Expression of the cobalamin transporters cubam and MRP1 in the canine ileum-Upregulation in chronic inflammatory enteropathy.

Chronic inflammatory enteropathy (CIE) in dogs, a spontaneous model of human inflammatory bowel disease (IBD), is associated with a high rate of cobalamin deficiency. The etiology of hypocobalaminemia in human IBD and canine CIE remains unknown, and compromised intestinal uptake of cobalamin resulting from ileal cobalamin receptor deficiency has been proposed as a possible cause. Here, we evaluated the intestinal expression of the cobalamin receptor subunits, amnionless (AMN) and cubilin (CUBN), and the basolateral efflux transporter multi-drug resistance protein 1 (MRP1) in 22 dogs with CIE in comparison to healthy dogs. Epithelial CUBN and AMN levels were quantified by confocal laser scanning microscopy using immunohistochemistry in endoscopic ileal biopsies from dogs with (i) CIE and normocobalaminemia, (ii) CIE and suboptimal serum cobalamin status, (iii) CIE and severe hypocobalaminemia, and (iv) healthy controls. CUBN and MRP1 expression was quantified by RT-qPCR. Receptor expression was evaluated for correlation with clinical patient data. Ileal mucosal protein levels of AMN and CUBN as well as mRNA levels of CUBN and MRP1 were significantly increased in dogs with CIE compared to healthy controls. Ileal cobalamin receptor expression was positively correlated with age, clinical disease activity index (CCECAI) score, and lacteal dilation in the ileum, inversely correlated with serum folate concentrations, but was not associated with serum cobalamin concentrations. Cobalamin receptor downregulation does not appear to be the primary cause of hypocobalaminemia in canine CIE. In dogs of older age with severe clinical signs and/or microscopic intestinal lesions, intestinal cobalamin receptor upregulation is proposed as a mechanism to compensate for CIE-associated hypocobalaminemia. These results support oral supplementation strategies in hypocobalaminemic CIE patients.

Culture of vibrating microtome tissue slices as a 3D model in biomedical research.

The basic idea behind the use of 3-dimensional (3D) tools in biomedical research is the assumption that the structures under study will perform at the best in vitro if cultivated in an environment that is as similar as possible to their natural in vivo embedding. Tissue slicing fulfills this premise optimally: it is an accessible, unexpensive, imaging-friendly, and technically rather simple procedure which largely preserves the extracellular matrix and includes all or at least most supportive cell types in the correct tissue architecture with little cellular damage. Vibrating microtomes (vibratomes) can further improve the quality of the generated slices because of the lateral, saw-like movement of the blade, which significantly reduces tissue pulling or tearing compared to a straight cut. In spite of its obvious advantages, vibrating microtome slices are rather underrepresented in the current discussion on 3D tools, which is dominated by methods as organoids, organ-on-chip and bioprinting. Here, we review the development of vibrating microtome tissue slices, the major technical features underlying its application, as well as its current use and potential advances, such as a combination with novel microfluidic culture chambers. Once fully integrated into the 3D toolbox, tissue slices may significantly contribute to decrease the use of laboratory animals and is likely to have a strong impact on basic and translational research as well as drug screening.

Feasibility of combined upper and lower gastrointestinal endoscopic biopsy in the common marmoset (Callithrix jacchus) to evaluate gastrointestinal diseases.

BACKGROUND: Chronic gastroenteropathies, including gluten sensitivity and marmoset wasting syndrome, frequently occur in captive colonies of common marmosets (Callithrix jacchus). Early identification and diagnosis of affected animals are desirable. Endoscopic examination of the colon in marmosets is described, but the small intestine can harbor significant mucosal lesions not representing those in the colon. Evaluating the small intestine currently requires invasive surgical biopsies due to the small patient size, carrying a risk of severe complications. METHODS: Endoscopic intubation and multisite biopsy of the duodenum/proximal jejunum are demonstrated in 10 marmosets under general anesthesia. RESULTS: Esophagogastroduodenoscopy with colonoscopy efficiently aid in examining the gastrointestinal tract and obtaining an antemortem histologic diagnosis in marmosets with chronic gastrointestinal signs. CONCLUSIONS: This minimally invasive technique is feasible in marmosets. Future investigations into the pathogenesis of chronic gastroenteropathies will benefit from these data, leading to improved animal welfare and better individual and colony health management.

