Helga Pfannkuche

Immunohistochemical evidence for the presence of calbindin containing neurones in the myenteric plexus of the guinea-pig stomach.

Using immunohistochemistry we studied the presence of calbindin in myenteric neurones of the guinea-pig stomach. A rabbit anti recombinant rat calbindin-D28k (CALB) stained 12, 12 and 25% of all myenteric neurones in the fundus, corpus and antrum, respectively. A rabbit anti recombinant human CALB stained 4, 4 and 16%, respectively. A mouse monoclonal antibody against the chicken intestinal CALB showed no labelling. In all regions most calbindin neurones were additionally choline acetyltransferase (ChAT) positive while only a small proportion exhibited nicotinamide adenosine dinucleatide phosphate (NADPH)-diaphorase-activity. Numerous calbindin-positive varicose nerve fibres were present within myenteric ganglia, rarely detectable in the muscle layers and virtually absent in the mucosa. This study demonstrated that a supopulation of cholinergic myenteric neurones in the stomach contain calbindin and suggested that many of these neurones fulfil interneuronal tasks.

Enkephalin-immunoreactive subpopulations in the myenteric plexus of the guinea-pig fundus project primarily to the muscle and not to the mucosa.

Abstract Enkephalin (ENK) immunoreactivity was localised in different neuronal subpopulations of the myenteric plexus in the guinea-pig gastric fundus using immunohistochemistry for neurone-specific enolase (NSE), ENK, choline acetyltransferase (ChAT), substance P (SP), neuropeptide Y (NPY), calretinin (CALRET), and somatostatin (SOM). NADPH-diaphorase staining was used to label nitric oxide synthase (NOS)-containing neurones. ENK was observed in 44% of the myenteric neurones. The major ENK-positive subpopulations were ChAT/ENK (35% of ENK-positive neurones), ChAT/SP/ENK (26%), NOS/NPY/ENK (22%) and ChAT/SP/ENK/CALRET (9%). The projection pathways of these ENK-positive subpopulations to the circular muscle and the mucosa were determined using retrograde labelling with DiI in organ culture followed by immunohistochemistry. Of myenteric neurones retrogradely labelled from the mucosa and the circular muscle, 13% and 48% exhibited ENK immunoreactivity, respectively. Three major ENK-positive subpopulations innervating the mucosa or circular muscle were identified: ascending ChAT/SP/ENK (7% of all mucosa neurones; 24% of all circular muscle neurones), ascending ChAT/ENK (4%; 15%) and descending NOS/NPY/ENK (1%; 8%) neurones. Only very few CALRET- or SOM-positive neurones projected to the mucosa or circular muscle. ChAT/SP/ENK and ChAT/ENK neurones might function as ascending excitatory muscle motor neurones, whereas NOS/NPY/ENK neurones are most likely descending inhibitory muscle motor neurones. The relatively few ENK-positive mucosa neurones do not favour a major involvement of ENK-positive myenteric neurones in the control of gastric mucosa activity.

Influence of isoform and DNP on butyrate transport across the sheep ruminal epithelium

Abstract. Short-chain fatty acids are absorbed in considerable amounts from the rumen. During transit through the epithelial layer, they are intensively metabolised. Interaction between intraepithelial metabolism and absorption, however, is hardly understood. The present study therefore compared the transepithelial transport of the easily metabolised n-butyrate with that of the more metabolism-resistant iso-butyrate both under in vivo conditions (isolated and washed reticulorumen) and in vitro conditions (Ussing chamber). Under in vivo conditions, net absorption of n-butyrate was significantly higher than that of iso-butyrate. The in vitro experiments showed that the higher net flux of n-butyrate was solely due to a higher mucosal-to-serosal flux, whereas the serosal-to-mucosal flux of butyrate was independent from the isoform. Blocking intraepithelial ATP delivery by 2,4-dinitrophenol abolished the net flux of n-butyrate. The study indicates that metabolism and/or ATP availability stimulates n-butyrate net absorption. By this, the metabolic activity of the epithelium may have a regulatory influence on absorption of n-butyrate.

Bicarbonate-dependent transport of acetate and butyrate across the basolateral membrane of sheep rumen epithelium

This study aimed to assess the role of HCO3− in the transport of acetate and butyrate across the basolateral membrane of rumen epithelium and to identify transport proteins involved.

