J Comp Physiol B (2014) 184:673–681 DOI 10.1007/s00360-014-0804-5
ORIGINAL PAPER
Expression of the ABC transport proteins MDR1 (ABCB1) and BCRP (ABCG2) in bovine rumen I. S. Haslam • N. L. Simmons
Received: 23 October 2013 / Revised: 7 January 2014 / Accepted: 13 January 2014 / Published online: 20 April 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Rumen fermentation of plant-based forage in bovines is the major site for generation and absorption of short-chain fatty acids. Consequentially, the rumen is also the site for initial exposure to toxins released from diet. Accordingly, we have investigated the expression of bovine ABC transporters in the rumen associated with cytoprotection against xenobiotic exposure, namely MDR1 (ABCB1), MRP2 (ABCC2) and BCRP (ABCG2). Bovine rumen samples from the ventral sac were obtained postmortem from a commercial slaughterhouse after humane killing. Rumen papilla samples were then prepared for total RNA isolation for RT-PCR, SDS-PAGE/Western blotting and immunohistochemistry. PCR products of the predicted size were observed for both MDR1 and BCRP, but not for MRP2 using bovine-specific primers. b-actin was used as a control transcript. Western blot analysis using C219 primary monoclonal antibody revealed MDR1 protein expression in bovine rumen (Mapp, of *170–180 kD). Immunolocalisation of MDR1 using UIC2 monoclonal antibody within cryosections of bovine rumen showed extensive membrane staining in the cells of the stratum granulosum, stratum spinosum and stratum basale. MDR1 expression was absent from outer stratum corneum. Protein
Communicated by H.V. Carey. I. S. Haslam Stopford Building, University of Manchester, Manchester M13 9PT, UK N. L. Simmons (&) Epithelial Research Group, Institute for Cell and Molecular Biosciences, Medical School, University of Newcastle Upon Tyne, Newcastle Upon Tyne NE2 4HH, UK e-mail: [email protected]
expression and immunolocalisation were also confirmed for BCRP, with prevalent staining in the stratum basale, becoming weaker in the stratum spinosum and stratum granulosum. Keywords Rumen ABC transporter MDR1 BCRP Rumen Xenobiotics
Introduction The bovine rumen is an organ optimised for microbial fermentation of ingested feed and the absorption of released nutrients. Large quantities of short-chain fatty acids (SCFA), including butyrate, propionate and acetate are absorbed across the stratified squamous epithelium that constitutes the leaf-like rumen papillae (Gabel and Sehested 1997). The functional organisation of this stratified epithelium has been described in some detail previously (Graham and Simmons 2005) and the expression of various members of the SLC family of transport proteins has since been confirmed (Graham et al. 2007). Transport studies had suggested that absorption of SCFAs was likely to be mediated by both passive, non-ionic diffusion and active transport via members of the SLC family, such as monocarboxylate transporters [MCTs; (Graham et al. 2007)]. Indeed, the basal localisation of MCT1 in stratum basale and stratum spinosum in the bovine rumen suggests a role for this transport protein in mediating efflux of SCFAs into the blood following diffusion through the outer layers of the rumen. In contrast, the Na: H exchangers, NHE3 and NHE1 are expressed mainly in the outer barrier-forming stratum granulosum where acid extrusion following SCFA uptake is coupled to Na? entry (Graham et al. 2007; Rabbani et al. 2011).
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In addition to the nutrients derived from both concentrated feed and natural forage, digestion and fermentation of ingested material is likely to release potentially harmful xenobiotic molecules as plant vacuoles are the cellular repository for detoxification of heavy metal conjugates, herbicides and toxins (Rea et al. 1998). Release of these toxins following ingestion could prove detrimental unless efficient detoxification mechanisms exist. Within the intestine of most mammalian species, efficient detoxification mechanisms are provided by the concerted actions of hydroxylation and conjugation (Phase I and Phase II metabolism) and active efflux (Phase III transport; active efflux) (Ritter 2007; Shugarts and Benet 2009; Thelen and Dressman 2009). Efflux transporters of the ATP-binding cassette (ABC) family are expressed in numerous locations throughout the body (e.g. kidney, liver, intestine, blood– brain barrier, blood–testis barrier) where they function as a barrier to the movement of a diverse array of chemically distinct molecules (Murakami and Takano 2008). This ‘‘multi-specificity’’ constitutes a prominent feature of their functionality, allowing for the recognition of an extremely wide variety of xenobiotics, including drug molecules, with a substantial degree of cross-recognition (Murakami and Takano 2008). This also allows for a certain amount of redundancy and/or compensation with regard to xenobiotic defence should the normal functioning of one protein be disturbed. We hypothesise that the expression of ABC transport proteins within the bovine rumen may provide a metabolically driven barrier to the movement of ingested xenobiotics. Previous research hints at this possibility, with plasma concentrations of ivermectin, an ABCB1 substrate (Brayden and Griffin 2008), increased co-administration of the P-glycoprotein inhibitor itraconazole after intra-ruminal administration of ivermectin in sheep, though the locus of this effect may result from actions in the small intestine (Ballent et al. 2007). The three ABC family efflux transporters expressed at the apical membrane of intestinal enterocytes are ABCB1 (multidrug resistance 1, MDR1; P-glycoprotein), ABCC2 (multidrug resistance related protein 2, MRP2; canalicular multispecific organic ion transporter) and ABCG2 (breast cancer resistance protein, BCRP) (Chan et al. 2004). The aim was, therefore, to establish whether expression of ABCB1, ABCC2 and ABCG2 occurred in bovine rumen.
Methods Tissue collection and preparation Four mature Holstein steers which were pasture grazed, with only grass hay in supplement, were humanely killed in
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a commercial slaughterhouse in accordance with UK law. Samples of bovine rumen from the ventral sac were obtained and prepared and transported as previously described (Graham and Simmons 2005). Epithelial tissue was then stripped from the underlying muscle layers and papillae samples collected for total RNA isolation for RT-PCR, SDS-PAGE/Western blotting and immunohistochemistry. RNA isolation and RT-PCR Rumen papillae for reverse-transcription (RT)-PCR were stored at 4 °C in RNAlater (Ambion). RNA was extracted from rumen papillae using RNAzol B reagent (Biogenesis). Briefly, tissue was ground in liquid nitrogen, followed by the addition of RNAzol B (2 mL 0.1 g of tissue). The resulting suspension was homogenised using a Polytron (CH-6010, Kinematica). 0.2 mL/mL chloroform was added to the suspension, which was shaken vigorously for 15 s followed by a 15 min incubation on ice. The samples were centrifuged at 20,000g for 30 min at 4 °C and the aqueous phase transferred to a clean tube. An equal volume of isopropanol was added and the sample incubated on ice for 15 min before centrifugation 20,000g for 60 min at 4 °C. The solvent was aspirated and the pellet washed with 85 % ethanol before a further centrifugation step at 20,000g for 15 min at 4 °C. The pellet was air-dried at room temperature and re-suspended in 10 mM Tris–HCl (pH 7.5). RNA was analysed by gel electrophoresis and found to be free of genomic DNA contamination. The ratio of 28:18 s ribosomal RNA was approximately 2:1, indicating intact RNA. RNA (2 lg) was reverse transcribed using Omniscript reverse transcriptase (Qiagen) according to the manufacturers guidelines. For each RNA sample, negative controls were included in which reverse transcriptase was omitted from the reaction. Hot-start Taq DNA polymerase (Qiagen) was used according to manufacturer’s protocol for amplification of 2 lL of reverse-transcribed RNA in a 50 lL reaction. Mg2? concentration was 1.5 mM, primer concentration was 0.5 lM and 2.5 U of enzyme were used per reaction. Enzyme activation was achieved with an initial heating step of 95 °C for 15 min. The annealing temperature for all primer pairs was set at 54–60 °C, as appropriate for primer pairs. Amplification was carried out over 35 cycles. Genespecific oligonucleotide primers for bovine ABCB1 (MDR1), ABCC2 (MRP2), ABCG2 (BCRP) and b-actin were designed from published bovine sequences (NCBI) incorporating exon-spanning regions of the nucleotide sequence. Expected product sizes were 792 base pairs (ABCB1), 750 base pairs (ABCC2), 1,161 base pairs (ABCG2) and 612 base pairs (b-actin)—Table 1. PCR products were analysed by agarose gel (1 %) electrophoresis, with products
