bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential regulation of cytochrome P-450 genes along rat intestinal crypt-villus axis
Authors: Traber P. G. , Wang W. , Yu L.
Published: December 2017

Mammalian small intestine contains cytochrome P-450-dependent monooxygenase enzymes that are capable of metabolizing a wide variety of xenobiotics and activating procarcinogens to mutagenic compounds. The epithelial cells lining the small intestine are separated into a proliferating undifferentiated compartment located in crypts and a nonproliferating differentiated compartment located on villi. The constitutive expression and induction by xenobiotics of genes that encode components of the cytochrome P-450-dependent mono-oxygenase system along the rat intestinal crypt-villus axis were investigated using isolated epithelial cells and in situ hybridization. For each gene examined, hybridization analysis of RNA obtained from isolated epithelial cells correlated with findings on in situ RNA hybridization. Cytochrome P-450IA1 mRNA (CYP1A1), the major aromatic hydrocarbon-inducible P-450, and cytochrome P-450IIB1 mRNA (CYP2B1), the major phenobarbital-inducible P-450, were constitutively expressed in villus cells with no detectable mRNA present in crypts. Treatment with several chemical inducers resulted in a marked increase in CYP1A1 mRNA in both crypt and villus cells. In contrast, although CYP2B1 mRNA was inducible in villus cells, CYP2B1 mRNA was not detected in crypts after treatment with chemical inducers. NADPH cytochrome P-450 reductase, a necessary component for the activity of all P-450 enzymes, was expressed constitutively at low levels only in villus cells. Treatment with dexamethasone induced reductase mRNA in both crypt and villus cells. Taken together, these results demonstrate that there is a complex gene-specific pattern of expression of the microsomal monooxygenase system along the crypt-villus axis of rat small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)
Expression and localization of GLUT-5 in Caco-2 cells, human small intestine, and colon
Authors: Mahraoui L. , Rousset M. , Dussaulx E. , Darmoul D. , Zweibaum A. , Brot-Laroche E.
Published: December 2017

The human colon carcinoma cell line Caco-2 was used as an enterocyte model to study the expression of the facilitative glucose transporters GLUT-1 and GLUT-2, and of the putative hexose transporter GLUT-5, which are expressed specifically in the gut. Northern blots indicate that Caco-2 cells express GLUT-1 and GLUT-5 mRNAs but not the mRNA coding for the basolateral glucose transporter GLUT-2. The level of GLUT-5 mRNA is growth dependent, being detectable only in postconfluent differentiated cells. In addition, the expression of GLUT-5 increases with the number of cell passages and is approximately 10 times higher in later passages (passage 184) than in early ones (passage 26). With the use of polyclonal antibodies directed against the COOH-terminus of GLUT-5, indirect immunofluorescence and Western blotting indicate that GLUT-5 is mainly localized to the brush border of Caco-2 cells. GLUT-5 is also found to be associated with the brush border of epithelial cells from fetal and normal adult human small intestine, but is absent from the colon.
cDNA cloning and localization of a band 3-related protein from ileum
Authors: Chow A. , Dobbins J. W. , Aronson P. S. , Igarashi P.
Published: December 2017

A Cl(-)-HCO3- exchanger in the brush-border membrane mediates active Cl- absorption and regulates intracellular pH in rabbit ileum. The molecular identity of the ileal Cl(-)-HCO3- exchanger has not been established. The best-characterized plasma membrane Cl(-)-HCO3- exchanger is erythroid band 3. Structurally related proteins in nonerythroid tissues comprise an anion exchanger (AE) family. We used the polymerase chain reaction to amplify and clone a cDNA encoding an ileal band 3-related protein (B3RP) from rabbit ileal enterocytes. The composite sequence is 3,909 bp and is predicted to encode a protein of 136 kDa. The deduced amino acid sequence is 95% identical to murine renal AE2, indicating that ileal B3RP is rabbit AE2. Antisera generated against a cytoplasmic fragment of ileal B3RP recognized a 160- to 170-kDa polypeptide in the brush-border membrane, but not the basolateral membrane, of ileal crypt and villus enterocytes. This correlates with previous studies indicating that a Cl(-)-HCO3- exchange is present in brush-border but not basolateral membrane vesicles from rabbit ileal enterocytes. We conclude that ileal B3RP is a product of the AE gene family, and is present in the brush-border of ileal enterocytes, where it may mediate Cl(-)-HCO3- exchange.
Pepsinogen secretion from streptolysin O-permeabilized chief cells from guinea pig stomach
Authors: Raffaniello R. D. , Raufman J. P.
Published: December 2017

To evaluate directly the actions of cellular mediators on pepsinogen secretion, a nearly homogeneous population of dispersed chief cells was permeabilized using the bacterial toxin streptolysin O (30 IU/ml for 10 min). This resulted in the release of > 60% of cellular lactate dehydrogenase activity, whereas cellular pepsinogen levels were not altered. Examination of permeabilized cells using light and electron microscopy revealed preservation of cellular polarity and organelles. Pepsinogen secretion from permeabilized chief cells could be stimulated with increasing concentrations of calcium (300 nM to 10 microM), adenosine 3',5'-cyclic monophosphate (cAMP; 1 microM to 1 mM), phorbol 12-myristate 13-acetate (10 nM to 1 microM), or by addition of carbamylcholine (0.1 mM) to the incubation solution. In the absence of calcium [0.4 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], activators of protein kinase C stimulated a two- to threefold increase in pepsinogen secretion, and potentiation of secretion was observed when these agents were combined with 0.3 and 1.0 microM calcium. In contrast to the effects of activators of protein kinase C, cAMP-induced pepsinogen secretion was observed only with calcium concentrations > or = 100 nM. Combinations of increasing concentrations of cAMP and calcium resulted in an additive secretory response. This study indicates the utility of streptolysin O-permeabilized chief cells for the study of signal transduction mechanisms that mediate pepsinogen secretion.
Rat intestinal angiotensin-converting enzyme: purification, properties, expression, and function
Authors: Erickson R. H. , Suzuki Y. , Sedlmayer A. , Song I. S. , Kim Y. S.
Published: December 2017

