Murine Stomach Research Articles

Synaptic specializations exist between enteric motor nerves and interstitial cells of Cajal in the murine stomach

Autonomic neurotransmission is thought to occur via a loose association between nerve varicosities and smooth muscle cells. In the gastrointestinal tract ultrastructural studies have demonstrated close apposition between enteric nerves and intramuscular interstitial cells of Cajal (ICC-IM) in the stomach and colon and ICC in the deep muscular plexus (ICC-DMP) of the small intestine. In the absence of ICC-IM, postjunctional neural responses are compromised. Although membrane specializations between nerves and ICC-IM have been reported, the molecular identity of these specializations has not been studied. Here we have characterized the expression and distribution of synapse-associated proteins between nerve terminals and ICC-IM in the murine stomach. Transcripts for the presynaptic proteins synaptotagmin, syntaxin, and SNAP-25 were detected. Synaptotagmin and SNAP-25-immunopositive nerve varicosities were concentrated in varicose regions of motor nerves and were closely apposed to ICC-IM but not smooth muscle. W/W(V) mice were used to examine the expression and distribution of synaptic proteins in the absence of ICC-IM. Transcripts encoding synaptotagmin, syntaxin, and SNAP-25 were detected in W/W(V) tissues. In the absence of ICC-IM, synaptotagmin and SNAP-25 were localized to nerve varicosities. Reverse transcriptase polymer chain reaction (RT-PCR) and immunohistochemistry demonstrated the expression of postsynaptic density proteins PSD-93 and PSD-95 in the stomach and expression levels of PSD-93 and PSD-95 were reduced in W/W(V) mutants. These data support the existence of synaptic specializations between enteric nerves and ICC-IM in gastric tissues. In the absence of ICC-IM, components of the synaptic vesicle docking and fusion machinery is trafficked and concentrated in enteric nerve terminals.

Neural regulation of slow-wave frequency in the murine gastric antrum

Gastric peristaltic contractions are driven by electrical slow waves modulated by neural and humoral inputs. Excitatory neural input comes primarily from cholinergic motor neurons, but ACh causes depolarization and chronotropic effects that might disrupt the normal proximal-to-distal spread of gastric slow waves. We used intracellular electrical recording techniques to study cholinergic responses in stomach tissues from wild-type and W/W(V) mice. Electrical field stimulation (5 Hz) enhanced slow-wave frequency. These effects were abolished by atropine and the muscarinic M(3)-receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide. ACh released from nerves did not depolarize antral muscles. At higher rates of stimulation (10 Hz), chronotropic effects were mediated by ACh and a noncholinergic transmitter and blocked by muscarinic antagonists and neurokinin (NK(1) and NK(2))-receptor antagonists. Neostigmine enhanced slow-wave frequency, suggesting that the frequency of antral pacemakers is kept low by efficient metabolism of ACh. Neostigmine had no effect on slow-wave frequency in muscles of W/W(v) mice, which lack intramuscular interstitial cells of Cajal (ICC-IM). These muscles also showed no significant chronotropic response to 5-Hz electrical field stimulation or the cholinergic agonist carbachol. The data suggest that the chronotropic effects of cholinergic nerve stimulation occur via ICC-IM in the murine stomach. The capacity of gastric muscles to metabolize ACh released from enteric motor neurons contributes to the maintenance of the proximal-to-distal slow-wave frequency gradient in the murine stomach. ICC-IM play a critical role in neural regulation of gastric motility, and ICC-IM become the dominant pacemaker cells during sustained cholinergic drive.

Identification of a d - glycero - d - manno -Heptosyltransferase Gene from Helicobacter pylori