J Comp Physiol B (2014) 184:673–681 Table 1 Oligonucleotides primer pairs used for identification of ABC family members in bovine rumen epithelium
Gene (alias)
675
Sequence
Product size (bp)
ABCB1 (MDR1, P-glycoprotein)
792
Forward
50 -GTGGGACAGGTCAGTTCATT-30
Reverse
50 -GCT CCT TGA TTC TGC CAT CT-30
ABCC2 (MRP2, CMOAT)
750
Forward
50 -TAC CAG CGA GTT CTG GAG G-30
Reverse
50 -AGG TCC CTC TGA GAG GAT G-30
ABCG2 (BCRP)
1,161
Forward
50 -CCC TTC GGC TTC CAA CAA C-30
Reverse
50 -CTG ACC TGC TGC TAT AGC C-30
b-Actin
612 0
Forward
5 -TCC ACG AAA CTA CCT TCA AT-3
Reverse
50 -TTT GGG AAG GCA AAG GAC-30
visualised using ethidium bromide staining and UV transillumination. Immunocytochemistry Full-thickness isolated rumen papillae were fixed in methanol at -20 °C or 3 % paraformaldehyde in PBS at 4 °C. They were then incubated for 24 h in 30 % sucrose in PBS at 4 °C before embedding in optimal temperature cutting compound (Tissue-Tek, Miles Laboratories, Naperville, IL) and cryo-sectioned. 5-lm sections were washed in PBS (3 times) and permeabilised with 0.1 % saponin in PBS for 20 min. Sections were blocked with 10 % serum (species-dependent on host animal used in the production of secondary antibodies) in PBS. Sections were incubated with primary antibodies (all monoclonal mouse IgG2a) overnight at 4 °C. Dilutions were 1:50, 1:50 and 1:100 for MDR1 (UIC2, Calbiochem), MRP2 (M2 III-6, Abcam) and BCRP (BXP-21, Abcam) antibodies, respectively. UIC2 recognises MDR1 in intact cells by an external epitope between transmembrane segments (TM) five and six of human MDR1 (GTTLVLSGEY) (Zhou et al. 1999) which is identical in 8/10 residues in bovine MDR1 (GTSLVLSKEY). UIC2 does not, however, recognise detergent solubilised protein (Mechetner and Roninson 1992). M2 III-6 was raised against the C-terminal of the human protein (residues 1339–1541) which is highly similar to the bovine protein with 180/202 identities by BLASTP (Altschul et al. 1997). BXP-21 was raised against residues 271–396 of the human protein and there are 87/125 identities with the bovine protein by BLASTP (Altschul et al. 1997). Sections were again washed three times with PBS before incubation with antimouse goat Alexa Fluor 488 secondary antibody (Life Technologies diluted 1:50 in 10 % serum) for 1 h at room temperature. Comparative immunostaining was also
0
performed for claudin-1, connexin-43 and the a-subunit of the Na–K ATPase as previously described (Graham and Simmons 2005). Nuclear staining was performed using ethidium homodimer 1 (1 mg/mL in water) at 1:1,000 dilution. Sections were imaged using confocal laser scanning microscopy (TCS-NT, Leica with Kr-Ar laser) using appropriate excitation and emission filter sets for dual fluorophore detection. Appropriate negative controls were performed where the primary antibody was omitted; these slides were used to set the background microscope intensity and gain for imaging. Western blotting Approximately 1 g of separated bovine rumen papillae was homogenised in 5 mL of lysis buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA) using a Polytron (CH6010, Kinematica). The homogenised tissue was centrifuged at 40009g for 10 min at 4 °C to pellet cell debris and nuclei. Protein was quantified using Bradford agent (Bio-Rad). Proteins were separated on 8 % SDS-polyacrylamide gels and transferred onto PVDF membranes (Millipore) overnight. Membranes were briefly washed with TBS-T (Tris-buffered saline and 0.1 % Tween 20, pH 7.6) and blocked in 3 % non-fat dried milk in TBS-T buffer. Membranes were probed overnight at 4 °C with the mouse monoclonal antibody C219 against ABCB1 (Calbiochem), M2 III-6 against ABCC2 (Abcam) and BXP-21 against ABCG2 (Abcam). The C-terminal human epitope recognised by the C219 antibody (VQEALD) is conserved in bovine MDR1. The antibodies were diluted 1:50 in TBST buffer. Following further washing, membranes were incubated with 1:10,000 HRP-labelled anti-mouse IgG (Abcam) and bands detected with the pico-chemiluminescence substrate (Pierce) following manufacturer’s instructions.
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Results The expression of ABCB1, ABCC2 and BCRP was investigated using bovine-specific primer pairs (Table 1), with RT-PCR of bovine rumen epithelium RNA. Expression of ABCB1 (MDR1) and ABCG2 (BCRP) is demonstrated by appropriately sized PCR products whilst there is absence of ABCC2 mRNA (Fig. 1). Expression of the housekeeping gene b-actin, as a positive control, was confirmed in all PCR reactions with RT-negative controls showing no contamination by genomic DNA. Data are representative of those for the four animals. ABCB1 protein expression was confirmed by Western blot analysis of rumen epithelium tissue using the mouse monoclonal antibody C219 (Fig. 2a), with a doublet of Mapp of *175 kD, corresponding to molecular weight of 170 kD seen in Caco-2 total protein used as a positive control (Fig. 2a). The human MDR1 protein is subject to
Fig. 1 RT-PCR products from bovine rumen mRNA, separated by agarose gel electrophoresis and visualised after ethidium bromide staining/UV-trans-illumination. Products of the predicted size for a MDR1 (792 base pairs) and c BCRP (1161 base pairs) are evident. No bands are visible for MRP2 b. b-Actin was present as a positive control with a PCR-negative control also included in each gel
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extensive N-glycosylation at three asparagine residues within the first extracellular loop between TM 1 and 2 (NITNRSDINDT) (Schinkel et al. 1993) which accounts for the difference in core Map (140 kDa) and the broad size distribution observed in SDS-PAGE (Greer and Ivey 2007). This part of the MDR1 sequence is poorly conserved between species; in the bovine sequence there are 31/48 non-identities with the human sequence. However, there are potentially 3 N-glycosylation sites (NITFPNTINGSDINDT) of the Asn/X/Thr or Ser in this extracellular loop. C219 does not give ABCB1 immunostaining in frozen-fixed bovine tissue so the alternative UIC2 antibody was used. Immunostaining with UIC2 is shown in Fig. 2b. Prominent staining is evident in the lateral and apical plasma membranes of epithelial cells reaching from the stratum basale to the stratum granulosum. Note that there is no immunostaining on the rumenfacing membrane of the stratum granulosum. No immunofluorescence is observed in keratinocytes of the stratum corneum. Figure 3 shows that MDR1 immunofluorescence is not restricted to the rumen papillary epithelium. Significant vascular MDR1 expression is seen associated with endothelial cells within central blood vessels (Fig. 3c). Immunofluorescence data for MDR1 and all other primary antibodies are representative for the four animals. ABCC2 protein expression was probed in bovine rumen epithelium using the mouse monoclonal antibody M2 III-6 (Fig. 4a). No bands were identified. A prominent, single band at *200 kD, likely resulting from the fully glycosylated form of ABCC2 (Zhang et al. 2005) was observed in Caco-2 cell extracts, known to express ABCC2. Immunocytochemical analysis of sections of bovine rumen using the same antibody did not display any specific fluorescence. Taken together with the lack of gene expression, confirmed by RT-PCR analysis, MRP2 can be seen to be absent in the bovine rumen epithelium. ABCG2 localisation was determined by immunocytochemistry using the mouse monoclonal antibody BXP-21 (Fig. 5). Immunofluorescence showed prominent lateral and apical membrane staining in the cells of the stratum basale and stratum spinosum, with decreasing intensity in the stratum granulosum. In positive cells there is vesicular cytoplasmic immunofluorescence. The stratum corneum was negative. BCRP immunofluorescence is also not restricted to the rumen papillary epithelium. Vascular endothelial cells are also seen to demonstrate BCRP immunofluorescence (Fig. 3d, e).