Possible influence of free fatty acid receptors on pH regulation in the ruminal epithelium of sheep.

High amounts of short-chain fatty acids (SCFAs) occur in the ovine rumen and constitute the animal's main energy source. However, they lead to an acidification of the ruminal epithelium. Therefore, effective intracellular pH (pHi ) regulation by transport proteins like monocarboxylate transporter 1 (MCT1) and Na+ /H+ exchangers (NHEs) is pivotal to ruminants to avoid epithelial damage. SCFAs might function not only as nutrients but also as signalling molecules by activating free fatty acid receptors (FFARs) in the ruminal epithelium and thus influence pHi regulation. FFARs work as nutrient sensors, transducing their information by modulating cyclic adenosine monophosphate (cAMP) levels. We hypothesized that (FFAR-modulated) decreases in cAMP levels stimulate the activity of MCT1 and NHEs in the ruminal epithelium of sheep. We detected two FFARs (GPR109A and FFAR2) immunohistochemically in the ovine ruminal epithelium. Administration of 10 mM butyrate to Ussing chamber-mounted epithelia provoked a significant reduction in intraepithelial cAMP levels. However, application of the GPR109A agonist niacin did not affect cAMP levels. MCT1 activity was analysed by measuring transepithelial 14 C-acetate fluxes, which were not inhibited by forskolin-induced increased cAMP levels. The recovery of pHi after acidification was assessed as an indicator of NHE activity in primary cultured ruminal epithelial cells. Recovery was significantly reduced when cells with increased cAMP levels were subjected to the NHE inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (10 µM). Nonetheless, with augmented cAMP levels alone, NHE activity tended to decline. We hypothesize that modulation of cAMP levels by butyrate is accomplished by FFAR2 activation, regulating NHE activity for pHi homoeostasis at least in part.

Glucose transport across lagomorph jejunum epithelium is modulated by AMP-activated protein kinase under hypoxia.

The gastrointestinal epithelium possesses adaptation mechanisms to cope with huge variations in blood flow and subsequently oxygenation. Since sufficient energy supply is crucial under hypoxic conditions, glucose uptake especially must be regulated by these adaptation mechanisms. Therefore, we investigated glucose transport under hypoxic conditions. Jejunal epithelia of rabbits were incubated in Ussing chambers under short-circuit current conditions. Hypoxia was simulated by gassing with 1% O2 instead of 100% O2. The activity of sodium-coupled glucose transporter-1 (SGLT-1) was assessed by measuring the increase of short circuit current ( Isc) after the addition of 2 mM glucose to the mucosal buffer solution. We observed decreased activity of SGLT-1 after hypoxia compared with control conditions. To investigate underlying mechanisms, epithelia were exposed to agonists and antagonists of AMP-activated protein kinase (AMPK) before assessment of SGLT-1-mediated transport and the pAMPK/AMPK protein ratio. Preincubation with the antagonist restored SGLT-1 activity under hypoxic conditions to the level of control conditions, indicating an involvement of AMPK in the downregulation of SGLT-1 activity under hypoxia, which was confirmed in Western blot analysis of pAMPK/AMPK. Transepithelial flux studies using radioactively labeled glucose, ortho-methyl-glucose, fructose, and mannitol revealed no changes after hypoxic incubation. Therefore, we could exclude a decreased transepithelial glucose transport rate and increased paracellular conductance under hypoxia. In conclusion, our study hints at a decreased activity of SGLT-1 under hypoxic conditions in an AMPK-dependent manner. However, transepithelial transport of glucose is maintained. Therefore, we suggest other transport mechanisms, especially glucose transporter 1 and/or 2 to substitute SGLT-1 under hypoxia. NEW & NOTEWORTHY To our knowledge, this is the first approach to simulate hypoxia and study its effects in the jejunal epithelium using the Ussing chamber technique. We were able show that AMPK plays a role in the downregulation of SGLT-1 and that there seems to be an upregulation of other glucose transport mechanisms in the apical membrane of lagomorph jejunum epithelium under hypoxia, securing the epithelial energy supply and thus integrity.