We have identified a Helicobacter pylori d-glycero-d-manno-heptosyltransferase gene, HP0479, which is involved in the biosynthesis of the outer core region of H. pylori lipopolysaccharide (LPS). Insertional inactivation of HP0479 resulted in formation of a truncated LPS molecule lacking an alpha-1,6-glucan-, dd-heptose-containing outer core region and O-chain polysaccharide. Detailed structural analysis of purified LPS from HP0479 mutants of strains SS1, 26695, O:3, and PJ1 by a combination of chemical and mass spectrometric methods showed that HP0479 likely encodes alpha-1,2-d-glycero-d-manno-heptosyltransferase, which adds a d-glycero-d-manno-heptose residue (DDHepII) to a distal dd-heptose of the core oligosaccharide backbone of H. pylori LPS. When the wild-type HP0479 gene was reintegrated into the chromosome of strain 26695 by using an "antibiotic cassette swapping" method, the complete LPS structure was restored. Introduction of the HP0479 mutation into the H. pylori mouse-colonizing Sydney (SS1) strain and the clinical isolate PJ1, which expresses dd-heptoglycan, resulted in the loss of colonization in a mouse model. This indicates that H. pylori expressing a deeply truncated LPS is unable to successfully colonize the murine stomach and provides evidence for a critical role of the outer core region of H. pylori LPS in colonization.

Reduced Insulin and IGF-I Signaling, not Hyperglycemia, Underlies the Diabetes-Associated Depletion of Interstitial Cells of Cajal in the Murine Stomach

Damage to interstitial cells of Cajal (ICC), pacemakers, and mediators of neuromuscular neurotransmission in the gastrointestinal tract contributes to the pathogenesis of diabetic gastroenteropathy in both patients and animal models. ICC depletion in diabetes may result from chronic hyperglycemia or lost/ineffective insulin signaling. Because independent control of insulin and glucose concentrations is difficult in chronic in vivo studies, we used long-term organotypic cultures to address this problem. Murine gastric muscles were cultured in normoglycemic or hyperglycemic basal media with or without insulin or IGF-I for 1-3 months, the time required for gastroparesis and ICC damage to develop in diabetic mice. ICC were assessed by c-Kit immunohistochemistry and quantitative analysis of c-kit expression. Electrical pacemaking was studied by intracellular recording of slow waves. ICC survived for at least 34 days in unsupplemented normoglycemic media, but their networks, c-kit expression, and slow waves were profoundly reduced after 68 days. These changes could be entirely prevented by insulin or IGF-I supplementation. ICC networks were completely resistant to hyperglycemia for at least 72 days. Thus, hyperglycemia is unlikely to be responsible for the diabetes-associated depletion of ICC. In contrast, maintenance of ICC requires insulin or IGF-I, which are reduced or ineffective in diabetes.

Regional variation in ICC distribution, pacemaking activity and neural responses in the longitudinal muscle of the murine stomach

Intramuscular interstitial cells of Cajal (ICC-IM) play a critical role in enteric neural regulation of the circular muscle layer in the stomach, but no studies have been performed on the longitudinal layer. Kit immunohistochemistry was used to examine ICC-IM in the longitudinal muscle layer of the murine corpus and antrum, and it revealed marked heterogeneity in the distribution of ICC-IM in longitudinal muscles. In the corpus, ICC-IM were found along the greater curvature near the fundus. ICC-IM decreased in density in the circumferential axis toward the lesser curvature and in the longitudinal axis toward the antrum. ICC-IM were absent from the longitudinal layer of the antrum. Double labelling with markers for specific classes of enteric motor neurones revealed that cholinergic and nitrergic motor neurones formed close contacts with ICC-IM in the corpus but not in the antrum. Enteric nerve stimulation evoked prominent cholinergic excitatory and nitrergic inhibitory responses in longitudinal muscles of the corpus, but not in the antrum of wild-type animals. Cholinergic and nitrergic nerves were also present in W/W(V) mice, but functional innervation of the longitudinal muscle layer by these nerves in the corpus and antrum were absent. The data show that cholinergic and nitrergic neurotransmission only occurs in the gastric longitudinal layer in regions where ICC-IM are present. In regions, such as the corpus, where ICC-IM are common, robust neural responses are present, but the reduced density of ICC-IM near the lesser curvature and in the distal stomach leads to reduced neural regulation in these gastric regions.