Discussion Although mammalian expression of ABC transporters along the intestinal tract is now well documented (Dietrich
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Fig. 2 MDR1 expression in rumen papilla epithelium and the colorectal adenocarcinoma cell line Caco-2. a Western blot of MDR1 displaying a double band of apparent molecular mass of *175 kD in the rumen and a single band of apparent molecular mass of *175 kD in Caco-2. b Immunolocalisation of MDR1 expression. Cells were co-stained with the nucleic acid marker ethidium
homodimer 1. The overlay combines images of MDR1 (green) and nucleic acid marker (red). Note the prominent lateral and apical staining of the cell membranes in layers reaching from the stratum basale to the stratum granulosum. The stratum corneum is negative. Scale bar 20 lm (colour figure online)
et al. 2003) including in cows (Lindner et al. 2013), little information is available for rumen. This study has, for the first time, confirmed the expression of members of the ABC family of transport proteins, in epithelial cells of the bovine rumen. Both ABCB1 (MDR1, P-glycoprotein) and ABCG2 (BCRP) were found to be expressed within several layers of the stratified rumen epithelium, showing overlapping but distinct localisations. MRP2 was not found at either the mRNA (RT-PCR) or protein (Western blot, IHC) level. Studies of the localisation of epithelial transporters and of the junctional protein, claudin 1, have emphasised the complexity of the rumen epithelial barrier (Fig. 6) (Graham et al. 2007; Graham and Simmons 2005). The tight junction proteins, ZO-1 and claudin 1, are expressed at the outer (lumen)-facing side of the stratum granulosum indicating that this cell layer forms the outer barrier of this stratified epithelium. The Na: H isoforms NHE1 and NHE3 are also expressed at this location, NHE3 also being present in the stratum spinosum with less in the stratum basale (Rabbani et al. 2011). A functional syncytium formed by connexin gap junctions between the cell layers underlying the stratum granulosum, comprising the stratum spinosum and the stratum basale, allows transepithelial transport of Na? to occur to blood from stratum basale cells by the concentration of Na–K ATPase expressed here (Graham and Simmons 2005). MCT1 is also expressed at the basal side of this syncytium likely facilitating export of SCFAs and ketone bodies to the blood (Graham et al. 2007). The presence of ABCB1 (MDR1) within the stratum basale,
spinosum and granulosum suggests that transport from these cell layers is directed at the interstitial space and not exclusively across the lumen-facing membrane of the stratum granulosum, as would be expected if transport was exclusively directed back towards the rumen lumen. Instead, transport function would act to prevent the syncytial cells themselves from being exposed to high concentrations of xenobiotics. Na–K ATPase (see Fig. 6), mitochondrial density and ATP supply are concentrated towards the base of cells in the stratum basale (Graham and Simmons 2005) and differ from MDR1. In the small intestine both metabolism (via mitochondrial CYP3A4) and transport via MDR1 combine to reinforce the oral barrier to xenobiotics (Zhang and Benet 2001). Export of parent compound or metabolite combines to maximise the metabolic capacity (Zhang and Benet 2001). Ultimately, transport of parent compound/metabolite via blood to liver will result in hepatocyte uptake, further metabolism and ultimately clearance via biliary excretion (again mediated by MDR1) to the intestine lumen. Recent evidence in another stratified epithelium, namely human skin, has also demonstrated expression of MDR1, predominantly in the basal cell layer and dermal appendages (Skazik et al. 2011). This partially confirms results from investigations in mdr1a/1b KO mice, in which MDR1 localisation was similar (Ito et al. 2008). An interesting observation (Ito et al. 2008) was the increased absorptive movement of the MDR1 substrate Rhodamine 123 in WT vs. mdr1a/b null animals. It is, therefore, possible to envisage a potential role for MDR1-mediated absorption of dietary components
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Fig. 3 Immunolocalisation of MDR1 and BCRP (both green) expression in the sub-epithelial connective and vascular tissue in bovine rumen papillae. Cells were co-stained with the nucleic acid marker ethidium homodimer-1 (red). a Transmitted-light photomicrograph of full-thickness papilla, scale bar 100 lm. b Fluorescence image of MDR1 immunofluorescence (green) of central papillary
blood vessels (arrowed), scale bar 100 lm. c High power image of endothelial MDR1-immunostaining, scale bar 20 lm. d Medium power image of BCRP-immunostaining (green), scale bar 20 lm. e High power image of endothelial BCRP-immunostaining, scale bar 20 lm (colour figure online)
in the bovine rumen. As has been suggested for both mouse and human skin (Ito et al. 2008), MDR1 could play a dual role in enhancing the absorption of certain molecular species whilst functioning in its traditional role of xenobiotic defence for the cells in which it is expressed. An additional feature of MDR1 expression in bovine rumen is the expression within endothelial cells within the papilla. MDR1 expression in the capillary endothelial cells of the blood–brain barrier and the blood–testes barrier is thought to isolate these tissues from blood-borne toxins and drugs (Cordoncardo et al. 1989). Whether expression in bovine blood vessel endothelium performs a role in aiding absorption or limiting access to the tissue from blood remains to be determined. Recently, cloned bovine BCRP expressed in MDCK cells has been investigated with regards to deposition to milk of flavonoids such as equol or quercetin and 19
commonly used veterinary drugs (Wassermann et al. 2013). Selected drugs included antibiotics, the avermectin/milbemycin anthelmintics, moxidectin and eprinomectin, and the benzimidazole anthelmintics albendazole and oxfendazole (Wassermann et al. 2013). The present data show BCRP expression in the bovine rumen which displays a similar pattern to MDR1, being present in the stratum basale and spinosum, but with concentration to the apical poles of the cells within the stratum spinosum consistent with limitation of xenobiotic uptake from the rumen. There is a marked decrease in immunofluorescence towards the stratum granulosum. This may indicate differing functionality compared with MDR1. BCRP has been reported to impact on the differentiation of certain mammalian cell populations, including stem cells, Langerhans cells and cultured cell lines (Bunting 2002; Prouillac et al. 2009) so the pattern oh high expression in stratum basale may indicate a
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Fig. 4 Lack of MRP2 expression in the bovine rumen. a Western blot for MRP2 displaying a single band of molecular mass *200 kDa in Caco-2 cell total protein extract, with the absence of a corresponding band in bovine rumen protein extract. b Immunolocalisation of MRP2 expression (green). Tissue was counterstained with the nucleic acid marker ethidium homodimer 1 (red). No MRP2 immunofluorescence was evident. Scale bar 20 lm (colour figure online)
Fig. 5 Immunolocalisation of BCRP expression in the bovine rumen. Cells were co-stained with the nucleic acid marker ethidium homodimer 1. The overlay combines images of BCRP (green) and nucleic acid marker (red). Note prominent lateral and apical staining of the cell membranes of the stratum basale and stratum spinosum with decreasing intensity towards the stratum granulosum. The stratum corneum is negative. Scale bar 20 lm (colour figure online)
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Fig. 6 Comparison of immunolocalisation of MDR1, BCRP, Na–K ATPase, connexion 43 and claudin 1 (all green) nucleic acid marker (red) in sections taken from an individual animal. L lumen, scale bars 20 lm (colour figure online)
similar action. As with MDR1 expression, BCRP protein was also observed in papillary dermis vasculature. BCRP has been reported in various human venous but not arterial endothelia (Maliepaard et al. 2001), providing a similar role to MDR1 in tissue defence, e.g. at the blood brain barrier (Eisenblatter et al. 2003). Whether expression in bovine blood vessel endothelium performs a dual role in aiding absorption or limiting access to the tissue from blood remains to be determined. Recent data from rat IEC-6 and human Caco-2 cells suggests that short-chain fatty acids such as butyric acid may act as substrates for BCRP (Goncalves et al. 2011). Given that SCFAs may reach substantial concentrations within bovine rumen (Gabel and Sehested 1997), especially after feeding with easily fermentable carbohydrates (Aschenbach et al. 2011), transport of SCFAs from the epithelial cells of the rumen may constitute a defensive function in rumen acidosis. Extreme loading of rumen epithelial cells with acid from SCFA uptake may result in tissue necrosis (Aschenbach et al. 2011).