Establishment and Characterization of an SV40 Large T Antigen-Transduced Porcine Colonic Epithelial Cell Line.

Continuous cell lines have become indispensable tools that have enabled investigations into cellular mechanisms by increasing experimental reproducibility and sample availability, and decreasing the use of experimental animals. To facilitate studies of epithelial barrier function of the porcine colon, we aimed to establish an epithelial cell line with an extended replicative capacity. Cells were isolated from the proximal colon of a 3-week-old piglet and transduced using a recombinant retroviral vector construct containing the simian virus 40 large T antigen (SV40 TAg). We established a clonal epithelial cell line, referred to as PoCo83-3, that stably expressed the SV40 TAg, verified at mRNA and protein levels. PoCo83-3 showed epithelial cell-specific features, such as cobblestone-like morphology, dome structure formation, the presence of apical microvilli, and the expression of keratin 18, E-cadherin and the tight junction-associated proteins zonula occludens-1, occludin, and claudin-1. To validate PoCo83-3 as an in vitro model in epithelial barrier research, proinflammatory cytokine-inducible alterations in barrier integrity were demonstrated by incubating the cells with TNF-α and IFN-γ for 48 h. These cytokine treatments promoted a decreased transepithelial electrical resistance. In summary, PoCo83-3 exhibited an extended life span and a differentiated phenotype while maintaining epithelial characteristics. Based on these results, we present this cell line as a valuable in vitro model for investigations of epithelial barrier function in the porcine colon.

Architecture and Chemical Coding of the Inner and Outer Submucous Plexus in the Colon of Piglets.

In the porcine colon, the submucous plexus is divided into an inner submucous plexus (ISP) on the epithelial side and an outer submucous plexus (OSP) on the circular muscle side. Although both plexuses are probably involved in the regulation of epithelial functions, they might differ in function and neurochemical coding according to their localization. Therefore, we examined expression and co-localization of different neurotransmitters and neuronal markers in both plexuses as well as in neuronal fibres. Immunohistochemical staining was performed on wholemount preparations of ISP and OSP and on cryostat sections. Antibodies against choline acetyltransferase (ChAT), substance P (SP), somatostatin (SOM), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), neuronal nitric oxide synthase (nNOS) and the pan-neuronal markers Hu C/D and neuron specific enolase (NSE) were used. The ISP contained 1,380 ± 131 ganglia per cm2 and 122 ± 12 neurons per ganglion. In contrast, the OSP showed a wider meshwork (215 ± 33 ganglia per cm2) and smaller ganglia (57 ± 3 neurons per ganglion). In the ISP, 42% of all neurons expressed ChAT. About 66% of ChAT-positive neurons co-localized SP. A small number of ISP neurons expressed SOM. Chemical coding in the OSP was more complex. Besides the ChAT/±SP subpopulation (32% of all neurons), a nNOS-immunoreactive population (31%) was detected. Most nitrergic neurons were only immunoreactive for nNOS; 10% co-localized with VIP. A small subpopulation of OSP neurons was immunoreactive for ChAT/nNOS/±VIP. All types of neurotransmitters found in the ISP or OSP were also detected in neuronal fibres within the mucosa. We suppose that the cholinergic population in the ISP is involved in the control of epithelial functions. Regarding neurochemical coding, the OSP shares some similarities with the myenteric plexus. Because of its location and neurochemical characteristics, the OSP may be involved in controlling both the mucosa and circular muscle.

Ontogenic Changes of Villus Growth, Lactase Activity, and Intestinal Glucose Transporters in Preterm and Term Born Calves with or without Prolonged Colostrum Feeding.

