies were obtained from abcam (Cambridge, UK). A monoclonal mouse antibody
directed against the α subunit of the sheep Na+/K+-ATPase (M7-PB-E9) was purchased from
Affinity Bioreagents (Golden, USA). Relevant secondary antibodies conjugated to Alexa
fluor 488 (Invitrogen, Darmstadt, Germany) were used for flow cytometry. For Western
blotting, a horseradish peroxidase (HRP)-conjugated antibody (sheep anti-mouse IgG)
obtained from Amersham Bioscience (Freiburg, Germany) was used.
Animals and experimental design. Twelve male castrated lambs (six months old) were fed
meadow hay ad libitum for one week. Thereafter, the animals were divided into two groups.
The control group received meadow hay ad libitum for another two weeks (h diet) and the
second group was fed with a mixed meadow hay ad libitum/concentrate diet (h/c diet) over
the same time period. The amount of the concentrate (10.2 MJ ME/kg, 16.0% crude protein,
3.2% crude fat, 9.5% crude fiber, 9.5% crude ash, 1.5% calcium, 0.5% phosphorus, 0.4%
sodium; ingredients of concentrate mixture: 15% sugar beet slices, 15% oat, 15% wheat bran,
12% wheat gluten food meal, 10% barley malt germ, 10% rape expeller, 8% corn, 6% sugar
beet molasses, 4.5% soybean solvent extracted oil meal, 3.2% CaCO3, 0.8% NaCl) was
stepwise increased from 150 to 1000 g/day and was given in two meals at 7 am and 2 pm.
Water was available ad libitum for both groups. Before starting the two-week experimental
period of the different feeding, sheep of both groups had a mean body weight of 43 ± 2 kg. At
the end of the experiment, body weight was not changed in h-fed sheep (0.7 ± 0.9 kg), but
h/c-fed sheep had gained weight (3.0 ± 0.5 kg; P < 0.05).
The experiments were conducted in accordance with German law for the care and use of
experimental animals as attested by the Animal Welfare and Ethics Representative of the
Leibniz Institute for Farm Animal Biology (FBN).
5
Sample preparation. Sheep were slaughtered at day 22 of the experiment between two and
three hours after the morning feeding.
Rumen fluid was taken from the perforated rumen immediately after slaughtering. The pH
of rumen fluid was measured with a glass electrode (N 1042A, pH meter CG 841, Schott,
Mainz, Germany) directly after sampling. The rumen fluid was then strained through 4 layers
of gauze and prepared for SCFA and ammonia analysis.
Rumen tissue pieces of at least 100 cm2 were taken from the atrium ruminis within 10 min
after slaughter and washed three to five times in ice-cold divalent-free phosphate-buffered
saline (PBS) containing penicillin-streptomycin. Then, after one wash in the same but
antibiotic-free solution, tissue pieces (1 cm2 surface) were fixed for morphometric studies, or
rumen papillae were frozen in liquid nitrogen for immunohistochemical analysis. The
remaining tissue was transferred into fresh ice-cold divalent-free PBS with penicillinstreptomycin, transported to the laboratory, and stored for one hour at 4°C before preparation
of rumen epithelial cells (REC).
Cell isolation and culture. Rumen papillae (Fig. 1A) were removed by scissors, washed
three times in divalent-free PBS with antibiotics, and then ones in antibiotic-free PBS without
Ca2+/Mg2+. Thereafter, fractional trypsination as described by Galfi et al. (10) was performed
in order to detach REC from the underlying connective tissue. Over a time period of 6 h cell
fractions were harvested every 30 min (fractions 1 to 10) and their composition was evaluated
by light microscopy. Fractions one and two (mostly consisting of cellular detritus and cells
from the stratum corneum; SC, Fig. 1B) were discarded. Starting with fraction 3, the cell
suspensions contained increasing numbers of small round cells known to represent cells from
stratum basale (SB) and stratum spinosum (SS) of the epithelium (Fig. 1B). However, more
differentiated cells from the upper strata (stratum granulosum; SG, SC, Fig. 1B) of the
epithelium were also present in all fractions. As the proportion of this functionally different
cell types seems to be relatively similar in fractions 3 to 5 (F3-5), 6 to 8 (F6-8), and 9/10
6
(F9/10), they were pooled. Also samples pooled from fractions 3 to 10 (F3-10) were prepared
for further analysis. All samples were washed two-times in DPBS containing 1% penicillinstreptomycin and re-suspended in 10 ml of antibiotic-free DPBS. Cell number and diameter
were determined by the use of a cell counter (Countess, Invitrogen, Darmstadt, Germany).
Samples of pooled fractions (F3-5, F6-8, F9/10, F3-10) were fixed with methanol (1x107
cells each) for flow-cytometric analysis for vH+-ATPase, Na+/K+-ATPase, basal cell
cytokeratin and cytokeratin 10 abundance or used to extract total protein or RNA. The protein
and RNA samples were stored at -80°C until analysis by Western blot and quantitative reverse
transcription with the polymerase chain reaction (qRT-PCR).
The remaining REC were grown in Medium 199 containing 15% FCS, 1.36 mM
glutamine, 20 mM HEPES and antibiotics (50 mg/l gentamycin, 100 mg/l kanamycin, 2.4x105
U/l nystatin) in an atmosphere of humidified air-5% CO2 at 38°C. From day 2 of culture, the
medium was nystatin-free and contained 10% FCS only. Then, 5-6 days after seeding,
fluorescence spectroscopic measurements of intracellular pH (pHi) were performed to
determine vH+-ATPase and NHE activity.
Rumen fluid analysis. For SCFA analysis, a mixture of 5 ml rumen fluid and 2 ml isocapronic acid (internal standard) was centrifuged at 3000 g at 4°C for 20 min. The supernatant
was then filtered (0.22 µm pore size) to measure the SCFA concentration by gas
chromatography (Shimadzu GC-14A, Shimadzu Corporation, Kyoto, Japan) on a capillary
column (Free Fatty Acid Phase, 25 m × 0.25 mm, Machery-Nagel GmbH & Co. KG, Düren,
Germany). Additionally, the ammonia concentration was determined by the microdiffusion
method.