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Functional data support a role for ABC transporters in the rumen. Plasma concentrations of the antihelminthic drug ivermectin attained lower, but detectable, levels when administered intra-ruminally (i.r.) compared to subcutaneous dosing, leading to lower antiparasitic activity (Prichard et al. 1985). Of note is the fact that ivermectin is now known to be an MDR1 substrate (Brayden and Griffin 2008; Schinkel et al. 1994). More recently, Ballent et al. (2007) followed up on these observations by investigating the impact of administering ivermectin i.r. and intravenously in the presence and absence of a Pgp inhibitor, itraconazole. Only i.r. co-dosing of ivermectin with the Pgp inhibitor had the effect of increasing plasma ivermectin concentrations. In addition, the level of ivermectin within abomasums tissues was increased relative to controls by *60 % by co-administration with itraconazole (Ballent et al. 2007). These data are compatible with reduced absorption of ivermectin due to MDR1 function. The results presented here have shown the expression and localisation of two members of the ABC family of
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transport proteins, ABCB1 (MDR1) and ABCG2 (BCRP). Both are traditionally concerned with xenobiotic defence which suggests a role in protecting bovines against ingested toxins and noxious metabolites produced during rumen fermentation. Direct functional investigations involving the use of MDR1 and BCRP-specific substrates are needed to elucidate the precise functional activity of these transport proteins within the intact bovine rumen. Acknowledgments Iain Haslam was supported by a studentship from the BBSRC (UK). This work was part funded by Pfizer Global Research Sandwich UK.
References Altschul SF et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402 Aschenbach JR, Penner GB, Stumpff F, Gabel G (2011) Ruminant nutrition symposium: role of fermentation acid absorption in the regulation of ruminal pH. J Anim Sci 89:1092–1107 Ballent M, Lifschitz A, Virkel G, Sallovitz J, Lanusse C (2007) Involvement of P-glycoprotein on ivermectin kinetic behaviour in sheep: itraconazole-mediated changes on gastrointestinal disposition. J Veterinary Pharmacol Ther 30:242–248 Brayden DJ, Griffin J (2008) Avermectin transepithelial transport in MDR1- and MRP-transfected canine kidney monolayers. Veterinary Res Commun 32:93–106 Bunting KD (2002) ABC transporters as phenotypic markers and functional regulators of stem cells. Stem Cells 20:11–20 Chan LMS, Lowes S, Hirst BH (2004) The ABCs of drug transport in intestine and liver: efflux proteins limiting drug absorption and bioavailability. Eur J Pharm Sci 21:25–51 Cordoncardo C et al (1989) Multidrug-resistance gene (P-Glycoprotein) is expressed by endothelial-cells at blood–brain barrier sites. Proc Natl Acad Sci USA 86:695–698 Dietrich CG, Geier A, Elferink RPJO (2003) ABC of oral bioavailability: transporters as gatekeepers in the gut. Gut 52:1788–1795 Eisenblatter T, Huwel S, Galla HJ (2003) Characterisation of the brain multidrug resistance protein (BMDP/ABCG2/BCRP) expressed at the blood–brain barrier. Brain Res 971:221–231 Gabel G, Sehested J (1997) SCFA transport in the forestomach of ruminants. Comp Biochem Physiol A Physiol 118:367–374 Goncalves P, Gregorio I, Martel F (2011) The short-chain fatty acid butyrate is a substrate of breast cancer resistance protein. Am J Physiol Cell Physiol 301:C984–C994 Graham C, Simmons NL (2005) Functional organization of the bovine rumen epithelium. Am J Physiol Regul Integr Comp Physiol 288:R173–R181 Graham C, Gatherar I, Haslam I, Glanville M, Simmons NL (2007) Expression and localization of monocarboxylate transporters and sodium/proton exchangers in bovine rumen epithelium. Am J Physiol Regul Integr Comp Physiol 292:R997–R1007 Greer DA, Ivey S (2007) Distinct N-glycan glycosylation of P-glycoprotein isolated from the human uterine sarcoma cell line MES-SA/Dx5. Biochim Biophys Acta 1770:1275–1282
681 Ito K et al (2008) P-Glycoprotein (Abcb1) is involved in absorptive drug transport in skin. J Control Release 131:198–204 Lindner S, Halwachs S, Wassermann L, Honscha W (2013) Expression and subcellular localization of efflux transporter ABCG2/ BCRP in important tissue barriers of lactating dairy cows, sheep and goats. J Vet Pharmacol Ther 36(6):562–570 Maliepaard M et al (2001) Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues. Cancer Res 61:3458–3464 Mechetner EB, Roninson IB (1992) Efficient inhibition of P-glycoprotein-mediated multidrug resistance with a monoclonal-antibody. Proc Natl Acad Sci USA 89:5824–5828 Murakami T, Takano M (2008) Intestinal efflux transporters and drug absorption. Expert Opin Drug Met 4:923–939 Prichard RK, Steel JW, Lacey E, Hennessy DR (1985) Pharmacokinetics of ivermectin in sheep following intravenous, intraabomasal or intraruminal administration. J Vet Pharmacol Ther 8:88–94 Prouillac C, Videmann B, Mazallon M, Lecoeur S (2009) Induction of cells differentiation and ABC transporters expression by a mycoestrogen, zearalenone, in human choriocarcinoma cell line (BeWo). Toxicology 263:100–107 Rabbani I, Siegling-Vlitakis C, Noci B, Martens H (2011) Evidence for NHE3-mediated Na transport in sheep and bovine forestomach. Am J Physiol Regul Integr Comp Physiol 301:R313–R319 Rea PA, Li ZS, Lu YP, Drozdowicz YM, Martinoia E (1998) From vacuolar Gs-X pumps to multispecific ABC Transporters. Annu Rev Plant Physiol Plant Mol Biol 49:727–760 Ritter JK (2007) Intestinal UGTs as potential modifiers of pharmacokinetics and biological responses to drugs and xenobiotics. Expert Opin Drug Metab 3:93–107 Schinkel AH, Kemp S, Dolle M, Rudenko G, Wagenaar E (1993) N-glycosylation and deletion mutants of the human MDR1 P-glycoprotein. J Biol Chem 268:7474–7481 Schinkel AH et al (1994) Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood–brain barrier and to increased sensitivity to drugs. Cell 77:491–502 Shugarts S, Benet LZ (2009) The role of transporters in the pharmacokinetics of orally administered drugs. Pharm Res 26:2039–2054 Skazik C et al (2011) P-glycoprotein (ABCB1) expression in human skin is mainly restricted to dermal components. Exp Dermatol 20:450–452 Thelen K, Dressman JB (2009) Cytochrome P450-mediated metabolism in the human gut wall. J Pharm Pharmacol 61:541–558 Wassermann L, Halwachs S, Lindner S, Honscha KU, Honscha W (2013) Determination of functional ABCG2 activity and assessment of drug-ABCG2 interactions in dairy animals using a novel MDCKII in vitro model. J Pharm Sci 102:772–784 Zhang YC, Benet LZ (2001) The gut as a barrier to drug absorption— combined role of cytochrome P450 3A and P-glycoprotein. Clin Pharmacokinet 40:159–168 Zhang P, Tian X, Chandra P, Brouwer KL (2005) Role of glycosylation in trafficking of Mrp2 in sandwich-cultured rat hepatocytes. Mol Pharmacol 67:1334–1341 Zhou Y, Gottesman MM, Pastan I (1999) The extracellular loop between TM5 and TM6 of P-glycoprotein is required for reactivity with monoclonal antibody UIC2. Arch Biochem Biophys 367:74–80
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ORIGINAL PAPER
Expression of the ABC transport proteins MDR1 (ABCB1) and BCRP (ABCG2) in bovine rumen I. S. Haslam • N. L. Simmons