Oral glucose supply is important for neonatal calves to stabilize postnatal plasma glucose concentration. The objective of this study was to investigate ontogenic development of small intestinal growth, lactase activity, and glucose transporter in calves (n = 7 per group) that were born either preterm (PT; delivered by section 9 d before term) or at term (T; spontaneous vaginal delivery) or spontaneously born and fed colostrum for 4 days (TC). Tissue samples from duodenum and proximal, mid, and distal jejunum were taken to measure villus size and crypt depth, protein concentration of mucosa and brush border membrane vesicles (BBMV), total DNA and RNA concentration of mucosa, mRNA expression and activity of lactase, and mRNA expression of sodium-dependent glucose co-transporter-1 (SGLT1) and facilitative glucose transporter 2 (GLUT2) in mucosal tissue. Additionally, protein expression of SGLT1 in BBMV and GLUT2 in crude mucosal membranes and immunochemical localization of GLUT2 in the enterocytes were determined. Villus height in distal jejunum was lower in TC than in T. Crypt depth in all segments was largest and the villus height/crypt depth ratio in jejunum was smallest in TC calves. Concentration of RNA was highest in duodenal mucosa of TC calves, but neither lactase mRNA and activity nor SGLT1 and GLUT2 mRNA and protein expression differed among groups. Localization of GLUT2 in the apical membrane was greater, whereas in the basolateral membrane was lower in TC than in T and PT calves. Our study indicates maturation processes after birth for mucosal growth and trafficking of GLUT2 from the basolateral to the apical membrane. Minor differences of mucosal growth, lactase activity, and intestinal glucose transporters were seen between PT and T calves, pointing at the importance of postnatal maturation and feeding for mucosal growth and GLUT2 trafficking.

Monocarboxylate transporters 1 and 4: expression and regulation by PPARα in ovine ruminal epithelial cells.

In the intact rumen epithelium, isoforms 1 and 4 of the monocarboxylate transporter (MCT1 and MCT4) are thought to play key roles in mediating transcellular and intracellular permeation of short-chain fatty acids and their metabolites and in maintaining intracellular pH. We examined whether both MCT1 and MCT4 are expressed at mRNA and protein levels in ovine ruminal epithelial cells (REC) maintained in primary culture and whether they are regulated by peroxisome proliferator-activated receptor-α (PPARα). Because both transporters have been characterized to function coupled to protons, the influence of PPARα on the recovery of intracellular pH after l-lactate exposure was evaluated by spectrofluorometry. MCT1 and MCT4 were detected using immunocytochemistry both at the cell margins and intracellularly in cultured REC. To test regulation by PPARα, cells were exposed to WY 14.643, a selective ligand of PPARα, for 48 h. The subsequent qPCR analysis resulted in a dose-dependent upregulation of MCT1 and PPARα target genes, whereas response of MCT4 was not uniform. Protein expression of MCT1 and MCT4 quantified by Western blot analysis was not altered by WY 14.643 treatment. l-Lactate-dependent proton export was blocked almost completely by pHMB, a specific inhibitor of MCT1 and MCT4. However, l-lactate-dependent, pHMB-inhibited proton export in WY 14.643-treated cells was not significantly altered compared with cells not treated with WY 14.643. These data suggest that PPARα is particularly regulating MCT1 but not MCT4 expression. Extent of lactate-coupled proton export indicates that MCT1 is already working on a high level even under unstimulated conditions.

Diet effects on glucose absorption in the small intestine of neonatal calves: importance of intestinal mucosal growth, lactase activity, and glucose transporters.

Colostrum (C) feeding in neonatal calves improves glucose status and stimulates intestinal absorptive capacity, leading to greater glucose absorption when compared with milk-based formula feeding. In this study, diet effects on gut growth, lactase activity, and glucose transporters were investigated in several gut segments of the small intestine. Fourteen male German Holstein calves received either C of milkings 1, 3, and 5 (d 1, 2, and 3 in milk) or respective formulas (F) twice daily from d 1 to d 3 after birth. Nutrient content, and especially lactose content, of C and respective F were the same. On d 4, calves were fed C of milking 5 or respective F and calves were slaughtered 2h after feeding. Tissue samples from duodenum and proximal, mid-, and distal jejunum were taken to measure villus size and crypt depth, mucosa and brush border membrane vesicles (BBMV) were taken to determine protein content, and mRNA expression and activity of lactase and mRNA expression of sodium-dependent glucose co-transporter-1 (SGLT1) and facilitative glucose transporter (GLUT2) were determined from mucosal tissue. Additionally, protein expression of SGLT1 in BBMV and GLUT2 in crude mucosal membranes and BBMV were determined, as well as immunochemically localized GLUT2 in the intestinal mucosa. Villus circumference, area, and height were greater, whereas crypt depth was smaller in C than in F. Lactase activity tended to be greater in C than in F. Protein expression of SGLT1 was greater in F than in C. Parameters of villus size, lactase activity, SGLT1 protein expression, as well as apical and basolateral GLUT2 localization in the enterocytes differed among gut segments. In conclusion, C feeding, when compared with F feeding, enhances glucose absorption in neonatal calves primarily by stimulating mucosal growth and increasing absorptive capacity in the small intestine, but not by stimulating abundance of intestinal glucose transporters.