Light microscopy and morphometry of rumen papillae. Samples from the atrium ruminis
were fixed in 4% neutral formaldehyde solution. After being rinsed with water, they were
dehydrated in a graded series of ethanol (30%, 50%, 70%, 90% and absolute ethanol), cleared
with benzene, and then saturated with and embedded in paraffin. At each sampling, sections
7
of 5 µm thickness were made of 30 papillae and stained with hematoxylin/eosin. The length
and width of papillae were determined by the computer-operated Image C image analysis
system (Imtronic GmbH, Berlin, Germany) and the IMES analysis program, by using a color
video camera (SONY 3 CCD, Sony Electronics Inc., Tokyo, Japan) and a light microscope
(Axiolab, Carl Zeiss Jena, Germany). The number of papillae per cm2 mucosa was estimated
by using a video camera equipped with an image analysis system. The total surface of papillae
per cm2 mucosa was determined as the length x width x 2, multiplied by the number of
papillae/cm2. In addition, the widths of the ruminal mucosa and of the SC were measured.
Immunohistochemistry. Rumen papillae were frozen in liquid nitrogen and cryosectioned
on a Leica CM3050 S (Leica, Bensheim, Germany). Sections (7 µm) were fixed in 4%
paraformaldehyde for 20 min and washed three times with PBS, permeabilized by incubation
for 20 min in PBS containing 0.1% Triton X-100, and blocked with 10% rabbit serum in
PBS+Triton X-100 for 15 min (all at room temperature). Subsequently, sections were
incubated 2 h at room temperature with primary antibody (diluted 1:50 with 2% serum in
PBS+Triton X-100) in a humidity chamber. After being washed three times, sections were
incubated for 45 min at room temperature in the dark with an appropriate secondary antibody
labeled with Alexa Fluor 488 (Molecular Probes, Eugene, OR) diluted 1:500 in PBS+Triton
X-100. Nuclei were usually counterstained with 1 µg/ml propidium iodide in PBS. Sections
were covered with MobiGLOW mounting medium (MoBiTec, Göttingen, Germany) and
appropriate cover-slips. Sections incubated with serum in PBS+Triton X-100 instead of
primary antibody were used as negative controls and showed no unspecific binding of
secondary antibodies in REC. Immunofluorescence was detected by using a Nikon Microphot
SA fluorescence microscope (Nikon Instruments Europe B.V., Netherlands) and an image
analysis system equipped with CELL^F image analysis software and a CC-12 high resolution
color camera (OSIS, Münster, Germany).
8
Detection of vH+-ATPase subunit B subunit E and GAPDH mRNA transcripts by qRTPCR. Total RNA was isolated by a standard procedure (Total RNA isolation kit and
manufacturer’s protocol, Macherey & Nagel, Düren, Germany). The concentration and
quality of the extracted RNA were measured by using a NanoDrop ND-1000
Spectrophotometer (Peqlab Biotechnology GmbH, Erlangen, Germany). The ratios of
absorbance at 260 and 280 nm of all preparations were about 2.0. The integrity of RNA was
checked by denaturing agarose gel electrophoresis and ethidium bromide staining. The
iScriptTM cDNA Synthesis Kit (Bio-Rad Laboratories GmbH, Munich, Germany) was used
to synthesize cDNA from 100 ng total RNA from each sample according to the
manufacturer’s instructions. A negative control, without reverse transcriptase, was processed
for each sample to detect possible contamination with genomic DNA or environmental DNA.
The abundance of mRNA for the ribosomal protein S18 (RPS18), for glyceraldehyde-3phosphate-dehydrogenase (GAPDH), and for the vH+-ATPase B and E subunit was quantified
by qRT-PCR by using an iCycler and the iQ-SYBR Green supermix (Bio-Rad Laboratories
GmbH, Munich, Germany) as described previously (48). Briefly, 1-μl aliquots of each RT
reaction (1/20 of total) were primed, in each 10-µl PCR, with gene-specific oligonucleotides
at a final concentration of 0.2 µM. The sequences of specific bovine primers used are
summarized in Table 1. The primers were designed to span a corresponding intron and to
anneal at 60°C to the published cDNA and gene sequences. PCR was performed over 40
cycles for 180 s at 94°C and 10 s at 94°C, followed by 30 s at 60°C and 45 s at 70°C. The
specificity of amplification was determined by melting curve analysis and agarose gel
electrophoresis in comparison with an oligonucleotide molecular mass ladder to confirm that
the calculated molecular mass of the cDNA corresponded to the produced cDNA. The cDNA
structure was checked by sequencing. Each cDNA was quantified in duplicate; the average
value of each sample value minus the corresponding negative control value was used to
calculate the cDNA product corresponding to the abundance of mRNA. The amounts of vH+-
9
ATPase B subunit, E subunit and GAPDH mRNA were normalized against the housekeeping
gene RPS18.
Western blot analysis. For Western blots, total protein from freshly isolated and washed
REC was extracted by using the Mammalian Protein Extraction Reagent (M-PER; Pierce,
Bonn, Germany), complemented with Halt™ protease inhibitor cocktail (Pierce, Bonn,
Germany). The protein concentration was determined by using the Bradford assay (Bio-Rad,
Munich, Germany). Protein samples (25 µg) were separated by SDS (12.5%)-polyacrylamide
gel electrophoresis and subsequently transferred to polyvinylidene fluoride (PVDF)
membranes (GE Healthcare, Munich, Germany). After transfer, membranes were blocked
with 3% non-fat dry milk in PBS (pH 7.5) containing 0.05% Tween 20 (PBS-T) for 2 h and
washed twice for 5 min in PBS-T. Thereafter, membranes were incubated at 4°C with the
primary antibodies (anti-vH+-ATPase: 1:600 dilution) overnight, washed three times (1 x 15
min, 2 x 5 min) with PBS-T, and incubated for 1 h with HRP-conjugated secondary antimouse IgG (1:10,000 dilution) antibody. Then, after three washes (1 x 10 min and 2 x 5 min)
in PBS-T, membranes were developed with ECL Western Blotting Substrate (Pierce, Bonn
Germany) on X-ray films for 3 min. For size comparison, Precision Plus Protein WesternC
Standard (161-0376, Bio-Rad, Munich, Germany) and the Precision Protein StrepTactin-HRP
Conjugate (161-0381, Bio-Rad, Munich, Germany) were used. The X-ray films were scanned,
and the density quantification was performed by the software ImageJ 1.41 (National Institutes
of Health).