Received: 23 October 2013 / Revised: 7 January 2014 / Accepted: 13 January 2014 / Published online: 20 April 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Rumen fermentation of plant-based forage in bovines is the major site for generation and absorption of short-chain fatty acids. Consequentially, the rumen is also the site for initial exposure to toxins released from diet. Accordingly, we have investigated the expression of bovine ABC transporters in the rumen associated with cytoprotection against xenobiotic exposure, namely MDR1 (ABCB1), MRP2 (ABCC2) and BCRP (ABCG2). Bovine rumen samples from the ventral sac were obtained postmortem from a commercial slaughterhouse after humane killing. Rumen papilla samples were then prepared for total RNA isolation for RT-PCR, SDS-PAGE/Western blotting and immunohistochemistry. PCR products of the predicted size were observed for both MDR1 and BCRP, but not for MRP2 using bovine-specific primers. b-actin was used as a control transcript. Western blot analysis using C219 primary monoclonal antibody revealed MDR1 protein expression in bovine rumen (Mapp, of *170–180 kD). Immunolocalisation of MDR1 using UIC2 monoclonal antibody within cryosections of bovine rumen showed extensive membrane staining in the cells of the stratum granulosum, stratum spinosum and stratum basale. MDR1 expression was absent from outer stratum corneum. Protein
Communicated by H.V. Carey. I. S. Haslam Stopford Building, University of Manchester, Manchester M13 9PT, UK N. L. Simmons (&) Epithelial Research Group, Institute for Cell and Molecular Biosciences, Medical School, University of Newcastle Upon Tyne, Newcastle Upon Tyne NE2 4HH, UK e-mail: [email protected]
expression and immunolocalisation were also confirmed for BCRP, with prevalent staining in the stratum basale, becoming weaker in the stratum spinosum and stratum granulosum. Keywords Rumen ABC transporter MDR1 BCRP Rumen Xenobiotics
Introduction The bovine rumen is an organ optimised for microbial fermentation of ingested feed and the absorption of released nutrients. Large quantities of short-chain fatty acids (SCFA), including butyrate, propionate and acetate are absorbed across the stratified squamous epithelium that constitutes the leaf-like rumen papillae (Gabel and Sehested 1997). The functional organisation of this stratified epithelium has been described in some detail previously (Graham and Simmons 2005) and the expression of various members of the SLC family of transport proteins has since been confirmed (Graham et al. 2007). Transport studies had suggested that absorption of SCFAs was likely to be mediated by both passive, non-ionic diffusion and active transport via members of the SLC family, such as monocarboxylate transporters [MCTs; (Graham et al. 2007)]. Indeed, the basal localisation of MCT1 in stratum basale and stratum spinosum in the bovine rumen suggests a role for this transport protein in mediating efflux of SCFAs into the blood following diffusion through the outer layers of the rumen. In contrast, the Na: H exchangers, NHE3 and NHE1 are expressed mainly in the outer barrier-forming stratum granulosum where acid extrusion following SCFA uptake is coupled to Na? entry (Graham et al. 2007; Rabbani et al. 2011).
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In addition to the nutrients derived from both concentrated feed and natural forage, digestion and fermentation of ingested material is likely to release potentially harmful xenobiotic molecules as plant vacuoles are the cellular repository for detoxification of heavy metal conjugates, herbicides and toxins (Rea et al. 1998). Release of these toxins following ingestion could prove detrimental unless efficient detoxification mechanisms exist. Within the intestine of most mammalian species, efficient detoxification mechanisms are provided by the concerted actions of hydroxylation and conjugation (Phase I and Phase II metabolism) and active efflux (Phase III transport; active efflux) (Ritter 2007; Shugarts and Benet 2009; Thelen and Dressman 2009). Efflux transporters of the ATP-binding cassette (ABC) family are expressed in numerous locations throughout the body (e.g. kidney, liver, intestine, blood– brain barrier, blood–testis barrier) where they function as a barrier to the movement of a diverse array of chemically distinct molecules (Murakami and Takano 2008). This ‘‘multi-specificity’’ constitutes a prominent feature of their functionality, allowing for the recognition of an extremely wide variety of xenobiotics, including drug molecules, with a substantial degree of cross-recognition (Murakami and Takano 2008). This also allows for a certain amount of redundancy and/or compensation with regard to xenobiotic defence should the normal functioning of one protein be disturbed. We hypothesise that the expression of ABC transport proteins within the bovine rumen may provide a metabolically driven barrier to the movement of ingested xenobiotics. Previous research hints at this possibility, with plasma concentrations of ivermectin, an ABCB1 substrate (Brayden and Griffin 2008), increased co-administration of the P-glycoprotein inhibitor itraconazole after intra-ruminal administration of ivermectin in sheep, though the locus of this effect may result from actions in the small intestine (Ballent et al. 2007). The three ABC family efflux transporters expressed at the apical membrane of intestinal enterocytes are ABCB1 (multidrug resistance 1, MDR1; P-glycoprotein), ABCC2 (multidrug resistance related protein 2, MRP2; canalicular multispecific organic ion transporter) and ABCG2 (breast cancer resistance protein, BCRP) (Chan et al. 2004). The aim was, therefore, to establish whether expression of ABCB1, ABCC2 and ABCG2 occurred in bovine rumen.
Methods Tissue collection and preparation Four mature Holstein steers which were pasture grazed, with only grass hay in supplement, were humanely killed in
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a commercial slaughterhouse in accordance with UK law. Samples of bovine rumen from the ventral sac were obtained and prepared and transported as previously described (Graham and Simmons 2005). Epithelial tissue was then stripped from the underlying muscle layers and papillae samples collected for total RNA isolation for RT-PCR, SDS-PAGE/Western blotting and immunohistochemistry. RNA isolation and RT-PCR Rumen papillae for reverse-transcription (RT)-PCR were stored at 4 °C in RNAlater (Ambion). RNA was extracted from rumen papillae using RNAzol B reagent (Biogenesis). Briefly, tissue was ground in liquid nitrogen, followed by the addition of RNAzol B (2 mL 0.1 g of tissue). The resulting suspension was homogenised using a Polytron (CH-6010, Kinematica). 0.2 mL/mL chloroform was added to the suspension, which was shaken vigorously for 15 s followed by a 15 min incubation on ice. The samples were centrifuged at 20,000g for 30 min at 4 °C and the aqueous phase transferred to a clean tube. An equal volume of isopropanol was added and the sample incubated on ice for 15 min before centrifugation 20,000g for 60 min at 4 °C. The solvent was aspirated and the pellet washed with 85 % ethanol before a further centrifugation step at 20,000g for 15 min at 4 °C. The pellet was air-dried at room temperature and re-suspended in 10 mM Tris–HCl (pH 7.5). RNA was analysed by gel electrophoresis and found to be free of genomic DNA contamination. The ratio of 28:18 s ribosomal RNA was approximately 2:1, indicating intact RNA. RNA (2 lg) was reverse transcribed using Omniscript reverse transcriptase (Qiagen) according to the manufacturers guidelines. For each RNA sample, negative controls were included in which reverse transcriptase was omitted from the reaction. Hot-start Taq DNA polymerase (Qiagen) was used according to manufacturer’s protocol for amplification of 2 lL of reverse-transcribed RNA in a 50 lL reaction. Mg2? concentration was 1.5 mM, primer concentration was 0.5 lM and 2.5 U of enzyme were used per reaction. Enzyme activation was achieved with an initial heating step of 95 °C for 15 min. The annealing temperature for all primer pairs was set at 54–60 °C, as appropriate for primer pairs. Amplification was carried out over 35 cycles. Genespecific oligonucleotide primers for bovine ABCB1 (MDR1), ABCC2 (MRP2), ABCG2 (BCRP) and b-actin were designed from published bovine sequences (NCBI) incorporating exon-spanning regions of the nucleotide sequence. Expected product sizes were 792 base pairs (ABCB1), 750 base pairs (ABCC2), 1,161 base pairs (ABCG2) and 612 base pairs (b-actin)—Table 1. PCR products were analysed by agarose gel (1 %) electrophoresis, with products