The G Protein-coupled Receptor P2Y14 Influences Insulin Release and Smooth Muscle Function in Mice

Background: The relevance of the widely expressed GPCR P2Y14 is only partially understood. Results: Analysis of P2Y14-KO mice revealed decreased gastrointestinal emptying, reduced glucose tolerance, and insulin release. Conclusion: P2Y14 function is required for proper intestine emptying and adequate glucose response. Significance: P2Y14 plays a role in smooth muscle function and maintaining energy homeostasis by influencing insulin release. 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.

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 cm2) and lowest in cattle (6 ± 1 ganglia per cm2). 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.

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.

Cholinergic and noncholinergic innervation of the smooth muscle layers in the bovine abomasum.

The intrinsic innervation of muscle layers in the mammalian gastrointestinal tract has been mainly studied in nonruminants. The aim of this study was to identify intrinsic motor neurones in the bovine abomasum that innervate the circular and longitudinal muscles. Circular (CMN) and longitudinal muscle motor neurones (LMN) were selectively labeled by application of the retrograde tracer 1,1′‐didodecyl‐3,3,3′,3′‐tetramethyl indocarbocyanine perchlorate (DiI) onto the muscle layers. The transmitter phenotype was determined by immunohistochemical detection of choline acetyltransferase (ChAT), nitric oxide synthase (NOS), and neurone‐specific enolase (NSE). On average, the myenteric ganglia contained 61 ± 19 NSE‐positive cell bodies, of which 89% were ChAT‐positive and 10% were NOS‐positive. Only 0.7% of NSE‐positive neurones (41 of 5,777) contained both ChAT and NOS. Application of DiI onto the circular and longitudinal muscles revealed on average 60 ± 27 (n = 4) and 68 ± 36 (n = 4), respectively, labeled cell bodies in the myenteric plexus. For the circular and longitudinal muscles the proportions of ascending to descending neurones were 76 : 24% and 54 : 46%, respectively. While most ascending CMN were ChAT‐positive (96%), 51% of the descending CMN were ChAT‐negative. All ascending and 95% of descending LMN were ChAT‐positive. It was concluded that cholinergic excitatory innervation is predominant in both muscle layers of the abomasum. Whereas the circular muscle receives cholinergic excitatory and nitrergic inhibitory innervation, the longitudinal muscle is only innervated by cholinergic pathways. This innervation pattern is different from that in gastric muscle layers in monogastric animals. Anat Rec 267:70–77, 2002.

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.

Neurochemical coding and projection patterns of gastrin-releasing peptide-immunoreactive myenteric neurone subpopulations in the guinea-pig gastric fundus.

The aim of this study was to characterise the projection and neurochemical coding patterns of gastrin-releasing peptide (GRP)-containing subpopulations of myenteric neurones in the guinea-pig gastric fundus. For this purpose, we used retrograde tracing with the dye DiI and immunohistochemistry against GRP, choline acetyltransferase (ChAT), enkephalin (ENK), substance P (SP) and neuropeptide Y (NPY). Cell counts revealed that 44% of the myenteric neurones were GRP-positive. Of the GRP-positive neurones, 92% were ChAT-positive and, hence, 8% were presumptively nitric oxide synthase positive (NOS). The GRP-positive subpopulations were ChAT/GRP (40% of all GRP neurones), ChAT/NPY/GRP (25%), ChAT/SP/GRP/+/-ENK (20%), ChAT/ENK/GRP (8%), NOS/NPY/GRP/+/-ENK (5%) and NOS/GRP (3%). The tracing experiments revealed the relative contributions of the various GRP-positive subpopulations to the innervation of the circular muscle and the mucosa. GRP immunoreactivity was detected in 46 and 38% of the DiI-labelled muscle and mucosa neurones, respectively. GRP was almost exclusively found in ascending ChAT-positive mucosa and muscle neurones. The populations encoded ChAT/SP/GRP/+/-ENK and ChAT/ENK/GRP projected predominantly to the circular muscle, whereas the ChAT/NPY/GRP and ChAT/GRP populations had primarily projections to the mucosa. GRP was colocalised with ChAT, ENK and/or SP in varicose nerve fibres innervating the circular muscle and the muscularis mucosae, whereas in the mucosal epithelium GRP was mainly present in nerve fibres containing ChAT and NPY. The data suggest that in the guinea-pig gastric fundus, the ChAT/SP/GRP/+/-ENK and ChAT/ENK/GRP neurones are ascending excitatory muscle motor neurones, whereas the ChAT/NPY/GRP and ChAT/GRP neurones are very likely involved in the regulation of mucosal functions.