bluuebunny/crossref_metadata_2025 huggingface dataset:
Transport of fluorescein methotrexate by multidrug resistance-associated protein 3 in IEC-6 cells
Authors: Li Tiesong , Ito Kousei , Horie Toshiharu
Published: March 2015

The transport characteristics of fluorescein methotrexate (F-MTX) were studied by using the rat intestinal crypt cell line IEC-6. Enhanced accumulation of F-MTX at 4°C suggests the existence of an active efflux system. MK-571, an inhibitor of the multidrug resistance-associated protein/ATP binding cassette C (MRP/ABCC) family, also enhanced the accumulation of F-MTX. Transcellular transport of F-MTX from the apical to the basolateral compartment was 2.5 times higher than the opposite direction. This vectorial transport was also reduced by MK-571, indicating the presence of Mrp-type transporter(s) on the basolateral membrane. Mrp3 mRNA was readily detectable, and the protein was localized on the basolateral membrane. Uptake of FMTX into membrane vesicles from IEC-6 cells and Spodoptera frugiperda-9 cells expressing rat Mrp3 were both ATP dependent and saturable as a function of the F-MTX concentration. Similar Kmvalues (11.0 ± 1.8 and 4.5 ± 1.1 μM) and inhibition profiles by MK-571, estradiol-17β-d-glucuronide, and taurocholate for the ATP-dependent transport of F-MTX into these vesicles were obtained. These findings suggest that the efflux of F-MTX is mediated by Mrp3 on the basolateral membrane of IEC-6 cells.
Endogenous interleukin-10 modulates fibrosis and regeneration in experimental chronic pancreatitis
Authors: Demols Anne , Van Laethem Jean-Luc , Quertinmont Eric , Degraef Chantal , Delhaye Myriam , Geerts Albert , Deviere Jacques
Published: March 2015

Interleukin (IL)-10, a potent anti-inflammatory cytokine, limits the severity of acute pancreatitis and downregulates transforming growth factor (TGF)-β release by inflammatory cells on stimulation. Proinflammatory mediators, reactive oxygen species, and TGF-β can activate pancreatic stellate cells and their synthesis of collagen I and III. This study evaluates the role of endogenous IL-10 in the modulation of the regeneration phase following acute pancreatitis and in the development of pancreatic fibrosis. IL-10 knockout (KO) mice and their C57BL/6 controls were submitted to repeated courses (3/wk, during 6 wk, followed by 1 wk of recovery) of cerulein-induced acute pancreatitis. TGF-β1 release was measured on plasma, and its pancreatic expression was assessed by quantitative RT-PCR and immunohistochemistry. Intrapancreatic IL-10 gene expression was assessed by semiquantitative RT-PCR, and intrapancreatic collagen content was assessed by picrosirius staining. Activated stellate cells were detected by immunohistochemistry. S phase intrapancreatic cells were marked using tritiated thymidine labeling. After repeated acute pancreatitis, IL-10 KO mice had more severe histological lesions and fibrosis (intrapancreatic collagen content) than controls. TGF-β1 plasma levels, intrapancreatic transcription, and expression by ductal and interstitial cells, as well as the number of activated stellate cells, were significantly higher. IL-10 KO mice disclosed significantly fewer acinar cells in S phase, whereas the opposite was observed for pseudotubular cells. Endogenous IL-10 controls the regeneration phase and limits the severity of fibrosis and glandular atrophy induced by repeated episodes of acute pancreatitis in mice.
High specific activity heme-Fe and its application for studying heme-Fe metabolism in Caco-2 cell monolayers
Authors: Follett Jennifer R. , Suzuki Yasushi A. , Lönnerdal Bo
Published: March 2015

Heme-Fe is an important source of dietary iron in humans. Caco-2 cells have been used extensively to study human iron absorption with an emphasis on factors affecting nonheme iron absorption. Therefore, we examined several factors known to affect heme iron absorption. Cells grown in bicameral chambers were incubated with high specific activity [59Fe]heme alone or with 1% globin, BSA, or fatty acid-free BSA (BSA-FA) to examine the effect of protein source on absorption. Heme iron absorption was enhanced by globin and inhibited by BSA and BSA-FA. Absorption of heme iron in cells pretreated for 7 days with serum-free medium containing 1, 25, 50, or 100 μM Fe was higher in the 1-μM-Fe pretreatment group than in all other groups ( P < 0.05), showing an effect of iron status. Increased heme concentrations resulted in decreased percent absorbed but increased total heme iron absorption and increased transport rate across the basolateral membrane. Finally, cells treated with 10 μM CdCl2, which induces heme oxygenase, demonstrated higher absorption of [59Fe]heme than control cells ( P < 0.05). Our results from Caco-2 cells are in agreement with human studies and make this a promising model for examining intestinal heme iron absorption.
Micellar distribution of cholesterol and phytosterols after duodenal plant stanol ester infusion
Authors: Nissinen Markku , Gylling Helena , Vuoristo Matti , Miettinen Tatu A.
Published: March 2015