J Comp Physiol B (2014) 184:673–681 Table 1 Oligonucleotides primer pairs used for identification of ABC family members in bovine rumen epithelium
Gene (alias)
675
Sequence
Product size (bp)
ABCB1 (MDR1, P-glycoprotein)
792
Forward
50 -GTGGGACAGGTCAGTTCATT-30
Reverse
50 -GCT CCT TGA TTC TGC CAT CT-30
ABCC2 (MRP2, CMOAT)
750
Forward
50 -TAC CAG CGA GTT CTG GAG G-30
Reverse
50 -AGG TCC CTC TGA GAG GAT G-30
ABCG2 (BCRP)
1,161
Forward
50 -CCC TTC GGC TTC CAA CAA C-30
Reverse
50 -CTG ACC TGC TGC TAT AGC C-30
b-Actin
612 0
Forward
5 -TCC ACG AAA CTA CCT TCA AT-3
Reverse
50 -TTT GGG AAG GCA AAG GAC-30
visualised using ethidium bromide staining and UV transillumination. Immunocytochemistry Full-thickness isolated rumen papillae were fixed in methanol at -20 °C or 3 % paraformaldehyde in PBS at 4 °C. They were then incubated for 24 h in 30 % sucrose in PBS at 4 °C before embedding in optimal temperature cutting compound (Tissue-Tek, Miles Laboratories, Naperville, IL) and cryo-sectioned. 5-lm sections were washed in PBS (3 times) and permeabilised with 0.1 % saponin in PBS for 20 min. Sections were blocked with 10 % serum (species-dependent on host animal used in the production of secondary antibodies) in PBS. Sections were incubated with primary antibodies (all monoclonal mouse IgG2a) overnight at 4 °C. Dilutions were 1:50, 1:50 and 1:100 for MDR1 (UIC2, Calbiochem), MRP2 (M2 III-6, Abcam) and BCRP (BXP-21, Abcam) antibodies, respectively. UIC2 recognises MDR1 in intact cells by an external epitope between transmembrane segments (TM) five and six of human MDR1 (GTTLVLSGEY) (Zhou et al. 1999) which is identical in 8/10 residues in bovine MDR1 (GTSLVLSKEY). UIC2 does not, however, recognise detergent solubilised protein (Mechetner and Roninson 1992). M2 III-6 was raised against the C-terminal of the human protein (residues 1339–1541) which is highly similar to the bovine protein with 180/202 identities by BLASTP (Altschul et al. 1997). BXP-21 was raised against residues 271–396 of the human protein and there are 87/125 identities with the bovine protein by BLASTP (Altschul et al. 1997). Sections were again washed three times with PBS before incubation with antimouse goat Alexa Fluor 488 secondary antibody (Life Technologies diluted 1:50 in 10 % serum) for 1 h at room temperature. Comparative immunostaining was also
0
performed for claudin-1, connexin-43 and the a-subunit of the Na–K ATPase as previously described (Graham and Simmons 2005). Nuclear staining was performed using ethidium homodimer 1 (1 mg/mL in water) at 1:1,000 dilution. Sections were imaged using confocal laser scanning microscopy (TCS-NT, Leica with Kr-Ar laser) using appropriate excitation and emission filter sets for dual fluorophore detection. Appropriate negative controls were performed where the primary antibody was omitted; these slides were used to set the background microscope intensity and gain for imaging. Western blotting Approximately 1 g of separated bovine rumen papillae was homogenised in 5 mL of lysis buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA) using a Polytron (CH6010, Kinematica). The homogenised tissue was centrifuged at 40009g for 10 min at 4 °C to pellet cell debris and nuclei. Protein was quantified using Bradford agent (Bio-Rad). Proteins were separated on 8 % SDS-polyacrylamide gels and transferred onto PVDF membranes (Millipore) overnight. Membranes were briefly washed with TBS-T (Tris-buffered saline and 0.1 % Tween 20, pH 7.6) and blocked in 3 % non-fat dried milk in TBS-T buffer. Membranes were probed overnight at 4 °C with the mouse monoclonal antibody C219 against ABCB1 (Calbiochem), M2 III-6 against ABCC2 (Abcam) and BXP-21 against ABCG2 (Abcam). The C-terminal human epitope recognised by the C219 antibody (VQEALD) is conserved in bovine MDR1. The antibodies were diluted 1:50 in TBST buffer. Following further washing, membranes were incubated with 1:10,000 HRP-labelled anti-mouse IgG (Abcam) and bands detected with the pico-chemiluminescence substrate (Pierce) following manufacturer’s instructions.
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Results The expression of ABCB1, ABCC2 and BCRP was investigated using bovine-specific primer pairs (Table 1), with RT-PCR of bovine rumen epithelium RNA. Expression of ABCB1 (MDR1) and ABCG2 (BCRP) is demonstrated by appropriately sized PCR products whilst there is absence of ABCC2 mRNA (Fig. 1). Expression of the housekeeping gene b-actin, as a positive control, was confirmed in all PCR reactions with RT-negative controls showing no contamination by genomic DNA. Data are representative of those for the four animals. ABCB1 protein expression was confirmed by Western blot analysis of rumen epithelium tissue using the mouse monoclonal antibody C219 (Fig. 2a), with a doublet of Mapp of *175 kD, corresponding to molecular weight of 170 kD seen in Caco-2 total protein used as a positive control (Fig. 2a). The human MDR1 protein is subject to
Fig. 1 RT-PCR products from bovine rumen mRNA, separated by agarose gel electrophoresis and visualised after ethidium bromide staining/UV-trans-illumination. Products of the predicted size for a MDR1 (792 base pairs) and c BCRP (1161 base pairs) are evident. No bands are visible for MRP2 b. b-Actin was present as a positive control with a PCR-negative control also included in each gel
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extensive N-glycosylation at three asparagine residues within the first extracellular loop between TM 1 and 2 (NITNRSDINDT) (Schinkel et al. 1993) which accounts for the difference in core Map (140 kDa) and the broad size distribution observed in SDS-PAGE (Greer and Ivey 2007). This part of the MDR1 sequence is poorly conserved between species; in the bovine sequence there are 31/48 non-identities with the human sequence. However, there are potentially 3 N-glycosylation sites (NITFPNTINGSDINDT) of the Asn/X/Thr or Ser in this extracellular loop. C219 does not give ABCB1 immunostaining in frozen-fixed bovine tissue so the alternative UIC2 antibody was used. Immunostaining with UIC2 is shown in Fig. 2b. Prominent staining is evident in the lateral and apical plasma membranes of epithelial cells reaching from the stratum basale to the stratum granulosum. Note that there is no immunostaining on the rumenfacing membrane of the stratum granulosum. No immunofluorescence is observed in keratinocytes of the stratum corneum. Figure 3 shows that MDR1 immunofluorescence is not restricted to the rumen papillary epithelium. Significant vascular MDR1 expression is seen associated with endothelial cells within central blood vessels (Fig. 3c). Immunofluorescence data for MDR1 and all other primary antibodies are representative for the four animals. ABCC2 protein expression was probed in bovine rumen epithelium using the mouse monoclonal antibody M2 III-6 (Fig. 4a). No bands were identified. A prominent, single band at *200 kD, likely resulting from the fully glycosylated form of ABCC2 (Zhang et al. 2005) was observed in Caco-2 cell extracts, known to express ABCC2. Immunocytochemical analysis of sections of bovine rumen using the same antibody did not display any specific fluorescence. Taken together with the lack of gene expression, confirmed by RT-PCR analysis, MRP2 can be seen to be absent in the bovine rumen epithelium. ABCG2 localisation was determined by immunocytochemistry using the mouse monoclonal antibody BXP-21 (Fig. 5). Immunofluorescence showed prominent lateral and apical membrane staining in the cells of the stratum basale and stratum spinosum, with decreasing intensity in the stratum granulosum. In positive cells there is vesicular cytoplasmic immunofluorescence. The stratum corneum was negative. BCRP immunofluorescence is also not restricted to the rumen papillary epithelium. Vascular endothelial cells are also seen to demonstrate BCRP immunofluorescence (Fig. 3d, e).