Developmental Changes in the Pattern of Ghrelin’s Acyl Modification and the Levels of Acyl-Modified Ghrelins in Murine Stomach

Ghrelin is an acylated peptide hormone secreted primarily from endocrine cells in the stomach. The major active form of ghrelin is a 28-amino acid peptide with an n-octanoyl modification at Ser(3) (n-octanoyl ghrelin), which is essential for its activity. In addition to n-octanoyl ghrelin, other forms of ghrelin peptide exist, including des-acyl ghrelin, which lacks an acyl modification, and other minor acylated ghrelin species, such as n-decanoyl ghrelin, whose Ser(3) residue is modified by n-decanoic acid. Multiple reports have identified various physiological functions of ghrelin. However, until now, there have been no reports that explore the process of ghrelin acyl modification, and only a few studies have compared the levels of des-acyl, n-octanoyl, and/or other minor populations of acylated ghrelin peptides. In this study we report that the amount of n-octanoyl ghrelin in murine stomachs increases gradually during the suckling period to a maximal level at 3 wk of age and falls sharply after the initiation of weaning. However, the concentration (picomoles per milligram of wet weight tissue) of total ghrelin, which includes des-acyl and all acylated forms of ghrelin peptides with intact C termini in murine stomach, remains unchanged across this suckling-weaning transition. Prematurely weaned mice exhibited a significant decrease in the amount of n-octanoyl or n-decanoyl ghrelin in the stomach. Orally ingested glyceryl trioctanoate, a medium-chain triacylglyceride rich in milk lipids, significantly increased the level of n-octanoyl-modified ghrelin in murine stomach. Fluctuations in the proportion of this biologically active, acyl-modified ghrelin could contribute to or be influenced by the change in energy metabolism during the suckling-weaning transition.

Influencing the between-feeding and endocrine responses of plasma ghrelin in healthy dogs

Ghrelin has recently been isolated from rat and human stomach as an endogenous ligand for the growth hormone (GH) secretagog receptor. Using beagle dogs, we investigated the distribution of ghrelin in the stomach and its possible role. We examined: (i) GH release in response to ghrelin injection (0.5 or 5 microg/kg, i.v.), (ii) gastric localization of ghrelin-immunostained cells, (iii) changes in daily food consumption after ghrelin injection (3, 10, and 20 microg/kg, i.v.), (iv) plasma ghrelin levels under regular, but restricted feeding conditions, and (v) variations in plasma ghrelin levels in relatively lean, normal and obese dogs. Administration of ghrelin to dogs promptly increased circulating GH concentrations, although this effect was transitory and was maintained for only 20 min. Ghrelin was localized in the stomach fundus and body, but none was detected in either the pylorus or cardia. Administration of ghrelin at a dose of 20 microg/kg increased the daily food intake of beagle dogs. Plasma ghrelin levels peaked just before meal times, and then returned to basal levels. Obese dogs had higher plasma ghrelin levels than did normal and lean dogs. These results indicate that ghrelin is a potent GH secretagog in dogs. The distribution of ghrelin-immunoreactive cells in the canine stomach resembles that of both the murine and human stomach. Ghrelin participates in the control of feeding behavior and energy homeostasis in dogs and may, therefore, be involved in the development of obesity.

Profiling of microdissected gastric epithelial cells reveals a cell type—specific response to Helicobacter pylori infection

Helicobacter pylori colonizes the epithelial lining of the human stomach and is associated with disorders ranging from chronic gastritis to peptic ulcers and gastric cancer. We have explored the transcriptional response of the epithelium globally by applying a whole-genome approach to a murine model of infection. The 3 major epithelial lineages of the stomach-the parietal, mucus-producing, and chief cells-were harvested from cryosections of infected and uninfected murine stomachs by laser microdissection and subjected to gene expression profiling. The localization and quantity of selected transcripts were verified by in situ hybridization and quantitative real-time reverse-transcription polymerase chain reaction. Each cell type is characterized by a transcriptional signature profile. The parietal cell profile is highly enriched for factors involved in mitochondrial energy generation, whereas the chief cell predominantly expresses digestive enzymes and glycosylation-associated proteins. In contrast, the mucus cell signature is distinguished by an abundance of cell-surface receptors, signaling molecules, and factors involved in antigen presentation. All of these indicate a role in sampling, sensing, and responding to environmental stimuli. In line with this biological function, we measured a strong transcriptional response to Helicobacter pylori infection only in this cell type. The genes that are differentially expressed upon infection are implicated in a proinflammatory and mucosal defense response as well as modulation of angiogenesis, iron availability, and tumor suppression. Laser microdissection-assisted transcriptional profiling is a useful tool to explore the biology of specific cell populations and is sensitive enough to measure the transcriptional response to bacterial infection in vivo.

Analysis of promoter region of X-linked pgk-1 gene polymorphisms: evidence for polyclonality of adult mouse gastric glands.