Flow cytometry. Methanol-fixed REC were incubated overnight at 4°C with anti-vH+ATPase (12.5 µg/ml) or anti-Na+/K+-ATPase (10 µg/ml) antibody dissolved in 10 mM PBS
with 0.2% BSA and 1 mM EDTA, pH 7.3. After being warmed to room temperature, cells
were washed twice in PBS-EDTA and incubated for 1 h in a 200-fold dilution (4 µg/ml) of
anti-mouse-IgGF(ab`)2 conjugated to Alexafluor 488 (Molecular Probes, Eugene, USA). The
primary antibodies were omitted from control incubations. After a further two washes in PBS-
10
EDTA, quantitative analysis of cellular fluorescence was carried out in an argon-laserequipped flow cytometer (Coulter-XL, Beckmann, Krefeld, Germany) to analyze the cells (3
x 10000 per sample) simultaneously according to size, granularity, and vH+- or Na+/K+ATPase abundance (portion of protein-expressing cells and relative fluorescence intensity per
cell). Cytokeratin 10 and basal cell cytokeratin abundance was determined in the same way,
but the incubation with the specific antibodies (7.5 µg/ml) was performed for 2 hours only,
and secondary fluorescein-isothiocanate-conjugated anti-mouse-IgG (4 µg/ml) antibodies
were employed.
Enzymatic ATPase activity assay. All steps were performed at 4°C. REC were immersed in
homogenate medium (in mM): 20 HEPES, 100 sucrose, and 0.25 EDTA, pH 7.4. They were
then homogenized for 20 s by using ceramic globules (∅ 1.4 mm; Peqlab, Erlangen,
Germany) and the FastPrep FP 120 Bio 101 cell disrupter at level 4. The homogenate was
centrifuged for 5 min at 1000 g and the resulting supernatant for 10 min at 10,000 g. After
determination of the protein concentration of the second supernatant by using the Bradford
assay (Bio-Rad Laboratories GmbH, Munich, Germany), the supernatant was stored at -80°C
until the ATPase assay.
The vH+-ATPase and Na+/K+-ATPase activity was measured by a modified coupledenzyme assay. To nine wells of a generic 8x12 well plate, we added 189 µl reaction buffer, 5
µl ATP-MgCl2 solution, and 2 µl lactate dehydrogenase (LDH)/pyruvate kinase (PK) mixture
(1230/ 986 units/ml). To three wells, either ouabain (1 mM) or foliomycin (2.4 µM) was
applied to determine Na+/K+- and vH+-ATPase-related activity. The final 200 µl assay
mixture contained (in mM) 125 Tris buffer, 1 EGTA, 12.5 KCl, 125 NaCl, 0.5 sodium azide,
2.5 phosphoenolpyruvate, 0.5 NADH, 5 ATP-MgCl2, and 2 units each of LDH and PK. The
reaction buffer was prepared in advance with NADH, sodium azide and phosphoenolpyruvate
being added on the day of assay. The assay was started by adding 4 µl cell homogenate
followed by orbital shaking of the plate for 1 s. The microplate was read at a wavelength of
11
340 nm and a temperature of 25°C in a victor3 multilabel counter (Perkin Elmer) at 12 s
intervals for 8 min. The ATPase activity was calculated by the following equation: ATPase
activity (µmol Pi/mg/h) = [slope (OD units/h) / 6.22 (OD units/ml/µmol)] x [0.2 ml/protein
(mg)].
Solutions for pHi measurements. Control experiments were performed in HCO3--free
HEPES-buffered measuring solution (in mM): 125 NaCl, 20 Na-butyrate, 5 KCl, 1 CaCl2, 2
MgCl2, 5 glucose, and 10 HEPES, pH 7.1. The osmolarity was adjusted to 280 mOsmol/kg by
using D-mannit. All experiments were performed in the nominal absence of CO2/HCO3- to
suppress Na+-HCO3- symporter related pHi regulation (34). Foliomycin (2 µM) and amiloride
(250 µM), respectively, were used to differentiate vH+-ATPase- and Na+/H+ exchanger
(NHE)-dependent H+-secretion.
Measurement of pHi by spectrofluorometry. For the determination of pHi, cells were loaded
with 1 µM BCECF-AM for 30 min and subsequently washed twice in DPBS. REC were
incubated for a further 30 min to allow complete de-esterification and washed twice before
measurement of fluorescence. Intracellular pH was detected by measuring the fluorescence of
the probe-loaded REC in a spectrofluorometer (LS-50 B, Perkin-Elmer, Rodgau, Germany)
equipped with a fast-filter accessory that allowed fluorescence to be measured at 20-ms
intervals with excitation for BCECF at 440 and 480 nm and emission at 515 nm. All
measurements were made at 37°C in a 3-ml cuvette containing 2 ml cell suspension (10%
cytocrit) under stirring. BCECF signals were calibrated for pH by placing the cells in medium
containing 135 mM KCl and the ionophore nigericin (10 µM) to equilibrate intra- and
extracellular [H+]. The procedure was repeated for various pH values between 6.0 and 8.0.
For data evaluation, 10-s data sets equivalent to 500 data points were each averaged at the
beginning of the measurement and then in 50-s intervals. The final pHi was determined as the
mean pHi of the last 10 s of the measurement.
Determination of REC proliferation and intracellular cAMP concentration. Sheep REC were
12
isolated (F3-10) from rumen material obtained at a local slaughter house and cultivated for 3
to 4 days as described above. Proliferation rate and intracellular cAMP concentration
([cAMP]i) were determined in REC (106 cells/ml) seeded in 96-well plates (100 µl per well)
and incubated for 18 hours in FCS-free, glutamine supplemented Medium 199 containing a
normal (5.5 mM) or strongly reduced (0.1 mM) glucose concentration. Thereafter, foliomycin
(2 µM) was added to respectively half of the wells with normal or reduced glucose
concentration. Then, after 30 min, REC [cAMP]i was measured by use of an enzyme-linked
immunoassay system (cAMP Biotrak EIA System, GE Healthcare, UK) according to the
protocol of the manufacturer.
To determine REC proliferative capacity, cells were provided with fresh media after the
18-hours incubation period and foliomycin (2 µM) was added as for [cAMP]i determinations.
However, after 30 min, 10 µl of the cell proliferation reagent WST-1 (Roche Diagnostics
GmbH, Mannheim, Germany) was added to each well and the absorbance (at 440 nm) of the
WST-1 formazan product was measured immediately. Afterwards, absorbance readings were
repeated every 30 min for 4 hours and the last measurement was performed 6 hours after
WST-1 application. The measured absorbance was corrected for background (blanks
containing the respective media only) and its development reflects the metabolic activity of
REC.
Statistical analysis. Unless not otherwise stated, data are presented as means ± standard
error (SE). Significance was determined by Student`s t-test or the paired t-test as appropriate.
P < 0.05 was considered to be significant. All statistical calculations were performed by using
SigmaStat (Jandel Scientific).