Discussion Although mammalian expression of ABC transporters along the intestinal tract is now well documented (Dietrich
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Fig. 2 MDR1 expression in rumen papilla epithelium and the colorectal adenocarcinoma cell line Caco-2. a Western blot of MDR1 displaying a double band of apparent molecular mass of *175 kD in the rumen and a single band of apparent molecular mass of *175 kD in Caco-2. b Immunolocalisation of MDR1 expression. Cells were co-stained with the nucleic acid marker ethidium
homodimer 1. The overlay combines images of MDR1 (green) and nucleic acid marker (red). Note the prominent lateral and apical staining of the cell membranes in layers reaching from the stratum basale to the stratum granulosum. The stratum corneum is negative. Scale bar 20 lm (colour figure online)
et al. 2003) including in cows (Lindner et al. 2013), little information is available for rumen. This study has, for the first time, confirmed the expression of members of the ABC family of transport proteins, in epithelial cells of the bovine rumen. Both ABCB1 (MDR1, P-glycoprotein) and ABCG2 (BCRP) were found to be expressed within several layers of the stratified rumen epithelium, showing overlapping but distinct localisations. MRP2 was not found at either the mRNA (RT-PCR) or protein (Western blot, IHC) level. Studies of the localisation of epithelial transporters and of the junctional protein, claudin 1, have emphasised the complexity of the rumen epithelial barrier (Fig. 6) (Graham et al. 2007; Graham and Simmons 2005). The tight junction proteins, ZO-1 and claudin 1, are expressed at the outer (lumen)-facing side of the stratum granulosum indicating that this cell layer forms the outer barrier of this stratified epithelium. The Na: H isoforms NHE1 and NHE3 are also expressed at this location, NHE3 also being present in the stratum spinosum with less in the stratum basale (Rabbani et al. 2011). A functional syncytium formed by connexin gap junctions between the cell layers underlying the stratum granulosum, comprising the stratum spinosum and the stratum basale, allows transepithelial transport of Na? to occur to blood from stratum basale cells by the concentration of Na–K ATPase expressed here (Graham and Simmons 2005). MCT1 is also expressed at the basal side of this syncytium likely facilitating export of SCFAs and ketone bodies to the blood (Graham et al. 2007). The presence of ABCB1 (MDR1) within the stratum basale,
spinosum and granulosum suggests that transport from these cell layers is directed at the interstitial space and not exclusively across the lumen-facing membrane of the stratum granulosum, as would be expected if transport was exclusively directed back towards the rumen lumen. Instead, transport function would act to prevent the syncytial cells themselves from being exposed to high concentrations of xenobiotics. Na–K ATPase (see Fig. 6), mitochondrial density and ATP supply are concentrated towards the base of cells in the stratum basale (Graham and Simmons 2005) and differ from MDR1. In the small intestine both metabolism (via mitochondrial CYP3A4) and transport via MDR1 combine to reinforce the oral barrier to xenobiotics (Zhang and Benet 2001). Export of parent compound or metabolite combines to maximise the metabolic capacity (Zhang and Benet 2001). Ultimately, transport of parent compound/metabolite via blood to liver will result in hepatocyte uptake, further metabolism and ultimately clearance via biliary excretion (again mediated by MDR1) to the intestine lumen. Recent evidence in another stratified epithelium, namely human skin, has also demonstrated expression of MDR1, predominantly in the basal cell layer and dermal appendages (Skazik et al. 2011). This partially confirms results from investigations in mdr1a/1b KO mice, in which MDR1 localisation was similar (Ito et al. 2008). An interesting observation (Ito et al. 2008) was the increased absorptive movement of the MDR1 substrate Rhodamine 123 in WT vs. mdr1a/b null animals. It is, therefore, possible to envisage a potential role for MDR1-mediated absorption of dietary components
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Fig. 3 Immunolocalisation of MDR1 and BCRP (both green) expression in the sub-epithelial connective and vascular tissue in bovine rumen papillae. Cells were co-stained with the nucleic acid marker ethidium homodimer-1 (red). a Transmitted-light photomicrograph of full-thickness papilla, scale bar 100 lm. b Fluorescence image of MDR1 immunofluorescence (green) of central papillary
blood vessels (arrowed), scale bar 100 lm. c High power image of endothelial MDR1-immunostaining, scale bar 20 lm. d Medium power image of BCRP-immunostaining (green), scale bar 20 lm. e High power image of endothelial BCRP-immunostaining, scale bar 20 lm (colour figure online)
in the bovine rumen. As has been suggested for both mouse and human skin (Ito et al. 2008), MDR1 could play a dual role in enhancing the absorption of certain molecular species whilst functioning in its traditional role of xenobiotic defence for the cells in which it is expressed. An additional feature of MDR1 expression in bovine rumen is the expression within endothelial cells within the papilla. MDR1 expression in the capillary endothelial cells of the blood–brain barrier and the blood–testes barrier is thought to isolate these tissues from blood-borne toxins and drugs (Cordoncardo et al. 1989). Whether expression in bovine blood vessel endothelium performs a role in aiding absorption or limiting access to the tissue from blood remains to be determined. Recently, cloned bovine BCRP expressed in MDCK cells has been investigated with regards to deposition to milk of flavonoids such as equol or quercetin and 19
commonly used veterinary drugs (Wassermann et al. 2013). Selected drugs included antibiotics, the avermectin/milbemycin anthelmintics, moxidectin and eprinomectin, and the benzimidazole anthelmintics albendazole and oxfendazole (Wassermann et al. 2013). The present data show BCRP expression in the bovine rumen which displays a similar pattern to MDR1, being present in the stratum basale and spinosum, but with concentration to the apical poles of the cells within the stratum spinosum consistent with limitation of xenobiotic uptake from the rumen. There is a marked decrease in immunofluorescence towards the stratum granulosum. This may indicate differing functionality compared with MDR1. BCRP has been reported to impact on the differentiation of certain mammalian cell populations, including stem cells, Langerhans cells and cultured cell lines (Bunting 2002; Prouillac et al. 2009) so the pattern oh high expression in stratum basale may indicate a
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Fig. 4 Lack of MRP2 expression in the bovine rumen. a Western blot for MRP2 displaying a single band of molecular mass *200 kDa in Caco-2 cell total protein extract, with the absence of a corresponding band in bovine rumen protein extract. b Immunolocalisation of MRP2 expression (green). Tissue was counterstained with the nucleic acid marker ethidium homodimer 1 (red). No MRP2 immunofluorescence was evident. Scale bar 20 lm (colour figure online)
Fig. 5 Immunolocalisation of BCRP expression in the bovine rumen. Cells were co-stained with the nucleic acid marker ethidium homodimer 1. The overlay combines images of BCRP (green) and nucleic acid marker (red). Note prominent lateral and apical staining of the cell membranes of the stratum basale and stratum spinosum with decreasing intensity towards the stratum granulosum. The stratum corneum is negative. Scale bar 20 lm (colour figure online)
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Fig. 6 Comparison of immunolocalisation of MDR1, BCRP, Na–K ATPase, connexion 43 and claudin 1 (all green) nucleic acid marker (red) in sections taken from an individual animal. L lumen, scale bars 20 lm (colour figure online)
similar action. As with MDR1 expression, BCRP protein was also observed in papillary dermis vasculature. BCRP has been reported in various human venous but not arterial endothelia (Maliepaard et al. 2001), providing a similar role to MDR1 in tissue defence, e.g. at the blood brain barrier (Eisenblatter et al. 2003). Whether expression in bovine blood vessel endothelium performs a dual role in aiding absorption or limiting access to the tissue from blood remains to be determined. Recent data from rat IEC-6 and human Caco-2 cells suggests that short-chain fatty acids such as butyric acid may act as substrates for BCRP (Goncalves et al. 2011). Given that SCFAs may reach substantial concentrations within bovine rumen (Gabel and Sehested 1997), especially after feeding with easily fermentable carbohydrates (Aschenbach et al. 2011), transport of SCFAs from the epithelial cells of the rumen may constitute a defensive function in rumen acidosis. Extreme loading of rumen epithelial cells with acid from SCFA uptake may result in tissue necrosis (Aschenbach et al. 2011).