Properties of the intestinal digestion of the dietary phytosterols, cholesterol and cholestanol, and the mechanisms by which phytosterols inhibit the intestinal absorption of cholesterol in healthy human subjects are poorly known. We have studied the hydrolysis of dietary plant sterol and stanol esters and their subsequent micellar solubilization by determining their concentrations in micellar and oil phases of the jejunal contents. Two liquid formulas with low ( formula 1) and high ( formula 2) plant stanol concentrations were infused via a nasogastric tube to the descending duodenum of 8 healthy human subjects, and intestinal contents were sampled for gas-liquid chromatographic sterol analysis 60 cm more distally. During the duodenal transit, phytosterol esters were hydrolyzed. This was especially profound for sitostanol, as its esterified fraction per milligram of sitosterol decreased 80% ( P < 0.001) in formula 1 and 61% ( P < 0.001) in formula 2. Contrary to that, esterified fraction of cholesterol per milligram of sitosterol was increased fourfold ( P < 0.001) in formula 1 and almost sixfold ( P < 0.001) in formula 2, whereas that of cholestanol remained unchanged. Percentages of esterified sterols and stanols in total intestinal fluid samples were higher after the administration of formula 2 than of formula 1. Esterified cholesterol and stanols accumulated in the oil phase, and free stanols replaced cholesterol in the micellar phase. At high intestinal plant stanol concentrations, cholesterol looses its micellar solubility possibly by replacement of its free fraction in the micellar phase by hydrolyzed plant stanols, which leads to a decreased intestinal absorption of cholesterol.
Sensory and biomechanical responses to ramp-controlled distension of the human duodenum
Authors: Gao Chunwen , Arendt-Nielsen Lars , Liu Weiming , Petersen Poul , Drewes Asbjørn Mohr , Gregersen Hans
Published: March 2015

The aim of this study was to develop a new method for investigation of the relationship among the mechanical stimulus, the biomechanical properties, and the visceral perception evoked by volume/ramp-controlled distension in the human duodenum in vivo. An impedance planimetric probe for balloon distension was placed in the third part of the duodenum in seven healthy volunteers. Distension of the duodenum was done at infusion rates of 10, 25, and 50 ml/min. The pump was reversed when level 7 was reached on a visual analog scale ranging from 0 to 10. Distensions were done with and without the administration of the antimuscarinic drug butylscopolamine. The total circumferential tension (Ttotal) and the passive circumferential tension (Tpassive) were determined from the distension tests without and with the administration of butylscopolamine, respectively. Ttotal and Tpassive showed an exponential behavior as a function of strain (a measure of deformation). The active circumferential tension (Tactive) was computed as Ttotal−Tpassive and showed a bell-shaped behavior as a function of strain. At low distension intensities, the intensity of sensation at 10 ml/min was significantly higher than that obtained at 25 and 50 ml/min. The coefficient of variation at the pain threshold for circumferential strain (average 4.34) was closer to zero compared with those for volume (8.72), pressure (31.22), and circumferential tension (31.55). This suggests that the mechanoreceptors in the gastrointestinal wall depend primarily on circumferential strain. The stimulus-response functions provided evidence for the existence of low- and high-threshold mechanoreceptors in the human duodenum. Furthermore, the data suggest that high-threshold receptors are nonadapting.
Time-dependent platelet-vessel wall interactions induced by intestinal ischemia-reperfusion
Authors: Cooper Dianne , Chitman Keith D. , Williams Matthew C. , Granger D. Neil
Published: March 2015

Platelets roll and adhere in venules exposed to ischemia-reperfusion (I/R). This platelet-endothelial adhesion may influence leukocyte trafficking because platelet depletion decreases I/R-induced leukocyte emigration. The objectives of this study were 1) to assess the time course of platelet adhesion in the small bowel after I/R and 2) to determine the roles of endothelial and/or platelet P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) in this adhesion. The adhesion of fluorescently labeled platelets was monitored by intravital microscopy in postcapillary venules exposed to 45 min of ischemia and up to 8 h of reperfusion. Peak platelet adhesion was observed at 4 h of reperfusion. To assess the contributions of platelet and endothelial cell P-selectin, platelets from P-selectin-deficient and wild-type mice were infused into wild-type and P-selectin-deficient mice, respectively. Platelets deficient in P-selectin exhibited low levels of adhesion comparable to that in sham-treated animals. In the absence of endothelial P-selectin, platelet adhesion was reduced by 65%. Treatment with a blocking antibody against PSGL-1 reduced adhesion by 57%. These results indicate that I/R induces a time-dependent platelet-endothelial adhesion response in postcapillary venules via a mechanism that involves PSGL-1 and both platelet and endothelial P-selectin, with platelet P-selectin playing a greater role.
Release of ATP during host cell killing by enteropathogenicE. coliand its role as a secretory mediator
Authors: Crane John K. , Olson Ruth A. , Jones Heather M. , Duffey Michael E.
Published: March 2015

Enteropathogenic Escherichia coli (EPEC) causes severe, watery diarrhea in children. We investigated ATP release during EPEC-mediated killing of human cell lines and whether released adenine nucleotides function as secretory mediators. EPEC triggered a release of ATP from all human cell lines tested: HeLa, COS-7, and T84 (colon cells) as measured using a luciferase kit. Accumulation of ATP in the supernatant medium was enhanced if an inhibitor of 5′-ectonucleotidase was included and was further enhanced if an ATP-regenerating system was added. In the presence of the inhibitor/regenerator, ATP concentrations in the supernatant medium reached 1.5–2 μM 4 h after infection with wild-type EPEC strains. In the absence of the inhibitor/regenerator system, extracellular ATP was rapidly broken down to ADP, AMP, and adenosine. Conditioned medium from EPEC-infected cells triggered a brisk chloride secretory response in intestinal tissues studied in the Ussing chamber (rabbit distal colon and T84 cell monolayers), whereas conditioned medium from uninfected cells and sterile filtrates of EPEC bacteria did not. The short-circuit current response to EPEC-conditioned medium was completely reversed by adenosine receptor blockers, such as 8-( p-sulfophenyl)-theophylline and MRS1754. EPEC killing of host cells releases ATP, which is broken down to adenosine, which in turn stimulates secretion via apical adenosine A2breceptors. These findings provide new insight into how EPEC causes watery diarrhea.
Isolation and characterization of human esophageal microvascular endothelial cells: mechanisms of inflammatory activation
Authors: Rafiee Parvaneh , Ogawa Hitoshi , Heidemann Jan , Li Mona S. , Aslam Mohammed , Lamirand Thomas H. , Fisher Pamela J. , Graewin Shannon J. , Dwinell Michael B. , Johnson Christopher P. , Shaker Reza , Binion David G.
Published: March 2015