The G protein-coupled receptor P2Y14 influences insulin release and smooth muscle function in mice.

UDP sugars were identified as extracellular signaling molecules, assigning a new function to these compounds in addition to their well defined role in intracellular substrate metabolism and storage. Previously regarded as an orphan receptor, the G protein-coupled receptor P2Y14 (GPR105) was found to bind extracellular UDP and UDP sugars. Little is known about the physiological functions of this G protein-coupled receptor. To study its physiological role, we used a gene-deficient mouse strain expressing the bacterial LacZ reporter gene to monitor the physiological expression pattern of P2Y14. We found that P2Y14 is mainly expressed in pancreas and salivary glands and in subpopulations of smooth muscle cells of the gastrointestinal tract, blood vessels, lung, and uterus. Among other phenotypical differences, knock-out mice showed a significantly impaired glucose tolerance following oral and intraperitoneal glucose application. An unchanged insulin tolerance suggested altered pancreatic islet function. Transcriptome analysis of pancreatic islets showed that P2Y14 deficiency significantly changed expression of components involved in insulin secretion. Insulin secretion tests revealed a reduced insulin release from P2Y14-deficient islets, highlighting P2Y14 as a new modulator of proper insulin secretion.

Cholinergic modulation of epithelial integrity in the proximal colon of pigs.

BACKGROUND: Within the gut, acetylcholine (ACh) is synthesised by enteric neurons, as well as by 'non-neuronal' epithelial cells. In studies of non-intestinal epithelia, ACh was involved in the generation of an intact epithelial barrier. In the present study, primary cultured porcine colonocytes were used to determine whether treatment with exogenous ACh or expression of endogenous epithelium-derived ACh may modulate epithelial tightness in the gastrointestinal tract. METHODS: Piglet colonocytes were cultured on filter membranes for 8 days. The tightness of the growing epithelial cell layer was evaluated by measuring transepithelial electrical resistance (TEER). To determine whether ACh modulates the tightness of the cell layer, cells were treated with cholinergic, muscarinic and/or nicotinic agonists and antagonists. Choline acetyltransferase (ChAT), cholinergic receptors and ACh were determined by immunohistochemistry, RT-PCR and HPLC, respectively. RESULTS: Application of the cholinergic agonist carbachol (10 µm) and the muscarinic agonist oxotremorine (10 µM) resulted in significantly higher TEER values compared to controls. The effect was completely inhibited by the muscarinic antagonist atropine. Application of atropine alone (without any agonist) led to significantly lower TEER values compared to controls. Synthesis of ACh by epithelial cells was proven by detection of muscarinic and nicotinic receptor mRNAs, immunohistochemical detection of ChAT and detection of ACh by HPLC. CONCLUSION: ACh is strongly involved in the regulation of epithelial tightness in the proximal colon of pigs via muscarinic pathways. Non-neuronal ACh seems to be of particular importance for epithelial cells forming a tight barrier.

Establishment and characterization of porcine colonic epithelial cells grown in primary culture.