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Functional data support a role for ABC transporters in the rumen. Plasma concentrations of the antihelminthic drug ivermectin attained lower, but detectable, levels when administered intra-ruminally (i.r.) compared to subcutaneous dosing, leading to lower antiparasitic activity (Prichard et al. 1985). Of note is the fact that ivermectin is now known to be an MDR1 substrate (Brayden and Griffin 2008; Schinkel et al. 1994). More recently, Ballent et al. (2007) followed up on these observations by investigating the impact of administering ivermectin i.r. and intravenously in the presence and absence of a Pgp inhibitor, itraconazole. Only i.r. co-dosing of ivermectin with the Pgp inhibitor had the effect of increasing plasma ivermectin concentrations. In addition, the level of ivermectin within abomasums tissues was increased relative to controls by *60 % by co-administration with itraconazole (Ballent et al. 2007). These data are compatible with reduced absorption of ivermectin due to MDR1 function. The results presented here have shown the expression and localisation of two members of the ABC family of
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transport proteins, ABCB1 (MDR1) and ABCG2 (BCRP). Both are traditionally concerned with xenobiotic defence which suggests a role in protecting bovines against ingested toxins and noxious metabolites produced during rumen fermentation. Direct functional investigations involving the use of MDR1 and BCRP-specific substrates are needed to elucidate the precise functional activity of these transport proteins within the intact bovine rumen. Acknowledgments Iain Haslam was supported by a studentship from the BBSRC (UK). This work was part funded by Pfizer Global Research Sandwich UK.
References Altschul SF et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402 Aschenbach JR, Penner GB, Stumpff F, Gabel G (2011) Ruminant nutrition symposium: role of fermentation acid absorption in the regulation of ruminal pH. J Anim Sci 89:1092–1107 Ballent M, Lifschitz A, Virkel G, Sallovitz J, Lanusse C (2007) Involvement of P-glycoprotein on ivermectin kinetic behaviour in sheep: itraconazole-mediated changes on gastrointestinal disposition. J Veterinary Pharmacol Ther 30:242–248 Brayden DJ, Griffin J (2008) Avermectin transepithelial transport in MDR1- and MRP-transfected canine kidney monolayers. Veterinary Res Commun 32:93–106 Bunting KD (2002) ABC transporters as phenotypic markers and functional regulators of stem cells. Stem Cells 20:11–20 Chan LMS, Lowes S, Hirst BH (2004) The ABCs of drug transport in intestine and liver: efflux proteins limiting drug absorption and bioavailability. Eur J Pharm Sci 21:25–51 Cordoncardo C et al (1989) Multidrug-resistance gene (P-Glycoprotein) is expressed by endothelial-cells at blood–brain barrier sites. Proc Natl Acad Sci USA 86:695–698 Dietrich CG, Geier A, Elferink RPJO (2003) ABC of oral bioavailability: transporters as gatekeepers in the gut. Gut 52:1788–1795 Eisenblatter T, Huwel S, Galla HJ (2003) Characterisation of the brain multidrug resistance protein (BMDP/ABCG2/BCRP) expressed at the blood–brain barrier. Brain Res 971:221–231 Gabel G, Sehested J (1997) SCFA transport in the forestomach of ruminants. Comp Biochem Physiol A Physiol 118:367–374 Goncalves P, Gregorio I, Martel F (2011) The short-chain fatty acid butyrate is a substrate of breast cancer resistance protein. Am J Physiol Cell Physiol 301:C984–C994 Graham C, Simmons NL (2005) Functional organization of the bovine rumen epithelium. Am J Physiol Regul Integr Comp Physiol 288:R173–R181 Graham C, Gatherar I, Haslam I, Glanville M, Simmons NL (2007) Expression and localization of monocarboxylate transporters and sodium/proton exchangers in bovine rumen epithelium. Am J Physiol Regul Integr Comp Physiol 292:R997–R1007 Greer DA, Ivey S (2007) Distinct N-glycan glycosylation of P-glycoprotein isolated from the human uterine sarcoma cell line MES-SA/Dx5. Biochim Biophys Acta 1770:1275–1282
681 Ito K et al (2008) P-Glycoprotein (Abcb1) is involved in absorptive drug transport in skin. J Control Release 131:198–204 Lindner S, Halwachs S, Wassermann L, Honscha W (2013) Expression and subcellular localization of efflux transporter ABCG2/ BCRP in important tissue barriers of lactating dairy cows, sheep and goats. J Vet Pharmacol Ther 36(6):562–570 Maliepaard M et al (2001) Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues. Cancer Res 61:3458–3464 Mechetner EB, Roninson IB (1992) Efficient inhibition of P-glycoprotein-mediated multidrug resistance with a monoclonal-antibody. Proc Natl Acad Sci USA 89:5824–5828 Murakami T, Takano M (2008) Intestinal efflux transporters and drug absorption. Expert Opin Drug Met 4:923–939 Prichard RK, Steel JW, Lacey E, Hennessy DR (1985) Pharmacokinetics of ivermectin in sheep following intravenous, intraabomasal or intraruminal administration. J Vet Pharmacol Ther 8:88–94 Prouillac C, Videmann B, Mazallon M, Lecoeur S (2009) Induction of cells differentiation and ABC transporters expression by a mycoestrogen, zearalenone, in human choriocarcinoma cell line (BeWo). Toxicology 263:100–107 Rabbani I, Siegling-Vlitakis C, Noci B, Martens H (2011) Evidence for NHE3-mediated Na transport in sheep and bovine forestomach. Am J Physiol Regul Integr Comp Physiol 301:R313–R319 Rea PA, Li ZS, Lu YP, Drozdowicz YM, Martinoia E (1998) From vacuolar Gs-X pumps to multispecific ABC Transporters. Annu Rev Plant Physiol Plant Mol Biol 49:727–760 Ritter JK (2007) Intestinal UGTs as potential modifiers of pharmacokinetics and biological responses to drugs and xenobiotics. Expert Opin Drug Metab 3:93–107 Schinkel AH, Kemp S, Dolle M, Rudenko G, Wagenaar E (1993) N-glycosylation and deletion mutants of the human MDR1 P-glycoprotein. J Biol Chem 268:7474–7481 Schinkel AH et al (1994) Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood–brain barrier and to increased sensitivity to drugs. Cell 77:491–502 Shugarts S, Benet LZ (2009) The role of transporters in the pharmacokinetics of orally administered drugs. Pharm Res 26:2039–2054 Skazik C et al (2011) P-glycoprotein (ABCB1) expression in human skin is mainly restricted to dermal components. Exp Dermatol 20:450–452 Thelen K, Dressman JB (2009) Cytochrome P450-mediated metabolism in the human gut wall. J Pharm Pharmacol 61:541–558 Wassermann L, Halwachs S, Lindner S, Honscha KU, Honscha W (2013) Determination of functional ABCG2 activity and assessment of drug-ABCG2 interactions in dairy animals using a novel MDCKII in vitro model. J Pharm Sci 102:772–784 Zhang YC, Benet LZ (2001) The gut as a barrier to drug absorption— combined role of cytochrome P450 3A and P-glycoprotein. Clin Pharmacokinet 40:159–168 Zhang P, Tian X, Chandra P, Brouwer KL (2005) Role of glycosylation in trafficking of Mrp2 in sandwich-cultured rat hepatocytes. Mol Pharmacol 67:1334–1341 Zhou Y, Gottesman MM, Pastan I (1999) The extracellular loop between TM5 and TM6 of P-glycoprotein is required for reactivity with monoclonal antibody UIC2. Arch Biochem Biophys 367:74–80
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