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 REFERENCES 1. Albrecht E, Kolisek M, Viergutz T, Zitnan R, Schweigel M. 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Dev Cell 17: 387-402, 2009. 54. Zitnan R, Kuhla S, Nürnberg K, Schönhusen U, Ceresnakova Z, Sommer A, Baran M, Greserova G, Voigt J. Influence of diet on the morphology of ruminal and intestinal mucosa and on intestinal carbohydrase levels in cattle. Vet Med. – Czech 48: 177-182, 2003. 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