13
RESULTS
Ruminal fluid analysis. Compared with the h-fed (control) group, the feeding of additional
concentrate led to an increased concentration of rumen fluid ammonia and total SCFA
accompanied by a reduced pH value (Table 2). The profile of SCFA changed to significantly
higher molar proportions of propionate and butyrate resulting in a decrease of the acetate to
propionate ratio in h/c-fed compared with h-fed sheep.
Epithelial morphology and REC characterization. Figure 1A shows the different
morphologies of rumen tissue samples taken from the atrium ruminis of both diet groups. An
increase of the length (P < 0.001) and width (P < 0.01) of the rumen papillae in h/c-fed (4.6 ±
0.2 and 2.2 ± 0.1 mm) vs. h-fed (3.4 ± 0.2 and 1.9 ± 0.1 mm) sheep was evident even at the
macroscopic level (Fig. 1A). The thickness of the metabolically active cell layers (SB to SG,
Fig. 1B) was not different between the feeding groups (Fig. 1C). However, compared with
the compact SC (28.5 ± 1.4 µM) seen in h-fed sheep, the rumen mucosa of sheep fed
additional concentrate was characterized by a predominant SC (44.5 ± 1.7 µM) containing
numerous so-called balloon cells (Figure 1B and 1C).
The proportion of cornified cells (REC not positive for Na+/K+-ATPase) was 5 ± 1% and
11 ± 3% in F3-10 from h- and h/c-fed sheep, respectively. Moreover, rumen epithelia from
h/c-fed sheep contained an elevated number of cytokeratin 10-positive and basal cytokeratinpositive cells (1.3 ± 0.2 x 107/ml and 3.0 ± 0.5 x107/ml) compared with that (0.4 ± 0.09
x107/ml and 1.1 ± 0.2 x107/ml) from h-fed sheep (Fig. 1D).
Protein expression of the vH+-ATPase B subunit in rumen epithelium. Western blot
analysis of REC revealed a ~60-kDa band representing the vH+-ATPase subunit B protein
(Fig. 2A). Also, by using flow cytometry (Fig. 2B), the vH+-ATPase protein was detected in
51 ± 3% and 59 ± 3% of REC from h-fed and h/c-fed sheep, respectively. The difference
between feeding groups resulted mainly from a higher percentage of vH+-ATPase-positive
14
REC in fractions 3 to 5 (62 ± 7 vs. 49 ± 7%) and 6 to 8 (60 ± 5% vs. 53 ± 3%) obtained from
h/c-fed sheep.
The vH+-ATPase-specific fluorescence intensity per single cell, a measure of the singlecell amount of vH+-ATPase subunit B protein, was 6.7 ± 1.0 arbitrary units (AU) in REC
from h-fed sheep and fell to 5.7 ± 0.6 AU after concentrate feeding (Fig. 2B). The reduction
was most prominent in F6-8 and F9/10 from h/c-fed (5.0 ± 1.0 AU and 6.4 ± 1.0 AU)
compared with h-fed (6.2 ± 1.5 AU and 8.6 ± 2.2 AU) sheep.
Figure 3A shows that the rumen epithelium of h/c-fed sheep contains a higher absolute
number of vH+-ATPase-positive REC with significant differences between feeding groups
(5.0 ± 0.4 x107/ml vs. 1.5 ± 0.5 x107/ml) in F9/10. Next, in REC homogenates from F9/10
vH+-ATPase and Na+/K+-ATPase activity were measured by an enzymatic assay showing an
approximately 9-fold higher enzymatic activity of the latter. The vH+-ATPase activity
amounted to 6.3 ± 3.8 and 13.0 ± 4.2 µM Pi / mg / h in REC homogenates from h-fed and h/cfed sheep, respectively, and accounted for 4.1 ± 1.6% of total ATPase activity under control
conditions and for 21.6 ± 5.9% in the h/c-fed group (Fig. 3B).
Differences between feeding groups were confirmed by Western blot analysis and
downstream quantification of the signal with ImageJ software (Fig. 4). Characteristic Western
blots of whole-cell protein extracts obtained from F3-5, F6-8 and F9/10 REC populations
from h-fed or h/c-fed sheep are given in figure 4 (inset). A stronger (76 ± 26%) expression of
the ~60-kDa band representing the vH+-ATPase subunit B protein in the F3-5 REC population
of h/c-fed sheep was observed. In contrast, the signal intensity was not different between the
F6-8 and F9/10 REC populations obtained from differently fed sheep. Additionally, the vH+ATPase B subunit protein was most abundant in F9/10 REC populations from both feeding
groups, however, a remarkable increase by 129 ± 17% from F3-5 to F9/10 was observed in
the h-fed group only.
15
Detection of vH+-ATPase B and E subunits mRNA. PCR revealed vH+-ATPase subunit B
transcripts in REC (Fig. 5A). The product obtained corresponded to the calculated base
number (249 bp) of the sequence produced by the primers. The results were confirmed by
sequencing the products. The sequence obtained was compared with the bovine sequence that
had been used for primer design, yielding an identity of 95%. Figure 5B summarizes results
concerning the expression of vH+-ATPase B subunit mRNA. The average concentrations of
vH+-ATPase B subunit mRNA were 0.183 ± 0.017 and 0.077 ± 0.011 pg per pg RPS18
mRNA control in the h-fed and h/c-fed REC populations, respectively, and were thus strongly
reduced (P < 0.001) in the latter. Also, as shown in Figure 5B (inset), there is a tendency for
vH+-ATPase E subunit mRNA to be reduced (0.26 ± 0.03 pg / pg RPS18 mRNA) in REC
from h/c-fed compared with that from h-fed sheep (0.34 ± 0.04 pg / pg RPS mRNA).
In h-fed sheep the mRNA content for the B and E subunits of vH+-ATPase increased by
25 and 39% from F3-5 to F9/10, respectively, and parallels the observed increase in vH+ATPase B subunit protein. In h/c-fed sheep, however, a 84% increase from F3-5 to F9/10 was
seen for the E subunit mRNA only, whereas for the B subunit a maximum mRNA level of
0.09 ± 0.03 pg / pg RPS18 mRNA was already found in F6-8. Compared to this value, the B
subunit mRNA concentration was down-regulated by 18% in 9/10.