BACKGROUND: Primary cultures of epithelial cells are suitable models for studying epithelial function and, in particular, the regulation of epithelial tightness in vitro. The aim of our study was to develop a protocol for the isolation and culture of porcine colonic epithelial cells and to establish transepithelial electrical resistance (TEER) as a functional parameter for epithelial tightness. METHODS: Epithelial cells were obtained from the proximal colon of piglets by enzymatic dispase digestion. Cells were cultured on collagen-coated membrane supports for 21 days. The epithelial origin of the cells was shown by immunohistochemical detection of cytokeratin and zonula occludens protein 1 (ZO-1). Scanning electron microscopy, transmission electron microscopy and confocal microscopy were used for further morphological characterization. The integrity and tightness of the artificial epithelium were determined by measuring TEER. RESULTS: The cultured epithelial cells were immunoreactive for cytokeratin and ZO-1. They showed dense microvilli on their apical membranes and expression of Na(+)/K(+)-ATPase on their basolateral membranes. Adjacent cells were connected by tight junctions. We observed TEER to continuously increase up to 870 ± 38 Ω·cm(2) during the culture period. TEER correlated with the amount of epithelial cells expressing ZO-1. CONCLUSIONS: The properties of primary cultured epithelial cells resemble the structural properties of polarized colonic epithelium in vivo. Measurement of TEER seems to be suitable for studying epithelial tightness in vitro. We suggest that these primary epithelial cultures be used to investigate the regulation of the epithelial barrier function.

Intrinsic ruminal innervation in ruminants of different feeding types.

According to their feeding habits, ruminants can be classified as grazers, concentrate selectors and those of intermediate type. The different feeding types are reflected in distinct anatomical properties of the forestomachs. The present study was designed to investigate whether the intrinsic innervation patterns of the rumen (the main part of the forestomach) differ between intermediate types and grazers. Myenteric plexus preparations from the rumen of goats (intermediate type), fallow deer (intermediate type), cattle (grazer) and sheep (grazer) were analysed by immunohistochemical detection of the following antigens: Hu-protein (HuC/D), choline acetyltransferase (ChAT), nitric oxide synthase (NOS), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), substance P (SP), calbindin (CALB) and somatostatin (SOM). Myenteric ganglia of cattle contained 73 +/- 6 neurons per ganglion, whereas the ganglia of sheep were significantly smaller (45 +/- 18 neurons per ganglion). The ganglion density of the myenteric plexus was highest in fallow deer (15 +/- 3 ganglia per cm(2)) and lowest in cattle (6 +/- 1 ganglia per cm(2)). All myenteric neurons were either ChAT or NOS positive. The proportion of NOS-positive neurons was significantly lower in sheep (29.5 +/- 8.2% of all neurons) than in goats (44.2 +/- 9.8%). In all species, additional analysis of the different neuropeptides revealed the following subpopulations in descending order of percentile appearance: ChAT/SP > NOS/VIP/NPY > ChAT/- > NOS/NPY. Expression of CALB was detected in a minority of the ChAT-positive neurons in all species. Somatostatin immunoreactive somata were found only in preparations obtained from fallow deer and sheep. These data suggest that the rumen of grazers is under stronger cholinergic control than the rumen of species belonging to the intermediate type, although most subpopulations of neurons are present in all species. However, whether the strong mixing patterns of low quality roughage during digestion are enabled by the prominent excitatory input of the rumen of grazers requires elucidation in further studies.

Infection of the enteric nervous system by Borna disease virus (BDV) upregulates expression of Calbindin D-28k.

Borna disease virus (BDV) is a neurotropic agent infecting distinct neuronal subpopulations in the central nervous system of various mammalian species possibly including humans. Horses, a major natural host for BDV, show gastrointestinal dysfunctions besides characteristic neurological symptoms. Therefore, we hypothesized that enteric neurons may be targets of BDV replication. The presence of BDV-specific antigen in subpopulations of the ENS was investigated. Four-week-old Lewis rats were infected intracerebrally and sacrificed 4-14 weeks post infection (p.i.). BDV-immunoreactive neurons were found in submucous and myenteric neurons of the proximal colon. Fourteen weeks p.i., the proportion of BDV-positive neurons was 44+/-17 and 24+/-7% in the submucous and myenteric plexus, respectively. The majority of BDV-positive myenteric neurons showed immunoreactivity for choline acetyltransferase. Expression of Calbindin D-28k (CALB) was found in 96% of submucous and 67% of myenteric BDV-immunoreactive neurons. Additionally, the number of CALB-immunoreactive neurons was significantly higher in the myenteric plexus of infected rats compared to controls. These data indicate that BDV infects specific subpopulations of enteric neurons. Therefore, the ENS might serve as a site for BDV replication and as an immunoprivileged reservoir for BDV. In addition, upregulation of CALB in neurons of the myenteric plexus is probably induced during BDV-infection.