Angiotensin-converting enzyme [ACE (peptidyl-dipeptidase A, EC 3.4.15.1)] was purified from a total cell membrane fraction of rat intestinal mucosa. A 4,500-fold purification was achieved after affinity chromatography with lisinopril-Sepharose and gel filtration. The final preparation was judged to be homogenous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 160,000. The purified protein is a glycoenzyme containing 12% N-linked carbohydrate. Purified ACE had a specific activity of 65 U/mg protein with benzoyl-Gly-His-Leu as substrate. A kinetic analysis showed that the enzyme had the maximal velocity with substrates containing proline at the COOH-terminal end. Inhibitor studies indicated that the enzyme is a metalloprotein. Along the proximal-distal axis of the small intestine, ACE activity is most predominant in the proximal to middle portions, decreasing toward the distal end. This pattern was also observed for ACE mRNA and protein, suggesting that ACE expression is controlled at the level of mRNA. Perfusion of benzoyl-Gly-His-Leu in vivo through a segment of intestinal jejunum demonstrated that ACE is an important intestinal dipeptidyl carboxypeptidase, participating in the digestion and assimilation of dietary peptides.
Effect of putrescine on S-adenosylmethionine decarboxylase in a small intestinal crypt cell line
Authors: Wang J. Y. , Viar M. J. , McCormack S. A. , Johnson L. R.
Published: December 2017

Two key enzymes in polyamine biosynthesis are ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC). SAMDC decarboxylates S-adenosylmethionine, which then donates aminopropyl groups for spermidine and spermine synthesis. The purpose of our study was to determine whether putrescine, taken up from medium or synthesized endogenously by ODC, alters SAMDC activity. Studies were conducted in the IEC-6 cell line derived from rat small intestinal crypt cells. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum (dFBS). They were deprived of serum for 24 h before experiments. Basal SAMDC activity was increased significantly by > or = 10(-4) M of putrescine. Lower doses had no significant effect. The same doses of putrescine decreased ODC activity to near zero. Asparagine at 10 mM or 5% dFBS not only stimulated ODC activity and the intracellular putrescine levels but also increased significantly SAMDC activity as well. ODC activity peaked at 3 h, and the maximum level of SAMDC occurred 3-4 h after exposure to asparagine or serum. Treatment with DL-alpha-difluoromethylornithine (DFMO), a specific ODC inhibitor, prevented the increases in both cellular putrescine levels and SAMDC activity in asparagine- and serum-treated cells. In the presence of DFMO, exogenous putrescine returned SAMDC activity toward control levels but had no effect on ODC. A very slight increase of SAMDC half-life in IEC-6 cells grown in the presence of putrescine was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)
Neuropeptides promote neutrophil adherence to endothelial cell monolayers
Authors: Zimmerman B. J. , Anderson D. C. , Granger D. N.
Published: December 2017

The objective of this study was to determine whether substance P and calcitonin gene-related peptide (CGRP), at physiologically relevant concentrations, affect leukocyte-endothelial cell adhesion. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) were incubated (40 min) with freshly isolated human neutrophils in the presence or absence of substance P or CGRP (10(-11) M). Both substance P and CGRP caused a significant increase (2-fold) in neutrophil adherence to HUVEC. Monoclonal antibodies (MAb) directed against the leukocyte adhesion glycoproteins CD11/CD18 (MAb IB4) and L-selectin (MAb DREG56) did not attenuate substance P-induced adhesion. Antibodies directed against the endothelial cell adhesion molecules E-selectin (MAb CL2) and ICAM-1 (MAb R6.5) were also without effect on substance P-induced neutrophil adhesion. Similar results were obtained when either MAb IB4, DREG56, CL2, or R6.5 was coincubated with CGRP-stimulated neutrophils and endothelial cells. Phorbol 12-myristate 13-acetate-stimulated neutrophil adherence was significantly attenuated by MAb IB4, indicating that CD11/CD18 participates in this adhesion process. The results of this study indicate that 1) the neuropeptides substance P and CGRP promote neutrophil adherence to venular endothelium and 2) the neuropeptide-induced adhesion is not mediated by the adhesion molecules CD11/CD18, L-selectin, E-selectin, or ICAM-1.
Biomechanical properties of duodenal wall and duodenal tone during phase I and phase II of the MMC
Authors: Gregersen H. , Orvar K. , Christensen J.
Published: December 2017