Gastroesophageal reflux disease is the most common malady of the esophagus, affecting 7% of the United States population. Histological assessment demonstrates classic inflammatory mechanisms including selective leukocyte recruitment and hemorrhage, suggesting a prominent role for the microvasculature. We isolated and characterized human esophageal microvascular endothelial cells (EC) (HEMEC), examined inflammatory activation in response to cytokines, LPS, and acidic pH exposure, and identified signaling pathways that underlie activation. HEMEC displayed characteristic morphological and phenotypic features including acetylated LDL uptake. TNF-α/LPS activation of HEMEC resulted in upregulation of the cell adhesion molecules (CAM) ICAM-1, VCAM-1, E-selectin, and mucosal addressin CAM-1 (MAdCAM-1), increased IL-8 production, and enhanced leukocyte binding. Both acid and TNF-α/LPS activation lead to activation of SAPK/JNK in HEMEC that was linked to VCAM-1 expression and U-937 leukocyte adhesion. Expression of constitutive inducible nitric oxide synthase in HEMEC was in marked contrast to intestinal microvascular endothelial cells. In this study, we demonstrate that HEMECs are phenotypically and functionally distinct from lower gut-derived endothelial cells and will facilitate understanding of inflammatory mechanisms in esophageal inflammation.
Trans-10,cis-12-conjugated linoleic acid inhibits Caco-2 colon cancer cell growth
Authors: Kim Eun J. , Holthuizen P. Elly , Park Hyun S. , Ha Yeong L. , Jung Kyeong C. , Park Jung H. Y.
Published: March 2015

A commercially available mixture of conjugated linoleic acid (CLA) isomers decreases colon cancer cell growth. We compared the individual potencies of the two main isomers in this mixture [ cis-9, trans-11 ( c9 t11) and trans-10, cis-12 ( t10 c12)] and assessed whether decreased cell growth is related to changes in secretion of insulin-like growth factor II (IGF-II) and/or IGF-binding proteins (IGFBPs), which regulate Caco-2 cell proliferation. Cells were incubated in serum-free medium with different concentrations of the individual CLA isomers. t10 c12 CLA dose dependently decreased viable cell number (55 ± 3% reduction 96 h after adding 5 μM t10 c12 CLA). t10 c12 CLA induced apoptosis and decreased DNA synthesis, whereas c9 t11 CLA had no effect. Immunoblot analysis of 24-h serum-free conditioned medium using a monoclonal anti-IGF-II antibody revealed that Caco-2 cells secreted both a mature 7,500 molecular weight ( M r) IGF-II and higher M r forms of IGF-II. The levels of the higher M r and the mature form of IGF-II were decreased 50 ± 3% and 22 ± 2%, respectively, by 5 μM t10 c12 CLA. c9 t11 CLA had no effect. Ligand blot analysis of conditioned medium using125I-labeled IGF-II revealed that t10 c12 CLA slightly decreased IGFBP-2 production; c9 t11 CLA had no effect. Exogenous IGF-II reversed t10 c12 CLA-induced growth inhibition and apoptosis. These results indicate that CLA-inhibited Caco-2 cell growth is caused by t10 c12 CLA and may be mediated by decreasing IGF-II secretion in Caco-2 cells.
Integrative control of rectoanal reflex in guinea pigs through lumbar colonic nerves
Authors: Yamanouchi Masaya , Shimatani Hidehiko , Kadowaki Makoto , Yoneda Satoshi , Nakagawa Tadashi , Fujii Hisao , Takaki Miyako
Published: March 2015

The aim of the present study was to analyze the neuromodulation of rectoanal reflex activity by lumbar sympathetic nerves in guinea pigs. The mechanical activities of the rectum were recorded with a balloon connected to a pressure transducer, and those of the internal anal sphincter (IAS) were recorded with a custom-made strain gauge force transducer. Gradual and sustained rectal distension evoked the rectoanal reflex, causing cholinergic contractions of the rectum and synchronous nitrergic relaxations of the IAS. Section of the lumbar colonic nerves enhanced both rectal contractions and IAS relaxations. Section of the 13th thoracic cord abolished both rectal contractions and IAS relaxations, but section of the lumbar colonic nerves restored them. Lumbar sympathectomy and pithing sacral cords greatly diminished these rectal contractions and IAS relaxations, but the intrinsic reflex component remained. N G-nitro-l-arginine methyl ester enhanced the intrinsic reflex-mediated contraction of the rectum and abolished reflex-mediated relaxation of the IAS and converted into cholinergic contractions. The present results indicate that the extrinsic lumbar inhibitory outflow causes marked inhibition of the rectoanal reflex via the lumbar colonic nerves.
I. PPARγ in the gastrointestinal tract: gain or pain?
Authors: Auwerx Johan
Published: March 2015

The peroxisome proliferator-activated receptor gamma (PPARγ) has recently been implicated in the pathogenesis of inflammatory bowel disease (IBD) and colon cancer. The observation that PPARγ agonists, through immune modulation, protect against inflammatory processes in the intestine justified their expedient evaluation in the clinical management of IBD. PPARγ agonists are reported to have both tumor-promoting and -inhibiting effects in models of colon cancer. These differences can, in part, be explained by PPARγ-independent effects of PPARγ agonists and by differences in the models used. Because it is still unclear how PPARγ impacts on colon cancer, careful monitoring of patients receiving PPARγ agonists and additional basic research is indicated before recommendations on the use of PPARγ ligands in colon cancer can be made.
Adenovirus-mediated gene transfer to nonparenchymal cells in normal and injured liver
Authors: Yu Qing , Que Loretta G. , Rockey Don C.
Published: March 2015