The clonality of gastric glands remains a controversial topic. Chimeric mouse studies suggested that all gastric glands were monoclonal. However, using the X-linked transgenic mouse model, we have suggested that most glands are polyclonal during development and that the fraction of monoclonal glands increases after birth. Nevertheless, a fraction of glands is perpetually polyclonal even in the adult murine stomach. To examine the existence of gastric polyclonal glands in the adult mouse, we studied an X-linked intrinsic polymorphic gene, pgk-1, in 6 week-old female mice heterozygous for the X-linked pgk-1a and pgk-1b. The sequence containing the seventh HpaII site in the promoter region of the gene and the polymorphic sites was utilized. Twenty-four of 225 fundic glands (10.7%) and 3 of 167 pyloric glands (1.8%) were clonally heterotypic. Only 0.6% of colonic crypts were heterotypic. Clonally heterotypic glands with inactive X-specific methylation were present in the adult murine stomach.

α-Helical structure in the C-terminus of vasoactive intestinal peptide: functional and structural consequences

The conformational properties of vasoactive intestinal peptide (VIP) include the N-terminal randomized structure and the C-terminal long α-helical structure. We have previously observed that the N-terminal random coil structure plays a crucial role in the receptor-selectivity. Here, to clarify how the formation of the α-helix plays a role in its biological functions, we chemically synthesized VIP analogues modified at the C-terminus, mid-chain, and N-terminus of the α-helical region, and evaluated the relationship between their α-helical contents and their biological activities including relaxant effects on murine stomach and receptor-binding activities. VIP and VIP-(1–27) showed equipotent biological activities with 48% and 50% α-helical content, respectively, each of which corresponds to 14 amino acid residues. VIP-(1–26) was 10% and threefold less potent in relaxant and binding activities, respectively, compared with VIP, and its 49% α-helical content resulted in 13 residues involved in the α-helix. Further truncation from 25 to 21 resulted in decrease in the α-helical content from 43% to 29%, corresponding residues from 11 to 6, the relaxant activity from 72% to 4%, and the affinity to the membrane from 60-fold to over 10 4-fold less potency. In addition, disruption of the mid-chain and the N-terminus in the α-helical stretch by oxidation of Met 17 and deletion of Thr 11 also inhibited biological activities. These findings suggest that the presence of α-helical structure forming in 14 amino acid residues between position 10 and 23 in VIP is essential to its biological functions and the C-terminal amino acid residues between position 24 and 27 are requisite for this α-helical formation.

The Transient Receptor Potential Protein Homologue TRPC4/5 as a Candidate for the Nonselective Cationic Channel Activated by Muscarinic Stimulation in the Murine Stomach

The transient receptor potential protein homologue TRPC5 was reported as a molecular identity for the muscarinic receptor-activated nonselective cationic channel (NSCC) in the murine stomach smooth muscle. The canonical, or classical, transient receptor potential proteins, TRPC4 and TRPC5, were suggested as members of the same subfamily of TRPC channels and to be coexpressed as a heteromultimer of both TRPC as well as a homotetramer of each TRPC protein. Thus, we investigated whether the TRPC4 channel is also responsible for the NSCC activated by acetylcholine (ACh) or carbachol (CCh) using electrophysiological techniques. The TRPC channels were expressed in HEK293 cells. When murine TRPC4 channels (mTRPC4) were expressed, the current–voltage relationship of mTRPC4 was also similar to that recorded in native murine gastric myocytes or mTRPC5-expressing HEK cells. With 0.2 mM GTPγS in the pipette solution, the currents in mTRPC4-expressing cells were activated transiently like those in NSCC in the murine stomach and the expressed mTRPC5. The currents recorded in mTRPC4-expressing cells were inhibited by 1 mM La3+ and 100 μM flufenamate. The currents recorded in mTRPC4-expressing cells depended on the extracellular calcium concentration. From the above results, we suggest that mTRPC4/5 might be candidates for the NSCC activated by ACh or CCh in the murine stomach.

TRPC5 as a candidate for the nonselective cation channel activated by muscarinic stimulation in murine stomach.