We used a new method, impedance planimetry, to look at variations in compliance, tone, and distension-induced peristaltic activity during phase I and phase II of the migrating myoelectric complex (MMC) in the human duodenum. A balloon was inflated stepwise with pressures up to 30 cmH2O in the duodenum, while the pressure and balloon cross-sectional area (CSA) were measured simultaneously. The biomechanical wall parameters were calculated from these measurements. Nine duodenal phase IIIs were recorded in six subjects. A balloon pressure of 20 cmH2O induced a smaller CSA in early phase I [266 (236-324) mm2] than in late phase II [385 (276-474) mm2] (P < 0.05). Balloon distensions elicited no contractions in phase I, whereas they increased contractile activity 60% (P < 0.05) proximal to the balloon and 4% distal to the balloon in late phase II. Step distensions in phase I with balloon pressures between 10 and 30 cmH2O increased the CSA from 40 (30-81) to 645 (603-704) mm2. It increased circumferential wall tension from 35 (28-63) to 429 (402-466) mm x cmH2O and the pressure elastic modulus from 9.7 (9.0-14.7) to 33.8 (27.6-33.8) cmH2O, respectively. Thus compliance differs from phase I to phase II. This is most likely caused by increased smooth muscle tone during phase I. Duodenal wall stiffness increases with the balloon pressure applied.
Uptake and metabolism of circulating fatty acids by rat intestine
Authors: Mansbach C. M. , Dowell R. F.
Published: December 2017

The present study was designed to investigate the uptake and metabolism of circulating fatty acids by the intestinal mucosa in rats actively absorbing glyceryl trioleate given intraduodenally to determine the plasma fatty acid contribution to mucosal triacylglycerol. Rats with duodenal, femoral vein, carotid artery, and mesenteric lymph duct cannulas were used. [3H]oleate was constantly infused into the femoral vein while glyceryl trioleate was infused into the duodenum (135 mumol/h). After 5 h of infusion, a mass and radioactive steady state existed in the plasma and mucosa. At 6 h of infusion, the plasma oleate specific activity was sixfold greater than mucosal oleate and 50 times greater than mucosal triacylglycerol oleate; 86% of the mucosal oleate disintegrations/minute were in triacylglycerol. Chylomicron triacylglycerol oleate specific activity was less than that of the mucosa. Furthermore, the percentage of mucosal triacylglycerol acyl groups composed of oleate was greater than the percentage of oleate in mucosal free fatty acids. The data indicate that fatty acids are taken up by the mucosa during active fat absorption and metabolized primarily to triacylglycerols by the mucosa. The triacylglycerols in the mucosa synthesized from circulating fatty acids are selected against as a precursor of chylomicron triacylglycerol. The results support our previous hypothesis suggesting that the mucosa has at least two pools of neutral lipid (J. Lipid Res. 23: 1009-1019, 1982) and that steady-state conditions as performed here yield different results from previous work using bolus tracer injection techniques.
Pharmacological basis of contractile effects of peptidoleukotrienes on rabbit colonic muscularis mucosae
Authors: Percy W. H. , Burton M. B. , Glaws W. R. , Rose K. , Burakoff R.
Published: December 2017

The present study was designed to determine the mechanism(s) underlying the excitatory effects of several sulfidopeptide leukotrienes (LTs) on the muscularis mucosae in three regions of the rabbit colon. Proximal colonic muscularis mucosae was refractory to LTs C4, D4, and E4. In addition, it exhibited no responses to prostaglandin (PG) E2 and only a minimal contractile response to PGF2 alpha. Mid and distal colonic muscularis mucosae each responded to LTs C4, D4, and E4 and PGs E2 and F2 alpha with concentration-dependent contractions. In both regions, responses to LTD4 and LTE4 were abolished by indomethacin (10(-6) M) pretreatment. LTC4-induced responses were reduced approximately 50% by this procedure. The residual contraction to LTC4 was resistant to both tetrodotoxin (10(-6) M) and atropine (10(-6) M). In separate experiments, responses to LTC4 were also reduced by approximately 50% if LTC4 conversion to LTD4 and LTE4 was first prevented by L-serine borate (45 mM) in combination with L-cysteine (10 mM). It is concluded that proximal colonic muscularis mucosae lacks the appropriate functional excitatory LT and PG receptors. On mid and distal colonic muscularis mucosae, the actions of LTD4 and LTE4 and, in part, LTC4 are the result of PG production, whereas LTC4 has an additional direct action, possibly mediated through a selective LTC4 receptor.
Cholera toxin-sensitive neurons in guinea pig submucosal plexus
Authors: Jiang M. M. , Kirchgessner A. , Gershon M. D. , Surprenant A.
Published: December 2017

Cholera toxin (CT) increases intestinal secretions by direct stimulation of mucosal enterocytes; enteric neurons also may play a role. We tested the latter possibility by retrograde labeling of mucosal terminals in guinea pig small intestine with the B subunit of CT (B-CT) and by intracellular recordings from submucosal neurons during superfusion with CT. All vasoactive intestinal peptide (VIP)-positive neurons, and only VIP-positive neurons, were labeled with B-CT. Fluorogold (FG) was used to retrogradely label nerve terminals in submucosal arterioles in preparations in which B-CT labeled mucosal terminals; colocalization of B-CT with FG was observed in neurons up to 3 mm from the site of FG application. CT selectively depolarized neurons known to contain VIP. We conclude that all VIP-containing neurons, and only VIP neurons, in guinea pig submucosal plexus possess B-CT binding sites and can be activated by CT. Some of these neurons provide a dual innervation to both arterioles and mucosa. We suggest that one functional consequence of CT may be to activate vasodilator nerves, thus increasing vascular perfusion of the mucosa to further stimulate intestinal secretions.
Molecular mechanism of two noncompetitive inhibitors of Na(+)-glucose cotransporter: comparison of DCCD and PCMB
Authors: Peerce B. E.
Published: December 2017