Adenovirus-mediated gene transfer has become an important tool with which to introduce genetic material into cells. Available data emphasize efficient adenoviral transduction of parenchymal liver cells (i.e., hepatocytes) in both in vitro and in vivo model systems, typically in normal cells. The aim of this study was to evaluate gene transfer to nonparenchymal (and parenchymal) cells of the normal and injured rat liver. Hepatocytes, stellate cells, and endothelial cells were isolated by standard methods. Liver injury was induced by bile duct ligation or carbon tetrachloride administration. Cells were transduced in vitro with an adenovirus encoding β-galactosidase (Ad.β-gal) over a range of viral titers, and transduced cells were identified by detection of X-gal. In vivo transduction efficiency was studied in normal and injured livers using cell isolation techniques. Nonparenchymal cells were transduced with greater frequency than hepatocytes at all adenoviral titers tested, both in vitro and in vivo. After liver injury, adenoviral transduction was reduced for all liver cell types compared with that for cells from normal livers (at all virus titers). Notably, transduction efficiency remained greater in nonparenchymal cells than in hepatocytes after liver injury. This work implies that, to achieve comparable gene expression in the injured liver, higher adenoviral titers may be required, an important consideration as gene therapy in disease states is considered.
Increasing cell membrane potential and GABAergic activity inhibits malignant hepatocyte growth
Authors: Sun D. , Gong Y. , Kojima H. , Wang G. , Ravinsky E. , Zhang M. , Minuk G. Y.
Published: March 2015

Increasing hepatocyte membrane potentials by augmenting GABAergic activity inhibits nonmalignant hepatocyte proliferative activity. The objectives of this study were to document 1) potential differences (PDs) of four malignant hepatocyte cell lines, 2) GABAAreceptor mRNA expression in the same cell lines, and 3) effects of restoring malignant hepatocyte PDs to levels approximating those of resting, nonmalignant hepatocytes. Hepatocyte PDs were documented in nonmalignant and malignant (Chang, HepG2, HuH-7, and PLC/PRF/5) hepatocytes with a fluorescent voltage-sensitive dye and GABAAreceptor expression by RT-PCR and Western blot analyses. Compared with nonmalignant human hepatocytes, all four malignant cell lines were significantly depolarized ( P < 0.0001, respectively). Only PLC/PRF/5 cells had detectable GABAA-β3receptor mRNA expression and all cell lines were negative for GABAA-β3receptor protein by Western blot analysis. Stable transfection of Chang cells with GABAA-β3receptor cDNA resulted in significant increases in PD and decreases in proliferative activity as manifest by decreased [3H]thymidine and bromodeoxyurieine incorporation rates, 4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate activity, a lower mitotic index, prolongation of cell-doubling times, and attenuated growth patterns compared with cells transfected with vector alone. Colony formation in soft agar and the number of abnormal mitoses were also significantly decreased in GABAA-β3receptor transfected cells. The results of this study indicate 1) relative to healthy hepatocytes, malignant hepatocytes are significantly depolarized, 2) GABAA-β3receptor expression is absent in malignant hepatocyte cell lines, and 3) increasing the PD of malignant hepatocytes is associated with less proliferative activity and a loss of malignant features.
Kininogen deficiency modulates chronic intestinal inflammation in genetically susceptible rats
Authors: Isordia-Salas Irma , Pixley Robin A. , Li Fengling , Sainz Irma , Sartor R. Balfour , Adam Albert , Colman Robert W.
Published: March 2015

Genetically susceptible Lewis rats injected in the intestinal wall with peptidoglycan-polysaccharide (PG-APS) polymers develop chronic granulomatous enterocolitis concomitant with activation of the kallikrein-kinin system. To elucidate the role of high-molecular-weight kininogen (HK) in chronic enterocolitis, we back crossed Brown-Norway rats having a HK deficiency with Lewis rats for five generations. Two new strains were produced, wild-type F5 (F5WT) and HK deficient (F5HKd), each with a ∼97% Lewis genome. The HK values of F5WT and F5HKd rat plasma were 0.62 ± 0.20 and 0.08 ± 0.03 U/ml, respectively. In PG-APS-injected rats, chronic inflammation was measured by using gross gut score, histological inflammation, liver granuloma, and white blood cell count. The mean gross gut scores were significantly lower in the F5HKd than in the F5WT rats. Plasma T-kininogen was significantly less in F5HKd. These results indicate the importance of the kallikrein-kinin system in this model of chronic enterocolitis and systemic inflammation.
Histamine is involved in gastric vasodilation during acid back diffusion via activation of sensory neurons
Authors: Rydning Astrid , Lyng Oddveig , Falkmer Sture , Grønbech Jon Erik
Published: March 2015

Protective vasodilation during acid back diffusion into the rat gastric mucosa depends on activation of sensory neurons and mast cell degranulation with histamine release. We hypothesized that these two mediator systems interact and that histamine partly exerts its effect via sensory nerves. Gastric blood flow (GBF) and luminal histamine were measured in chambered stomachs, and mast cell numbers were assessed by morphometry. Ablation of sensory neurons and depletion of mast cells were produced by pretreatment with capsaicin or dexamethasone, respectively. Mucosal exposure to 1.5 M NaCl and then to pH 1.0 saline in ablated and control rats caused increased luminal histamine and reduced numbers of mast cells. Enterochromaffin-like cell marker pancreastatin remained unchanged. Only control rats responded with an increase in GBF. Capsaicin stimulation (640 μM) of the undamaged mucosa induced identical increase in GBF and unchanged mast cell mass in normal and dexamethasone-treated rats. Increase in GBF after topical exposure to histamine (30 mM) in rats pretreated with capsaicin or a calcitonin gene-related peptide (CGRP)1antagonist human CGRP8–37or exposed to the calcium pore blocker ruthenium red was less than one-half of that in control rats. These data suggest that mast cell-derived histamine is involved in gastric vasodilatation during acid back diffusion partly via sensory neurons.
Metabolic acidosis in rats increases intestinal NHE2 and NHE3 expression and function
Authors: Lucioni Alvaro , Womack Christopher , Musch Mark W. , Rocha Flavio L. , Bookstein Cres , Chang Eugene B.
Published: March 2015