REC intracellular pH (pHi) in sheep fed the h- or h/c diet. BCECF-loaded REC from h- or
h/c-fed sheep were suspended in HEPES-buffered Na-medium with Na-butyrate and,
subsequently, the pHi was measured over a 10-min period. The initial and end pHi measured
in REC from h/c-fed sheep were 6.91 ± 0.03 and 7.16 ± 0.03 and thus, significantly higher
than those (6.74 ± 0.05 and 6.94 ± 0.04) from h-fed control sheep (Fig. 6). In both groups, a
butyrate-induced pHi recovery was observed that amounted to 0.24 ± 0.03 and 0.31 ± 0.06 pH
units in control and h/c-fed REC, respectively.
Role of vH+-ATPase in regulating the pHi in sheep fed the h- or h/c diet. The pHi of
BCECF-loaded REC from h- or h/c-fed sheep was measured in either HEPES-buffered Na-
16
butyrate medium (control) or in the same medium containing foliomycin, amiloride or both
inhibitors over a 10-min period. Characteristic original traces showing the time course of REC
pHi for control conditions and after foliomycin or amiloride treatment are shown in figure
7A. Figure 7B presents a summary of the pHi reduction induced by foliomycin or amiloride
alone or by a combination of both inhibitors. In Na+-containing media, a Na+/H+ exchanger
(NHE) is known to be active in REC (9). Therefore, amiloride was used to determine this
NHE-related pHi component. As shown in figure 7B, the pHi of foliomycin-treated REC was
reduced by 0.16 ± 0.02 pH units in control REC. In addition, the application of amiloride
induced a strong pHi decrease amounting to 0.57 ± 0.04 pH units. The effects of both
inhibitors (-0.70 ± 0.06 pH units) were additive in REC from h-fed sheep. In contrast, the
inhibitory effect of foliomycin was negligible (-0.02 ± 0.04 pH units), and the application of
amiloride (-0.38 ± 0.04 pH units) or of both inhibitors (-0.45 ± 0.05 pH units) was less
effective in REC from h/c-fed sheep.
Effect of foliomycin on REC proliferation and intracellular cAMP concentration ([cAMP]i). A
direct link between vH+-ATPase and REC proliferative activity has never been shown.
Therefore, we here performed a first in vitro experiment and investigated the effect of energy
availability and of the vH+-ATPase inhibitor foliomycin on REC proliferation and [cAMP]i.
Lowering the media glucose concentration from 5.5 mM (control) to 0.1 mM reduced the
production of WST-1 formazan and thus, the absorbance increase seen over the 6-hrs
measuring period (Fig. 8A). This increase reflects the metabolic activity/proliferation rate of
REC and was reduced from 0.56 ± 0.17 units under control conditions to 0.21 ± 0.03 units in
low glucose media. As shown in figure 8B, foliomycin not only decreased the proliferation
rate (control: -26 ± 5%, low glucose: -32 ± 4%) but also the [cAMP]i (control: -25 ± 1%, low
glucose: -74 ± 9%) under both conditions.
17
DISCUSSION
The adaptation of the rumen epithelium to feeding a mixed hay/concentrate diet has been
verified by the characteristic morphological changes, such as the increased length (109%) and
width (79%) of rumen papillae and increased width (55%) of the SC, as has also been found
by other investigators (12, 44, 54). Together, such changes should help to increase the
absorptive capacity, in particular for ruminally produced SCFA (12, 43), and to ensure the
protective function of the epithelium.
It has long been known that factors derived from rumen fluid, particularly SCFA, are
important signals contributing to the initiation of diet-induced morphological transformations
(11, 54). On concentrate feeding, rumen fluid butyrate and propionate concentrations are
increased, as shown in this and other studies (15, 44). In vivo, butyrate acts as a reversible
mitosis inhibitor, thereby initiating and accelerating the process of REC differentiation ending
up with keratinization (11). The more rapid differentiation of REC from sheep fed the h/c diet
is reflected by an elevated number of cells positive for the differentiation marker cytokeratin
10 appearing in cells from the upper SS to SG (8). Moreover, intraruminal infusion of
butyrate induces REC proliferation in vivo (37); this is an indirect effect, most probably via
growth promotors such as insulin and insulin-like growth-factor-1 (IGF-1) and/or modulation
of the expression of ruminal IGF-binding proteins (2, 41, 45).
At the cell level, the rumen epithelium of sheep fed the h/c diet show an elevated number
of basal cytokeratin-positive cells indicating their high proliferation and/or survival rate
during adaptation to a high-energy diet (15). Whereas the number of REC with
basal/parabasal phenotype was nearly constant in F3-5, F6-8 and F9/10 of h-fed sheep (1.1 ±
0.2 x107/ml), it increased from 0.9 ± 0.2 x107/ml in F3-5 to 4.3 ± 0.5 x107/ml in F9/10 of h/cfed sheep and shows a high correlation (correlation coefficient of 0.998; P < 0.05) with the
total number of vH+-ATPase B subunit positive REC. When compared with REC isolates (F3-
18
10) from the rumen epithelium of h-fed sheep, those from h/c-fed sheep are characterized by
an increased (2.3-fold) total number of cells (4.1 ± 0.4 x107/ml vs. 1.8 ± 0.3 x107/ml; P <
0.01) expressing vH+-ATPase B subunit protein. The difference between feeding groups is
significant for F9/10 and, in agreement with this, a 2-fold elevation of total vH+-ATPase
enzymatic activity was found in F9/10 REC homogenates from h/c- compared with h-fed
sheep. These results suggest that the pump is mainly expressed in undifferentiated, basal
cytokeratin-positive cells.
To verify the epithelial localization of the vH+-ATPase B subunit, we performed
immunohistochemical experiments (Fig. 9). Indeed, in both feeding groups the vH+-ATPase
signal mostly occurs in the cell membrane and cytoplasm of basal and parabasal REC with
only minor signal in the upper layers of the epithelium. In agreement with these results, we
demonstrate that vH+-ATPase B and E subunits mRNA are expressed throughout all REC
populations, although at lower levels in REC that were at higher stages of differentiation (F35 REC populations). Protein expression was investigated for the B subunit only. In
accordance with its mRNA expression, vH+-ATPase B subunit protein expression peaked in
F9/10 in both feeding groups.
The increased expression of vH+-ATPase mRNA and protein in REC with basal/parabasal
phenotype, the marked increase of this cell type in the rumen epithelium of h/c-fed sheep and
the predominant localization of the vH+-ATPase signal in REC originating from the
germinative cell layers, suggest a role in the regulation of rumen epithelium proliferation and
regression processes.
Cell division is an energetically demanding process that can executed only if cells have
sufficient resources to support doubling of their mass. However, to date, there has been little
advance in describing the molecular basis and functional mechanisms linking rumen
epithelium proliferation processes with energy (ATP) and/or substrate availability during diet
adaptation.