The effects of noncompetitive inhibitors of Na(+)-dependent glucose uptake, p-chloromercuribenzoate N,N'-dicyclohexylcarbodiimide (DCCD), on substrate-induced cotransporter conformational changes were examined using fluorescein isothiocyanate (FITC) and tryptophan fluorescence. p-chloromercuribenzoate (PCMB) inhibited both substrate-induced conformational changes with similar concentration required for 50% quenching/enhancement of tryptophan or FITC fluorescence. In contrast, DCCD inhibited the Na(+)-induced conformational change with an apparent concentration resulting in 50% inhibition (K0.5) of 18 microM and the glucose-induced conformational change with an apparent K0.5 of 5 microM. DCCD slightly increased the apparent K0.5 for the Na+ concentration required for Na(+)-induced conformational change. DCCD and PCMB altered tryptophan accessibility to quench reagents in all three conformations. Tryptophan residues on the PCMB-treated cotransporter were more Cs+ than I- sensitive in contrast to the unlabeled cotransporter. The PCMB-treated cotransporter had a reduced response to Na+, suggesting that the mode of PCMB inactivation of cotransporter activity resulted from conformational changes in the substrate-free cotransporter. DCCD had a smaller effect on cotransporter tryptophan quench reagent susceptibility. However, DCCD-labeled cotransporter was equally accessible to I- and Cs+, and the DCCD-labeled cotransporter did not respond to substrates. Loss of charge distribution around cotransporter tryptophans correlated with loss of substrate-induced conformational changes.
Paracrine control of gastric epithelial cell growth in culture by transforming growth factor-alpha
Authors: Chen M. C. , Lee A. T. , Karnes W. E. , Avedian D. , Martin M. , Sorvillo J. M. , Soll A. H.
Published: December 2017

Studying primary cultures of replicating canine oxyntic mucosal cells, we found evidence for modulation of cell growth by endogenous factors. [3H]thymidine incorporation into DNA was rapid with cells cultured in medium free of serum or added growth factors, and growth rates of these cultures were markedly dependent on plating density, indicating mitogenic control by soluble endogenous growth factors. Data indicated that endogenous transforming growth factor-alpha (TGF-alpha) exerted mitogenic control under the following conditions. 1) TGF-alpha was detected in the cultured cells by radioimmunoassay and immunohistochemistry. 2) TGF-alpha-like immunoreactivity and receptor reactivity were present in the medium in concentrations sufficient to exert mitogenic control. 3) Receptors for TGF-alpha and epidermal growth factor (EGF) were present in the cultures. 4) Immunoabsorption by a TGF-alpha-specific antisera reduced [3H]thymidine incorporation. TGF-alpha was localized to parietal cells by immunohistochemistry and cell separation. In contrast, combined [3H]thymidine autoradiography and immunohistochemistry with anti-TGF-alpha did not detect TGF-alpha in dividing cells. We conclude that parietal cell TGF-alpha exerts paracrine control of mucosal cell growth in vitro, and we speculate that this is an important paracrine mechanism in vivo.
Prostaglandin E2 downregulates Kupffer cell production of IL-1 and IL-6 during hepatic regeneration
Authors: Goss J. A. , Mangino M. J. , Callery M. P. , Flye M. W.
Published: December 2017

The mammalian liver possesses the ability to regenerate to its original size after a 70% partial hepatectomy (PHx). The capacity of rat Kupffer cells (KC) isolated at specific intervals after PHx to produce interleukin (IL)-1, IL-6, and prostaglandin E2 (PGE2) in response to endotoxin [lipopolysaccharide (LPS)] stimulation was evaluated in standard RPMI 1640 (1,200 microM L-arginine) and arginine-depleted RPMI 1640 (< 10 microM L-arginine) media. Because KC function in an environment in which high arginase activity results in negligible L-arginine levels, the 10 microM L-arginine RPMI 1640 was used to simulate the hepatic microenvironment. Regenerating liver KC 12-120 h after PHx responded to LPS with a significantly greater (P < 0.05) production of IL-1 and IL-6 in standard RPMI 1640. This enhancement of regenerating liver KC to produce IL-1 and IL-6 was increased (P < 0.05) by placing these same KC in 10 microM arginine RPMI 1640 culture media. During the same time period, regenerating liver KC produced significantly elevated (P < 0.01) PGE2, again with greater differences in the low-arginine media. In vivo KC PGE2 blockade by indomethacin (5 mg/kg) significantly (P < 0.05) inhibited hepatic regeneration. When the cyclooxygenase inhibitor indomethacin (10 microM) was added to cultures, the production of PGE2 by KC was prevented, and in arginine-depleted cultures, IL-1 and IL-6 production was upregulated (P < 0.05). We conclude that during hepatic regeneration, KC IL-1 and IL-6 production is elevated and is controlled in an autoregulatory fashion by elevated KC PGE2 production.
Mechanism of action of capsaicin on submucosal arterioles in the guinea pig ileum
Authors: Vanner S.
Published: December 2017

Vasomotor neurons in the enteric nervous system release acetylcholine to dilate submucosal arterioles, but it is not known whether sensory nerves that project to these vessels also can provide a vasodilator innervation. This possibility was examined by determining the mechanism of action of capsaicin on guinea pig ileal submucosal arterioles in vitro. Capsaicin dilated all vessels that had been preconstricted with prostaglandin F2 alpha; mean effective concentration was 11 nM, and maximal dilation occurred at 60-200 nM. The vasodilation showed marked desensitization upon repeated applications of capsaicin. Tetrodotoxin blocked the capsaicin-induced vasodilation but not the desensitization observed upon repeated application. Muscarinic receptor antagonists did not affect the actions of capsaicin. Capsaicin did not dilate arterioles whose extrinsic sensory afferent fibers had been surgically removed. Substance P and human calcitonin gene-related peptide II dilated arterioles; these dilations were not inhibited after desensitization of the capsaicin-induced vasodilation. Thus capsaicin dilates submucosal arterioles by selectively activating extrinsic afferent fibers that release vasodilator transmitter substances onto these vessels.
Timed photoaffinity labeling and characterization of bile acid binding and transport proteins in rat ileum
Authors: Lin M. C. , Mullady E. , Wilson F. A.
Published: December 2017