We investigated which transient receptor potential (TRP) channel is responsible for the nonselective cation channel (NSCC) activated by carbachol (CCh) in murine stomach with RT-PCR and the electrophysiological method. All seven types of TRP mRNA were detected in murine stomach with RT-PCR. When each TRP channel was expressed, the current-voltage relationship of mTRP5 was most similar to that recorded in murine gastric myocytes. mTRP5 showed a conductance order of Cs(+) > K(+) > Na(+), similar to that in the murine stomach. With 0.2 mM GTPgammaS in the pipette solution, the current was activated transiently in both NSCC in the murine stomach and the expressed mTRP5. Both NSCC activated by CCh in murine stomach and mTRP5 were inhibited by intracellularly applied anti-G(q/11) antibody, PLC inhibitor U-73122, IICR inhibitor 2-aminoethoxydiphenylborate, and nonspecific cation channel blockers La(3+) and flufenamate. There were two other unique properties. Both the native NSCC and mTRP5 were activated by 1-oleoyl-2-acetyl-sn-glycerol. Without the activation of NSCC by CCh, the NSCC in murine stomach was constitutively active like mTRP5. From the above results, we suggest that mTRP5 might be a candidate for the NSCC activated by ACh or CCh in murine stomach.

Intramucosal Helicobacter pylori in the Human and Murine Stomach: Its Relationship to the Inflammatory Reaction in Human Helicobacter pylori Gastritis

Intramucosal Helicobacter pylori (H. pylori) has been described in biopsy tissues and culture systems. However, the association of intramucosal H. pylori with histopathologic features has not been evaluated. The purpose of this study is to investigate the relationship between intramucosal H. pylori and inflammatory reactions in H. pylori infection. In 113 randomly selected human gastric biopsies and 20 murine stomachs, which were inoculated with SSI every day for a week, immunohistochemical analysis for intramucosal H. pylori was done and correlated with histologic parameters. Electron microscopic examination was done on murine stomachs. H. pylori infection was present in 104 gastric biopsies and 17 murine stomachs. Intraepithelial immunopositivity for H. pylori was detected in 27 of 104 (26%) biopsies and in 11 of 17 (65%) murine stomachs. Lamina proprial immunopositivity for H. pylori was present in 51 of 104 (48%) biopsies. Neutrophil-associated immunopositivity for H. pylori was observed in 22 of 90 (24%) biopsies with H. pylori chronic active gastritis. Lamina proprial and neutrophil-associated immunopositivity for H. pylori correlated significantly with the density of H. pylori and the grade of acute inflammatory reaction in H. pylori gastritis. Intramucosal location of H. pylori itself or its antigen is closely associated with acute inflammatory reactions and may play an important role in establishing a persistent infection in chronic H. pylori gastritis. Furthermore, lamina proprial and/or neutrophil-associated H. pylori appears to be more important than intraepithelial H. pylori in acute inflammatory reactions of H. pylori gastritis.

Regional variation in contribution of myenteric and intramuscular interstitial cells of Cajal to generation of slow waves in mouse gastric antrum.

When intracellular recordings were made from the antral region of murine stomach, cells with three different patterns of electrical activity were detected. One group of cells generated follower potentials, the second group generated pacemaker potentials and the third group generated slow waves that consisted of primary and secondary components. Slow waves recorded in different regions of the gastric antrum had similar amplitudes but different characteristic shapes. At the greater curvature, slow waves had large initial components. Midway between the greater and lesser curvature, the amplitude of the initial component was reduced and at the lesser curvature an initial component was difficult to detect. When the distributions of myenteric (ICC-MY) and intramuscular interstitial cells of Cajal (ICC-IM) were determined, using an antibody to Kit, ICC-MY were found to be present at the greater curvature but were greatly reduced in density at the lesser curvature. In contrast, ICC-IM were found in the circular layer of each region. When recordings were made from the antrum of W/W(V) mice, which lack ICC-IM, incomplete slow waves were detected and their amplitudes fell from the greater to the lesser curvature. Again, a corresponding fall in the density of ICC-MY was detected. The observations indicate that the contribution of ICC-MY and ICC-IM to the generation of slow waves varies in different regions of the mouse gastric antrum.

An aspartic proteinase expressed in the yolk sac and neonatal stomach of the mouse.