19
In addition to other factors, a high pHi is prerequisite for cell proliferation (16) and linked
to energy and substrate metabolism (7). Inactivation of REC ATP production and glycolytic
flux reduced vH+-ATPase activity and pHi (28), a condition that has been shown to inhibit
REC proliferation (13, Fig. 8A of this study). In contrast, feeding with a concentrate diet leads
to an increase in rumen SCFA and glucose oxidation rates (18) and, in agreement with a
growth promoting role of pHi, REC from h/c-fed sheep regulate their pHi at a significantly
higher level (6.91 ± 0.03) than h-fed control sheep (6.74 ± 0.05).
Unexpectedly, vH+-ATPase-related, foliomycin-sensitive H+ secretion does not directly
contribute to this effect and was markedly reduced by 88% in REC from h/c-fed sheep.
Nevertheless, we found that REC proliferation rate and [cAMP]i were reduced after
foliomycin-induced vH+-ATPase inhibition. Our results are in agreement with a recent report
(7), proposing that metabolically induced pHi changes affect the vH+-ATPase assembly state
followed by downstream inhibition or activation of the cAMP-dependent protein kinase A
(PKA) pathway known to be involved in growth control. An association of cAMP-dependent
PKA catalytic subunit beta with ruminal tissue maintenance has been found by Taniguchi et
al. (46). However, in further studies it has to be shown more directly if vH+-ATPasedependent stimulation of cAMP signaling is important for the initiation and/or maintenance of
the high proliferative capacity observed in sheep adapting to high-concentrate diets.
Interestingly, in a cell model, Wang et al. (49) has been shown that SCFA (acetate, propionate
and butyrate) are able to stimulate cAMP accumulation; an effect that was inhibited by overexpression of the bovine G protein-coupled receptors (GPR) 41 and 43, also known as free
fatty acid receptor 3 and 2, respectively.
In sheep and cattle, regulation of the actin cytoskeleton is one of the most effected
pathways during adaptation to high-concentrate diet (46) and cytoskeleton-related proteins
have been shown to be up-regulated after feeding a concentrate-supplemented diet (5). Via
direct binding to F-actin (51), vH+-ATPase subunits B and C can modulate actin cytoskeleton
20
remodeling an essential process for normal proliferation (4, 22). An increased content of Factin can lead to hyperproliferation (22).
The vH+-ATPase E subunit has been shown to regulate EGF-dependent cellular DNA
synthesis and mitogen-activated protein kinase activation via binding to the guanine
nucleotide exchange factor mSos1 (33). Our results on vH+-ATPase E subunit mRNA
expression showing increasing amounts from F3 to F9/10 in both feeding groups give rise to
the hypothesis, that it could thus modulate the sensitivity of functional different REC types to
known growth promotors such as EGF, Insulin and IGF-1 (2, 41, 44, 45).
The reduction of apoptosis is another important factor that can increase cell accumulation
in rumen mucosa (32, 36; 45). For example, by down-regulation of IGFBP3 butyrate is
thought to slow apoptosis thereby encouraging rumen tissue growth (32, 45). In various cell
types inhibition of the vH+-ATPase were accompanied by a higher degree of apoptosis (23,
52). Moreover, an anti-apoptotic function for the B subunit of vH+-ATPase has been proposed
by various groups (16, 29). In our previous study (28), the expression of the regulative vH+ATPase B subunit protein was drastically reduced or absent in rumen epithelium protein
extracts from sheep with prolonged energy deficiency. Here, when compared with h-fed
sheep, we found a higher total number of vH+-ATPase B subunit expressing cells and, in F3-5
REC populations, also a higher amount of vH+-ATPase B subunit protein, which could mean
a lower rate of apoptosis. Although we have not investigated the apoptotic index of REC
populations from differently fed sheep in this study, we hypothesize that by changing the
number of rumen vH+-ATPase B subunit expressing REC in the epithelium the apoptotic rate
could be modulated.
In this context it is interestingly to note that in our study a significant decrease of the
amount of GAPDH mRNA (0.068 ± 0.009 pg per pg RPS18 mRNA vs. 0.098 ± 0.011 pg per
pg RPS18 mRNA) has been found in REC from h/c- compared with REC from h-fed sheep.
In addition to its role in glycolysis, GAPDH acts as apoptosis activator (17). A decrease of
21
GAPDH mRNA has also been reported in the liver of cows fed 120% of the predicted energy
requirements and in hyperinsulinemic cows (40) making it possible that, in addition to the
factors named above, the enzyme is involved in the regulation of rumen epithelium
proliferative capacity by influencing the degree of apoptosis.
Interestingly, as for Na+/K+-ATPase (27), a reduction of the vH+-ATPase B and E subunit
mRNA abundance and of the vH+-ATPase B subunit protein amount per single cell has been
found in REC populations from h/c-fed sheep. Moreover, despite a marked increase in rumen
epithelium cell and protein mass in h/c-fed sheep, vH+-ATPase B subunit protein expression
over all cell fractions was not significantly different from h-fed sheep.
In addition, the Na+/H+ exchanger (NHE)- and vH+-ATPase-related H+ secretion was
markedly reduced by 33% and 88%, respectively, in REC from h/c-fed sheep. Under our
experimental conditions (HCO3--free, butyrate-containing medium) NHE and vH+-ATPase
have been shown to be the major components of pHi regulation (9) but here, this has been
confirmed for the condition of h-feeding only. By use of ouabain, a specific Na+/K+-ATPase
inhibitor, Albrecht et al. (1) have demonstrated that NHE activity is related to Na+/K+ATPase. A down regulation of Na+/K+-ATPase-binding sides (25) can thus explain the
reduced amiloride-sensitive component of H+ secretion seen in h/c-fed sheep. This is
supported by results from our recent study (27) showing that concentrate feeding induced a
decrease of the Na+/K+-ATPase protein amount per cm2 cell membrane surface area, a
decrease that equals the reduction of the amiloride effect seen after h/c-feeding in this study
(-31% and -33%). In the concentration used, foliomycin mainly affects vH+-ATPases
localized in the cell membrane (21) and thus, we hypothesize that the proportion of vH+ATPase localized in the cytosol or in intracellular organelles is increased in REC from h/c-fed
compared with h-fed sheep. The reduced expression/activity of two major energy-consuming
transport components can be assumed as a mechanism to provide more energy for covering
the cost of the increase in protein synthesis and cell proliferation (31, 39).
22
At this time point, the variety of the possible functions of the vH+-ATPases and its subunits
makes it difficult to define its exact function(s) in REC and in diet response of ruminal
epithelium.