Rat ileal enterocytes were radiolabeled by flash photolysis with a photolabile derivative of taurocholate (7,7-azo-[3H]TC) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximal labeling of the bile acid binding proteins (BABPs) was achieved between 15 and 90 s. When enterocytes were pulsed with 7,7-azo-[3H]TC for 2 min, and then 0.5 mM TC was added to chase the radiolabel, the radioactivity in the BABPs was displaced by 50% after 2 min. The 99-kDa brush-border membrane (BBM) protein had the highest initial labeling rate, followed by 43-kDa actin, 35- and 14-kDa cytosolic proteins, 54-kDa basolateral membrane (BLM) protein, 59-kDa BLM-associated protein, and 20-kDa microsomal protein. When a mixed microsomal and cytosolic fraction was photolabeled with 7,7-azo-[3H]TC and then separated, the 20-kDa microsomal protein was labeled. However, if the microsomal fraction alone was photolabeled, the 20-kDa protein was not labeled, suggesting this protein required a cytosolic cofactor for labeling. Using Triton X-114 phase separation and EDTA extraction, the BABPs were separated into amphiphilic integral membrane proteins (99- and 54-kDa proteins) and hydrophilic proteins (14-, 35-, 43-, and 59-kDa proteins). From these data, a model is proposed for transcellular bile acid transport in rat ileal enterocytes.
Cardiac function and responsiveness to beta-adrenoceptor agonists in rats with obstructive jaundice
Authors: Jacob G. , Nassar N. , Hayam G. , Ben-Haim S. , Edoute Y. , Better O. S. , Bomzon A.
Published: December 2017

The function and role of the heart and the contribution of cardiac beta-adrenoceptors in the pathogenesis of circulatory failure in obstructive jaundice were studied in the 3-day bile duct-ligated (BDL) rat using three different techniques to measure cardiac function and beta-adrenoceptor activity, number, and affinity. The techniques were the pithed rat preparation, the isolated working heart preparation, and a competitive radioligand binding assay for beta-adrenoceptors. The results of these experiments were compared with those obtained in 3-day bile duct-manipulated (sham operated; SO) rats. Impaired indexes of basal cardiac contractility were observed in the BDL pithed rats and isolated working hearts. In these two preparations, responsiveness to norepinephrine and the beta-adrenoceptor agonists, isoproterenol and dobutamine, was unaffected by bile duct ligation. The affinity and number of cardiac beta-adrenoceptors in membranes from the hearts of SO and BDL rats were not significantly different from each other. These experiments have established for the first time that the 3-day BDL rat has a cardiac myopathy associated with intact responsiveness to beta-adrenoceptor agonists, a normal unchanged affinity and number of cardiac beta-adrenoceptors.
Effect of maternal sialoadenectomy on ontogenic response of rat gastric mucosa to luminal H+
Authors: Tepperman B. L. , Vozzolo B. L. , Soper B. D.
Published: December 2017

Epidermal growth factor (EGF) originating from salivary glands has been shown to affect the development and integrity of rodent gastrointestinal mucosa. Because newborn rats receive EGF from maternal milk, we have examined the effect of sialoadenectomy as a method of depleting EGF in milk on the resistance of neonatal rat mucosa to luminal H+. Rat dams were sialoadenectomized (SALX) or sham operated 5 days after parturition. Experiments were performed on newborns 10-22 days old. Mucosal permeability responses to intraluminal HCl (300 mM) were examined in terms of luminal appearances of Na+, K+, and protein and H+ loss. EGF levels in maternal milk were determined by immunoassay. In rat pups from control litters, luminal HCl resulted in an age-associated increase in cation and protein flux across the gastric mucosa. Luminal cation and protein fluxes observed in 10- to 18-day-old rat pups from SALX dams were not significantly different from similarly aged rats from control dams. However, in 19- to 22-day-old rats from SALX dams, the permeability responses to luminal HCl were exacerbated compared with similarly aged neonates from control dams. These responses were reduced by treatment with EGF. EGF levels in milk from sham-operated and SALX dams were not significantly different in the 10- to 16-day lactational period. However, in SALX dams EGF was significantly reduced at 19 and 22 days. Chromatographic elution pattern of milk EGF was dissimilar to the pattern exhibited by submandibular gland EGF.(ABSTRACT TRUNCATED AT 250 WORDS)
TGF-alpha is a potent mitogen for primary cultures of guinea pig gastric mucous epithelial cells
Authors: Rutten M. J. , Dempsey P. J. , Solomon T. E. , Coffey R. J.
Published: December 2017

Transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) are thought to be important in gastric epithelial proliferation and repair. It was therefore of interest to determine if TGF-alpha and EGF promoted the growth of an in vitro primary culture system of guinea pig gastric mucous epithelial cells (MEC). MEC were isolated from guinea pig stomachs and cultured in 24-well Primaria plates with DMEM with or without 10% fetal calf serum (FCS). Growth of MEC was determined by changes in [3H]thymidine uptake, cell counts, protein, and DNA. The sources of peptides were human recombinant TGF-alpha (recTGF-alpha) and human recombinant EGF (recEGF). Both recTGF-alpha and recEGF were used at equipotent doses as determined by competing activity in a 125I-labeled TGF-alpha radioreceptor binding assay using A-431 cells. Basal growth (no peptides) of MEC in 10% FCS was dependent on the initial plating density. Under serum-free conditions, [3H]thymidine uptake increased up to 17-fold at 24 h with recTGF-alpha (0.1-10.0 nM) compared with only a 4-fold increase using rec-EGF (0.1-10.0 nM) at this same time period. Under serum-free conditions, recTGF-alpha (0.01-10.0 nM) increased cell counts up to 4.9-fold over control cultures, whereas similar does of recEGF produced a 2.5-fold increase in cell counts. Administration of recEGF 1 ng/ml) resulted in a 1.9-fold increase in the 4.8-kb TGF-alpha mRNA transcript, and TGF-alpha protein immunoreactivity was found in both 24-h conditioned media and cell lysates.(ABSTRACT TRUNCATED AT 250 WORDS)
Gastric ulcer induced by submucosal injection of ET-1: role of potent vasoconstriction and intraluminal acid
Authors: Lazaratos S. , Kashimura H. , Nakahara A. , Fukutomi H. , Osuga T. , Urushidani T. , Miyauchi T. , Goto K.
Published: December 2017

To investigate whether submucosally applied endothelin-1 (ET-1) can induce gastric ulcer, ET-1 (62.5, 125, 250, and 500 pmol/kg) was injected in the submucosal layer of the rat gastric body. Twenty-four hours later, gastric ulcer (ulcer area: 10.31 +/- 5.13 mm2, mean +/- SE, at 500 pmol/kg, n = 8) was induced. The mucosal damage induced by the two highest doses was present even at 2 wk after their injection. Measurement of the mucosal blood flow at the injected area with three different methods (laser-Doppler flowmetry, hydrogen gas clearance, and reflectance spectrophotometry) revealed that injected ET-1 produced an extremely long-lasting vasoconstriction. Pretreatment with nicardipine, a Ca(2+)-channel blocker (1 mg/kg iv), significantly attenuated the ET-1-induced mucosal damage as well as the decrease in mucosal blood flow. Pretreatment with omeprazole (5-40 mumol/kg) also, significantly attenuated the ET-1-induced mucosal damage. Combined pretreatment with omeprazole (40 mumol/kg) and nicardipine almost abolished the ET-1-induced damage. The present study shows that a novel model for experimental ulcers can be induced by submucosal injection of ET-1.
HNF-1 alpha and HNF-1 beta expression in mouse intestinal crypts
Authors: Serfas M. S. , Tyner A. L.
Published: December 2017

Hepatocyte nuclear factors 1 alpha and 1 beta (HNF-1 alpha and HNF-1 beta) are homologous homeodomain-containing transcription factors that form homodimers or heterodimers that bind the same consensus sequence in the promoters of several genes expressed in liver. Many of these genes, including the mouse alpha-fetoprotein (AFP) gene, are also expressed in the intestinal epithelium. AFP expression is restricted to the enteroendocrine cell lineage in the adult small intestine, and we have investigated the distribution of HNF-1 alpha and HNF-1 beta mRNA in the mouse intestine to determine whether expression of either of these regulatory factors colocalizes with AFP. We found that transcripts encoding both factors are expressed at the highest levels in crypts of small and large intestine. Quantitation shows that the relative ratio of HNF-1 alpha to HNF-1 beta mRNAs appears lower in the colon, where AFP is not expressed. Alterations in the ratio of HNF-1 alpha to HNF-1 beta along the length of the intestine may influence HNF-1 dimer formation and expression of target genes. Although HNF-1 is necessary for transcription of the AFP gene, other factors must be involved in eliciting the cell type-specific pattern of AFP expression in the intestine.
Propionate activates multiple ion transport mechanisms in the HT29-18-C1 human colon cell line
Authors: Rowe W. A. , Blackmon D. L. , Montrose M. H.
Published: December 2017

Short-chain fatty acids (SCFAs) are the major solutes and the major anions in the colonic lumen. We studied the response of suspended HT29-18-C1 cells (an epithelial cell line derived from a human colon carcinoma) to SCFA exposure. Cellular response was evaluated by measurement of cell volume (Coulter counter), intracellular pH [pHi; measured fluorometrically with 2',7'-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein (BCECF)], and intracellular Na+, K+, and Cl- content (flame photometry and chloride titrator). Exposure to 130 mM propionate in isosmotic medium causes a rapid decrease in pHi and activates pHi recovery via amiloride-sensitive Na-H exchange. In the presence of propionate, Na-H exchange also causes cell swelling to a peak volume 11% above control cells and causes a 2.8-fold increase in intracellular Na+ content. After peak swelling, a regulatory-volume decrease (RVD) significantly reduced volume and intracellular Na+ returned to baseline. Other SCFAs (acetate, butyrate, and valerate) also elicit swelling and RVD. Activation of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) is required to return Na+ to normal levels and to indirectly provide ion gradients required for propionate-induced RVD, but Na(+)-K(+)-ATPase activity does not directly mediate RVD. When 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) is added in the presence of propionate, RVD was inhibited and cell Na+ content increased. Cl- depletion inhibited propionate-induced RVD and diminished the effect of SITS.
PAP, a pancreatic secretory protein induced during acute pancreatitis, is expressed in rat intestine
Authors: Iovanna J. L. , Keim V. , Bosshard A. , Orelle B. , Frigerio J. M. , Dusetti N. , Dagorn J. C.
Published: December 2017