Chronic metabolic acidosis increases intestinal Na absorption, although through undefined mechanisms. Whether this occurs through enhanced expression and/or function of the brush-border Na+/H+exchangers (NHE)2 and NHE3 is unknown. Metabolic acidosis was induced in rats by feeding ammonium chloride through their drinking water. Intestinal NHE activities were measured using brush-border22Na+uptake. Western and Northern blots measured changes in protein and mRNA expression, respectively. Acidosis occurred within 2 days of ammonium chloride feedings but increased after 6 days. NHE2 and NHE3 activities, protein expression, and mRNA levels increased in acidotic rats compared with controls. In contrast, basolateral NHE1 expression was not affected. Brush-border alkaline phosphatase showed no effect of metabolic acidosis on cellular differentiation. This study demonstrated a direct effect of metabolic acidosis on NHE2 and NHE3 activity, expression, and gene transcription. Metabolic acidosis is one of the few circumstances shown to affect NHE2 function and expression, thus providing insights into the role of NHE2 on intestinal physiology.
Regulation of TGF-α-induced activation of AP-1 in the aging gastric mucosa
Authors: Xiao Zhi-Qiang , Li Jianling , Majumdar Adhip P. N.
Published: March 2015

Although the age-related activation of EGF receptor (EGFR) in the gastric mucosa of Fischer 344 rats is associated with increased DNA binding activity of activator protein-1 (AP-1), little is known about the EGFR signaling cascades that regulate this process. The primary objective of this investigation was to determine the role of signaling pathways initiated by EGFR in regulating the transforming growth factor-α (TGF-α)-induced activation of AP-1 in the gastric mucosa in aged rats. Freshly isolated gastric mucosal cells from male young (4–5 mo) and aged (22–24 mo) rats were used. We have observed that although exposure of mucosal cells from young (4–5 mo) and old (22–24 mo) rats to 1 nM TGF-α for 20 min stimulates the DNA binding activity of AP-1 in both age groups, the magnitude of stimulation is substantially higher in aged (131%) than in young (35%) rats. This stimulation in the aged is associated with a concomitant activation of MEKs and ERKs, but not JNKs and p38. The TGF-α induction of AP-1 transcriptional activity in gastric mucosal cells from aged rats could be totally abrogated by either PD153035, a specific inhibitor of EGFR tyrosine kinase, or PD98059, a specific inhibitor of MEKs, but not by Wortmannin, which inhibits phosphatidylinositol kinase. PP2, a specific inhibitor of Src kinase, produces a 50% inhibition of the TGF-α-induced activation of AP-1 transcriptional activity. Our results suggest that the TGF-α-induced stimulation of DNA binding activity of AP-1 in the gastric mucosa of aged rats is primarily through a signaling pathway involving MEKs and ERKs, whereas Src kinase pathways play a minor role.
Effects of peptides derived from dietary proteins on mucus secretion in rat jejunum
Authors: Claustre Jean , Toumi Férial , Trompette Aurélien , Jourdan Gérard , Guignard Henri , Chayvialle Jean Alain , Plaisancié Pascale
Published: March 2015

The hypothesis that dietary proteins or their hydrolysates may regulate intestinal mucin discharge was investigated in the isolated vascularly perfused rat jejunum using an enzyme-linked immunosorbent assay for rat intestinal mucins. On luminal administration, casein hydrolysate [0.05–5% (wt/vol)] stimulated mucin secretion in rat jejunum (maximal response at 417% of controls). Lactalbumin hydrolysate (5%) also evoked mucin discharge. In contrast, casein, and a mixture of amino acids was without effect. Chicken egg albumin and its hydrolysate or meat hydrolysate also did not modify mucin release. Interestingly, casein hydrolysate-induced mucin secretion was abolished by intra-arterial TTX or naloxone (an opioid antagonist). β-Casomorphin-7, an opioid peptide released from β-casein on milk ingestion, induced a strong mucin secretion (response at 563% of controls) that was inhibited by naloxone. Intra-arterial β-casomorphin-7 also markedly increased mucin secretion (410% of controls). In conclusion, two enzymatic milk protein hydrolysates (casein and lactalbumin hydrolysates) and β-casomorphin-7, specifically, induced mucin release in rat jejunum. The casein hydrolysate-induced mucin secretion is triggered by a neural pathway and mediated by opioid receptor activation.
Aging impairs endothelium-dependent vasodilation in rat skeletal muscle arterioles
Authors: Muller-Delp Judy M. , Spier Scott A. , Ramsey Michael W. , Delp Michael D.
Published: March 2015