Our results are most consistent with a role of vH+-ATPase in feed-induced adaptation of the
rumen epithelium most probably by acting as a cell intrinsic sensor for metabolically induced
pHi changes that couples nutrient flux to cellular signaling pathways involved in the
regulation of cell proliferation and maintenance. Therefore, the vH+-ATPase may help to
coordinate REC proliferation and apoptosis relative to their ability to take up and metabolize
specific nutrients (glucose, butyrate) and to produce both ATP and macromolecules.
Interestingly, the mRNA and protein expression of the vH+-ATPase as well as its ATPconsuming functional activity were reduced during adaptation from h- to h/c-feeding, which
could
be
seen
as
an
energy
saving
mechanism
promoting
proliferation.
23
ACKNOWLEDGEMENTS
We gratefully acknowledge the valuable technical assistance of H. Pröhl, R. Brose and K.
Marquardt (FBN Dummerstorf). We also thank the animal husbandry staff of the FBN
Dummerstorf and of the Faculty of Agriculture and Environment at the University of Rostock.
The authors wish to express their gratitude to Dr. T. Jones for linguistic corrections.
This study was supported by the DFG (M. Röntgen, SCHW 652).
24
AUTHOR CONTRIBUTIONS
Kuzinski, Judith
• performed experiment including sampling
• experimental work (qRT-PCR, Western Blot, pHi measurement, ATPase activity
assay)
• data evaluation and interpretation
• manuscript writing
Zitnan, Rudolf
• performed experiment including sampling
• experimental work (rumen fluid analysis, morphometry, histology,
immunohistochemistry)
• data evaluation
• manuscript writing: part of “methods”
Albrecht, Elke
• tissue sampling
• experimental work (method improvement - immunohistochemistry)
• data interpretation
• manuscript writing: part of “methods”
Viergutz, Torsten
• experimental work (flow cytometry, qRT-PCR)
• data evaluation and interpretation
25
Röntgen, Monika
• project development and study design
• performed experiment including sampling
• data evaluation and interpretation
• manuscript writing and editing
26
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33
FIGURE LEGENDS
Fig. 1. Rumen epithelial morphology and cell characteristics in sheep fed hay ad libitum (h
diet) or hay ad libitum + concentrate (h/c diet). A: Different morphology of rumen
tissue samples taken from the atrium ruminis of the two diet groups. An increase of
the length and width of the rumen papillae in h/c-fed sheep is visible at the
macroscopic level. B/C: Transmitted light photomicrographs showing hematoxylineosin stained cross sections of the multilayered rumen mucosa. In sheep fed the h/c
diet a predominant and thicker stratum corneum (SC) (45 µM vs. 29 µM) containing
numerous so-called balloon cells was found. SC: stratum corneum; SG: stratum
granulosum; SS: stratum spinosum; SB: stratum basale. D: Flow cytometric
determination of the number of basal cytokeratin and cytokeratin 10 (differentiation
marker) positive rumen epithelial cells. *P < 0.05 vs. control (cell number in h-fed
sheep).
Fig. 2. Expression of the vH+-ATPase B subunit protein in REC obtained from sheep fed hay
ad libitum (h diet) or hay ad libitum + concentrate (h/c diet) for 14 days. A: Typical
whole immunoblot of the vH+-ATPase B subunit showing a band at ~60 kDa. B:
Percentage of vH+-ATPase-positive REC and vH+-ATPase-specific mean fluorescence
intensity per cell, a measure of the vH+-ATPase B subunit protein amount, in both
treatment groups as determined by flow cytometry. Values are means ± SE; n = 6 per
diet. *P < 0.05 between feeding groups.
Fig. 3. Expression of the vH+-ATPase B subunit protein and activity in REC obtained from
sheep fed hay ad libitum (h diet) or hay ad libitum + concentrate (h/c diet) for 14 days.
A: The figure shows the total number of vH+-ATPase-positive REC for pooled REC
34
fractions 3-5, 6-8 and 9/10 of both treatments. Values are means ± SE; n = 6 per diet.
*P < 0.05 between feeding groups; aP = 0.01 vs. control (F3-5 of h/c-fed sheep). B:
Relative vH+-ATPase enzyme activity of F9/10 of the two diets in an enzyme-coupled
assay. Values are means ± SE; n = 4 per diet; *P < 0.05 between feeding groups.
Fig. 4. Densitometric quantification of vH+-ATPase B subunit protein by ImageJ software.
Results of Western blotting analysis are given as change of density compared with h
diet. In the inset characteristic immunoblots for both feeding groups are shown, also
illustrating an increase in vH+-ATPase B subunit protein amount from fractions 3-5 to
fractions 9/10 (significant for the h-fed group, P < 0.05). Values are means ± SE; n = 4
sheep per diet; *P < 0.05 between feeding groups.
Fig. 5. Detection of vH+-ATPase B subunit mRNA in differently fed sheep. A: SYBR Goldstained 2% agarose gel of RT-PCR products for DNA size ladder (bp, lane L),
negative control (lane n, without reverse transcriptase), and vH+-ATPase B subunit
(lane p) are shown. The product obtained with the vH+-ATPase B subunit-specific
primer has the expected size of 249 bp. B: Expression of vH+-ATPase B subunit
mRNA in REC isolates (fractions 3-10) of sheep fed hay ad libitum (h diet) or hay ad
libitum + concentrate (h/c diet) for 14 days. Values have been normalized to the
RPS18 signal and are shown as means ± SE; n = 4 per diet; **P = 0.003. Inset:
Expression of vH+-ATPase E subunit mRNA in REC isolates (fractions 3-10) of sheep
fed hay ad libitum (h diet) or hay ad libitum + concentrate (h/c diet) for 14 days.
Values have been normalized to the RPS18 signal and are shown as means ± SE; n = 2
per diet.
35
Fig. 6. Intracellular pH (pHi) in REC obtained from sheep fed hay ad libitum (h diet) or hay
ad libitum + concentrate (h/c diet) for 14 days. The pHi measured at the start and the
end of the measurement (after 10 min) is shown for both feeding groups. Values are
means ± SE; n = 4 per diet; *P < 0.05 and **P < 0.01 between feeding groups.
Fig. 7. Vacuolar-type H+-ATPase and Na+/H+ exchanger functional activity in sheep fed hay
ad libitum (h diet) or hay ad libitum + concentrate (h/c diet) for 14 days. The effect of
foliomycin (2 µM), amiloride (250 µM), and of the combination of both on REC
(pooled F3-10) pHi is shown. Measurements were made in HEPES-buffered HCO3-free Na-butyrate medium with blockers as indicated. A: Original traces from one
experiment. B: To summarize the inhibitor effects, the mean pHi reduction from the
pHi measured in respective control medium without inhibitor(s) has been calculated
and is shown for each condition. Values are means ± SE; n = 4 per diet; *P < 0.05
between feeding groups.