The pancreatitis-associated protein (PAP) is a lectin-related secretory protein present in small amounts in the rat pancreas and rapidly overexpressed during the acute phase of pancreatitis. We demonstrate in this report that PAP is also expressed in rat intestine. A cDNA library from rat jejunum was probed with pancreatic PAP cDNA. The inserts of the selected recombinant clones corresponded to a transcript whose nucleotide sequence was identical to that of pancreatic PAP mRNA. The transcript was detected in duodenum, jejunum, ileum, and colon. A protein with same molecular mass (16 kDa) and pI (8.2) as pancreatic PAP was actually immunodetected in ileum homogenate after separation by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Intestinal PAP was immunolocalized to the epithelial cells of the lower part of the villi. The protein accounted respectively for 0.02, 0.05, and 0.1% of soluble proteins in duodenum, jejunum, and ileum homogenates, as measured by enzyme-linked immunosorbent assay, and could not be detected in stomach and colon. Influence of fasting and feeding on PAP mRNA concentration was analyzed in ileum. Concentration decreased by 81 and 94% after animals were fasted for 24 and 48 h, respectively. Feeding restored the initial content within 6 h. On the other hand, intestinal PAP mRNA concentration was not altered during acute pancreatitis
Interstitial cells in deep muscular plexus of canine small intestine may be specialized smooth muscle cells
Authors: Torihashi S. , Kobayashi S. , Gerthoffer W. T. , Sanders K. M.
Published: December 2017

The fine structures and properties of cells between the inner and outer circular muscle layers in the canine small intestine were studied by transmission electron microscopy (TEM), immunocytochemistry, and scanning electron microscopy (SEM). A nerve plexus (deep muscular plexus) supported by enteroglial cells, fibroblasts around blood vessels, macrophages, and thin and branched cells previously identified as interstitial cells of Cajal was observed. The interstitial cells of the deep muscular plexus (IC-DMP) were rich in mitochondria, dense bodies, and caveolae, and they were closely associated with nerve fibers. The IC-DMP had incomplete basal laminae. These cells also had numerous interconnecting gap junctions, and they also formed gap junctions with the surrounding smooth muscle cells of the outer circular muscle layer. IC-DMP were rich in myofilaments, which were primarily actin thin filaments, but myosin thick filaments, identified with anti-myosin light-chain antibodies, were also apparent. IC-DMP and circular smooth muscle cells both expressed immunoreactivity to anti-smooth muscle actin antisera, but these two types of cells differed in their intermediate filament proteins: IC-DMP featured vimentin immunopositive filaments, and circular smooth muscle cells featured desmin immunoreactivity. SEM showed that IC-DMP had thin and flat cell bodies with numerous branching processes. These cells came into close contact with nerve fibers and circular smooth muscle cells. The findings that IC-DMP cells contained myosin thick filaments and were immunopositive for anti-smooth muscle actin suggest that they may be more properly categorized as a type of smooth muscle cell.
Nutrient feedback inhibition of gastric emptying plays a larger role than osmotically dependent duodenal resistance
Authors: Lin H. C. , Elashoff J. D. , Gu Y. G. , Meyer J. H.
Published: December 2017

The slowing of gastric emptying by hyperosmolar solutions has been postulated to result from the triggering of duodenal osmoreceptor feedback on the stomach. We tested the idea that the inhibition of gastric emptying by a hyperosmolar solution depended on the duodenal resistance and the triggering of nutrient-specific feedback by tracking gastric emptying of 300 and 1,200 mosmol/kgH2O test solutions in 12 dogs in which duodenal resistance was either removed (by temporarily diverting chyme from uncorked duodenal fistula) or preserved (by keeping duodenal fistula corked). Mannitol was used to test osmolality alone, and glucose was used to examine the combined effects of osmolality and nutrient-specific inhibitory feedback. We found that: 1) the slowing effect of hyperosmolality was more marked with the duodenal resistance preserved (P < 0.05; analysis of variance), 2) the slowing effect of glucose was greater than that of mannitol for all conditions (P = 0.01; analysis of variance), and 3) the inhibitory effect of mannitol was localized to the duodenum. We conclude that inhibition of gastric emptying by hyperosmolar mannitol depended primarily on duodenal resistance, while the inhibitory effect of hyperosmolar glucose depended on nutrient-specific feedback on the stomach more than duodenal resistance.
Involvement of phospholipase D in caerulein-induced phosphatidylcholine hydrolysis in rat pancreatic acini
Authors: Rydzewska G. , Rossignol B. , Morisset J.
Published: December 2017