Blood flow capacity in skeletal muscle declines with age. Reduced blood flow capacity may be related to decline in the maximal vasodilatory capacity of the resistance vasculature. This study tested the hypothesis that aging results in impaired vasodilatory capacity of first-order (1A) arterioles isolated from rat-hindlimb locomotory muscle: 1A arterioles (90–220 μm) from gastrocnemius and soleus muscles of young (4 mo) and aged (24 mo) Fischer-144 rats were isolated, cannulated, and pressurized via hydrostatic reservoirs. Vasodilatory responses to increasing concentrations of ACh (10−9to 10−4M), adenosine (ADO, 10−10to 10−4M), and sodium nitroprusside (SNP, 10−10to 10−4M) were evaluated at a constant intraluminal pressure of 60 cmH2O in the absence of flow. Flow-induced vasodilation was also evaluated in the absence of pressure changes. Responses to ADO and SNP were not altered by age. Endothelium-dependent vasodilation induced by flow was significantly reduced in arterioles from both gastrocnemius and soleus muscles. In contrast, endothelium-dependent vasodilation to ACh was reduced only in soleus muscle arterioles. These results indicate that aging impairs vasodilatory responses mediated through the endothelium of resistance arterioles from locomotory muscle, whereas smooth muscle vasodilatory responses remain intact with aging. Additionally, ACh-induced vasodilation was altered by age only in soleus muscle arterioles, suggesting that the mechanism of age-related endothelial impairment differs in arterioles from soleus and gastrocnemius muscles.
Antioxidants attenuate myocyte apoptosis and improve cardiac function in CHF: association with changes in MAPK pathways
Authors: Qin Fuzhong , Shite Junya , Liang Chang-seng
Published: March 2015

Antioxidant vitamins reduce cardiac oxidative stress and cardiomyocyte apoptosis produced by exogenous norepinephrine (NE) and attenuate cardiac dysfunction in animals with pacing-induced congestive heart failure (CHF). This study was carried out to determine whether the mitogen-activated protein kinase (MAPK) signal transduction pathways are involved in oxidative stress-induced myocyte apoptosis. Rabbits with rapid pacing-induced CHF and sham operation were randomized to receive either a combination of antioxidant vitamins (β-carotene, ascorbic acid, and α-tocopherol), α-tocopherol alone, or placebo for 8 wk. Compared with sham-operated animals, CHF animals exhibited increased oxidative stress as evidenced by decreased myocardial reduced-to-oxidized glutathione (GSH/GSSG) ratio (27 ± 7 vs. 143 ± 24, P < 0.05), myocyte apoptosis (77 ± 18 vs. 17 ± 4 apoptotic nuclei/10,000 cardiomyocytes, P < 0.05), increased total and phosphorylated c-Jun NH2-terminal protein kinase (p-JNK; 1.95 ± 0.14 vs. 1.04 ± 0.04 arbitrary units, P < 0.05) and phosphorylated p38 kinase (p-p38), and decreased phosphorylated extracellular signal-regulated kinase (p-ERK). Administration of antioxidant vitamins and α-tocopherol attenuated oxidative stress, myocyte apoptosis, and cardiac dysfunction, with reversal of the changes of total JNK, p-JNK, and p-ERK in CHF. Furthermore, because NE infusion produced changes of JNK, p-p38, and p-ERK similar to those in CHF, we conclude that NE may play an important role in the production of oxidative stress, MAPK activation, and myocyte apoptosis in CHF.
P38 MAPK: critical molecule in thrombin-induced NF-κB-dependent leukocyte recruitment
Authors: Kaur Jaswinder , Woodman Richard C. , Kubes Paul
Published: March 2015

Thrombin-stimulated endothelium synthesizes numerous adhesion molecules to recruit leukocytes; however, it is unknown which intracellular pathways are responsible for this event. A recent report from our laboratory has shown that thrombin induces E-selectin expression and that blocking nuclear factor-κB (NF-κB) activity partially blocked both E-selectin expression (60%) and leukocyte recruitment. In this study, we systematically assessed the importance of p38 MAPK in thrombin-induced NF-κB activation and E-selectin-dependent leukocyte recruitment. Thrombin caused phosphorylation of p38 MAPK, its substrate ATF-2, and JNK MAPK, but not ERK MAPK. The p38 MAPK inhibitors, SKF86002 and SB-203580 only reduced ATF-2 activity. We treated human umbilical vein endothelial cells with SKF86002, 1 h before thrombin stimulation, and noted inhibition of NF-κB mobilization and complete inhibition of leukocyte rolling and adhesion in a laminar flow chamber. Significant inhibition of leukocyte recruitment and E-selectin expression was also observed with SB-203580. SKF86002 did not affect other systems, including tumor necrosis factor-α-induced E-selectin-dependent leukocyte recruitment. Moreover, thrombin-induced rapid mobilization of P-selectin from Weibel Palade bodies was not p38 MAPK dependent. These data suggest that thrombin induces p38 MAPK activation, which leads to NF-κB mobilization to the nucleus and causes the upregulation of E-selectin and subsequent leukocyte recruitment.
Obesity lowers hyperglycemic threshold for impaired in vivo endothelial nitric oxide function
Authors: Bohlen H. G. , Nase Geoffrey P.
Published: March 2015

Obesity is a risk for type II diabetes mellitus and increased vascular resistance. Disturbances of nitric oxide (NO) physiology occur in both obese animals and humans. In obese Zucker rats, we determined whether a protein kinase C-βII (PKC-βII) mechanism may lower the resting NO concentration ([NO]) and predispose endothelial NO abnormalities at lower glucose concentrations than occur in lean rats. NO was measured with microelectrodes touching in vivo intestinal arterioles. At rest, the [NO] in obese Zucker rats was 60 nm less than normal or about a 15% decline. After local blockade of PKC-βII with LY-333531, the [NO] increased ∼90 nm in obese rats but did not change in lean rats. In lean rats, administration of 300 mg/dl d-glucose for 45 min depressed endothelium-dependent dilation; only 200 mg/dl was required in obese animals. These various observations indicate that resting [NO] is depressed in obese rats by a PKC-βII mechanism and the hyperglycemic threshold for endothelial NO suppression is reduced to 200 mg/dl d-glucose.
Endothelium-dependent responses in coronary arteries are changed with puberty in male pigs
Authors: Chatrath Ritu , Ronningen Karen L. , Severson Sandra R. , LaBreche Peter , Jayachandran Muthuvel , Bracamonte Margarita P. , Miller Virginia M.
Published: March 2015