Fig. 8. Glucose deficiency and/or vH+-ATPase inhibition affect REC proliferation and
intracellular cAMP concentration. Cells were incubated and measured in media
containing a normal (5.5 mM) or strongly reduced (0.1 mM) glucose concentration.
A: Glucose deficiency induced a strong decrease of REC proliferation rate. Values are
means ± SE; n = 6 per condition; *P < 0.05; B: Effect of the specific vH+-ATPase
inhibitor foliomycin (2 µM) on REC proliferation rate and intracellular cAMP
concentration ([cAMP]i). The mean reduction of proliferation rate and [cAMP]i in
relation to respective measures under control conditions (without inhibitor) has been
calculated and is shown for each condition. Values are means ± SE; n = 3 to 6 per
condition; *P < 0.05.
36
Fig. 9. Immunolocalization of the vH+-ATPase B subunit (green) in the atrium ruminis of
sheep fed hay ad libitum (h diet) or hay ad libitum + concentrate (h/c diet) for 14 days.
Cells were co-stained with propidium iodide (red) to label nuclei. The vH+-ATPase
(arrows) was found in the cell membrane and cytoplasm of basal and suprabasal REC.
A/B: Scale bar = 50 µm. C: Scale bar = 20 µm.
37
TABLE LEGENDS
Table 1. Sequences of primer sets used for amplification of specific cDNA. Primers were
constructed according to data from Gene Bank (Acc. No. accession number; bp
number of base pairs; vH+-ATPase B subunit, vH+-ATPase E subunit, RPS18, and
GAPDH denote the genes encoding vH+-ATPase B subunit, vH+-ATPase E subunit
ribosomal protein S18 (RPS18), and GAPDH, respectively).
Table 2. Rumen fluid parameters determined from sheep fed hay ad libitum (h diet) or hay ad
libitum + concentrate (h/c diet) for 14 days. Values are means ± SE; n = 6.
Figure 1
h diet
h/c diet
A
D
B
number of positive cells (x107/ml)
4
SC
SC
SG
SS
SG
SS
SB
SB
C
100 µM
29 µm
119 µm
129 µm
45 µm
*
3
2
*
1
0
cytokeratin 10 basal cytokeratin
Figure 2
A
MW (kDa)
150
100
75
50
37
B
70
60
vH+-ATPase-positive REC (%)
mean fluorescence intensity
per single REC (AU)
*
50
40
30
25
20
20
15
10
0
h diet
h/c diet
6
5
h diet
h/c diet
*
a
B
30
*
25
vH+-ATPase activity (%)
A
number of vH+-ATPase-positive REC (x107/ml)
Figure 3
4
3
2
1
0
20
15
10
5
0
F3-5
F6-8
F9/10
h diet
h/c diet
Figure 4
120
Change of density (%)
100
*
h diet
MW
F6-8 F9/10
(kDa) F3-5
75
50
80
60
75
50
40
h/c diet
20
0
-20
F3-5
F6-8
F9/10
bp
B
h diet
n
p
1500
850
400
200
50
249 bp
0.25
vH+-ATPase E subunit mRNA
(pg) / RPS mRNA 18 (pg)
A
vH+-ATPase B subunit mRNA (pg)/RPS18 mRNA (pg)
Figure 5
0.20
0.15
0.10
0.40
0.35
0.30
0.25
0.20
0.15
0.10
h diet
**
0.05
0.00
h diet
h/c diet
h/c diet
Figure 6
7.4
initial pHi
pHi (pH units)
7.2
**
pHi after 10 min
*
7.0
6.8
6.6
6.4
6.2
6.0
h diet
h/c diet
Figure 7
h/c diet
h diet
A
7.2
7.0
pHi
6.8
6.6
6.4
control
6.2
foliomycin
amiloride
6.0
5.8
0
B
100 200 300 400 500 600
time (s)
0
100 200 300 400 500 600
time (s)
h/c diet
h diet
change of pHi (pH units)
0.0
*
-0.2
-0.4
*
-0.6
-0.8
foliomycin
amiloride
foliomycin + amiloride
Figure 8
1.0
A
B
decrease (%)
absorbance
-20
0.8
0.6
-40
*
-60
*
0.4
5.5 mM glucose
0.1 mM glucose
-80
intracellular [cAMP]
cell proliferation rate
*
0.2
0
0.5 1
1.5 2 2.5
3 3.5
time (h)
4
4.5
5 5.5
6
5.5 mM
0.1 mM
media glucose concentration
Figure 9
A
B
C
h diet
50 µm
50 µm
20 µm
h/c diet
Table 1
Transcript
Forward primer 5′-3′
Reverse primer 5′-3′
Acc. no.
vH+-ATPase
B1 subunit
bovine (249 bp)
642-665
GGA CTA TCA TGA TGA CAA CTT TGC
890-867
TAG GAA CTC ATG TCC GTC AGT ATG
NM_176654
vH+-ATPase
E1 subunit
bovine (182 bp)
56-79
TTT TAT TGA ACA AGA AGC CAA TGA
237-214
GAT TCA TCA AAT TGG ACA TCT GAA
NM_174810
GAPDH
bovine (332 bp)
232-256
TAC ATG GTC TAC ATG TTC CAG TAT G
563-540
CAG GAG GCA TTG CTG ACA ATC TTG
XM_001252511
RPS 18
bovine (218 bp)
293-316
CTT AAA CAG ACA GAA GGA CGT GAA
510-487
CCA CAC ATT ATT TCT TCT TGG ACA
NM_001033614
Table 2
P-value
h diet
h/c diet
6.69 ± 0.07
5.58 ± 0.07
≤ 0.001
SCFA [mmol/l]
81.71 ± 6.34
157.74 ± 10.42
≤ 0.001
NH3 [mmol/l]
11.22 ± 0.52
17.02 ± 1.40
0.003
acetate [Mol %]
70.35 ± 1.05
63.28 ± 0.90
≤ 0.001
propionate [Mol %]
16.96 ± 0.85
23.24 ± 1.31
0.002
8.41 ± 0.38
10.97 ± 0.17
0.002
2.78 ± 0.20
0.001
pH value
butyrate [Mol %]
acetate:propionate
4.21 ± 0.25