A murine aspartic proteinase, described herein, is intermediate in amino acid sequence identity between the placentally produced pregnancy-associated glycoproteins (PAGs) and gastric pepsins. While PAGs are secreted products of placental trophoblast tissue of ungulates and most are not believed to function proteolytically, pepsins are digestive enzymes. The cDNA for this aspartic proteinase was amplified by reverse transcription-polymerase chain reaction from RNA extracted from murine placentas and neonatal stomachs. The open reading frame encoded a 387-amino acid polypeptide with a 15-residue signal sequence. The enzyme most resembled pepsinogen F (a protein identified in the stomachs of neonatal rabbits and rats) and PAG-like proteins cloned from equine and feline placentae. In the stomach, both its mRNA and protein were expressed in gastric chief cells of preweaned neonates. Within the placenta, its mRNA was present in both the parietal and visceral yolk sacs. However, the protein was most prevalent in the visceral yolk sac, with little detectable in the parietal yolk sac. The recombinant protein was expressed in Escherichia coli. This protein was capable of self-activation and exhibited proteolytic activity toward casein. The presence of this enzyme in two organs involved in the selective transcellular transport of proteins suggests that it has specialized digestive functions.

SS1 Helicobacter pylori disrupts the paracellular barrier of the gastric mucosa and leads to neutrophilic gastritis in mice

Helicobacter pylori induces severe neutrophilic infiltration in the lamina propria of the stomach, which leads to gastritis in humans. The possible involvement of a paracellular route for bacterial nutrients and etiologic agents that may play an important role in colonization of the bacteria and cause gastritis has been suggested. To study the functions of the paracellular barrier of gastric surface epithelium, SS1, a strain of H. pylori adapted to the murine stomach, was inoculated into the stomachs of C57BL/6 mice. At 4 months after inoculation, SS1 had achieved a high level of colonization (10(6)-10(7) colony-forming units/g tissue) associated with neutrophilic infiltration in the lamina propria of the junctional zone. Disruption of the paracellular barrier was observed in the SS1-infected stomachs, as revealed by the invasion of a lanthanum tracer into the paracellular space of the surface epithelium. Only 2% of junctions were permeable in control stomachs, whereas 72% of the paracellular barrier was disrupted in the SS1-infected gastric epithelia. Furthermore, distribution of tight junction-related molecules such as 7H6 antigen, occludin, and cortical actin was affected in the surface epithelium by SS1 infection. The linear expression pattern of occludin was disrupted and became irregular or punctuated. The 7H6 antigen accumulated as aggregates in the apical portion of the surface epithelium and cortical actin became irregular and punctuated. Taken together, these results indicate that infection by SS1 directly or indirectly caused an increase in paracellular permeability and altered the localization of tight junction-related molecules of the gastric surface epithelium. This observation suggests that the paracellular pathway may play a significant role in establishing H. pylori-induced gastritis in the clinical setting.

Progress in Fetal Pathology, Abstract 252–253, Symposium

IntramucosalHelicobacter pylori(H. pylori) has been described in biopsy tissues and culture systems. However, the association of intramucosalH. pylori with histopathologic features has not been evaluated. The purpose of this study is to investigate the relationship between intramucosal H. pylori and inflammatory reactions inH. pylori infection. In 113 randomly selected human gastric biopsies and 20 murine stomachs, which were inoculated with SS1 every day for a week, immunohistochemical analysis for intramucosal H. pylori was done and correlated with histologic parameters. Electron microscopic examination was done on murine stomachs. H. pylori infection was present in 104 gastric biopsies and 17 murine stomachs. Intraepithelial immunopositivity for H. pylori was detected in 27 of 104 (26%) biopsies and in 11 of 17 (65%) murine stomachs. Lamina proprial immunopositivity for H. pylori was present in 51 of 104 (48%) biopsies. Neutrophil-associated immunopositivity for H. pylori was observed in 22 of 90 (24%) biopsies with H. pylori chronic active gastritis. Lamina proprial and neutrophil-associated immunopositivity for H. pylori correlated significantly with the density of H. pylori and the grade of acute inflammatory reaction in H. pylori gastritis. Intramucosal location of H. pylori itself or its antigen is closely associated with acute inflammatory reactions and may play an important role in establishing a persistent infection in chronic H. pylori gastritis. Furthermore, lamina proprial and/or neutrophil-associated H. pylori appears to be more important than intraepithelialH. pylori in acute inflammatory reactions of H. pylori gastritis.