Calbindin-immunoreactive neurones in the ovine rumen.

In small laboratory animals, such as guinea pigs, immunoreactivity for the calcium-binding protein calbindin (CALB) can be used to distinguish functionally different classes of myenteric neurones. The rumen of sheep is a highly specialized gastrointestinal region, and the control of its functions requires specific intrinsic innervation patterns. The aim of this study was to neurochemically identify and characterize CALB-positive myenteric neurones of the ovine rumen. Therefore, we performed quadruple immunohistochemistry against CALB, substance P (SP), vasoactive intestinal peptide (VIP), and nitric oxide synthase (NOS) using whole-mount preparations of the ruminal myenteric plexus. On average, 3 +/- 2 and 1 +/- 0.4 myenteric neurones/ganglion were CALB-immunoreactive in suckling lambs and adult sheep, respectively. These neurones had Dogiel type-I morphology. Most of them (89.2% +/- 8.7% and 71.7% +/-44.8% in suckling lambs and adult sheep, respectively) did not colocalize any of the other antigenes. Since it has been shown in previous studies that ruminal myenteric neurones are immunoreactive for either choline acetyltransferase (ChAT) or NOS, we defined neurones which were CALB-positive and NOS-negative as CALB/ChAT. The other CALB-positive neurones were encoded CALB/NOS/+/-VIP (10.3% +/- 9.3% and 26.7% +/- 46.2% in suckling lambs and adult sheep, respectively) or CALB/ChAT/SP (0.5% +/- 1.0% and 1.7% +/- 1.9% in suckling lambs and adult sheep, respectively). We used cryostat sections of the ruminal wall to analyze the projections of the CALB-positive neurones. CALB-immunoreactive somata were exclusively located within the myenteric plexus. CALB-immunoreactive nerve fibers were found primarily in the lamina propria of the ruminal papillae. We conclude that CALB-positive myenteric neurones within the ovine rumen project to the epithelium; however, their functional role remains to be investigated.

Intrinsic innervation patterns of the smooth muscle in the rumen and reticulum of lambs.

The rumen and reticulum of sheep serve as a fermentation chamber. Both compartments exhibit specific motility patterns. With developmental changes, the size of the reticulorumen dramatically increases when newborn lambs mature to adult sheep. This makes it possible to investigate the intrinsic innervation of the reticuloruminal muscles in lambs by taking the entire reticulum and rumen into account. The aim of the study was to analyse the projections and neurochemistry of myenteric neurons in the rumen and reticulum, which project to the inner or outer muscle layer, respectively. Therefore, we applied retrograde tracing with the fluorescent dye 1,1'-didodecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) and subsequent immunohistochemical detection of choline acetyltransferase (ChAT), substance P (SP) and vasoactive intestinal peptide (VIP). In both compartments innervation of both the inner and the outer muscle layer consisted mainly of cholinergic neurons (65-70%). The majority of them co-localized SP. The non-cholinergic neurons projecting to the muscle expressed immunoreactivity for VIP. Polarized innervation of the muscle layers was found neither in the rumen nor in the reticulum. Consequently, intrinsic innervation patterns for the smooth muscle layers in the rumen and reticulum differ from all gastrointestinal regions examined thus far.

Age-associated plasticity in the intrinsic innervation of the ovine rumen.