In humans, cardiovascular disease begins in young adulthood and is more prevalent in males than females. However, little is known about vascular function during transition to adulthood in males. The aim of this study was to define changes in production of endothelium-derived nitric oxide (NO) and coronary arterial responses during puberty. Plasma was collected from juvenile (2–3 mo of age) and adult (5–6 mo of age) male pigs ( n = 8/group) for measurement of NO, and aortic endothelial cells were collected for measurement of mRNA and protein for endothelial NO synthase (eNOS). Although plasma NO was higher in juvenile (67.0 ± 25.6 μM) than in adult (15.0 ± 7.1 μM) male pigs, eNOS protein was similar in both groups. However, levels of mRNA for eNOS were lower in aortic endothelial cells from juvenile pigs. In rings of coronary arteries suspended in organ chambers for measurement of isometric force and contracted with PGF2α, relaxations to an α2-adrenergic agonist were significantly inhibited by indomethacin only in juvenile pigs [EC50 (–log M), 6.7 ± 0.3 with indomethacin and 7.7 ± 0.3 under control conditions]. NG-monomethyl-l-arginine (l-NMMA) inhibited relaxations in both groups. On the contrary, in the presence of indomethacin, relaxations to bradykinin were inhibited by l-NMMA only in arteries from adult pigs [EC50 (–log M), 8.9 ± 0.3 with indomethacin and 8.6 ± 0.3 with addition of l-NMMA]. These results suggest that hormonal changes associated with sexual maturity may affect posttranscriptional and/or translational regulation of eNOS protein and result in lower plasma NO in adult male pigs. In addition, endothelium-derived inhibitory cyclooxygenase products seem to predominate in juveniles.
ADP-induced pial arteriolar dilation in ovariectomized rats involves gap junctional communication
Authors: Xu H. L. , Santizo R. A. , Baughman V. L. , Pelligrino D. A.
Published: March 2015

It was previously shown that, despite the loss of nitric oxide (NO) dependence, ADP-induced pial arteriolar dilation was not attenuated in estrogen-depleted [i.e., ovariectomized (Ovx)] rats. Additional evidence suggested that the NO was replaced by an endothelium-dependent hyperpolarizing factor (EDHF)-like mechanism. To further characterize the nascent EDHF role in Ovx females, the current study was undertaken to test whether, in Ovx rats, ADP-induced pial arteriolar dilation retained its endothelial dependence and whether gap junctions are involved in that response. A closed cranial window and intravital microscopy system was used to monitor pial arteriolar diameter changes in anesthetized rats. The endothelial portion of the ADP-induced dilation was evaluated using light dye endothelial injury (L/D). The study was organized around three experimental approaches. First, the responses of pial arterioles to ADP before and after L/D exposure in intact and Ovx female rats were tested. L/D reduced the ADP response by 50–70% in both groups, thereby indicating that the endothelium dependence of ADP-induced vasodilation is not altered by chronic estrogen depletion. Second, the NO synthase inhibitor Nω-nitro-l-arginine (l-NNA) and the prostanoid synthesis inhibitor indomethacin (Indo) were coapplied. In intact females, l-NNA-Indo attenuated the response to ADP by 50%, with no further changes upon the addition of L/D. On the other hand, l-NNA-Indo did not affect ADP reactivity in Ovx rats, but subsequent L/D exposure reduced the ADP response by >50%. The NO-prostanoid-independent, but endothelium-dependent, nature of the response in Ovx females is a hallmark of EDHF participation. Third, gap junctional inhibition strategies were applied. A selective inhibitor of gap junctional function, Gap 27, did not affect ADP reactivity in intact females but reduced the the ADP response by 50% in Ovx females. A similar result was obtained following application of a connexin43 antisense oligonucleotide. These findings suggest that the nascent EDHF dependency of ADP-induced pial arteriolar dilation in Ovx females involves connexin43-related gap junctional communication.
Attenuation of activity-induced increases in cerebellar blood flow in mice lacking neuronal nitric oxide synthase
Authors: Yang Guang , Zhang Yi , Ross M. Elizabeth , Iadecola Costantino
Published: March 2015

We used mice deficient in neuronal nitric oxide (NO) synthase (nNOS) to specifically investigate the role of neuronal NO in the increase of cerebellar blood flow (BFcrb) produced by neural activation. Crus II, a region of the cerebellar cortex that receives trigeminal sensory afferents, was activated by low-intensity stimulation of the upper lip (5–25 V, 4–16 Hz) in anesthetized mice. BFcrbwas recorded in Crus II by using a laser-Doppler flow probe. In wild-type mice, upper lip stimulation increased BFcrbin the Crus II by 28 ± 3% (25 V, 10 Hz, n = 6). The rise in BFcrbwas attenuated by 73 ± 3% in nNOS-/-mice ( P < 0.05, n = 6). The increases in BFcrbproduced by superfusion of Crus II with glutamate or by systemic administration of harmaline were also attenuated in nNOS-/-mice ( P < 0.05). In contrast, the increases in BFcrbproduced by topical superfusion of Crus II with acetylcholine or adenosine and the increase in BFcrbproduced by hypercapnia were not affected ( P > 0.05). The field potentials evoked in the Crus II by upper lip stimulation did not differ between wild-type and nNOS-null mice. These data provide the first nonpharmacological evidence that nNOS-derived NO is a critical link between glutamatergic synaptic activity and blood flow in the activated cerebellum.
Sole activation of three luminal adenosine receptor subtypes in different parts of coronary vasculature
Authors: Rubio Rafael , Ceballos Guillermo
Published: March 2015