Effect of tissue non-specific alkaline phosphatase in maintenance of structure of murine colon and stomach

The gastrointestinal tract of mammals secretes a phospholipid-rich membrane that is enriched in alkaline phosphatase (AP) and surfactant proteins (surfactant-like particle, SLP). The production of this particle is stimulated in the small intestine by fat feeding and in cultured cells in vitro by transfection with intestinal alkaline phosphatase (IAP). To test whether tissue non-specific alkaline phosphatase (TNAP) was a factor in stimulating surfactant-like particle production in stomach and colon (tissues expressing TNAP), mice lacking this enzyme were studied. Mice were harvested at 8 days of life, when body weight of homozygous animals (TNAP -/-) was about half that of congenic controls (TNAP +/+) or heterozygotes (TNAP +/-), but before seizures had begun. No difference in content of the major SLP protein (65 kDa) by Western blotting or immunocytochemistry was seen in stomach or colon of TNAP -/- vs. TNAP +/+ animals, but the content was only about half in the IAP-expressing small bowel. Transmission electron microscopy of the TNAP -/- small bowel showed large dilated lysosomes and residual bodies. Colonocytes and gastric surface epithelial cells from the same animals showed mitochondria containing homogeneous dense inclusions, consistent with neutral lipid. In the underweight homozygous animals, there was a decrease in the neuronal content of submucosal ganglia in the jejunum and ileum and of myenteric ganglia in the jejunum of TNAP -/- animals. These findings suggest that (1) TNAP is not important in maintaining surfactant-like particle content of tissues that express TNAP, (2) normal fat absorption is important in maintaining SLP content in the small intestine, and (3) TNAP is important in the maintenance of some intestinal structures, and perhaps their function.

Functional genomics of Helicobacter pylori: identification of a beta-1,4 galactosyltransferase and generation of mutants with altered lipopolysaccharide.

A previously annotated open reading frame (ORF) (HP0826) from Helicobacter pylori was cloned and expressed in Escherichia coli cells and determined to be a beta-1,4-galactosyltransferase that used GlcNAc as an acceptor. Mutational analysis in H. pylori strains demonstrated that this enzyme plays a key role in the biosynthesis of the type 2 N-acetyl-lactosamine (LacNAc) polysaccharide O-chain backbone, by catalysing the addition of Gal to GlcNAc. To examine the potential role of this O-chain structure in bacterial colonization of the host stomach, the mutation was introduced into H. pylori strain SS1 which is known to be capable of colonizing the gastric mucosa of mice. Compared with the parental strain, mutated SS1 was less efficient at colonizing the murine stomach.

Effect of tissue non-specific alkaline phosphatase in maintenance of structure of murine colon and stomach.

The gastrointestinal tract of mammals secretes a phospholipid-rich membrane that is enriched in alkaline phosphatase (AP) and surfactant proteins (surfactant-like particle, SLP). The production of this particle is stimulated in the small intestine by fat feeding and in cultured cells in vitro by transfection with intestinal alkaline phosphatase (IAP). To test whether tissue non-specific alkaline phosphatase (TNAP) was a factor in stimulating surfactant-like particle production in stomach and colon (tissues expressing TNAP), mice lacking this enzyme were studied. Mice were harvested at 8 days of life, when body weight of homozygous animals (TNAP -/-) was about half that of congenic controls (TNAP +/+) or heterozygotes (TNAP +/-), but before seizures had begun. No difference in content of the major SLP protein (65 kDa) by Western blotting or immunocytochemistry was seen in stomach or colon of TNAP -/- vs. TNAP +/+ animals, but the content was only about half in the IAP-expressing small bowel. Transmission electron microscopy of the TNAP -/- small bowel showed large dilated lysosomes and residual bodies. Colonocytes and gastric surface epithelial cells from the same animals showed mitochondria containing homogeneous dense inclusions, consistent with neutral lipid. In the underweight homozygous animals, there was a decrease in the neuronal content of submucosal ganglia in the jejunum and ileum and of myenteric ganglia in the jejunum of TNAP -/- animals. These findings suggest that (1) TNAP is not important in maintaining surfactant-like particle content of tissues that express TNAP, (2) normal fat absorption is important in maintaining SLP content in the small intestine, and (3) TNAP is important in the maintenance of some intestinal structures, and perhaps their function.