The rumen of adult sheep functions as a large fermentation chamber. In the newborn suckling ruminant, the rumen is bypassed and milk enters the abomasum directly. It was the aim of our study to investigate whether the transmitter content of intrinsic nerves changes with the developmental stage. The neurochemical code of myenteric neurons in the rumen from suckling lambs, fattened lambs and adult sheep was determined by using quadruple immunohistochemistry against choline-acetyltransferase (ChAT), nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). Three neurochemically distinct subpopulations were identified within the rumen. They expressed the code ChAT/-, ChAT/SP and NOS/VIP. The number of ChAT/SP neurons did not change during development. It was 62% in the newborn lamb and remained stable in fattened lambs (63%) and adult sheep (63%). By contrast, the number of ChAT/- neurons decreased significantly from 20% in suckling lambs to 11% and 7% in fattened lambs and adult sheep, respectively. Simultaneously, the proportion of NOS/VIP neurons increased from 16% in suckling lambs to 29% in adult sheep. The increase in the proportion of NOS/VIP immunoreactive neurons indicates an adaptation to large volumes of ingesta at the beginning of roughage intake and rumination. We conclude that the age-associated changes in neurochemical code of myenteric neurons in the forestomach are related to the adaption of the rumen to different functional properties during development.

Reticular groove and reticulum are innervated by myenteric neurons with different neurochemical codes.

The reticulum and the reticular groove are functional distinct compartments within the ovine forestomach. While the reticulum takes part in various motor functions, such as mixing, retaining, and rejecting the forestomach ingesta, the reticular groove serves mainly as a bypass between the esophagus and the abomasum. To accomplish these different tasks, the compartments develop specific motility patterns that are controlled by intrinsic neural circuits. In this study the intrinsic innervation by myenteric neurons was analyzed by quadruple immunohistochemistry against cholineacetyl transferase (ChAT), nitric oxide synthase (NOS), substance P (SP), and vasoactive intestinal peptide (VIP). Four neurochemically different subpopulations of myenteric neurons were found in the reticulum and the floor of the reticular groove: ChAT/-, ChAT/SP, NOS/-, and NOS/VIP. The neuronal proportions were calculated relative to all myenteric neurons. Neurons of the reticulum were mostly immunoreactive for ChAT (89% +/- 3%), whereas neurons adjacent to the reticular groove predominantly expressed a nitrergic phenotype (62% +/- 4%). ChAT-positive neurons were also immunoreactive for SP (ChAT/SP: 64% +/- 3% reticulum; 25% +/- 1% reticular groove) or were purely cholinergic (ChAT/-: 25% +/- 4% reticulum; 13% +/- 3% reticular groove). NOS-positive neurons colocalized VIP (NOS/VIP: 10% +/- 3% reticulum; 46% +/- 1% reticular groove) or none of the other neurotransmitters (NOS/-: 1% +/- 1% reticulum; 17% +/- 3% reticular groove). Analysis of the soma sizes revealed that in both compartments the nitrergic neurons were significantly larger than the cholinergic neurons. It is suggested that the specific neurochemical code in combination with a specific morphology leads to a precise regulation of the specialized tasks of the reticulum and reticular groove by subpopulations of myenteric neurons.

Transport of ketone bodies and lactate in the sheep ruminal epithelium by monocarboxylate transporter 1.

Due to intensive intracellular metabolism of short-chain fatty acids, ruminal epithelial cells generate large amounts of D-beta-hydroxybutyric acid, acetoacetic acid, and lactic acid. These acids have to be extruded from the cytosol to avoid disturbances of intracellular pH (pH(i)). To evaluate acid extrusion, pH(i) was studied in cultured ruminal epithelial cells of sheep using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Extracellular addition of D-beta-hydroxybutyrate, acetoacetate, or lactate (20 mM) resulted in intracellular acidification. Vice versa, removing extracellular D-beta-hydroxybutyrate, acetoacetate, or lactate after preincubation with the respective monocarboxylate induced an increase of pH(i). Initial rate of pH(i) decrease as well as of pH(i) recovery was strongly inhibited by pCMBS (400 microM) and phloretin (20 microM). Both cultured cells and intact ruminal epithelium were tested for the possible presence of proton-linked monocarboxylate transporter (MCT) on both the mRNA and protein levels. With the use of RT-PCR, mRNA encoding for MCT1 isoform was demonstrated in cultured ruminal epithelial cells and the ruminal epithelium. Immunostaining with MCT1 antibodies intensively labeled cultured ruminal epithelial cells and cells located in the stratum basale of the ruminal epithelium. In conclusion, our data indicate that MCT1 is expressed in the stratum basale of the ruminal epithelium and may function as a main mechanism for removing ketone bodies and lactate together with H+ from the cytosol into the blood.