In isolated guinea pig hearts saline perfused at constant flow, adenosine A1, A2A, and A3(Ax) agonists covalently bound to a large polymer (Pol; ≫2,000 kDa) were intracoronarily administered, and three effects were studied: dromotropic, vascular and inotropic. The rank order of potencies were the following: dromotropic (Pol-A2A≪≪Pol-A1>Pol-A3) and vascular and inotropic (Pol-A2A≥Pol-A1≫Pol-A3), where the rank order of potency for Pol-Axdepends on the part of the coronary vascular network involved; i.e., there is a vascular heterogeneity. The large size of Pol-Axprevents extravascular diffusion and causes it to act solely in the endothelial luminal surface. This implies their cardiac effects are due to endothelial mediators. Inhibition of nitric oxide (NO) and prostaglandin (PG) synthesis with NG-nitro-l-arginine methyl ester and indomethacin, respectively, show that the three cardiac effects of Pol-A1were mediated by NO and PG, whereas for Pol-A2Aand Pol-A3the mediator was mainly NO but not PG. These results suggest that if Pol-Axactivated the corresponding endothelial Ax-receptor subtype, a different mediator would be produced.
Altered dose response to β-agonists in SERCA1a-expressing hearts ex vivo and in vivo
Authors: Huke Sabine , Prasad Vikram , Nieman Michelle L. , Nattamai Kalpana J. , Grupp Ingrid L. , Lorenz John N. , Periasamy Muthu
Published: March 2015

H965, 2002; 10.1152/ajpheart.00078.2002. —In this study we evaluated the contractile characteristics of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)1a-expressing hearts ex vivo and in vivo and in particular their response to β-adrenergic stimulation. Analysis of isolated, work-performing hearts revealed that transgenic (TG) hearts develop much higher maximal rates of contraction and relaxation than wild-type (WT) hearts. Addition of isoproterenol only moderately increased the maximal rate of relaxation (+20%) but did not increase contractility or decrease relaxation time in TG hearts. Perfusion with varied buffer Ca2+concentrations indicated an altered dose response to Ca2+. In vivo TG hearts displayed fairly higher maximal rates of contraction (+ 25%) but unchanged relaxation parameters and a blunted but significant response to dobutamine. Our study also shows that the phospholamban (PLB) level was decreased (−40%) and its phosphorylation status modified in TG hearts. This study clearly demonstrates that increases in SERCA protein level alter the β-adrenergic response and affect the phosphorylation of PLB. Interestingly, the overall cardiac function in the live animal is only slightly enhanced, suggesting that (neuro)hormonal regulations may play an important role in controlling in vivo heart function.
Impact of aging on myocardial metabolic response to dobutamine
Authors: Soto Pablo F. , Herrero Pilar , Kates Andrew M. , Dence Carmen S. , Ehsani Ali A. , Dávila-Román Victor , Schechtman Kenneth B. , Gropler Robert J.
Published: March 2015

In humans, under resting conditions there is an age-related decrease in myocardial fatty acid utilization (MFAU) and oxidation (MFAO) and a relative increase in myocardial glucose utilization (MGU). The impact of age on an individual's myocardial metabolic response to catecholamines is not well defined. Sixteen younger (mean age, 26 ± 5 yr) and 14 older (mean age, 69 ± 4 yr) volunteers underwent positron emission tomography to measure myocardial blood flow, myocardial oxygen consumption (MV̇o2), MFAU, MFAO, and MGU both under resting conditions and during dobutamine infusion. In response to dobutamine administration, the rate-pressure product, myocardial blood flow, and MV̇o2measurements increased by similar amounts in both groups. No age-related differences were noted in the responses of plasma insulin, glucose, fatty acid, or lactate levels to dobutamine. With dobutamine infusion, MFAU and MFAO increased by a similar extent in both younger and older volunteers (age/dobutamine interactions, P = 0.62 and 0.75, respectively). In contrast, MGU increased with dobutamine administration in the younger (from 149 ± 71 to 209 ± 78 nmol · g–1· min–1; P = 0.04) but not in the older (from 235 ± 147 to 176 ± 84 nmol · g–1· min–1; P = 0.23; age/dobutamine interaction, P = 0.03) group. With dobutamine infusion, hearts in both younger and older volunteers responded by increasing their MFAU and MFAO values. Whereas younger hearts also responded with an increase in MGU, older hearts did not. Although the clinical significance of these findings awaits further study, these results may partially explain the impaired contractile reserve and the increased incidence of cardiovascular disease in older individuals.
Effect of passive myocardium on the compliance of porcine coronary arteries
Authors: Hamza Leila H. , Dang Quang , Lu Xiao , Mian Ayesha , Molloi Sabee , Kassab Ghassan S.
Published: March 2015

The objective of this study was to determine the effect of passive myocardium on the coronary arteries under distension and compression. To simulate distension and compression, we placed a diastolic-arrested heart in a Lucite box, where both the intravascular pressure and external (box) pressure were varied independently and expressed as a pressure difference (ΔP = intravascular pressure – box pressure). The ΔP-cross-sectional area relationship of the first several generations of porcine coronary arteries and the ΔP-volume relationship of the coronary arterial tree (vessels >0.5 mm in diameter) were determined using a video densitometric technique in the range of +150 to –150 mmHg. The vasodilated left anterior descending (LAD) coronary artery of six KCl-arrested hearts were perfused with iodine and 3% Cab-O-Sil. The intravascular pressure was varied in a triangular pattern, whereas the absolute cross-sectional area of each vessel and the total arterial volume were calculated using video densitometry under different box pressures (0, 50, 100, and 150 mmHg). In the range of positive ΔP, we found that the compliance of the proximal LAD artery in situ (4.85 ± 3.8 × 10–3 mm2/mmHg) is smaller than that of the same artery in vitro (16.5 ± 6 × 10–3 mm2/mmHg; P = 0.009). Hence, the myocardium restricts the compliance of the epicardial artery under distension. In the negative ΔP range, the LAD artery does not collapse, whereas the same vessel readily collapses when tested in vitro. Hence, we conclude that myocardial tethering prevents collapse of large blood vessel under compression.