Phosphatidylcholine (PC) metabolism stimulated by caerulein (Cae), a cholecystokinin analogue, was investigated in rat pancreatic acini prelabeled with [3H]choline or [3H]-myristic acid. Both labels were incorporated mostly into PC. An inhibition of choline incorporation into PC was first observed in response to Cae (100 and 500 pM) stimulation, as indicated by reduced [3H]choline incorporation into trichloroacetic acid-precipitable material. Whereas choline incorporation was reduced in PC, Cae (500 pM) significantly increased [3H]choline metabolites release in the incubation medium. Separation of these metabolites by thin-layer chromatography showed that approximately 90% of the labeled products released into the medium was phosphocholine; however, Cae caused significant increases of [3H]choline release after 5, 15, and 30 min. In response to Cae, manoalide, a phospholipase C (PLC) inhibitor, totally prevented phosphocholine release into the medium but did not affect choline release. Staurosporine, a protein kinase C inhibitor, did not influence basal and Cae-induced choline release. In cells prelabeled with [3H]myristic acid, Cae stimulated within 5 min a rapid increase in intracellular [3H]phosphatidic acid (PA) levels in the presence of the PA phosphohydrolase inhibitor, propranolol; this PA production was further increased after 15 and 30 min of stimulation. The time course of [3H]PA formation in the presence of propranolol was similar to that of choline release in the medium. Staurosporine partially blocked PA accumulation stimulated by Cae after 30 min. In contrast, manoalide significantly reduced basal PA accumulation but did not prevent its production in response to Cae. In the presence of ethanol, Cae also significantly stimulated above control values the formation of [3H]phosphatidylethanol. These data indicate that Cae-induced PC hydrolysis in rat pancreatic acini is mediated mostly by phospholipase D (PLD) to produce PA and choline; they suggest a direct action of Cae on PLD activation, an effect independent of PLC activation
Role of histamine2 receptor in increased expression of rat gastric H(+)-K(+)-ATPase alpha-subunit induced by omeprazole
Authors: Tari A. , Yamamoto G. , Sumii K. , Sumii M. , Takehara Y. , Haruma K. , Kajiyama G. , Wu V. , Sachs G. , Walsh J. H.
Published: December 2017

Omeprazole is a specific inhibitor in vivo of the functioning gastric acid pump, the H(+)-K(+)-adenosinetriphosphatase (ATPase), in the secretory canaliculus of the parietal cell. It has been shown previously that omeprazole in rats led to an increase in the mRNA for the alpha-subunit of the H(+)-K(+)-ATPase. Omeprazole causes a marked increase in circulating gastrin in this species, which in turn stimulates release of histamine from the enterochromaffin-like cell. The possible role of this pathway was investigated by the in vivo administration of famotidine, a potent H2 receptor antagonist. A single intraperitoneal dose of famotidine, 200 mg/kg, produced a transient hypergastrinemia peaking at 3 h and normalizing at 12 h, inhibition of secretion that lasted for 12 h, but no change in the level of the alpha-subunit mRNA or of beta-actin mRNA. In contrast, a single dose of omeprazole,xs 100 mg/kg, inhibited acid secretion and produced hypergastrinemia, peaking at 12 h, both effects lasting for the 24-h observation period. Omeprazole elevated the alpha-subunit mRNA transiently by more than threefold at 3 h, with normal levels being restored at 24 h. The administration of famotidine 1 h after omeprazole did not change the effects of omeprazole on acid secretion but elevated the gastrin levels further. There was now no elevation of the alpha-subunit mRNA for the first 6 h, but a small increase at 12 h and a further increase to approximately 2.5-fold at 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)
Coordination of Cl- secretion and contraction by a histamine H2-receptor agonist in guinea pig distal colon
Authors: Cooke H. J. , Wang Y. Z. , Rogers R.
Published: December 2017

Short-circuit current (Isc) was measured simultaneously with muscle tension recorded in the longitudinal or circumferential axis from strain-gauge transducers sutured to the serosal surface of whole thickness segments of distal colon from guinea pigs. Isc and the amplitude and frequency of small-amplitude phasic contractions were stable for several hours. The histamine H2-receptor agonist dimaprit (5-10 microM) evoked recurrent increases in Isc at a frequency of 0.32-0.37 cycles/min. Associated with each cyclical Isc response was an increase in muscle tension indicative of large-amplitude phasic contractions. Recurrent cycles of Isc and associated large-amplitude phasic contractions were abolished by 10 microM cimetidine. In the presence of dimaprit, severing the neural connections between the myenteric and submucosal plexuses abolished the large-amplitude phasic contractions but not cyclical Isc. The results suggest that coordination of cyclical secretion and large-amplitude contraction during chronic activation of histamine H2 receptors requires intact neural connections between the submucosal and myenteric plexuses. The findings provide potential mechanisms to explain altered motility and ion transport during inflammatory diseases or allergic responses to food antigens.
Muscarinic cholinergic receptor density following small intestinal transplantation in rats
Authors: Zhang Z. , Koch T. R. , Mustin E. , Walgenbach-Telford S. , Telford G. L.
Published: December 2017

After small intestinal transplantation, intestinal isografts can organize migrating myoelectric complexes, and we have shown that migrating myoelectric complex frequency in the fasted state was reduced compared with controls after transplantation of the distal 50% of small intestine. We hypothesized that changes in motor activity after transplantation were related to alteration of cholinergic nerve activity or receptor density. With use of standard microsurgical techniques, the distal 50% of small intestine was orthotopically transplanted in a Lewis-to-Lewis donor-recipient combination. Resection controls were prepared by resecting the proximal 50% of small intestine, and sham controls were prepared by performing a sham laparotomy. Two months after surgery, small intestine was harvested. Choline acetyltransferase activity among the three groups was similar, suggesting that intrinsic cholinergic nerves remained intact. There was a strong trend toward decreased acetylcholinesterase activity [analysis of variance (ANOVA), P = 0.16] after transplantation, consistent with loss of extrinsic vagal nerve fibers. There were no differences in histochemical distribution of acetylcholinesterase among these groups. Muscarinic receptor density, as determined by binding to [N-methyl-3H]scopolamine, was decreased after transplantation (ANOVA, P = 0.02). There was a trend toward decreased receptor density in animals with resected small intestine. Surgical interruption of intrinsic nerve pathways rather than ischemia or extrinsic denervation might be the mechanism for diminished receptor density after transplantation, and reduced small bowel motor activity may be related to decreased density of muscarinic cholinergic receptors.